KR20150129033A - Low acidic species compositions and methods for producing and using the same - Google Patents

Abstract

The present invention relates to a low acidic (AR) composition comprising a protein, for example an antibody or antigen-binding portion thereof, and a method for producing such a low AR composition, for example a cell culture and / . Also provided is a method for using such compositions to treat disorders, e. G., Disorders where TNFa is deleterious.

Description

TECHNICAL FIELD [0001] The present invention relates to a low acidic species composition and a method for producing the same,

Related application

The present application claims priority from International Patent Application Serial No. PCT / US2013 / 031485, filed March 14, 2013, and International Patent Application Serial No. PCT / US2013 / 031681, filed March 14, Quot ;, each of which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

The preparation of a composition comprising a protein for biopharmaceutical application can be carried out using an upstream process technology (e.g., cell culture), which is known to produce a protein that exhibits varying levels of protein variants and impurities in the composition, And the use of downstream process technology (e. G., Protein purification). Such protein variants include, but are not limited to, the presence of acidic species, including process-related impurities. For example, in a monoclonal antibody (mAb) formulation, the acidic species may be purified by ion exchange chromatography, for example, WCX-10 HPLC (weak cation exchange chromatography) or IEF (isoelectric focusing) It can be detected by various methods like the above. Because of their similar chemical properties to the desired antibody product molecules, the reduction of acidic species is a challenge in the production of monoclonal antibodies.

Reduction of acidic species has the potential to affect a number of product characteristics including, but not limited to, product stability, product safety, and product efficacy, so reduction of acidic species can be achieved using commercially available recombinant biological therapeutics biotherapeutics). Thus, there remains a need in the art for low-acidic species compositions and protein compositions with low levels of acidic species, for example, high-efficiency methods of producing antibodies.

SUMMARY OF THE INVENTION

The present invention relates to the identification of upstream and downstream process technologies for the production of proteins, for example for the production of antibodies or antigen-binding portions thereof, leading to the production of compositions comprising proteins comprising a low percentage of acidic species, It is based on optimization. As evidenced herein, these low acid species compositions can be used to improve therapeutic efficacy and improve biological properties, for example, to increase cartilage tissue penetration and / Increase the protection against the onset of arthritis when measured by an arthritic score and / or a histopathology score, or to reduce / prevent cartilage destruction, reduce synovial proliferation and / or reduce bone erosion and / Reduce / inhibit cell infiltration, decrease proteoglycan loss and / or decrease cartilage cell death and / or increase TNF alpha affinity.

Thus, in one embodiment, the present invention provides a low acid species (low AR) composition comprising an antibody or antigen-binding portion thereof, wherein the composition comprises an antibody or antigen-binding portion thereof (Low AR) compositions comprising the same. In one aspect of this embodiment, the low AR composition comprises up to about 14% AR, up to 13% AR, up to 12% AR, up to 11% AR, up to 10% AR, up to 9% AR of less than 8%, AR of less than 7%, AR of less than 6%, AR of less than 5%, AR of less than 4.5%, AR of less than 4%, AR of less than 3.5%, AR of less than or equal to 3% AR of less than 2%, AR of less than 1.9%, AR of less than 1.8%, AR of less than 1.7%, AR of less than 1.6%, AR of less than 1.5%, AR of less than 1.4%, AR of less than 1.3% AR, AR of less than 1.2%, AR of less than 1.1%, AR of less than 1%, AR of less than 0.9%, AR of less than 0.8%, AR of less than 0.7%, AR of less than 0.6%, AR of less than 0.5% Up to 0.4% AR, up to 0.3% AR, up to 0.2% AR, up to 0.1% AR, or 0.0% AR, and ranges within one or more of the above ranges. In one aspect of this embodiment, the invention provides a low AR composition comprising an antibody or antigen-binding portion thereof, wherein the composition comprises from about 0.0% to about 10% AR, from about 0.0% to about 5% AR, from about 0.0% To about 4% AR, from about 0.0% to about 3% AR, from about 0.0% to about 2% AR, from about 3% to about 5% AR, from about 5% to about 8% AR, or from about 8% AR, or from about 10% to about 15% AR, and ranges within at least one of the above, or an antigen-binding portion thereof.

In one embodiment, the low AR composition comprises a first acidic species region AR1 and a second acidic species region AR2. In one aspect of this embodiment, the low AR composition comprises less than about 0.1% AR1 and less than about 3% AR2, or less than about 0.0% AR1 and less than about 1.4% AR2. In a related embodiment, the low AR composition comprises up to about 15% AR1, or up to 14% AR1, up to 13% AR1, up to 12% AR1, up to 11% AR1, up to 10% AR1, AR1 less than 8%, AR1 less than 7%, AR1 less than 6%, AR1 less than 5%, AR1 less than 4.5%, AR1 less than 4%, less than 3.5% AR1 less than 3% AR1 up to 2.5%, AR1 up to 2%, AR1 up to 1.9%, AR1 up to 1.8%, AR1 up to 1.7%, AR1 up to 1.6%, AR1 up to 1.5%, AR1 up to 1.4% AR1 less than 1.3%, AR1 less than 1.2%, AR1 less than 1.1%, AR1 less than 1%, AR1 less than 0.9%, AR1 less than 0.8%, AR1 less than 0.7%, AR1 less than 0.6% Or less of AR1, 0.4% or less of AR1, 0.3% or less of AR1, 0.2% or less of AR1, 0.1% or less of AR1, or 0.0% of AR1 and ranges within one or more of the above ranges. In one aspect of this embodiment, the invention provides a low AR composition comprising an antibody or antigen-binding portion thereof, wherein the composition comprises from about 0.0% to about 10% AR1, from about 0.0% to about 5% From about 0.0% to about 4% AR1, from about 0.0% to about 3% AR1, from about 0.0% to about 2% AR1, from about 3% to about 5% AR1, from about 5% to about 8% An AR1 comprising about 8% to about 10% AR1, or about 10% to about 15% AR1, and ranges within at least one of the above. do.

In one aspect of this embodiment, the low AR composition comprises up to about 15% AR2, up to 14% AR2, up to 13% AR2, up to 12% AR2, up to 11% AR2, up to 10% AR2 of up to 9%, AR2 of less than 8%, AR2 of less than 7%, AR2 of less than 6%, AR2 of less than 5%, AR2 of less than 4.5%, AR2 of less than 4%, AR2 of less than 3.5% AR2 of not more than 2.5%, AR2 of not more than 2%, AR2 of not more than 1.9%, AR2 of not more than 1.8%, AR2 of not more than 1.7%, AR2 of not more than 1.6%, AR2 of not more than 1.5% AR2 of not more than 1.3%, AR2 of not more than 1.2%, AR2 of not more than 1.1%, AR2 of not more than 1%, AR2 of not more than 0.9%, AR2 of not more than 0.8%, AR2 of not more than 0.7% Not more than 0.5% AR2, not more than 0.4% AR2, not more than 0.3% AR2, not more than 0.2% AR2, not more than 0.1% AR2, or 0.0% AR2. In one aspect of this embodiment, the invention provides a low AR composition comprising an antibody or antigen-binding portion thereof, wherein the composition comprises about 0.0% to about 10% AR2, about 0,0% to about 5% AR2 AR2, about 0.0% to about 3% AR2, about 0.0% to about 2% AR2, about 3% to about 5% AR2, about 5% to about 8% AR2 , Or from about 8% to about 10% AR2, or from about 10% to about 15% AR2, and ranges within at least one of the above, or an antigen-binding portion thereof .

In another embodiment, a low AR composition, for example, a low AR composition of adalimumab, contains up to about 1.4% AR. For example, in one aspect of this embodiment, the low AR composition, such as a low AR composition of, for example, less than about 1.4% AR, comprises less than about 0.01% AR1 and less than about 1.4% AR2 . ≪ / RTI >

In another aspect, the present invention provides a composition comprising an antibody or antigen-binding portion thereof, wherein the composition comprises, for example, a host cell protein (HCP), a host nucleic acid, a chromatographic material, and / Binding portion of an antibody or antigen-binding portion thereof that is substantially free of acidic species and other process-related impurities, including product-related impurities such as aggregates.

In one embodiment, the antibody or antigen-binding portion thereof of the compositions disclosed herein is an anti-TNFa antibody or antigen-binding portion thereof. For example, in one aspect of this embodiment, the anti-TNFa antibody or antigen-binding portion thereof has a K _d of about 1 x 10 ^-8 M or less and a K _off rate constant of 1 x 10 ^-3 S ^-1 or less Lt; / RTI > In another aspect of this embodiment, the anti-TNFa antibody or antigen-binding portion thereof comprises a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and an amino acid sequence of SEQ ID NO: A light chain variable region (LCVR) having a CDR3 domain comprising; And a heavy chain variable region (HCVR) having a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDR3 domain comprising the amino acid sequence of SEQ ID NO:

In another aspect of this embodiment, the anti-TNFa antibody or antigen-binding portion thereof comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2 . In another aspect of this embodiment, the anti-TNFa antibody or antigen-binding portion thereof is adalimumab or an antigen-binding portion thereof.

In one embodiment, the low AR composition of the present invention is ah otherwise contain a free MAB and, currently approved and described in the literature [ "Highlights of Prescribing Information" for HUMIRA ® (adalimumab) Injection (Revised Jan. 2008)] otherwise formulated ah as HUMIRA ^® has a percentage of the AR is not equal to the percent of AR existing in the non-MAB, the contents of the above documents are incorporated by reference herein.

In another embodiment, the low AR composition of the present invention is ah otherwise contain a free MAB and, currently approved and described in the literature [ "Highlights of Prescribing Information" for HUMIRA ® (adalimumab) Injection (Revised Jan. 2008)] otherwise formulated ah as HUMIRA ^® has a percentage of more than the percentage of the low AR AR existing in the non-MAB, the contents of the above documents are incorporated by reference herein.

In another embodiment, the invention provides a light chain variable region comprising a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR3 domain comprising the amino acid sequence of SEQ ID NO: LCVR); And a heavy chain variable region (HCVR) comprising a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDR3 domain comprising the amino acid sequence of SEQ ID NO: TNFa antibody or antigen-binding portion thereof, wherein the composition comprises less than about 10% AR. In one aspect of this embodiment, the anti-TNF [alpha] antibody or antigen-binding portion thereof comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2 , Wherein the composition comprises less than about 10% AR. In another aspect of this embodiment, the anti-TNF [alpha] antibody or antigen-binding portion thereof is adalimumab or an antigen-binding portion thereof, and the composition comprises less than about 10% AR. In one aspect of this embodiment, a low AR composition comprising an anti-TNF [alpha] antibody or antigen-binding portion thereof comprises less than about 0.1% AR1 and less than about 3% AR2, or less than about 0.0% AR1 and less than about 1.4% AR2.

In one embodiment, an acidic species in a low AR composition comprising an antibody or antigen-binding portion thereof (e.g., an anti-TNF alpha antibody or antigen binding portion thereof, for example, adalimumab) Structural variants, and fragment variants (see, for example, Figure 188).

For example, in one aspect of this embodiment, the charge mutant in the low AR composition is selected from the group consisting of AR1 species, for example, a deamidated variant, a glycation variant, an afucosylation variant, (MGO) variants or citric acid variants. For example, when the low AR composition comprises adalimumab, the deamidated variant may be a deacetylated residue derived from an asparagine residue comprising Asn393 and Asn329 of adalimumab and a glutamine residue comprising Gln3 and Gln6 Amidation. ≪ / RTI > In another aspect of this embodiment, where the low AR composition comprises adalimumab, the glycosylation variant may result from glycosylation occurring in Lys98 and Lys151 of adalimumab.

In another aspect of this embodiment, a structural variant in a low AR composition comprising an antibody or antigen-binding portion thereof (e.g., an anti-TNF [alpha] antibody or antigen binding portion thereof, For example, glycosylation variants or acetone variants.

In another aspect of this embodiment, a fragmented variant in a low AR composition comprising an antibody or antigen-binding portion thereof (e.g., an anti-TNF [alpha] antibody or antigen binding portion thereof, AR1 species, such as Fab fragment variants, C-terminal truncation variants, or variants in which the heavy chain variable domain has been lost.

In another embodiment, the acidic species in the low AR composition, including the antibody or antigen-binding portion thereof (e. G., An anti-TNFa antibody or antigen binding portion thereof, A charge mutant, for example, a deamidated mutant or a saccharified mutant. For example, when the low AR composition comprises adalimumab, the deamidated variant may be a deacetylated residue derived from an asparagine residue comprising Asn393 and Asn329 of adalimumab and a glutamine residue comprising Gln3 and Gln6 Amidation. ≪ / RTI > In another aspect of this embodiment, where the low AR composition comprises adalimumab, the glycosylation variant may result from glycosylation occurring in Lys98 and Lys151 of adalimumab.

In one embodiment, the percentage of acid species in the low AR composition is determined using ion exchange chromatography, for example, WCX-10 HPLC. In another aspect of this embodiment, the percentage of acid species in the low AR composition is measured using isoelectric point electrophoresis (IEF).

In one embodiment, the low AR composition of the present invention comprises product-inducing acidic species. For example, in one aspect of this embodiment, the acidic species is a cell culture-induced acidic species. In another aspect of this embodiment, the acidic species of the low AR composition is a storage-derived acidic species that is predominantly produced during storage under process conditions, intermediate conditions, or shelf storage conditions prior to use.

In another embodiment, the present invention provides a low AR composition further comprising a pharmaceutically acceptable carrier.

In another aspect, the invention provides a method of treating a subject having a deleterious disorder of TNFa by administering to a subject having a deleterious disorder of TNFa a low AR composition of the invention, e. A method for treating a subject having a deleterious disorder is provided. In one aspect of this embodiment, the disorder wherein TNFa is harmful is selected from the group consisting of rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, ankylosing spondylitis, pediatric idiopathic arthritis (JIA), ulcerative colitis, and Crohn's disease.

In one aspect, an upstream method for making the low AR composition of the present invention is included. In one embodiment, a method for producing a low acidic species composition comprising an antibody or antigen-binding portion thereof comprises contacting an amino acid concentration in a cell culture medium used to produce a non-acidic species composition comprising an antibody or antigen- Culturing a cell expressing an antibody or an antigen-binding portion thereof in a cell culture medium containing an amino acid selected from the group consisting of arginine, lysine, ornithine and histidine, or a combination thereof at an increased concentration, . In another aspect of this embodiment, the amino acid concentration in the culture medium is about 0.025 to 20 g / L.

In another embodiment, a method for producing a low acidic species composition comprising an antibody or antigen-binding portion thereof comprises contacting a cell with a calcium concentration in a cell culture medium used to produce a non- low acid species composition comprising an antibody or antigen- And culturing cells expressing the antibody or antigen-binding portion thereof in a cell culture medium containing an increased concentration of calcium, In one aspect of this embodiment, the calcium concentration is about 0.005 to 5 mM. In another aspect of this embodiment, the cell culture medium comprises an increased concentration of an arginine, an arginine, or a combination thereof, as compared to the amino acid concentration in the cell culture medium used to produce the non-acidic species composition comprising the antibody or antigen- Lysine, ornithine and histidine, or a combination thereof.

In yet another embodiment, a method for preparing a low acidic species composition comprising an antibody or antigen-binding portion thereof comprises contacting a niacin in a cell culture medium used to produce a non-acidic species composition comprising an antibody or antigen- Culturing the cells expressing the antibody or antigen-binding portion thereof in a cell culture medium containing increased concentrations of niacinamide, calcium, and one or more amino acids, as compared to the concentrations of amide, calcium, and amino acid. In one aspect of this embodiment, the at least one amino acid is selected from the group consisting of arginine, lysine, ornithine and histidine, and combinations thereof.

In another embodiment, a method for preparing a low acid species composition comprising an antibody or antigen-binding portion thereof comprises culturing cells expressing an antibody or antigen-binding portion thereof in a cell culture medium having a pH of about 7.1 to 6.8 .

In yet another embodiment, a method for preparing a low acid species composition comprising an antibody or antigen-binding portion thereof comprises the steps of: exchanging a cell culture medium used to produce a non-acidic species composition comprising an antibody or antigen- Culturing the cells expressing the antibody or antigen-binding portion thereof in a cell culture medium having a modified exchange rate as compared with the culture medium.

In another embodiment, a method for preparing a low acidic species composition comprising an antibody or antigen-binding portion thereof comprises culturing cells expressing an antibody or antigen-binding portion thereof, extracting the clarified collection from the cell culture And adding at least one amino acid to the purified collection. In one aspect of this embodiment, the one or more amino acids are selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid and leucine, and combinations thereof.

In another embodiment, a method for preparing a low acid species composition comprising an antibody or antigen-binding portion thereof comprises culturing cells expressing the antibody or antigen-binding portion thereof, extracting the purified collection from the cell culture, And adjusting the pH of the purified collection to 4.5 to 6.5.

In another aspect of the present invention, an upstream method for preparing a low AR composition of the present invention is included. In one embodiment, the present invention provides a method for the detection of an immune response comprising: contacting a first sample comprising an antibody or antigen-binding portion thereof with a chromatographic medium, wherein said contacting occurs in connection with a loading buffer; Washing the chromatographic medium with wash buffer substantially equal to the loading buffer; And collecting the chromatographic medium, wherein the chromatographic sample comprises a composition of an antibody or antigen-binding portion thereof containing less than about 10% of an acidic species, whereby the antibody or antigen- Acidic species composition comprising an antibody or antigen-binding portion thereof, wherein the acidic species composition is prepared. In one aspect of this embodiment, the bound antibody material is eluted with a buffer having a different composition than the wash buffer. In another aspect of this embodiment, the chromatographic medium is selected from the group consisting of an anion exchange adsorbent material, a cation exchange adsorbent material, a mixed mode medium, a cation exchange mixed mode medium, and an anion exchange mixed mode medium. In one embodiment, the chromatography medium is a mixed-mode medium comprising cation exchange (CEX) and hydrophobic interaction functional groups. In another embodiment, the chromatography medium is a mixed mode medium comprising an anion exchange (AEX) and a hydrophobic interaction functional group. For example, the mixed type medium may be Capto MMC resin, CEX resin may be Poros XS resin, and AEX resin may be Poros 50HQ resin.

In one embodiment, the chromatographic medium is a CEX adsorbing material or mixed media, and the pH of the loading and washing buffer is lower than the isoelectric point of the antibody. In another embodiment, the chromatographic sample contains a reduced level of antibody fragment as compared to the first sample. In another embodiment, the chromatographic sample contains a reduced level of host cell protein compared to the first sample. In another embodiment, the chromatographic sample comprises a reduced level of a charge mutant (e. G., A deamidated variant, a glycosyl variant, an afacosylated variant, an MGO variant or a citrate variant) (For example, glycosylation mutants or acetone mutants) or fragmentation mutants (for example, variants in which a Fab fragment mutant, C-terminal truncation mutant or heavy chain variable domain is lost).

In one embodiment, a method for preparing a low acid species composition comprising an antibody or antigen-binding portion thereof comprises contacting a first sample comprising an antibody or antigen-binding portion thereof with an affinity chromatography medium (e. G., A protein A resin) and eluting the sample from the affinity chromatography medium as a first eluted sample; Contacting the first eluted sample with an anion exchange (AEX) chromatography adsorbent material (e.g., a Poros 50HQ resin) in a loading buffer and eluting the sample from the AEX chromatographic adsorbent material as a second eluted sample step; And contacting the second eluted sample with a cation exchange (CEX) chromatography adsorbent material (e.g., Poros XS resin) in a loading buffer and eluting the sample from the CEX chromatographic adsorbent material as a third eluted sample Wherein the third eluted sample comprises a composition of an antibody or antigen-binding portion thereof containing less than 3% of an acidic species, whereby a low acid species composition comprising an antibody or antigen-binding portion thereof, . In one embodiment, the second eluted sample is contacted with CEX chromatography at one or more additional times. In one embodiment, the method further comprises performing virus filtration on the third eluted sample to obtain a filtered sample. In another embodiment, the method further comprises filtering the filtered sample using ultrafiltration / dialysis filtration (UF / DF).

In another aspect, the invention provides a method for producing a low acid species composition comprising an antibody or antigen-binding portion thereof, the method comprising contacting a sample comprising an antibody or antigen-binding portion thereof with an anion exchange ) Chromatographic adsorbent material, a cation exchange (CEX) chromatography adsorbent material, a mixed mode medium, a cation exchange mixed medium, and an anion exchange mixed medium, Eluting said sample from a CEX chromatographic adsorbent material, a mixed mode medium, a cation exchange mixed mode medium, or an anion exchange mixed mode medium, wherein said eluted sample contains less than about 3% Or an antigen-binding portion thereof, whereby the antibody or antigen-binding portion thereof It provides a method for producing a low-acid species composition comprising an antibody or antigen-binding thereof, that are acidic species composition prepared comprising the joint. In one aspect of this embodiment, the method further comprises contacting the eluted sample with a hydrophobic interaction chromatography (HIC) medium.

The present invention is further described by the following detailed description and claims.

1Shows the effect of total arginine concentration on the viable cell line 2, medium 1, on the viable cell density (n = 2).
2Shows the effect of total arginine concentration on the viability of viable cell line 2, medium 1 (n = 2).
3Shows the effect of total arginine concentration on adalimumab productivity cell line 2, Medium 1, on the harvest titer (n = 2).
4Shows the effect of total arginine concentration on Adalimumab-producing cell line 2, Medium 1, on the WCX-10 profile total acid area on day 10 (n = 2).
5Shows the effect of total arginine concentration on Adil Mammob Productive cell line 2, Medium 1, on the WCX-10 profile total acid area on day 12 (n = 2).
6(N = 2) shows the effect of total arginine concentration in medium 1 on medium 3, medium 1, on viable cell density.
7Shows the effect of total arginine concentration on Adil Mammab Productive Cell Line 3, Medium 1 on viability (n = 2).
8Shows the effect of total arginine concentration on Adalimumab productivity cell line 3, medium 1, on the harvesting activity (n = 2).
9(N = 2) the effect of total arginine concentration on Adil Mammob Productive Cell Line 3, Medium 1, on the WCX-10 profile total acid area.
10(N = 2) shows the effect of total arginine concentration on Adalimumab-producing cell line 1, medium 1, for the WCX-10 profile total acidic area.
11Shows the effect of arginine addition on adalimumab productivity cell line 1, medium 2 for the WCX-10 profile total acid area (n = 2).
12(N = 2) of the addition of arginine to medium 3 of Adalimumab-producing cell line 2, medium 3 for the total acid region of the WCX-10 profile.
13Shows the effect of total arginine concentration on the mAb1 productive cell line for the WCX-10 profile total acidic region (n = 1).
14Shows the effect of total arginine concentration on the mAb2 productive cell line for the WCX-10 profile total acidic region (n = 2).
15(N = 1) the effect of carboxypeptidase digestion of the product from the adalimumab productivity cell line 3, Medium 1 experiment on the WCX-10 profile total acidic region.
16Shows the effect of carboxypeptidase digestion of the product from the mAb2 productive cell line to the WCX-10 profile total acidic region (n = 2).
17Shows the effect of the total lysine concentration on medium 2, medium 1, on the viable cell count for viable cell density (n = 2).
18(N = 2) shows the effect of total lysine concentration on Adil Mammava Productive Cell Lines 2, Medium 1 on viability.
19Shows the effect of the total lysine concentration on Adil Mammob Productive cell line 2, medium 1, relative to collection potency (n = 2).
20Shows the effect of total lysine concentration (n = 2) on Adil Mammob Productive cell line 2, medium 1, for the WCX-10 profile total acid area.
21Shows the effect of total lysine concentration (n = 2) on medium 3, medium 1, of the unmodulated productive cell line 3 against viable cell density.
22Shows the effect of total lysine concentration on the viability of the agar-mamab productive cell line 3, medium 1 (n = 2).
23Shows the effect of the total lysine concentration on Adalimumab productivity cell line 3, Medium 1, on the harvesting potency (n = 2).
24Shows the effect of total lysine concentration (n = 2) on Adalimumab-producing cell line 3, Medium 1, for the WCX-10 profile total acid area.
25Shows the effect of total lysine concentration (n = 2) on Adalimumab-producing cell line 1, medium 1 for the WCX-10 profile total acidic area.
26Shows the effect of lysine addition on adalimumab-producing cell line 1, medium 2 for the WCX-10 profile total acid area (n = 2).
27Shows the effect of lysine addition on adalimumab-producing cell line 2, medium 3 for the WCX-10 profile total acidic domain (n = 2).
28Shows the effect of total lysine concentration on the mAb1 productive cell line for the WCX-10 profile total acidic region (n = 1).
29Shows the effect of the total lysine concentration on the mAb2 productive cell line to the total acidic region of the WCX-10 profile (n = 2).
30Shows the effect of carboxypeptidase digestion of the product from cell line 3, Medium 1, on the WCX-10 profile total acidic region (n = 1).
31Shows the effect of carboxypeptidase digestion of the product from the mAb2 productive cell line to the WCX-10 profile total acidic region (n = 2).
32(N = 2) shows the effect of total histidine concentration on medium 2, medium 1, on the viable cell line 2 relative to viable cell density.
33(N = 2) the effect of total histidine concentration on medium 2, medium 1, on viability.
34Shows the effect of total histidine concentration on Adalimumab productivity cell line 2, medium 1, relative to collection potency (n = 2).
35(N = 2) the effect of total histidine concentration on Adalimumab-producing cell line 2, Medium 1, on the WCX-10 profile total acid area.
36Shows the effect of total histidine concentration on medium 3, medium 1, on the viable cell line 3 relative to viable cell density (n = 2).
37Shows the effect of total histidine concentration on Adil Mammava Productive Cell Line 3, Medium 1, on viability (n = 2).
38Shows the effect of total histidine concentration on Adal Mammob Productive cell line 3, Medium 1, on the harvesting titers (n = 2).
39Shows the effect of total histidine concentration on Adalimumab-producing cell line 3, Medium 1, on the WCX-10 profile total acid area (n = 2).
40(N = 2) shows the effect of total histidine concentration on Adal Mammob Productive Cell Line 1, Medium 1 for the WCX-10 profile total acid area.
41(N = 2) shows the effect of histidine addition to Adalimumab-producing cell line 1, Medium 2, on the WCX-10 profile total acidic region.
42Shows the effect of histidine addition to Adalimumab-producing cell line 2, Medium 3 on the WCX-10 profile total acidic region (n = 2).
43Shows the effect of total histidine concentration on the mAb1 productive cell line for the WCX-10 profile total acidic region (n = 1).
44Shows the effect of total histidine concentration on the mAb2 productive cell line for the WCX-10 profile total acidic region (n = 2).
45Shows the effect of carboxypeptidase digestion of the product from cell line 3, Medium 1, on the WCX-10 profile total acidic region (n = 1).
46Shows the effect of carboxypeptidase digestion of the product from the mAb2 productive cell line to the WCX-10 profile total acidic region (n = 2).
47(N = 2) shows the effect of total ornithine concentration on the viable cell line 2, medium 1, on the viable cell density.
48(N = 2) shows the effect of total ornithine concentration on the viability of the adalimumab-producing cell line 2, Medium 1.
49Shows the effect of total ornithine concentration on Adil Mammob Productive Cell Lines 2, Medium 1 on collection potency (n = 2).
50Shows the effect of total ornithine concentration on Adalimumab-productive cell line 2, Medium 1, on the WCX-10 profile total acid area.
51(N = 2) shows the effect of total ornithine concentration on medium 1, medium 2, on viable cell line 3, Medium 1, on viable cell density.
52Shows the effect of total ornithine concentration on the viability of agar medium-free cell line 3, medium 1 (n = 2).
53Shows the effect of total ornithine concentration on adalimumab productivity cell line 3, medium 1, relative to collection potency (n = 2).
54Shows the effect of total ornithine concentration (n = 2) on medium 1, adalimumab-producing cell line 3, medium 1 for the WCX-10 profile total acid area.
55Shows the effect of total ornithine concentration (n = 2) on Adalimumab-producing cell line 1, medium 1 for the WCX-10 profile total acid area.
56Shows the effect of ornithine addition on adalimumab productivity cell line 1, medium 2 for the WCX-10 profile total acid area (n = 2).
57Shows the effect of ornithine addition on adalimumab productivity cell line 2, medium 3 for the WCX-10 profile total acid area (n = 2).
58Shows the effect of total ornithine concentration on the mAb1 productive cell line for the WCX-10 profile total acidic region (n = 1).
59Shows the effect of total ornithine concentration on the mAb2 productive cell line for the WCX-10 profile total acidic region (n = 2).
60Shows the effect of carboxypeptidase digestion of the product from the cell line 3, medium 1 experiment on the WCX-10 profile total acidic region (n = 1).
61Shows the effect of carboxypeptidase digestion of the product from the mAb2 productive cell line to the WCX-10 profile total acidic region (n = 2).
62(N = 2) of the addition of multiple amino acids to Adalimumab-producing cell line 2, Medium 1, for the WCX-10 profile total acidic region.
62(N = 2) of the addition of multiple amino acids to Adalimumab-producing cell line 2, Medium 1, for the WCX-10 profile total acidic region.
63(N = 3) the effect of increased arginine and lysine concentration on medium 1, medium 1, of the unmodulated productive cell line 1 against viable cell density.
Fig. 64(N = 3) the effect of increased arginine and lysine concentrations on medium 1, medium 1, and the viability of the agar-mamab productive cell line.
65Shows the effect of increased arginine and lysine concentrations on medium 1 in adalimumab-producing cell line 1 relative to incubation titer (n = 3).
66(N = 2) the effect of increased arginine and lysine concentrations on Adil Mammob Productive Cell Line 1, Medium 1 for the WCX-10 profile total acid area.
67Shows the effect of arginine, lysine and pH adjustments on medium 1, medium 1, medium to multiply to viable cell density (n = 2).
68Shows the effect of arginine, lysine and pH adjustment on Adil Mammob Productive cell line 1, medium 1 for viability (n = 2).
69Shows the effect of arginine, lysine and pH adjustment on Adil Mammava Productive Cell Line 1, Medium 1 on culture titer (n = 2).
70Shows the effect of arginine, lysine and pH adjustment on Adil Mammob Productive cell line 1, medium 1 for the WCX-10 profile total acid area (n = 2).
71(N = 2) shows the effect of total calcium concentration on medium 2, medium 1, on the viable cell line 2 relative to viable cell density.
72Shows the effect of total calcium concentration on the viability of the agarmamov productivity cell line 2, medium 1 (n = 2).
73Shows the effect of total calcium concentration on Adalimumab-producing cell line 2, Medium 1, on the harvesting potency (n = 2).
74(N = 2) shows the effect of total calcium concentration on Adalimumab-producing cell line 2, Medium 1, on the WCX-10 profile total acid area.
75Shows the effect of the total calcium concentration on the viable productive cell line 3, Medium 1, on the viable cell density (n = 2).
76Shows the effect of total calcium concentration on Adil Mammob Productive cell line 3, Medium 1, on viability (n = 2).
77Shows the effect of total calcium concentration on Adalimumab productivity cell line 3, Medium 1, on the harvesting activity (n = 2).
78(N = 2) shows the effect of total calcium concentration on Adalimumab-producing cell line 3, Medium 1, on the WCX-10 profile total acid area.
79(N = 2) shows the effect of total calcium concentration on Adalimumab-producing cell line 1, medium 1 for the WCX-10 profile total acid area.
80Shows the effect of calcium addition on adalimumab productivity cell line 1, medium 2 for the WCX-10 profile total acid area (n = 2).
81Shows the effect of calcium addition on adalimumab-producing cell line 2, medium 3 for the WCX-10 profile total acid area (n = 2).
82Shows the effect of total calcium concentration on the mAb1 productive cell line for the WCX-10 profile total acidic region (n = 2).
83Shows the effect of total calcium concentration on the mAb2 productive cell line for the WCX-10 profile total acidic region (n = 2).
84A to 84B(A) the total anticipated plots, b) the anticipated plots for each additive (n = 2), the effect of multiple amino acid additions on cell line 1, medium 1 for the WCX-10 profile total acidic area.
85(N = 2) of the addition of niacinamide to medium 1, medium 1, of the unmodulated productive cell line 1 against viable cell density.
86(N = 2) of the addition of niacinamide to medium 1, Medium 1 of Adalmumab productivity cell line for viability.
87Shows the effect of niacinamide addition on adalimumab productivity cell line 1, medium 1, relative to collection potency (n = 2).
88(N = 2) of the addition of niacinamide to Adalimumab-producing cell line 1, medium 1, on the WCX-10 profile total acidic area at day 11.
89(N = 2) of the addition of niacinamide to Adalimumab-producing cell line 1, medium 1 for the total acid area of the WCX-10 profile at day 12.
90Shows the effect of niacinamide addition on the mAb2 productive cell line, Media 1, on live cell density (n = 2).
91Shows the effect of addition of niacinamide to medium 1, an mAb2 productive cell line to survival (n = 2).
92Shows the effect of addition of niacinamide to the mAb2 productive cell line, medium 1, relative to the harvesting activity (n = 2).
93Shows the effect of adding niacinamide to the mAb2 productive cell line, Medium 1 for the WCX-10 profile total acidic region (n = 2).
94A to 94D(1) to CD medium GIA-1D in adalimumab-producing CHO cell line # 1 against (a) culture growth, (b) culture viability, (c) acidic species, and ≪ / RTI >
95Shows the effect of the pH adjustment of the agar medium-productivity cell line 1, Medium 1 on the viable cell density.
96Shows the effect of pH adjustment on the viability of Adalimumab productivity cell line 1, medium 1.
97Shows the effect of pH adjustment of Adalimumab-producing cell line 1, medium 1 on the collection potency.
98Shows the effect of pH adjustment of Adalimumab-producing cell line 1, medium 1 on the WCX-10 profile total acidic area.
99Shows the effect of pH adjustment on the viable cell line 1, medium 2 for the viable cell density.
100Shows the effect of the pH regulating portion of medium 2 on the viability of the agar medium-productivity cell line 1, Medium 2.
101Shows the effect of pH adjustment of Adalimumab productivity cell line 1, Medium 2 on collection potency.
102Shows the effect of pH adjustment of Adalimumab-producing cell line 1, medium 2 on the WCX-10 profile total acidic area.
103Shows the effect of pH adjustment of medium 1 on medium cell viability with medium 2 on viable cell density.
104Shows the effect of the pH adjustment of Adil Mammobi Productive Cell Lines 3, Medium 1 on viability.
105Shows the effect of pH adjustment of Adalimumab-producing cell line 3, Medium 1 on the collection potency.
106Shows the effect of pH adjustment of Adalimumab-producing cell line 3, medium 1 on the WCX-10 profile total acidic area.
107Shows the effect of dissolved oxygen adjustment of adalimumab-producing cell line 1, Medium 2 at 35 占 폚 against live cell density.
108Shows the effect of dissolved oxygen adjustment of Adalimumab-producing cell line 1, Medium 2 at 35 ° C for viability.
109Shows the effect of dissolved oxygen adjustment of Adalimumab-producing cell line 1, Medium 2 at 35 ° C for viability.
110Shows the effect of dissolved oxygen adjustment of adalimumab-producing cell line 1, Medium 2 at 35 占 폚 for the total acid region of the WCX-10 profile.
111Shows the effect of dissolved oxygen adjustment of adalimumab productivity cell line 1, Medium 2 at 33 캜 against live cell density.
112Shows the effect of dissolved oxygen adjustment of adalimumab-producing cell line 1, Medium 2 at 33 ° C for viability.
Figure 113Shows the effect of dissolved oxygen adjustment of adalimumab-producing cell line 1, medium 2 at 33 占 폚 against the collection potency.
114Shows the effect of dissolved oxygen adjustment of adalimumab-producing cell line 1, Medium 2 at 33 ° C for the total acidic region of the WCX-10 profile.
115Shows the effect of dissolved oxygen adjustment of adalimumab-producing cell line 1, medium 1 at 35 占 폚 against the viable cell density.
116Shows the effect of dissolved oxygen adjustment of Adalimumab-producing cell line 1, Medium 1 at 35 ° C for viability.
117Shows the effect of dissolved oxygen adjustment of Adalimumab-producing cell line 1, medium 1 at 35 占 폚 against the collection potency.
118Shows the effect of dissolved oxygen adjustment of the adalimumab-producing cell line 1, medium 1 on the WCX-10 profile total acidic area.
119Shows the effect of dissolved oxygen adjustment on the viable cell line 3, medium 1, on the viable cell density.
120Shows the effect of dissolved oxygen control on the viability of Adalimumab-producing cell line 3, Medium 1.
121Shows the effect of dissolved oxygen adjustment of the adalimumab-producing cell line 3, medium 1 on the harvesting potency.
122Shows the effect of dissolved oxygen adjustment of the adalimumab-producing cell line 3, medium 1 on the WCX-10 profile total acid area.
123Shows the effect of dissolved oxygen adjustment of medium 1, an mAb2-producing cell line on viable cell density.
Figure 124Shows the effect of the dissolved oxygen regulator of medium 1 on the mAb2 productive cell line to viability.
125Shows the effect of the dissolved oxygen adjustment of the mAb2 productive cell line, Medium 1, on the harvesting potency.
126Shows the effect of dissolved oxygen adjustment of mAb2-producing cell line, medium 1 on the WCX-10 profile total acidic area.
Figure 127Shows an acidification sample preparation scheme.
128Lt; / RTI > shows the arginine sample preparation scheme.
129Lt; / RTI > shows the histidine sample preparation scheme.
130Shows the lysine sample preparation scheme.
131Lt; / RTI > shows the methionine sample preparation scheme.
132Lt; / RTI > shows the amino acid sample preparation scheme.
133Shows a CDM purified collection sample preparation scheme.
134Shows the acid forming pH study sample preparation scheme.
135Illustrate the effect of low pH treatment on subsequent neutralization to initial acidic variant content.
136Illustrate the effect of low pH treatment on subsequent neutralization to the rate of acidic variant formation.
Figure 137Shows the effect of the sample preparation method on the initial acidic variant content.
138Shows the effect of the sample preparation method on the initial acidic variant content.
Figure 139Shows the dose-dependent effect of arginine on the reduction of the rate of acidic variant formation.
140Shows the effect of histidine concentration on the initial acidic variant content.
Figure 141Shows the effect of histidine concentration on the rate of acidic variant formation.
142Shows the effect of lysine on the initial acidic variant content.
143Shows the effect of lysine on the rate of acidic variant formation.
144Shows the effect of methionine on the initial acidic variant content.
145Shows the effect of methionine on the rate of acidic variant formation.
146Shows the effect of amino acids on the initial acidic variant content.
147Shows the effect of amino acids on the rate of acidic variant formation.
148Illustrate the effect of an alternative additive on the initial acidic variant content.
Fig. 149Illustrate the effect of an alternative additive on the rate of acidic variant formation.
150Shows the effect of low pH / arginine treatment on the initial acidic variant content of adalimumab CDM.
151Illustrate the effect of low pH / arginine treatment on the rate of formation of acidic variants of adalimumab CDM.
152Shows the effect of low pH / arginine treatment on the initial acidic mutant content of mAb B hydrolyzate.
153Illustrate the effect of low pH / arginine treatment on the rate of mAb B hydrolyzate acidic variant formation.
154Shows the effect of low pH / arginine treatment on mAb C hydrolyzate initial acidic variant content.
155Shows the effect of low pH / arginine treatment on mAb C hydrolyzate acid mutant formation rate.
156Shows the effect of acid type / pH on acidic variant content.
Figure 157Shows the effect of acid concentration on the acidic variant content.
158Shows the effect of acid concentration on the acidic variant content.
159Shows the effect of neutralization on the acidic variant content.
160Shows the effect of neutralization on the acidic variant content.
Figure 161Show the rate of medium exchange for total acidic species reduction and the effect of amino acid supplementation of arginine and lysine.
Figure 162Of an exemplary antibody expressed in association with the cell culture conditions of the present invention, comprising a formulation of specific mass traces for both modified and unmodified peptides to accurately quantify the total amount of MGO variants, MS < / RTI > peptide mapping analysis. Mass spectra are also analyzed for specific regions of the chromatogram to identify peptide identity.
163Shows a chromatogram in which the total acid species associated with the expression of adalimumab are divided into a first acidic species region AR1 and a second acidic species region AR2.
Figure 164Show AR growth at 25 [deg.] C of low and high AR containing samples.
Figure 165Shows the process chromatogram of pH gradient elution with respect to AEX chromatography.
166Shows the process chromatogram of linear concentration gradient elution by increasing the anion concentration with respect to AEX chromatography.
167Shows the process chromatogram of a fractionation of 300 g / L load and wash with respect to AEX chromatography.
Figure 168Shows the effect of pH on AR reduction with respect to AEX chromatography.
169Show process chromatograms at different salt (cation) concentrations with respect to CEX chromatography.
170Shows the recovery to AR reduction with respect to the CEX purification of adalimumab.
171Shows the WCX-10 profile of glycated load material and CEX eluate.
172Shows the WCX-10 profile of the MGO modified load material and eluate from the CEX column using Toyo Pearl MX TRP 650M resin.
173Illustrate the change in lysine distribution during CEX chromatography, highlighting the effect of Tris concentration.
Figure 174Illustrate the effect of pH and conductivity on the dalmalum AR reduction and recovery yields with respect to MM chromatography.
Figure 175≪ / RTI > shows the AR reduction achieved with the corresponding protein recovery with respect to MM chromatography.
176Flow Through, and Total Agilabum Protein Concentration Levels and AR Levels During Wash.
177Shows total mAb B protein concentration levels and AR levels during perfusion and washing in conjunction with MM chromatography.
178Shows total mAb C protein concentration levels and AR levels during perfusion and washing in conjunction with MM chromatography.
179Shows cumulative AR breakthrough (%) of mAb C for different MM resins.
180Illustrate the effect of pH-pI and conductivity on the hydra-amide AR reduction with respect to MM chromatography.
181Illustrate the effect of pH-pI and conductivity on mAb B AR reduction with respect to MM chromatography.
182Illustrate the effect and trend of pH-pI on mAb C AR reduction with respect to MM chromatography.
183Illustrate the effect of pH and conductivity on AR reduction and yield with respect to MM chromatography.
184Shows the AR reduction and protein recovery versus pH for MM chromatography.
185Illustrate the effect of pH, conductivity and protein loading on AR reduction and yield.
186Show the effect of pH, conductivity and protein loading on AR reduction and yield.
187Shows the effect of AEX adsorption pKa on mAb B to several different AEX adsorbents for different pKa values, run with acetate / Tris buffer at pH 9.1.
188Is a schematic representation of exemplary AR1 and AR2 present in a composition comprising an exemplary antibody. Manufacture-derived ARs and storage-derived ARs are shown.
189Shows cumulative AR reduction as a function of yield for various formic acid concentrations.
190Illustrate exemplary flow paths for the preparation of low AR compositions.191Shows an experimental scheme for a "continuous chromatography" process to produce low AR compositions.
Fig. 192Shows the AR percentage in each cycle of the continuous MM process.
193Illustrate chromatograms in which acidic and basic species in adalimumab are identified and various fractions are described.
194a To 194b(A) mean arthritic score and (b) growth-related weight gain of the mice to which the low AR composition, the AR1 composition, the Lys-1/2 composition, and the control AR composition were administered.
195Shows the mean arthritic score (area under the curve) of the mice to which the low AR composition, the AR1 composition, the Lys-1/2 composition, and the control AR composition were administered.
196a To 196b(A) mean trough serum drug levels and (b) mean trophe serum ADA levels for the mice to which the low AR composition, the AR1 composition, the Lys-1/2 composition, and the control AR composition were administered.
Figure 197Show the average PK and ADA profiles (area under the curve) for the mice to which the low AR composition, the AR1 composition, the Lys-1/2 composition, and the control AR composition were administered.
Figure 198Shows the compound TNF level (area under the curve), and the cumulative serum of Adalimumab for mice administered with low AR composition, AR1 composition, Lys-1/2 composition, and control AR composition for a treatment period of 10 weeks Concentration values were highest for the low AR and control AR compositions and lowest for the AR1 fraction.
199Shows chondrocyte apoptosis, synovial proliferation, proteoglycan loss, cartilage destruction, and bone erosion in mice administered with low AR composition, AR1 composition, Lys-1/2 composition, and control AR composition.
200a To 200d(A) a low AR composition; (b) a control AR composition; (c) AR1 composition; And (d) the mean drug levels of various tissues (feet, lymph nodes, spleen, skin, knee and serum) for the mice to which the Lys-1/2 composition is administered.
201a To 201d(A) a low AR composition; (b) a control AR composition; (c) AR1 composition; And (d) the mean ADA level of various tissues (foot, lymph node, spleen, skin, knee and serum) for the mice to which the Lys-1/2 composition was administered.
202a To 202dShows the results of micro CT analysis of vertebrae and femurs obtained from TNF-Tg197 gene transfer mice administered with placebo, low AR composition, control (normal) AR composition, AR1 composition, and Lys-1/2 composition. The graphs show: (a) spinal bulb volume; (b) trabecular number of vertebrae; (c) spinal bone soak thickness; And (d) the effect of the administered composition on the spinal canal space.
203a To 203dShows the results of micro CT analysis of vertebrae and femurs obtained from TNF-Tg197 gene transfer mice administered with placebo, low AR composition, control (normal) AR composition, AR1 composition, and Lys-1/2 composition. The graphs show: (a) vertebral body loss; (b) number of vertebrae; (c) spinal bone soak thickness; And (d) the effect of the administered composition on the spinal canal space.
204a To 204dShows the results of micro CT analysis of vertebrae and femurs obtained from TNF-Tg197 gene transfer mice administered with placebo, low AR composition, control (normal) AR composition, AR1 composition, and Lys-1/2 composition. The graphs are: (a) total volume at vertebral body volume / femoral metaphysis; (b) vertebral bone number when the thigh bone is simple; (c) vertebral bone thickness at the thigh bone; And (d) the effect of the administered composition on vertebral bone separation in the femoral osteotomy.
205(Group 7), AR1 composition (containing only AR1 acidic mutants) (group 8), and the following compositions: naive, vehicle (control), low AR composition (group 5), low host cell protein And micro-CT images of the vertebra from each of the six groups of mice to which the Lys-1/2 composition (containing only Lys 1 and Lys 2 variants) (Group 9) was administered.
206(Group 7), AR1 composition (containing only AR1 acidic mutants) (group 8), and Lys- CT images of the femur from each of the six groups of mice to which the 1/2 composition (containing only Lys 1 and Lys 2 variants) (Group 9) was administered.

The present invention relates to a method for producing a protein, e.g., a protein, which results in the production of a protein composition comprising a low percentage of acidic species (AR) and / or low levels of process-related impurities (e.g., host cell proteins and media components) For example, it is based on the identification and optimization of upstream and downstream process technologies for the production of antibodies or antigen-binding portions thereof.

As demonstrated herein, the compositions of the present invention exhibit increased therapeutic efficacy upon administration to a subject. For example, a composition comprising an anti-TNFa antibody or antigen-binding portion thereof, including a low AR, may be administered to a subject in need of such treatment, wherein the TNFa inhibits a deleterious disorder such as rheumatoid arthritis (RA), childhood idiopathic arthritis (JIA) Can increase the therapeutic efficacy in the treatment and prevention of arthritis, ankylosing spondylitis, Crohn's disease, and ulcerative colitis. Accordingly, the present invention provides compositions comprising a protein comprising low AR and / or low level process-related impurities, and methods of making and using the same.

In one embodiment, the low AR composition of the present invention comprises up to about 15% AR, up to 14% AR, up to 13% AR, up to 12% AR, up to 11% AR, up to 10% % AR, AR less than 8%, AR less than 7%, AR less than 6%, AR less than 5%, AR less than 4.5%, AR less than 4%, AR less than 3.5%, AR less than 3% Of AR, less than 2.5% AR, less than 2% AR, less than 1.9% AR, less than 1.8% AR, less than 1.7% AR, less than 1.6% AR, less than 1.5% AR, less than 1.4% AR AR of less than 1.3%, AR of less than 1.2%, AR of less than 1.1%, AR of less than 1%, AR of less than 0.9%, AR of less than 0.8%, AR of less than 0.7%, AR of less than 0.6% Or less AR, 0.4% or less AR, 0.3% or less AR, 0.2% or less AR, 0.1% or less AR, or 0.0% In one aspect of this embodiment, a low AR composition of the invention comprises from about 0.0% to about 10% AR, from about 0.0% to about 5% AR, from about 0.0% to about 4% AR, From about 3% to about 5% AR, from about 5% to about 8% AR, or from about 8% to about 10% AR, or from about 10% To about 15% AR, and ranges within one or more of the above. In one embodiment, the composition of the present invention is not a composition comprising 2.4% or 2.5% AR, for example , an arabidumab composition.

In another embodiment, the low AR composition comprises a first acidic species (AR1) and a second acidic species (AR2). In one aspect of this embodiment, the low AR composition comprises up to about 0.1% AR1 and up to about 3% AR2. In one aspect of this embodiment, the low AR composition comprises about 0.0% AR1 and up to about 1.4% AR2.

In another aspect of this embodiment, the low AR composition comprises up to about 15% AR1, up to 14% AR1, up to 13% AR1, up to 12% AR1, up to 11% AR1, up to 10% AR1 less than 8%, AR1 less than 7%, AR1 less than 6%, AR1 less than 5%, AR1 less than 4.5%, AR1 less than 4%, AR1 less than 3.5%, less than 3% AR1 of less than 2.5%, AR1 of not more than 2%, AR1 of not more than 1.9%, AR1 of not more than 1.8%, AR1 of not more than 1.7%, AR1 of not more than 1.6%, AR1 of not more than 1.5%, AR1 of not more than 1.4% , AR1 of not more than 1.3%, AR1 of not more than 1.2%, AR1 of not more than 1.1%, AR1 of not more than 1%, AR1 of not more than 0.9%, AR1 of not more than 0.8%, AR1 of not more than 0.7%, AR1 of not more than 0.6% Or less of AR1, 0.4% or less of AR1, 0.3% or less of AR1, 0.2% or less of AR1, 0.1% or less of AR1, or 0.0% of AR1 and ranges within one or more of the above ranges. In one embodiment, the composition of the present invention is not a composition comprising 0.2% AR1, for example , an arabidumab composition.

In another aspect of this embodiment, the low AR composition comprises up to about 15% AR2, up to 14% AR2, up to 13% AR2, up to 12% AR2, up to 11% AR2, up to 10% AR2 of up to 9%, AR2 of less than 8%, AR2 of less than 7%, AR2 of less than 6%, AR2 of less than 5%, AR2 of less than 4.5%, AR2 of less than 4%, AR2 of less than 3.5% AR2 of not more than 2.5%, AR2 of not more than 2%, AR2 of not more than 1.9%, AR2 of not more than 1.8%, AR2 of not more than 1.7%, AR2 of not more than 1.6%, AR2 of not more than 1.5% AR2 of not more than 1.3%, AR2 of not more than 1.2%, AR2 of not more than 1.1%, AR2 of not more than 1%, AR2 of not more than 0.9%, AR2 of not more than 0.8%, AR2 of not more than 0.7% Not more than 0.5% AR2, not more than 0.4% AR2, not more than 0.3% AR2, not more than 0.2% AR2, not more than 0.1% AR2, or 0.0% AR2. In one aspect of this embodiment, a low AR composition of the invention comprises from about 0.0% to about 10% AR2, from about 0.0% to about 5% AR2, from about 0.0% to about 4% AR2, About 3% to about 5% AR2, about 5% to about 8% AR2, or about 8% to about 10% AR2, or about 10% AR2, To about 15% AR2, and ranges within one or more of the above. In one embodiment, the composition of the present invention is not a composition comprising 2.2% AR2, for example , an arabidumab composition.

In another embodiment, a low AR composition, for example , a low AR composition of adalimumab, contains up to about 1.4% AR. For example, in one aspect of this embodiment, a low AR composition, such as a low AR composition of, for example , less than 1.4% AR, comprises less than about 0.0% AR1 and less than about 1.4% AR2 .

In one embodiment, the protein is an antibody or antigen-binding portion thereof, for example, adalimumab or an antigen-binding portion thereof.

I. Justice

In order that the present invention may be more readily understood, certain terms are first defined.

As used herein, the terms "acidic species "," acidic region ", and "AR" refer to variants of a protein, e.g., an antibody or antigen- binding portion thereof, characterized by a full acidic charge. For example, for monoclonal antibody (mAb) preparations, such acidic species may be purified by various methods, such as ion exchange, for example, WCX-19 HPLC (weak cation exchange chromatography) or IEF ). ≪ / RTI > As shown in Figure 188, the acidic species of the antibody may include a charge mutant, a structural mutant, and / or a fragment variant. Exemplary charge mutants include, but are not limited to, deamidated variants, afacosylated variants, methylglyoxal (MGO) variants, glycosylated variants, and citric acid variants. Exemplary structural variants include, but are not limited to, glycosylation variants and acetone variants. Exemplary fragmented variants include Fc and Fab fragments, Fab fragments, Fab fragment fragments, heavy chain variable domain deleted fragments, C-terminal truncation variants, variants with excision of the N-terminal Asp in the light chain, But not limited to, any cleaved protein species from the target molecule due to dissociation, enzymatic and / or chemical modification of the peptide chain. Other acidic species variants include non-paired disulfides, host cell proteins, and variants containing host nucleic acids, chromatographic materials, and media components.

In certain embodiments, the protein composition may comprise more than one type of acidic variant. For example and not by way of limitation, the total acid species can be divided based on the chromatographic retention time of the peaks, for example, as seen on the WCX-10 weak cation exchange HPLC of the protein preparations. Figure 163 shows a non-limiting example of this segmentation, in which the total acidic species associated with the expression of adalimumab is divided into a first acidic species region AR1 and a second acidic species region AR2.

As depicted schematically in Figure 188, AR1 can be, for example, a mutant, such as a deamidated variant, an MGO modified species, a glycosyl variant, and a citric acid variant, a structural variant such as a glycosylated Variants and acetone variants, and / or fragmented variants. In another embodiment, AR2 may include, for example, a charge mutant, such as a glycosylated and a deamidated variant.

In particular, with respect to adalimumab (and antibodies that share certain structural characteristics of adalimumab, for example, one or more CDRs of adalimumab and / or heavy and light chain variable regions), the AR1 charge variant But are not limited to, deamidated variants, glycosylated variants, afacosylated variants, MGO ( e.g., MGO variants in the residues shown in Table 5 below), or citric acid variants. In one embodiment, the deamidated mutants result from the deamidation occurring at the asparagine residue comprising Asn393 and Asn329 and at the glutamine residue comprising Gln3 and Gln6. In another embodiment, the glycosylation variants result from glycation occurring in Lys98 and Lys151. AR1 structural variants can include, but are not limited to, glycosylated variants or acetone variants.

AR1 fragmentation variants can be obtained by introducing Fc and Fab fragments, Fab-lost fragments, fragments with loss of heavy chain variable domains, C-terminal truncation mutants, variants with ablation of N-terminal Asp in light chains, And < / RTI >

The AR2 charge variant can include, but is not limited to, a deamidated variant or a glycosyl variant, wherein the deamidated variant has an asparagine residue comprising Asn393 and Asn329 and a glutamine residue comprising Gln3 and Gln6 And the glycosylation variant can be attributed to glycation caused by Lys98 and Lys151.

The term "acid species" does not include process-related impurities. As used herein, the term "process-related impurities " refers to impurities that are present in a composition comprising a protein but are not derived from the protein itself. Process-related impurities include, but are not limited to, host cell proteins (HCPs), host cell nucleic acids, chromatographic materials, and media components. As used herein, "low process-related impurity composition" refers to a composition comprising a reduced level of process-related impurities as compared to a composition in which impurities have not been reduced. For example, the low process-related impurity composition may be applied to a process-related impurity composition at a concentration of about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% Related impurities. In one embodiment, the low process-related impurity composition does not comprise process-related impurities or is substantially free of process-related impurities.

The acidic species may be the result of the production of the product (also referred to herein as a "manufacture-derived acidic species") or the result of storage (also referred to as "storage-derived acidic species"). Production-derived acidic species are acidic species that are formed during the manufacture (upstream and / or downstream processing) of proteins, e. G. , Antibodies or antigen-binding portions thereof. For example, production-induced acidic species can be formed during cell culture ("cell culture-induced acidic species"). Storage-derived acidic species are acidic species that are formed or produced during storage of the sample, although they may or may not be present in a population of proteins directly after manufacture. The type and amount of storage-derived acidic species may vary based on the formulation of the sample. The formation of a storage-induced acidic species can be partially or completely inhibited if the agent is stored under certain conditions. For example, aqueous formulations may be stored at certain temperatures to partially or completely inhibit AR formation. For example, the formed or storage-derived AR can be partially inhibited in aqueous formulations stored at about 2 ° C to 8 ° C and completely inhibited when stored at -80 ° C. In addition, the low AR composition may be lyophilized or freeze-dried to partially or completely inhibit the formation of the storage-induced AR.

As used herein, the term "low acidic species composition" or "low AR composition" refers to a composition comprising an antibody or antigen-binding portion thereof, wherein said composition contains less than about 15% acidic species. As used herein, the AR percentage in a low AR composition refers to the weight of an acidic species in a sample compared to the total weight of the antibody contained in the sample. For example, the AR percentage can be calculated using, for example, a weak cation exchange chromatography such as WCX-10 described in Example 1 below.

In one embodiment, the low AR composition of the present invention comprises up to about 15% AR, up to 14% AR, up to 13% AR, up to 12% AR, up to 11% AR, up to 10% % AR, AR less than 8%, AR less than 7%, AR less than 6%, AR less than 5%, AR less than 4.5%, AR less than 4%, AR less than 3.5%, AR less than 3% Of AR, less than 2.5% AR, less than 2% AR, less than 1.9% AR, less than 1.8% AR, less than 1.7% AR, less than 1.6% AR, less than 1.5% AR, less than 1.4% AR AR of less than 1.3%, AR of less than 1.2%, AR of less than 1.1%, AR of less than 1%, AR of less than 0.9%, AR of less than 0.8%, AR of less than 0.7%, AR of less than 0.6% Or less of AR, 0.4% or less of AR, 0.3% or less of AR, 0.2% or less of AR, 0.1% or less of AR, or 0.0% of AR and ranges within one or more of the above ranges. In addition, the low AR composition of the present invention may contain from about 0.0% to about 10% AR, from about 0.0% to about 5% AR, from about 0.0% to about 4% AR, from about 0.0% to about 3% From about 0.0% to about 2% AR, from about 3% to about 5% AR, from about 5% to about 8% AR, or from about 8% to about 10% AR, or from about 10% to about 15% , And ranges within one or more of the above.

The low AR composition of the present invention comprises up to about 15% AR1, up to 14% AR1, up to 13% AR1, up to 12% AR1, up to 11% AR1, up to 10% AR1, up to 9% AR1 less than 8%, AR1 less than 7%, AR1 less than 6%, AR1 less than 5%, AR1 less than 4.5%, AR1 less than 4%, AR1 less than 3.5%, AR1 less than 3% AR1 less than 2%, AR1 less than 1.9%, AR1 less than 1.8%, AR1 less than 1.7%, AR1 less than 1.6%, AR1 less than 1.5%, AR1 less than 1.4%, less than 1.3% AR1, up to 1.2% of AR1, up to 1.1% of AR1, up to 1% of AR1, up to 0.9% of AR1, up to 0.8% of AR1, up to 0.7% of AR1, up to 0.6% of AR1, 0.4% or less of AR1, 0.3% or less of AR1, 0.2% or less of AR1, 0.1% or less of AR1, or 0.0% of AR1 and ranges within one or more of the above ranges. The low AR composition of the present invention may also contain about 0.0% to about 10% AR1, about 0.0% to about 5% AR1, about 0.0% to about 4% AR1, about 0.0% to about 3% From about 0.0% to about 2% AR1, from about 3% to about 5% AR1, from about 5% to about 8% AR1, or from about 8% to about 10% AR1, or from about 10% to about 15% , And ranges within one or more of the above.

In addition, the low AR composition of the present invention comprises up to about 15% AR2, up to 14% AR2, up to 13% AR2, up to 12% AR2, up to 11% AR2, up to 10% AR2 up to 9% AR2 less than 8%, AR2 less than 7%, AR2 less than 6%, AR2 less than 5%, AR2 less than 4.5%, AR2 less than 4%, AR2 less than 3.5%, AR2 less than 3% AR2 of not more than 2.5%, AR2 of not more than 2%, AR2 of not more than 1.9%, AR2 of not more than 1.8%, AR2 of not more than 1.7%, AR2 of not more than 1.6%, AR2 of not more than 1.5%, AR2 of not more than 1.4% AR2 of not more than 1.2%, AR2 of not more than 1.1%, AR2 of not more than 1%, AR2 of not more than 0.9%, AR2 of not more than 0.8%, AR2 of not more than 0.7%, AR2 of not more than 0.6% AR2, no more than 0.4% AR2, no more than 0.3% AR2, no more than 0.2% AR2, no more than 0.1% AR2, or 0.0% AR2 and ranges within one or more of the above. In addition, the low AR composition of the present invention may comprise from about 0.0% to about 10% AR2, from about 0.0% to about 5% AR2, from about 0.0% to about 4% AR2, from about 0.0% to about 3% From about 0.0% to about 2% AR2, from about 3% to about 5% AR2, from about 5% to about 8% AR2, or from about 8% to about 10% AR2, or from about 10% to about 15% , And ranges within one or more of the above.

In one embodiment, the low AR composition comprises about 0.0% to about 3% AR1. In another embodiment, the low AR composition comprises from about 0.0% to about 3% AR2. In another embodiment, the low acid species composition comprises less than about 3% AR2.

In another embodiment, the low AR composition comprises about 1.4% or less AR. For example, in one embodiment, the composition comprises about 1.4% AR2 and about 0.0% AR1.

In one embodiment, the low AR composition of the invention comprises at least about 15% of at least one of a deamidated variant, an afacosylated variant, an MGO variant, a glycosyl variant, a citric acid variant, a glycosylated variant, an acetone variant, or a fragment variant, , Less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5% Or less, 3.5% or less, 3% or less, 2.5% or less, 2% or less, 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2% , No more than 1%, no more than 0.9%, no more than 0.8%, no more than 0.7%, no more than 0.6%, no more than 0.5%, no more than 0.4%, no more than 0.3%, no more than 0.2%, no more than 0.1% Or more. In one aspect of this embodiment, the low AR composition of the present invention further comprises one of a deamidated variant, an afacosylated variant, an MGO variant, a glycosyl variant, a citric acid variant, a glycosylated variant, an acetone variant, or a fragment variant , About 0.0% to about 5%, about 0.0% to about 4%, about 0.0% to about 3%, about 0.0% to about 2%, about 3% From about 5% to about 8%, or from about 8% to about 10%, or from about 10% to about 15%, and ranges within one or more of the foregoing. For example, a low AR composition of the invention may comprise less than 15% deamidated variants, while each of the other acid variants, alone or in combination, is present in a percentage greater than 15%.

The term "non-low acid species composition" as used herein refers to a composition comprising an antibody or antigen-binding portion thereof, which contains more than about 16% of an acidic species. For example, the non-acidic species composition may comprise at least about 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24% % ≪ / RTI > acid species. In one embodiment, the non-acidic species composition comprises about 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% It may contain at least 25% AR1. In another embodiment, the non-acidic species composition comprises about 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% 25% or more AR2, and ranges within one or more of the above.

In one embodiment, the low AR composition is administered to a subject in need thereof, as compared to a non-low acidic composition, to a subject having a biological and functional disorder, including increased efficacy in the treatment or prevention of a disorder, e. Properties. In one embodiment, the low AR composition comprises an anti-TNF [alpha] antibody or antigen-binding portion thereof, e.g., adalimumab or a fragment thereof. For example, in one embodiment, a low AR composition comprising an antibody or antigen-binding portion thereof, when administered to a subject suffering from a disorder where the TNFa activity is detrimental, may be administered in combination with a non- Compared to the plant composition, it exhibits increased cartilage penetration, decreased bone erosion, and / or reduced cartilage destruction.

The term " increased cartilage penetration " as used herein refers to increased penetration of cartilage in vivo by a low AR composition, as compared to a non-low AR composition comprising the same antibody or antigen binding portion thereof.

The term " reduced cartilage destruction " as used herein refers to a measure of the breakdown of cartilage tissue in vivo by a low AR composition, as compared to a non-low AR composition comprising the same antibody or antigen- It is possible reduction. As used herein, the term " reduced bone erosion "refers to the ability of a low AR composition to determine in vivo the erosion of bone tissue as compared to a non-low acid species composition comprising the same antibody or antigen- . For example, an in vivo model of a disease or disorder in which TNFa activity is deleterious, such as a mouse model of arthritis, may be used to treat cartilage infiltration, bone erosion, and / or osteoporosis by a composition comprising an anti- TNFa antibody or antigen- Or cartilage destruction can be measured. One non-limiting example of a mouse model of arthritis that is recognized in the art is the human TNF gene transfer 197 mouse model of arthritis (TNF-Tg197) (Keffer, J. et al ., EMBO J (1991) 10 : 4025-4031, the contents of which are expressly incorporated herein by reference for further details of the TNF-Tg197 model of arthritis).

In another embodiment, a low AR composition comprising an antibody or antigen-binding portion thereof, when administered to an animal model of arthritis, for example , a TNF-Tg197 model of arthritis, Score, and / or histopathology score. ≪ / RTI > As used herein, "arthritis score" refers to signs and symptoms of arthritis in an animal model of arthritis. The term " histopathology score "as used herein refers to radiologic damage not only to local inflammation but also to cartilage and bone.

In another embodiment, a low AR composition comprising an antibody or antigen-binding portion thereof has reduced synovial proliferation, decreased cell invasion, decreased chondrocyte apoptosis, and / or reduced synovial cell proliferation compared to a non- Proteoglycan loss. In another embodiment, a low AR composition comprising an anti-TNF [alpha] antibody or antigen-binding portion thereof exhibits increased TNF [alpha] affinity compared to a non-low acid species composition.

As used herein, the term "a disorder wherein TNFa activity is deleterious" means that the presence of TNF [alpha] in a subject suffering from the disorder is found to be involved in a factor contributing to the pathogenesis of the disorder or the deterioration of the disorder, Is contemplated to include diseases and other disorders that are suspected of being involved in factors that contribute to the pathophysiology of the disorder or to the deterioration of the disorder. Thus, disorders in which TNF [alpha] activity is detrimental are disorders in which inhibition of TNF [alpha] activity is expected to alleviate the symptoms and / or progression of the disorder. Such a disorder can be evidenced, for example , by an increase in the concentration of TNF [alpha] in the biological fluid of a subject suffering from the disorder ( e.g., serum, plasma, or synovial fluid of the subject) RTI ID = 0.0 > anti-TNFa < / RTI > antibodies. There are numerous examples of disorders in which TNF [alpha] activity is detrimental. In one embodiment, the disorder wherein the TNFa activity is detrimental is an autoimmune disorder. In one embodiment, the autoimmune disorder is selected from the group consisting of rheumatoid arthritis, childhood idiopathic arthritis, rheumatoid spondylitis, ankylosing spondylitis, psoriasis, osteoarthritis, gouty arthritis, allergies, multiple sclerosis, psoriatic arthritis, autoimmune diabetes, autoimmune uveitis, And is selected from the group consisting of pediatric rheumatoid arthritis, Crohn's disease, ulcerative colitis, active axial spondyloarthritis (active axSpA) and non-radiographic axial spondyloarthritis (nr-axSpA) . TNFα activity is detrimental is a disorder is present, even in US Patent No. 6,090,382, and, the literature [the "Highlights of Prescribing Information" for HUMIRA ® (adalimumab) Injection (Revised Jan. 2008)], the contents of which incorporated herein by . The use of the TNFa antibodies and antibody portions obtained using the methods of the invention for the treatment of certain disorders is discussed in further detail below.

The term "antibody" includes immunoglobulin molecules consisting of four polypeptide chains of two heavy (H) chains and two light (L) chains interlinked by a disulfide bond. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains: CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain of CL. The VH and VL regions can be further subdivided into hypervariable regions termed complementarity determining regions (CDRs) interposed between more conserved regions termed framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino-terminus to the carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The term "antigen-binding portion" (or "antibody portion") of an antibody includes a fragment of an antibody that retains the ability to specifically bind to an antigen (for example, hTNFa in the case of adalimumab). It has been found that the antigen-binding function of the antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments comprised within the "antigen-binding portion" of the antibody include (i) a Fab fragment that is a monovalent fragment comprising the VL, VH, CL, and CH1 domains; (ii) a F (ab ') 2 fragment that is a bivalent fragment comprising two Fab fragments linked by disulfide bridging in the hinge region; (iii) an Fd fragment comprising the VH and CH1 domains; (iv) a Fv fragment comprising the VL and VH domains of a single arm of the antibody, (v) a dAb fragment comprising the VH domain [Ward et al ., (1989) Nature 341: 544-546, The teaching of which is incorporated herein by reference; And (vi) an isolated complementarity determining region (CDR). In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they use a recombination method in which the VL and VH regions are paired to form a monovalent molecule (known as single chain Fv (scFv) See, for example, Bird et al . (1988) Science 242: 423-426 and Huston et al . (1988) Proc. Natl. Acad Sci USA 85: 5879-5883, Quot; is incorporated herein by reference). ≪ / RTI > Such single chain antibodies are also intended to be included within the term " antigen-binding portion "of the antibody. Other forms of single chain antibodies, e. G. Diabodies, are also included. The diabodies are VH and VL, except that the domains are paired with the complementary domains of the other chain by using a linker that is too short to allow pairing between the two domains on the same chain to create two antigen binding sites domain is a bivalent bispecific antibody that is expressed on a single polypeptide chain (for example, in [Holliger, P., et al ( 1993) Proc Natl Acad Sci USA 90:..... 6444-6448; Poljak , RJ, et al . (1994) Structure 2: 1121-1123, the teachings of which are incorporated herein by reference). Further, further, the antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to produce tetrameric scFv molecules (Kipriyanov, SM, et al . (1995) Human Antibodies and Hybridomas 6: 93-101, , And the use of cysteine residues, marker peptides and C-terminal polyhistidine tags to produce divalent biotinylated scFv molecules (Kipriyanov, SM, et al . (1994) Mol. Immunol. 31: 1047-1058, the teachings of which are incorporated herein by reference). Antibody moieties, such as Fab and F (ab ') 2 fragments, can be prepared from whole antibodies using the prior art, for example, papain or pepsin digestion of whole antibodies, respectively. In addition, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques as described herein. In one aspect, the antigen binding portion is a complete domain or a complete pair of domains.

The terms "CABARN numbering "," CABAT DEFINITION "and" CABAT LABELING "are used interchangeably herein. These terms, which are recognized in the art, refer to a numbering system of amino acid residues that are more variable ( i.e. , hypervariable) than other amino acid residues in the heavy and light chain variable regions of the antibody or antigen-binding portion thereof (see Kabat et al . (1971) Ann. NY Acad, Sci. 190: 382-391 and Kabat, EA, et al . (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, the teachings of which are incorporated herein by reference). For the heavy chain variable region, the hypervariable region is the amino acid position 31 to 35 relative to CDR1, the amino acid position 50 to 65 for CDR2, and the amino acid position 95 to 102 for CDR3. For the light chain variable region, the hypervariable region is the amino acid position 24 to 34 for CDR1, the amino acid position 50 to 56 for CDR2, and the amino acid position 89 to 97 for CDR3.

The term "human antibody" is described in Kabat, et al . (1991) Sequences of proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242. ≪ Desc / Clms Page number 2 > The term " human antibody " Human antibodies of the present invention can be used to detect human gonadal immunoglobulin sequences ( e. G., By random or site-specific mutagenesis in vitro or somatic mutations in vivo) within the CDRs and in particular CDR3 Lt; RTI ID = 0.0 > (e. G., Mutations introduced by < / RTI > Mutations can be introduced using the "selective mutagenesis approach ". Human antibodies can have one or more positions that are replaced by amino acid residues, e. G., Activity enhancing amino acid residues that are not encoded by human gonadal immunoglobulin sequences. Human antibodies can have no more than 20 positions replaced by amino acid residues that are not part of the human gonadal immunoglobulin sequence. In other embodiments, no more than ten, no more than five, no more than three, or no more than two positions. In one embodiment, these substitutions are within the CDR region. However, the term "human antibody" as used herein is not intended to encompass antibodies that have been CDR sequences derived from the gonads of other mammalian species, e. G. Mice, transcribed onto human framework sequences.

The phrase "recombinant human antibody" means a human antibody produced by recombinant means, expressed, produced or isolated, for example, an antibody expressed using a recombinant expression vector transfected into a host cell, recombinant combinatorial human antibody the antibody isolated from the library, an animal transgenic for human immunoglobulin genes of an antibody isolated from (e.g., a mouse) (for example, literature [Taylor, LD, et al. (1992) Nucl. Acids Res. 20 : 6287-6295, the teachings of which are incorporated herein by reference), or by any other means, including splicing of human immunoglobulin gene sequences to other DNA sequences Produced, expressed, generated, or isolated from an animal. Such recombinant human antibodies have variable and constant regions derived from human gonadal immunoglobulin sequences (see Kabat, EA, et al . (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services , NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies are administered in vitro If a transgenic animal is used for the mutagenesis (or human Ig sequences in vivo The amino acid sequence of the VH and VL regions of the recombinant antibody can be naturally present in the in vivo human antibody germline repertoire, derived from the human germline VH and VL sequences and associated with the human germline VH and VL sequences There is no sequence. However, in certain embodiments, such recombinant antibodies are the result of a selective mutagenesis approach or a back-mutation or both.

An "isolated antibody" is an antibody that is substantially free of other antibodies having different antigen specificity ( for example , an isolated antibody that specifically binds to hTNFa is an antibody that specifically binds to an antigen other than hTNFa Substantially free of the antibody). Isolated antibodies that specifically bind to hTNF [alpha] can bind to TNF [alpha] molecules from other species. In addition, the isolated antibody may be substantially free of other cellular materials and / or chemicals. A suitable anti-TNFa antibody is adalimumab.

Under the trade name HUMIRA ^® As used herein, the term is well known as a (AbbVie) "Oh otherwise free MAB" is refers to a person IgG ₁ antibodies that bind to human tumor necrosis factor α (TNFα). Generally, the heavy chain constant domain 2 (CH2) of the dalyalmumab IgG-Fc region is glycosylated through covalent attachment of the oligosaccharide at asparagine 297 (Asn-297). The light chain variable region of adalimumab is provided herein as SEQ ID NO: 1 and the heavy chain variable region of adalimumab is provided herein as SEQ ID NO: 2. Adalimumab comprises a light chain variable region comprising CDR1 of SEQ ID NO: 7, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 3. Adalimumab comprises a heavy chain variable region comprising CDR1 of SEQ ID NO: 8, CDR2 of SEQ ID NO: 6, and CDR3 of SEQ ID NO: The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO: 9. The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 10. The full length amino acid sequence of the light chain is shown in SEQ ID NO: 11 and the full length amino acid sequence of the heavy chain is shown in SEQ ID NO: Adalmumab is described in U. S. Patent No. 6,090, 382; 6,258,562; 6,509,015; 7,223, 394; 7,541,031; 7,588,761; 7,863, 426; 7,919,264; 8,197,813; 8,206,714; 8,216,583; 8,420,081; 8,092,998; 8,093,045; 8,187, 836; 8,372, 400; 8,034,906; 8,436,149; 8,231, 876; 8,414,894; No. 8,372,401, the entire contents of each of which are expressly incorporated herein by reference in their entirety. Further, unlike non-ah MAB it has been described in the literature [ "Highlights of Prescribing Information for HUMIRA ® (adalimumab) Injection (Revised Jan. 2008)], the contents of which documents are incorporated herein by reference.

In one embodiment, O is otherwise free MAB, and dissociation from both surface plasmon resonance as measured by, one TNFα with a K _off of less than 1x10 ^-8 M of K _d and 1x10 ^-3 s ^-1 or less, the standard In vitro L929 assays neutralize human TNFα cytotoxicity with an IC50 of 1x10-M or less. In another embodiment, the non-contrast MAB is O, dissociates from human TNFα with a K _off of less than 5x10 ^-4 s ^-1 or 1x10 K _off of less than ^-4 s ^-1. In another embodiment, Adalimumab neutralizes human TNF alpha cytotoxicity in an in vitro L929 assay in an IC50 of ¹ x ^10-8 M or less, an IC50 of 1 x ^10-9 M or less, or an IC50 of 1 x ^{10 -10} M or less .

Generally, the heavy chain constant domain 2 (CH2) of the dalyalmumab IgG-Fc region is glycosylated through covalent attachment of the oligosaccharide at asparagine 297 (Asn-297). Analysis of adalimumab has revealed that it has three major basic variants ( i . E., Lys 0, Lys 1, and Lys 2) that are referred to herein as "lysine mutant species ". The term "lysine variant species " as used herein refers to an antibody or antigen-binding portion thereof, comprising a heavy chain having zero, one, or two C-terminal lysines. For example, a "Lys 0" variant comprises an antibody or heavy chain-binding portion thereof having a heavy chain that does not comprise a C-terminal lysine. A "Lys 1" variant includes an antibody having one heavy chain comprising C-terminal lysine or an antigen-binding portion thereof. "Lys 2" variants include antibodies having two heavy chains, including C-terminal lysine. Lysine variants can be detected, for example, by weak cation exchange chromatography (e. G., WCX-10) of expression products of host cells expressing the antibody or antigen-binding portion thereof. For example, but not by way of limitation, FIGS. 163 and 193 illustrate the WCX-10 assay of Adalimumab, wherein the three lysine variants and the two acid species regions are degraded from each other.

The compositions of the present invention may comprise more than one lysine variant species of the antibody or antigen-binding portion thereof. For example, in one embodiment, the composition may comprise an Lys 2 variant of an antibody or antigen-binding portion thereof. The composition may comprise an Lys 1 variant of the antibody or antigen-binding portion thereof. The composition may comprise an Lys 0 variant of the antibody or antigen-binding portion thereof. In another embodiment, the composition may comprise Lys 1 and Lys 2 of the antibody or antigen-binding portion thereof, or Lys 1 and Lys 0, or both Lys 2 and Lys 0 mutants. In another embodiment, the composition may comprise all three lysine variants of the antibody or antigen-binding portion thereof, i.e., Lys 0, Lys 1 and Lys 2.

The term "upstream process technology" as used herein with respect to proteins, e. G., Antibody preparations, refers to the ability of a protein ( e. G. , Antibody) from a cell Production and collection. The term "cell culture " as used herein refers to methods and techniques for generating and maintaining a population of host cells capable of producing the desired recombinant protein, and for optimizing production and collection of the desired protein . For example, once an expression vector has been introduced into an appropriate host, the host can be maintained under conditions suitable for expression of the relevant nucleotide coding sequence, and collection and purification of the desired recombinant protein.

When the cell culture technique of the present invention is used, the desired protein may be produced in the cell or in the surrounding cytoplasmic space, or may be directly secreted into the medium. In embodiments where the desired protein is produced intracellularly, particulate debris ( e. G., Due to homogenization) that is a host cell or a lysed cell includes, but is not limited to, centrifugation or ultrafiltration It can be removed by various means. When the desired protein is secreted into the medium, the supernatant from such an expression system can first be concentrated using a commercial protein concentration filter, for example , an Amicon ™ or Millipore Pellicon ™ ultrafiltration unit.

The term "downstream process technology " as used herein refers to one or more techniques used after upstream process techniques to purify the desired protein, e.g., an antibody. For example, downstream process techniques can be performed using affinity chromatography, including, for example, protein A affinity chromatography, ion exchange chromatography, for example, anion or cation exchange chromatography, hydrophobic interaction chromatography, Or purification of the protein product using displacement chromatography.

A nucleic acid encoding the antibody or antibody portion (e.g., VH, VL, CDR3), such as the phrase "isolated nucleic acid molecule" as used herein in connection with binding to hTNFa, Encoding nucleic acid sequence comprises a nucleic acid molecule that does not contain another nucleotide sequence encoding an antibody or antibody portion that binds to an antigen other than hTNF [alpha] that the other sequence can naturally flank the nucleic acid in the human genomic DNA do. Thus, for example , the isolated nucleic acid of the invention encoding the VH region of an anti-TNF [alpha] antibody does not contain any other sequence encoding another VH region that binds to an antigen other than, for example, hTNF [alpha]. Also, the phrase "isolated nucleic acid molecule" is intended to include a sequence encoding a divalent bi-specific antibody, such as a diabody, wherein the VH and VL regions do not contain sequences other than the diabody sequence.

The phrase "recombinant host cell" (or simply "host cell") includes cells into which a recombinant expression vector has been introduced. It is to be understood that these terms are intended to represent progeny of such cells as well as the particular subject cells. This offspring may still not actually be identical to the mother cell, but is still included within the scope of the term "host cell " as used herein, as mutations or environmental effects may cause certain strains in subsequent generations.

The term "recombinant protein " as used herein refers to a protein produced as a result of transcription and translation of a gene carried out with a recombinant expression vector introduced into a host cell. In certain embodiments, the recombinant protein is an antibody, for example , a chimeric antibody, a humanized antibody, or a fully human antibody. In certain embodiments, the recombinant protein is an isotype of antibody selected from the group consisting of IgG ( e.g. , IgGl, IgG2, IgG3, IgG4), IgM, IgAl, IgA2, IgD, or IgE. In certain embodiments, the antibody molecule is a full length antibody ( e. G., An IgG1 or IgG4 immunoglobulin) or, alternatively, the antibody may be a fragment ( e. G. , An Fc fragment or a Fab fragment).

The phrase " purified collection "refers to a cell culture that has undergone centrifugation to remove large solid particles from a liquid material containing the desired antibody, and subsequent filtration to remove finer solid particles and impurities, Refers to a liquid material containing the desired protein, for example, the desired monoclonal antibody, extracted from a fermentation bioreactor, such as the desired antibody.

The term "preparative scale " as used herein refers to the scale of a refining operation that can be easily scaled-up and performed with large scale manufacturing while still providing the desired separation. For example, the art skilled, in the laboratory, for example, 0.5cm (id) x 20cm ( L) using a column by developing the process, for example, a 30cm (id) x 20cm packed with the same resin (L) column can be run with the same set of buffers, the same linear flow rate (or residence time), and a buffer volume to move them to large scale production. In manufacturing scale separation, the column layer height is typically less than about 30 cm and the column pressure drop is less than about 5 bar.

II. The low acid species composition of the present invention

The present invention provides a low AR composition comprising a protein, for example , an antibody such as a dalyma mab or an antigen-binding portion thereof, wherein the composition comprises up to about 15% AR, up to 14% AR, 13 % AR, 12% AR, 11% AR, 10% AR, 9% AR, 8% AR, 7% AR, 6% AR, 5% Of AR, AR of less than 4.5%, AR of less than 4%, AR of less than 3.5%, AR of less than 3%, AR of less than 2.5%, AR of less than 2%, AR of less than 1.9%, AR of less than 1.8% , AR of less than 1.7%, AR of less than 1.6%, AR of less than 1.5%, AR of less than 1.4%, AR of less than 1.3%, AR of less than 1.2%, AR of less than 1.1%, AR of less than or equal to 1% % AR,% AR,% AR,% AR,% AR,% AR,% AR,% AR,% AR, AR, or 0.0% AR, and ranges within one or more of the above. In addition, the low AR composition of the present invention may contain from about 0.0% to about 10% AR, from about 0.0% to about 5% AR, from about 0.0% to about 4% AR, from about 0.0% to about 3% From about 0.0% to about 2% AR, from about 3% to about 5% AR, from about 5% to about 8% AR, or from about 8% to about 10% AR, or from about 10% to about 15% , And ranges within one or more of the above.

In one embodiment, the low AR composition of the present invention comprises up to about 15% AR1, up to 14% AR1, up to 13% AR1, up to 12% AR1, up to 11% AR1, up to 10% AR1 less than 8%, AR1 less than 7%, AR1 less than 6%, AR1 less than 5%, AR1 less than 4.5%, AR1 less than 4%, AR1 less than 3.5%, less than 3% AR1 of less than 2.5%, AR1 of not more than 2%, AR1 of not more than 1.9%, AR1 of not more than 1.8%, AR1 of not more than 1.7%, AR1 of not more than 1.6%, AR1 of not more than 1.5%, AR1 of not more than 1.4% , AR1 of not more than 1.3%, AR1 of not more than 1.2%, AR1 of not more than 1.1%, AR1 of not more than 1%, AR1 of not more than 0.9%, AR1 of not more than 0.8%, AR1 of not more than 0.7%, AR1 of not more than 0.6% Or less of AR1, 0.4% or less of AR1, 0.3% or less of AR1, 0.2% or less of AR1, 0.1% or less of AR1, or 0.0% of AR1 and ranges within one or more of the above ranges. The low AR composition of the present invention may also contain about 0.0% to about 10% AR1, about 0.0% to about 5% AR1, about 0.0% to about 4% AR1, about 0.0% to about 3% From about 0.0% to about 2% AR1, from about 3% to about 5% AR1, from about 5% to about 8% AR1, or from about 8% to about 10% AR1, or from about 10% to about 15% , And ranges within one or more of the above.

In another embodiment, the low AR composition of the present invention also comprises less than about 15% AR2, less than 14% AR2, less than 13% AR2, less than 12% AR2, less than 11% AR2, less than 10% AR2 less than 9%, AR2 less than 8%, AR2 less than 7%, AR2 less than 6%, AR2 less than 5%, AR2 less than 4.5%, AR2 less than 4%, AR2 less than 3.5% AR2 of not more than 3%, AR2 of not more than 2.5%, AR2 of not more than 2%, AR2 of not more than 1.9%, AR2 of not more than 1.8%, AR2 of not more than 1.7%, AR2 of not more than 1.6%, AR2 of not more than 1.5% AR2 of not more than 1.3%, AR2 of not more than 1.2%, AR2 of not more than 1.1%, AR2 of not more than 1%, AR2 of not more than 0.9%, AR2 of not more than 0.8%, AR2 of not more than 0.7%, AR2 of not more than 0.6% AR2, up to 0.5% AR2, up to 0.4% AR2, up to 0.3% AR2, up to 0.2% AR2, up to 0.1% AR2, or up to 0.02 AR2, have. In addition, the low AR composition of the present invention may comprise from about 0.0% to about 10% AR2, from about 0.0% to about 5% AR2, from about 0.0% to about 4% AR2, from about 0.0% to about 3% From about 0.0% to about 2% AR2, from about 3% to about 5% AR2, from about 5% to about 8% AR2, or from about 8% to about 10% AR2, or from about 10% to about 15% , And ranges within one or more of the above.

As demonstrated herein, these low AR compositions improved the biological properties (see Example 13). For example, the low AR compositions of the present invention can be used in a variety of applications including, but not limited to, increased cartilage tissue infiltration, reduced cartilage destruction, reduced synovial proliferation, reduced bone erosion, arthritic score and / Increased protection against the onset of score, reduced cellular infiltration, reduced proteoglycan loss, reduced chondrocyte apoptosis, and / or increased TNF affinity. In addition, the compositions of the present invention exhibit increased therapeutic efficacy upon administration to a subject.

In one embodiment, the protein in the low AR composition of the invention is an antibody or antigen-binding portion thereof. For example, the antibody or antigen-binding portion thereof may be an anti-TNFa antibody or an antigen-binding portion thereof, for example, adalimumab or an antigen-binding portion thereof. In one aspect of this embodiment, the antibody or antigen-binding portion thereof may comprise a light chain variable region comprising the sequence set forth in SEQ ID NO: 1, and a heavy chain variable region comprising the sequence set forth in SEQ ID NO: In another aspect of this embodiment, the antibody comprises a light chain variable region comprising CDRl having the sequence set forth in SEQ ID NO: 7, CDR2 having the sequence set forth in SEQ ID NO: 5, and CDR3 having the sequence set forth in SEQ ID NO: 3 . In another aspect of this embodiment, the antibody comprises a heavy chain RKUS region comprising CDRl having the sequence set forth in SEQ ID NO: 8, CDR2 having the sequence set forth in SEQ ID NO: 6, and CDR3 having the sequence set forth in SEQ ID NO: 4 .

The antibody or antigen-binding portion thereof used in the AR composition of the present invention may be a human antibody, a humanized antibody, or a chimeric antibody.

Antibodies that can be used in the low AR compositions of the present disclosure can be prepared by conventional monoclonal antibody methods, for example , by the standard antigenic determinant of the desired antigen according to the standard somatic cell hybridization technique of Kohler and Milstein (1975) Nature 256: 495 Lt; / RTI > may be produced by various techniques, including immunization of animals. Somatic cell hybridization procedures can be used. In principle, other techniques for producing monoclonal antibodies, including viral or tumorigenic transformation of B lymphocytes, can also be used.

One exemplary animal system for producing hybridomas is the murine system. Hybridoma production is a very well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners ( e . G., Murine myeloma cells) and fusion procedures are also known.

The antibody used in the low AR composition of the present invention may be a human antibody, a chimeric antibody, or a humanized antibody. The chimeric or humanized antibodies used in the low AR compositions of the invention can be prepared based on the sequences of non-human monoclonal antibodies prepared as described above. DNA encoding the heavy and light chain immunoglobulins can be obtained from the desired non-human hybridoma and manipulated to contain non-murine ( e.g. , human) immunoglobulin sequences using standard molecular biology techniques . For example, to generate chimeric antibodies, murine variable regions can be linked to human constant regions using methods known in the art ( see, for example , US Patent No. 4,816, 567 to Cabilly et al . ). To generate humanized antibodies, murine CDR regions can be inserted into human frameworks using methods known in the art ( see, for example , Winter Patent No. 5,225,539 and Queen et al . U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

In one non-limiting embodiment, the antibody used in the low AR composition of the invention is a human monoclonal antibody. Such human monoclonal antibodies may be produced using transgenic or transchromosomic mice having a portion of the human immune system rather than the mouse system. These gene transfer or chromosomal transfer mice include mice represented herein as HuMAb Mouse (Medarex, Inc.), KM Mouse (Medarex, Inc.), and XenoMouse (Amgen). In addition, the antibody or antigen-binding portion thereof used in the low AR composition of the present invention can be used in the U.S. Can be prepared using the method described in patent 6,090,382, the entire contents of which are expressly incorporated herein by reference.

In addition, alternative chromosome transferring animal systems expressing human immunoglobulin genes are available in the art and can be used to generate antibodies of the present disclosure. For example, mice having both human heavy chain transit chromosome and human light chain transit chromosome, designated as "TC mouse" can be used; Such mice are described in Tomizuka et al . (2000) Proc. Natl. Acad. Sci. USA 97: 722-727. In addition, cows with human heavy and light chain transfer chromosomes have been described in the art (see, for example, Kuroiwa et al . (2002) Nature Biotechnology 20: 889-894 and PCT Application WO 2002/092812) Lt; / RTI > of the antibody.

Recombinant human antibodies for use in the low AR compositions of the invention can be obtained by screening scFv phage display libraries prepared using recombinant combinatorial antibody libraries, for example , human VL and VH cDNAs prepared from mRNA derived from human lymphocytes Lt; / RTI > Methods for making and screening such libraries are known in the art. Commercially available kits for generating phage display libraries (e.g., Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP ™ phage display kit is, Cat. No. 240 612, the entire teachings of these are included in the present application) Examples of methods and reagents that are particularly easy to use in generating and screening antibody display libraries are described, for example, in Ladner et al . US Patent No. 5,223, 409; Kang et al . PCT Publication No. WO 92/18619; Dower et al . PCT Publication No. WO 91/17271; Winter et al . PCT Publication No. WO 92/20791; Markland et al . PCT Publication No. WO 92/15679; Breitling et al . PCT Publication No. WO 93/01288; McCafferty et al . PCT Publication No. WO 92/01047; Garrard et al . PCT Publication No. WO 92/09690; Fuchs et al . (1991) Bio / Technology 9: 1370-1372; Hay et al . (1992) Hum Antibody Hybridomas 3: 81-85; Huse et al . (1989) Science 246: 1275-1281; McCafferty et al ., Nature (1990) 348: 552-554; Griffiths et al . (1993) EMBO J 12: 725-734; Hawkins et al . (1992) J Mol Biol 226: 889-896; Clackson et al . (1991) Nature 352: 624-628; Gram et al . (1992) PNAS 89: 3576-3580; Garrard et al . (1991) Bio / Technology 9: 1373-1377; Hoogenboom et al . (1991) Nuc Acid Res 19: 4133-4137; And Barbas et al . (1991) PNAS 88: 7978-7982; The entire teachings of these documents are incorporated herein.

In addition, the human monoclonal antibody used in the low AR composition of the present invention can be produced so that a human antibody reaction can be generated upon immunization using a SCID mouse in which human immune cells have been reconstituted. Such mice are described, for example, in Wilson et al., U.S. Pat. 5,476,996 and 5,698,767. ≪ / RTI >

In certain embodiments, the human antibodies used in the low AR compositions of the present invention comprise an anti-TNF alpha antibody and its antibody portion, an anti-TNF alpha related antibody and antibody portion, and equivalent properties to the anti-TNF alpha antibody, Is a human antibody and antibody portion having high affinity and high neutralizing ability to bind to hTNF? Having low dissociation dynamics. In one aspect, the invention, both as determined by surface plasmon resonance, from about 1 x 10 ^-8 M or less for Kd and x 10 ^-3 s ^-1 or less of 1 is dissociated from hTNFα with a Koff rate constant, 0.0 > AR < / RTI > composition containing an isolated human antibody or antigen-binding portion thereof. In certain non-limiting embodiments, the anti-TNF [alpha] antibodies used in the low AR compositions of the present invention competitively inhibit binding of adalimumab to TNF [alpha] under physiological conditions. In one embodiment, the low AR composition of the invention comprises adalimumab or an antigen-binding fragment thereof.

The antibody or fragment thereof used in the low AR composition of the present invention can be varied wherein the constant region of the antibody is modified such that one or more constant region-mediated biological effector functions are reduced in association with the unmodified antibody. In order to modify the antibody of the present invention to exhibit reduced binding to the Fc receptor, immunoglobulin constant region fragments of the antibody can be mutated in certain regions necessary for Fc receptor (FcR) interactions ( see, for example , And Lund et al . (1991) J. of Immunol. 147: 2657-2662, the entire teachings of which are incorporated herein by reference. . In addition, a reduction in the FcR binding capacity of the antibody may also reduce other effector functions dependent on FcR interaction, such as opsonization and phagocytosis, and cytotoxicity of antibody-dependent cells have.

III. Upstream  Preparation of low AR compositions using process technology

A low AR composition of the invention comprising a protein, e. G. , An antibody or antigen binding portion thereof, e. G., Dalidimab, may be prepared by adjusting conditions during upstream protein production, such as cell culture. In one embodiment, the methods of the present invention comprise the step of administering an acidic species variant or antigenic determinant that is expressed by a host cell that produces the desired protein, including an antibody or antigen-binding portion thereof, during upstream process technology ( e.g., during cell culture) And reducing process-related impurities. The upstream process technology may be used alone or in conjunction with the downstream process technology described in Section IV and Example 10 below.

In one embodiment, one or more of the upstream process techniques described herein may comprise up to 15% AR, up to 14% AR, up to 13% AR, up to 12% AR, up to 11% AR, % AR, AR less than 9%, AR less than 8%, AR less than 7%, AR less than 6%, AR less than 5%, AR less than 4.5%, AR less than 4%, less than 3.5% Of AR, less than 3% AR, less than 2.5% AR, less than 2% AR, less than 1.9% AR, less than 1.8% AR, less than 1.7% AR, less than 1.6% AR, less than 1.5% AR , AR of 1.4% or less, AR of 1.3% or less, AR of 1.2% or less, AR of 1.1% or less, AR of 1% or less, AR of 0.9% or less, AR of 0.8% or less, AR of 0.7% % AR, less than 0.5% AR, less than 0.4% AR, less than 0.3% AR, less than 0.2% AR, less than 0.1% AR, or 0.0% AR, To produce a low AR composition comprising an antibody or antigen-binding portion thereof. In one aspect of this embodiment, a low AR composition of the invention comprises from about 0.0% to about 10% AR, from about 0.0% to about 5% AR, from about 0.0% to about 4% AR, From about 3% to about 5% AR, from about 5% to about 8% AR, or from about 8% to about 10% AR, or from about 10% To about 15% AR, and ranges within one or more of the above.

In other embodiments, one or more of the upstream process techniques described herein may comprise up to 15% AR1, up to 14% AR1, up to 13% AR1, up to 12% AR1, up to 11% AR1 less than 9%, AR1 less than 8%, AR1 less than 7%, AR1 less than 6%, AR1 less than 5%, AR1 less than 4.5%, AR1 less than 4%, less than 3.5% AR1 of not more than 3%, AR1 of not more than 2.5%, AR1 of not more than 2%, AR1 of not more than 1.9%, AR1 of not more than 1.8%, AR1 of not more than 1.7%, AR1 of not more than 1.6% AR1 of not more than 1.4%, AR1 of not more than 1.3%, AR1 of not more than 1.2%, AR1 of not more than 1.1%, AR1 of not more than 1%, AR1 of not more than 0.9%, AR1 of not more than 0.8%, AR1 of not more than 0.7% % AR1, not more than 0.5% AR1, not more than 0.4% AR1, not more than 0.3% AR1, not more than 0.2% AR1, not more than 0.1% AR1 or 0.0% AR1, To produce a low AR composition comprising an antibody or antigen-binding portion thereof. In one aspect of this embodiment, a low AR composition of the invention comprises from about 0.0% to about 10% AR1, from about 0.0% to about 5% AR1, from about 0.0% to about 4% AR1, About 3% to about 5% AR1, about 5% to about 8% AR1, or about 8% to about 10% AR1, or about 10% To about 15% of AR1, and ranges within one or more of the above.

In yet another embodiment, one or more of the upstream process techniques described herein may comprise up to 15% AR2, up to 14% AR2, up to 13% AR2, up to 12% AR2, up to 11% AR2 less than 10%, AR2 less than 9%, AR2 less than 8%, AR2 less than 7%, AR2 less than 6%, AR2 less than 5%, AR2 less than 4.5%, AR2 less than 4% AR2 of not more than 3%, AR2 of not more than 2.5%, AR2 of not more than 2%, AR2 of not more than 1.9%, AR2 of not more than 1.8%, AR2 of not more than 1.7%, AR2 of not more than 1.6% AR2 of not more than 1.4%, AR2 of not more than 1.3%, AR2 of not more than 1.2%, AR2 of not more than 1.1%, AR2 of not more than 1%, AR2 of not more than 0.9%, AR2 of not more than 0.8% AR2 of not more than 0.6%, AR2 of not more than 0.5%, AR2 of not more than 0.4%, AR2 of not more than 0.3%, AR2 of not more than 0.2%, AR2 of not more than 0.1%, or AR2 of not more than 0.0% Lt; RTI ID = 0.0 > AR < / RTI > In one aspect of this embodiment, a low AR composition of the invention comprises from about 0.0% to about 10% AR2, from about 0.0% to about 5% AR2, from about 0.0% to about 4% AR2, AR2, from about 0.0% to about 2% AR2, from about 3% to about 5% AR2, from about 5% to about 8% AR2, or from about 8% to about 10% AR2, About 15% AR2, and ranges within one or more of the above.

Some embodiments of the invention include culturing host cells to express the desired protein under conditions that limit the amount of acidic species expressed by the cells. Some embodiments of the invention include culturing host cells under conditions that limit the conversion of a product to an acidic variant.

Cell culture conditions can be modified in comparison to conditions during production of a non-acidic species composition comprising the same protein. In one embodiment, a low acid species composition is produced by culturing cells expressing an antibody or antigen-binding portion thereof in a cell culture medium comprising an increased concentration of one or more amino acids. In another embodiment, a low acid species composition is produced by culturing cells expressing an antibody or antigen-binding portion thereof in a cell culture medium comprising an increased concentration of calcium ( e.g., calcium chloride dihydrate). In another embodiment, a low acid species composition is produced by culturing cells expressing an antibody or antigen-binding portion thereof in a cell culture medium comprising an increased concentration of niacinamide. In certain embodiments, the methods described herein comprise culturing cells in a medium supplemented with one or more amino acids, calcium ( e.g. , calcium chloride dihydrate) and / or niacinamide, and combinations thereof. do.

In certain embodiments, the hypoallergenic seed composition is adapted to reduce and / or reduce the amount of acid species produced by the host cell, for example, where process parameters such as pH or dissolved oxygen (DO) are adjusted, Lt; RTI ID = 0.0 > cultured < / RTI >

In addition, low AR can be obtained using continuous or perfusion techniques. In certain embodiments, the reduction of acid species is obtained by adjusting the medium exchange during cell culture.

In other embodiments, one or more of the supplements and variants may be combined and used during cell culture of a single protein, e. G., Antibody, composition.

In order to express the antibody or antigen-binding portion thereof used in the low AR composition of the present invention, a DNA encoding a protein, for example, a DNA encoding a partial or full-length light chain and a heavy chain in the case of an antibody, Is inserted into one or more expression vectors to be operatively linked to translation control sequences. (See, for example, US Pat. No. 6,090,382, the entire teachings of which are incorporated herein by reference.) In this regard, the term "operably linked" Transcription and translation control sequences in the vector are ligated into the vector to perform their intended function of regulating transcription and translation of the gene. The expression vector and expression control sequence are selected to be compatible with the expression host cell used. In certain embodiments, the desired protein will comprise multiple polypeptides, e. G., The heavy and light chains of the antibody. Thus, in certain embodiments, genes encoding multiple polypeptides, such as antibody light chain genes and antibody heavy chain genes, may be inserted into separate vectors, or, more typically, the genes are inserted into the same expression vector. The gene is inserted into the expression vector by standard methods ( for example , ligation of the vector with complementary restriction sites on the gene fragment, or blunt end ligation in the absence of restriction sites). Prior to inserting the gene or genes, the expression vector may already have additional polypeptide sequences, such as, but not limited to, antibody constant region sequences. For example, one approach to convert anti-TNF [alpha] antibodies or anti-TNF [alpha] antibody-associated VH and VL sequences into full-length antibody genes is to convert these sequences to a VH fragment operatively linked to the CH fragment (s) Linking and inserting the heavy chain constant and light chain constant regions into the encoded vectors, respectively, so that the VL fragment is operatively linked to the CL fragment in the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that enables secretion of the protein from the host cell. The gene can be cloned into a vector such that the signal peptide is linked in-frame to the amino terminus of the gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide ( i . E., A signal peptide derived from a non-immunoglobulin protein).

In addition to the protein coding gene, the recombinant expression vector may have one or more regulatory sequences that control the expression of the protein coding gene in the host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements ( e. G. , Polyadenylation signals) that control transcription or translation of a protein encoding gene. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990), the entire teachings of which are incorporated herein by reference. It will be understood by those skilled in the art that the design of an expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of host cell to be transformed, the level of expression of the desired protein, and the like. Suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells such as cytomegalovirus (CMV) (e.g., the CMV promoter / enhancer) 40 (SV40) (e.g., the SV40 promoter / enhancer), adenovirus ( e.g. , adenovirus major late promoter (AdMLP)) and promoters and / or enhancers derived from polyoma. For the viral regulatory elements, and further description of its sequence, for example, in [US patent by Stinski claim 5,168,062 arc, US patent by the US Patent No. 4,510,245 No. and Schaffner et al. By Bell et al. The No. 4,968,615, the entire teachings of which are incorporated herein by reference.

The recombinant expression vector may also contain one or more additional sequences, for example, a sequence that controls the replication of the vector ( e. G. , The origin of replication) in the host cell, and / have. Selectable marker genes enable the selection of host cells into which the vector is introduced ( see, for example , US Pat. Nos. 4,399,216, 4,634,665 and 5,179,017 both by Axel et al . The entire teachings of which are incorporated herein by reference). For example, typically selectable marker genes confer resistance to drugs such as G418, hygromycin or methotrexate against vector-introduced host cells. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (when used in dhfr-host cells with methotrexate selection / amplification) and the neo gene (in the case of G418 selection).

The antibody or antibody portion used in the low AR composition of the present invention can be prepared by recombinant expression of an immunoglobulin light chain and heavy chain gene in a host cell. In order to recombinantly express the antibody, the host cell is transfected with one or more recombinant expression vectors having a DNA fragment encoding the immunoglobulin light chain and the heavy chain of the antibody, such that the light and heavy chains are expressed in the host cell, Is secreted into a culture medium from which the antibody can be recovered. Antibody heavy and light chain genes are obtained using standard recombinant DNA methods, these genes are incorporated into recombinant expression vectors, and these vectors are cloned into the vector according to the method of Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989), Ausubel et al . (Eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and US Pat. Nos. 4,816,397 & 6,914,128, the entire teachings of which are incorporated herein by reference .

For expression of proteins, e. G., Light and heavy chains of the antibody, the expression vector (s) encoding the protein is transfected into host cells by standard techniques. The various forms of the term "transfection" refer to a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium-phosphate precipitation, and DEAE-dextran Transfection, and the like. Although it is theoretically possible to express a protein of the invention in a prokaryotic or eukaryotic host cell, it is possible, for example, that such a eukaryotic cell, and in particular a mammalian cell, assembles an appropriately folded immunologically active protein Because of the greater tendency to ring and secrete, antibody expression in eukaryotic cells, such as mammalian host cells, is appropriate. Procaryotic expression of protein genes has been reported to be ineffective in producing high yield of active protein (Boss and Wood (1985) Immunology Today 6: 12-13, the entire teachings of which are incorporated herein by reference).

Suitable host cells for cloning or expressing DNA in the vectors herein are prokaryotes, yeast, or higher eukaryotic cells as described above. Suitable prokaryotes for this purpose include true bacteria, for example, gram-negative or gram-positive organisms such as intestinal bacteria such as Escherichia, for example , For example, E. coli, Enterobacter, Erwinia, Kelbsiella, Proteus, Salmonella such as Salmonella typhimurium, Serratia, such as Serratia marcescans, and Shigella, and Bacilli, for example, Serratia marcescans, B. subtilis and rain. B. licheniformis ( e.g., B. licheniformis 41P disclosed in DD 266,710, published April 12, 1989), Pseudomonas, such as P. licheniformis, P. aeruginosa, and Streptomyces. this. E. coli B, E. coli X1776 (ATCC 31,537), and E. coli. Other strains such as E. coli W3110 (ATCC 27,325) are suitable, but one suitable strain. The E. coli cloning host is E. coli. Coli 294 (ATCC 31,446). These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for polypeptide coding vectors. Saccharomyces cerevisiae, or conventional baker's yeast, is most commonly used among low eukaryotic host microorganisms. However, a number of other genera, species, and strains such as, for example, Schizosaccharomyces pombe; Kluyveromyces hosts, e. G., K. K. lactis, K. et al. K. fragilis (ATCC 12, 424), Kay. K. bulgaricus (ATCC 16,045), Kay. K. wickeramii (ATCC 24,178), Kay. K. waltii (ATCC 56,500), Kay. K. drosophilarum (ATCC 36,906), Kay. K. thermotolerans, and K. et al. K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; And filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as, for example, E. coli. A. nidulans and A. nidulans. A. niger is commonly available and is useful herein.

Suitable host cells for glycosylated proteins, such as glycosylated antibodies, are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains and variants, and strains of Spodoptera frugiperda (Caterpillar), Aedes aegypti (mosquitoes), Aedes albopictus ( Mosquito), Drosophila melanogaster (Drosophila), and Bombyx mori have been identified as the corresponding permissive insect host cells. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Spring Bixmory NPV, are publicly available, As a virus according to the present invention, in particular, for the transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

Although mammalian cells can be used for the expression and production of recombinant proteins used in the low AR compositions of the present invention, other eukaryotic cell types may also be used in connection with the present invention. See, for example, Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987). Suitable mammalian host cells for expressing recombinant proteins according to the present invention include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) PNAS USA 77: 4216-4220) Used in conjunction with DHFR selectable markers as described, for example, in Kaufman and Sharp (1982) Mol. Biol. 159: 601-621, the entire teachings of which are incorporated herein by reference , NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding a protein gene is introduced into a mammalian host cell, the antibody is incubated with the host for a sufficient time to allow expression of the antibody in the host cell or secretion of the antibody to the culture medium in which the host cell is grown Cells. Other examples of useful mammalian host cell lines include the monkey kidney CV1 cell line (COS-7, ATCC CRL 1651) transformed by SV40; Human embryonic kidney cell lines 293, or 293 cells subcloned for growth in suspension culture, Graham et al ., J. Gen Virol. 36: 599 (1977)); Fetal hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al ., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); Mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); Monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); Human cervical carcinoma cells (HELA, ATCC CCL 2); Canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); Human liver cells (Hep G2, HB 8065); Mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al ., Annals NY Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; And human hepatocellular carcinoma cell line (Hep G2), the entire teachings of which are incorporated herein by reference.

The host cell may be transformed with the above-described expression or cloning vector for protein production, transformed into a conventional nutrient medium suitably modified to induce a promoter, select a transformant, or amplify a gene encoding the desired sequence Lt; / RTI >

The host cells used to produce the protein can be cultured in various media. (Sigma), minimum essential medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle medium (DMEM) . ≪ / RTI > Also see Ham et al ., Meth. Enz. 58: 44 (1979), Barnes et al ., Anal. Biochem. 102: 255 (1980), US Pat. 4,767,704; 4,657,866; 4,927, 762; 4,560,655; Or 5,122, 469; WO 90/03430; WO 87/00195; Or US Pat. My Re. 30,985] can be used as a culture medium for host cells, and the entire teaching of the above-mentioned documents is incorporated herein by reference. Any of these media can be supplemented with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (Defined as inorganic compounds that are present at a final concentration in the micromolar range, for example), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamycin drugs) , And glucose or an equivalent energy source. In addition, any other necessary replenishments may be included at the appropriate concentrations known to those skilled in the art. The culture conditions, e.g., temperature, and pH, etc., are conditions previously used with the host cell selected for expression and will be apparent to those skilled in the art.

In addition, host cells can be used to produce fragments of an intact protein, e. G., An antibody, comprising Fab fragments or scFv molecules. It is understood that changes to the above procedures are within the scope of the present invention. For example, in certain embodiments, it may be desirable to transfect a host cell with DNA encoding a light or heavy chain (but not both) of the antibody. Also, using recombinant DNA technology, some or all of the DNA encoding either or both of the light and heavy chains that are not required for binding to the antigen can be removed. Molecules expressed from such truncated DNA molecules are also included in the antibody of the present invention. Also, a bifunctional antibody, wherein one heavy chain and one light chain are antibodies of the present invention and the other heavy and light chains are specific for an antigen other than the target antibody, can be prepared by a standard chemical cross-linking method according to the specificity of the antibody of the present invention To the second antibody by cross-linking the antibody of the present invention.

In a system suitable for the recombinant expression of a protein, e. G., An antibody or antigen-binding portion thereof, a recombinant expression vector encoding both proteins, for example, antibody heavy and antibody light chains, is produced by calcium phosphate- CHO cells. Using the recombinant expression vector, the protein gene (s) is operatively linked to the CMV enhancer / AdMLP promoter regulatory elements, respectively, to drive the transcription of a high level of gene (s). In addition, the recombinant expression vector has a DHFR gene that allows selection of CHO cells transfected with a vector using methotrexate selection / amplification. Selected transformant host cells are cultured to allow the expression of proteins, e. G., Antibody heavy and light chains, and intact proteins, e. G., Antibodies, are recovered from the culture medium. Recombinant expression vectors are prepared using standard molecular biology techniques, host cells are transfected, transformants are selected, host cells are cultured, and proteins are recovered from the culture medium.

When using recombinant techniques, a protein, e. G., An antibody or antigen-binding portion thereof, can be produced in the cell, in the surrounding cytoplasmic space, or secreted directly into the medium. In one aspect, where the protein is produced intracellularly, as a first step, the host cell or dissolved debris of microbial populations ( e.g., resulting from homogenization) can be separated, for example , by centrifugation or ultrafiltration Can be removed by filtration. When the protein is secreted into the medium, the supernatant from such an expression system can first be concentrated using a commercial protein concentration filter, for example , an Amicon ™ or Millipore Pellicon ™ ultrafiltration unit.

Some antibodies can be secreted directly from the cells into the surrounding growth medium; Other antibodies are produced intracellularly. With respect to antibodies produced in cells, the first step of the purification process typically involves dissolution of the cells, which can be accomplished by a variety of methods including mechanical shear, osmotic shock, or enzymatic treatment. This destruction releases the entire content of cells into the homogenate and also produces subcellular fragments that are difficult to remove due to their small size. These are generally removed by fractional centrifugation or by filtration. When the antibody is secreted, the supernatant from such an expression system is first concentrated, typically using a commercial protein concentration filter, such as an Amicon ™ or Millipore Pellicon ™ ultrafiltration unit. When the antibody is secreted into the medium, the recombinant host cell can also be isolated from the cell culture medium by, for example , tangential flow filtration. Antibodies can be recovered from the culture medium using the antibody purification method of the present invention.

Adjustment of amino acid concentration to control acidic species (AR)

In certain embodiments, the amount of one or more amino acids in the medium is adjusted ( e.g. , increased or decreased) to produce a low acidic species composition of the invention (see Examples section below). Such an increase or decrease in the amount of one or more amino acids may be about 1%, 5%, 10%, 15%, 20%, 25% or more of the original amount used during cell culture in which the non- , 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% Or more.

In certain embodiments, the cell culture medium will comprise one or more of the amino acids or other compositions described herein as reducing the acidic species. Thus, the amount present in the medium can be calculated prior to supplementing by adjusting the amount of amino acid or other composition to be supplemented.

In some embodiments, the cell culture medium contains about 0.025 to 20 g / L, or about 0.05 to 15 g / L, or about 0.1 to 14 g / L, or about 0.2 to 13 g / L, or about 0.25 to 12 g / Or from about 1 to about 10 g / L, or from about 1.5 to about 9.5 g / L, or from about 2 to about 9 g / L, or from about 2.5 to 8.5 g / L, or from about 3 to 8 g / To about 7.5 g / L, or about 4 to 7 g / L, or about 4.5 to 6.5 g / L, or about 5 to 6 g / L. In some embodiments, the cell culture medium is provided with one or more cells in an amount of about 0.25 g / L, or about 0.5 g / L, or about 1 g / L, or about 2 g / L, or about 4 g / Amino acids are supplemented.

In some embodiments, the cell culture medium contains up to about 15% AR, up to 14% AR, up to 13% AR, up to 12% AR, up to 11% AR, up to 10% AR, up to 9% AR of less than 8%, AR of less than 7%, AR of less than 6%, AR of less than 5%, AR of less than 4.5%, AR of less than 4%, AR of less than 3.5%, AR of less than or equal to 3% AR of less than 2.5%, AR of less than 2%, AR of less than 1.9%, AR of less than 1.8%, AR of less than 1.7%, AR of less than 1.6%, AR of less than 1.5%, AR of less than 1.4%, 1.3 AR, AR of less than 1.2%, AR of less than 1.1%, AR of less than 1%, AR of less than 0.9%, AR of less than 0.8%, AR of less than 0.7%, AR of less than 0.6%, less than 0.5% Of AR, not more than 0.4% AR, not more than 0.3% AR, not more than 0.2% AR, not more than 0.1% AR, or 0.0% AR, ≪ / RTI > in an effective amount.

In another embodiment, the cell culture medium contains up to about 15% AR1, up to 14% AR1, up to 13% AR1, up to 12% AR1, up to 11% AR1, up to 10% AR1, up to 9% AR1 less than 8%, AR1 less than 7%, AR1 less than 6%, AR1 less than 5%, AR1 less than 4.5%, AR1 less than 4%, AR1 less than 3.5%, AR1 less than 3% AR1 less than 2%, AR1 less than 1.9%, AR1 less than 1.8%, AR1 less than 1.7%, AR1 less than 1.6%, AR1 less than 1.5%, AR1 less than 1.4%, AR1 less than 1.4% AR1 of not more than 1.2%, AR1 of not more than 1.1%, AR1 of not more than 1%, AR1 of not more than 0.9%, AR1 of not more than 0.8%, AR1 of not more than 0.7%, AR1 of not more than 0.6%, not more than 0.5% Of AR1, up to 0.4% of AR1, up to 0.3% of AR1, up to 0.2% of AR1, up to 0.1% of AR1, or of 0.0% of AR1, ≪ / RTI > in an effective amount.

In yet another embodiment, the cell culture medium contains up to about 15% AR2, up to 14% AR2, up to 13% AR2, up to 12% AR2, up to 11% AR2, up to 10% AR2, up to 9% AR2 less than 8%, AR2 less than 7%, AR2 less than 6%, AR2 less than 5%, AR2 less than 4.5%, AR2 less than 4%, less than 3.5% AR2 less than 3% AR2 less than 2.5%, AR2 less than 2%, AR2 less than 1.9%, AR2 less than 1.8%, AR2 less than 1.7%, AR2 less than 1.6%, AR2 less than 1.5%, AR2 less than 1.4% AR2 of not more than 1.3%, AR2 of not more than 1.2%, AR2 of not more than 1.1%, AR2 of not more than 1%, AR2 of not more than 0.9%, AR2 of not more than 0.8%, AR2 of not more than 0.7%, AR2 of not more than 0.6% Of AR2, up to 0.4% of AR2, up to 0.3% of AR2, up to 0.2% of AR2, up to 0.1% of AR2, or of up to 0.02% of AR2, One or more amino acids are supplemented in an amount effective to:

In another embodiment, the cell culture medium contains a percentage of acidic species in the protein or antibody composition of about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5% , 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% At least one amino acid is supplemented in an amount effective to reduce by at least 1%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the above.

In some embodiments, at least one amino acid used to supplement the cell culture medium is a basic amino acid. In certain embodiments, the at least one amino acid is any combination of arginine, lysine, histidine, ornithine, or any combination of arginine or lysine with ornithine, or all four amino acids. In certain embodiments, the amino acid is a single peptide, a dipeptide, a tripeptide, or a longer oligopeptide. In certain embodiments, the di-, tri-, and / or oligopeptides are individually composed of a single amino acid, while in alternative embodiments the di-, tri-, and / or oligopeptides are individually Amino acids. In certain embodiments, the amount of amino acid is supplemented to the cell culture to about 0 to about 9 g / L for arginine, about 0 to about 11 g / L for lysine, about 0 to about 11 g / L for histidine, 0.0 > g / L < / RTI > for tin. About 0 to about 30 g / L for arginine, about 0 to about 30 g / L for lysine, about 0 to about 30 g / L for histidine, and about 0 to about 30 g / L for ornithine, The present invention is not limited thereto.

For example, if arginine is supplemented to the production medium used in the examples to achieve a total concentration of arginine of 9 g / L, as described in Example 1 below, The total amount of acidic species present in adalimumab was reduced from 19.7% of the control sample to 12.2% of the purified sample from cells incubated with arginine supplemented medium. Similarly, the production medium used in the examples was supplemented with lysine, or histidine, or ornithine to achieve a total concentration of 11 g / L lysine, 10 g / L ornithine or 10 g / L histidine, respectively, Were reduced by 11.5%, 10.4% and 10.9%, respectively, as compared to the control sample.

In certain embodiments, the cell culture medium is supplemented, for example, at the start of the culture, or in a fed-batch, or in a continuous mode. The feed rate can be calculated to achieve a predetermined concentration based on off-line or on-line measurements. The supplements may be immobilized by means of multimers, e.g. , arg-arg, his-his, arg-his-orn, and / or chemical variants of, for example , amino acid or analogues of amino acids, salt forms of amino acids, , And / or as a completely or partially dissolved form. The addition of one or more replenishers may be based on a measured amount of acid species. The obtained medium can be used in various culture methods including, but not limited to, batch, fed-batch, chemostat and perfusion, and in a shaking flask, a spinner flask, a stirrer But are not limited to, bioreactors, airlift bioreactors, membrane bioreactors, reactors using cells immobilized / trapped on a solid support or as in microporous beads, and any other configuration suitable for optimal growth and productivity of the desired cell line But not limited to, cell culture equipment.

To control acidic species (AR) CaCl ₂  And / or adjustment of the niacinamide concentration

In certain embodiments, the cell culture medium is supplemented with calcium ( e.g., calcium chloride dihydrate) to a concentration of about 0.05 to 2.5 mM, or about 0.05 to 1 mM, or about 0.1 to 0.8 mM, or about 0.15 to 0.7 mM, or A calcium concentration of about 0.2 to 0.6 mM, or about 0.25 to 0.5 mM, or about 0.3 to 0.4 mM.

In some embodiments, the cell culture medium contains up to about 15% AR, up to 14% AR, up to 13% AR, up to 12% AR, up to 11% AR, up to 10% AR, up to 9% AR, less than 8% AR, less than 7% AR, less than 6% AR, less than 5% AR, less than 4.5% AR, less than 4% AR, less than 3.5% AR, less than 3% AR, AR of less than 2.5%, AR of less than 2%, AR of less than 1.9%, AR of less than 1.8%, AR of less than 1.7%, AR of less than 1.6%, AR of less than 1.5%, AR of less than 1.4% AR of less than 1.2%, AR of less than 1.1%, AR of less than 1%, AR of less than 0.9%, AR of less than 0.8%, AR of less than 0.7%, AR of less than 0.6%, AR of less than or equal to 0.5% AR, no more than 0.4% AR, no more than 0.3% AR, no more than 0.2% AR, no more than 0.1% AR, or no more than 0.0% AR, Supplement calcium with an effective amount ( for example , calcium chloride dihydrate).

In another embodiment, the cell culture medium contains up to about 15% AR1, up to 14% AR1, up to 13% AR1, up to 12% AR1, up to 11% AR1, up to 10% AR1, up to 9% AR1 less than 8%, AR1 less than 7%, AR1 less than 6%, AR1 less than 5%, AR1 less than 4.5%, AR1 less than 4%, AR1 less than 3.5%, AR1 less than 3% AR1 less than 2%, AR1 less than 1.9%, AR1 less than 1.8%, AR1 less than 1.7%, AR1 less than 1.6%, AR1 less than 1.5%, AR1 less than 1.4%, AR1 less than 1.4% AR1 of not more than 1.2%, AR1 of not more than 1.1%, AR1 of not more than 1%, AR1 of not more than 0.9%, AR1 of not more than 0.8%, AR1 of not more than 0.7%, AR1 of not more than 0.6%, not more than 0.5% Of AR1, up to 0.4% of AR1, up to 0.3% of AR1, up to 0.2% of AR1, up to 0.1% of AR1, or 0.0% of AR1, Supplement calcium with an effective amount ( for example , calcium chloride dihydrate).

In yet another embodiment, the cell culture medium contains up to about 15% AR2, up to 14% AR2, up to 13% AR2, up to 12% AR2, up to 11% AR2, up to 10% AR2, up to 9% AR2 less than 8%, AR2 less than 7%, AR2 less than 6%, AR2 less than 5%, AR2 less than 4.5%, AR2 less than 4%, less than 3.5% AR2 less than 3% AR2 less than 2.5%, AR2 less than 2%, AR2 less than 1.9%, AR2 less than 1.8%, AR2 less than 1.7%, AR2 less than 1.6%, AR2 less than 1.5%, AR2 less than 1.4% AR2 of not more than 1.3%, AR2 of not more than 1.2%, AR2 of not more than 1.1%, AR2 of not more than 1%, AR2 of not more than 0.9%, AR2 of not more than 0.8%, AR2 of not more than 0.7%, AR2 of not more than 0.6% Or less of AR2, less than or equal to 0.4% AR2, less than or equal to 0.3% AR2, less than or equal to 0.2% AR2, less than or equal to 0.1% AR2, or 0.0% AR2, calcium in an amount effective supplements (e.g., calcium chloride dihydrate).

In another embodiment, the cell culture medium contains about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4% , 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% %, 80%, 85%, and calcium in an amount effective to 90%, 95%, 100%, and reduced by the range within one or more of the supplements (e.g., calcium chloride dihydrate).

For example, as described in Example 1 below, when calcium ( for example , calcium chloride dihydrate) is supplemented at a concentration of 1.05 mM to the production medium used in the examples , The total amount of acidic species of adalimumab present in the post-cell culture sample was reduced from 23.2% of the control sample to 16.5% of the purified sample from the cells cultured with the calcium supplemented medium.

In certain embodiments, the cell culture may supplement calcium, for example , a combination of CaCl ₂ and one or more basic amino acids, as described above. In certain embodiments, the amount of the basic amino acid concentration in the combination with calcium in the cell culture is about 0 to about 9 g / L for arginine, about 0 to about 11 g / L for lysine, about 0 to 11 g / L, and about 0 to about 11 g / L for ornithine. But are not limited to, about 0 to about 30 g / L of arginine, about 0 to 30 g / L of lysine, about 0 to about 30 g / L of histidine, and about 0 to about 30 g / The range is also within the scope of the present invention.

In certain embodiments, the cell culture medium is supplemented with niacinamide to achieve a niacinamide concentration of about 0.2 to 3.0 mM, or about 0.4 to 3.0 mM, or about 0.8 to 3.0 mM.

In some embodiments, the cell culture medium contains a heterogeneity of acidic species in the protein or antibody sample of less than about 15% AR, less than 14% AR, less than 13% AR, less than 12% AR, 11 % AR, less than 10% AR, less than 9% AR, less than 8% AR, less than 7% AR, less than 6% AR, less than 5% AR, less than 4.5% AR, less than 4% Of AR, less than 3.5% of AR, less than 3% of AR, less than 2.5% of AR, less than 2% of AR, less than 1.9% of AR, less than 1.8% of AR, less than 1.7% of AR, AR of less than 1.5%, AR of less than 1.4%, AR of less than 1.3%, AR of less than 1.2%, AR of less than 1.1%, AR of less than 1%, AR of less than 0.9%, AR of less than 0.8% % AR, up to 0.6% AR, up to 0.5% AR, up to 0.4% AR, up to 0.3% AR, up to 0.2% AR, up to 0.1% AR, or up to 0.0% AR, Supplementing the niacinamide in an amount effective to reduce by at least one or more ranges.

In another embodiment, the cell culture medium contains up to about 15% AR1, up to 14% AR1, up to 13% AR1, up to 12% AR1, up to 11% AR1, up to 10% AR1, up to 9% AR1 less than 8%, AR1 less than 7%, AR1 less than 6%, AR1 less than 5%, AR1 less than 4.5%, AR1 less than 4%, AR1 less than 3.5%, AR1 less than 3% AR1 less than 2%, AR1 less than 1.9%, AR1 less than 1.8%, AR1 less than 1.7%, AR1 less than 1.6%, AR1 less than 1.5%, AR1 less than 1.4%, AR1 less than 1.4% AR1 of not more than 1.2%, AR1 of not more than 1.1%, AR1 of not more than 1%, AR1 of not more than 0.9%, AR1 of not more than 0.8%, AR1 of not more than 0.7%, AR1 of not more than 0.6%, not more than 0.5% Of AR1, up to 0.4% of AR1, up to 0.3% of AR1, up to 0.2% of AR1, up to 0.1% of AR1, or of 0.0% of AR1, Lt; RTI ID = 0.0 > niacinamide < / RTI >

In yet another embodiment, the cell culture medium contains up to about 15% AR2, up to 14% AR2, up to 13% AR2, up to 12% AR2, up to 11% AR2, up to 10% AR2, up to 9% AR2 less than 8%, AR2 less than 7%, AR2 less than 6%, AR2 less than 5%, AR2 less than 4.5%, AR2 less than 4%, less than 3.5% AR2 less than 3% AR2 less than 2.5%, AR2 less than 2%, AR2 less than 1.9%, AR2 less than 1.8%, AR2 less than 1.7%, AR2 less than 1.6%, AR2 less than 1.5%, AR2 less than 1.4% AR2 of not more than 1.3%, AR2 of not more than 1.2%, AR2 of not more than 1.1%, AR2 of not more than 1%, AR2 of not more than 0.9%, AR2 of not more than 0.8%, AR2 of not more than 0.7%, AR2 of not more than 0.6% Of AR2, up to 0.4% of AR2, up to 0.3% of AR2, up to 0.2% of AR2, up to 0.1% of AR2, or of up to 0.02% of AR2, The niacinamide is supplemented with an effective amount as follows.

For example, as described in the following example 1, when the production medium used in the examples is supplemented with niacinamide at a concentration of 1.6 mM, The total amount of marbled acidic species was reduced from 19.9% of the control sample to 15.9% of the purified sample from the cells cultured with the niacinamide supplemented medium. In a separate example, when supplemented with 3 mM niacinamide in the medium, the total amount of acidic species of adalimumab present in the cell culture sample after purification was from 27.0% of the control sample to cells incubated with the niacinamide supplemented medium To 19.8% of the purified sample.

In certain embodiments, the cell culture may be supplemented with niacinamide, calcium, for example, CaCl _2, and / or one or more combinations of basic amino acid. In certain embodiments, the amount of basic amino acid concentration (after supplementation) in the combination with calcium in the cell culture is about 0 to about 9 g / L for arginine, about 0 to about 11 g / L for lysine, About 0 to about 11 g / L for ornithine, and about 0 to about 11 g / L for ornithine. But are not limited to, about 0 to about 30 g / L of arginine, about 0 to 30 g / L of lysine, about 0 to about 30 g / L of histidine, and about 0 to about 30 g / The range is also within the scope of the present invention.

In certain embodiments, one or more amino acids, calcium, and / or niacinamide may be included in the medium at the start of the culture, or added in a fed-batch or continuous mode. The feed rate can be calculated to achieve a predetermined concentration based on off-line or on-line measurements. The addition of the supplement can be based on the measured amount of the acidic species. Other salts of certain replenishers, such as calcium, for example, calcium nitrate, may also be used. The resulting medium can be used in a variety of culture methods including, but not limited to, batch, fed-batch, chemostatin and perfusion, and in a shaking flask, a spinner flask, a sterilized bioreactor, an air lift bioreactor But are not limited to, reactors, membrane bioreactors, reactors using cells immobilized / trapped, such as in microporous beads, held on a solid support, and any other configuration suitable for optimal growth and productivity of the desired cell line Can be used with cell culture equipment.

In certain embodiments, a low AR composition is prepared by replenishing purified water. For example, but not by way of limitation, this purified collection may be supplemented (as with, for example, calcium, niacinamide, and / or basic amino acids or combinations thereof) as described above to reduce AR formation (See Example 3).

Adjustment of process parameters to control acid species (AR)

In certain embodiments, the low AR composition is prepared by adjusting the dissolved oxygen (DO) concentration and / or the pH of the cell culture run. In certain embodiments, such an adjustment comprises increasing the DO concentration of the cell culture or decreasing the pH of the cell culture. This increase in DO concentration or decrease in pH can be reduced by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% And may be in the range of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%

In certain embodiments, the cell culture comprises more than about 15%, greater than about 20%, greater than about 30%, or about 15% to about 80%, about 30% to about 50%, or about 80% Lt; RTI ID = 0.0 > DO < / RTI >

In certain embodiments, the pH is decreased or increased to increase or decrease the amount of acid species and / or the rate at which such acid species are formed. For example, but not by way of limitation, a pH reduction from a control pH of about 7.1 to about 6.7 can be used to reduce the rate of formation of acidic species and acid species associated with purified water during cell culture.

In certain embodiments, the DO concentration and / or pH is less than or equal to about 15% AR, less than or equal to 14% AR, less than or equal to 13% AR, less than or equal to 12% AR, less than or equal to 11% , Less than 9% AR, less than 8% AR, less than 7% AR, less than 6% AR, less than 5% AR, less than 4.5% AR, less than 4% AR, less than 3.5% AR, % AR, less than 2.5% AR, less than 2% AR, less than 1.9% AR, less than 1.8% AR, less than 1.7% AR, less than 1.6% AR, less than 1.5% AR, less than 1.4% AR Of AR, AR of less than 1.3%, AR of less than 1.2%, AR of less than 1.1%, AR of less than 1%, AR of less than 0.9%, AR of less than 0.8%, AR of less than 0.7%, AR of less than 0.6% , Less than 0.5% AR, less than 0.4% AR, less than 0.3% AR, less than 0.2% AR, less than 0.1% AR, or 0.0% AR, Lt; / RTI >

In other embodiments, the DO concentration and / or pH is less than or equal to about 15% AR1, less than 14% AR1, less than 13% AR1, less than 12% AR1, less than 11% AR1, less than 10% AR1 less than 9%, AR1 less than 8%, AR1 less than 7%, AR1 less than 6%, AR1 less than 5%, AR1 less than 4.5%, AR1 less than 4%, AR1 less than 3.5% AR1 less than 2.5%, AR1 less than 2%, AR1 less than 1.9%, AR1 less than 1.8%, AR1 less than 1.7%, AR1 less than 1.6%, AR1 less than 1.5%, less than 1.4% AR1 AR1, up to 1.3% of AR1, up to 1.2% of AR1, up to 1.1% of AR1, up to 1% of AR1, up to 0.9% of AR1, up to 0.8% of AR1, up to 0.7% of AR1, A low AR composition comprising not more than 0.5% AR1, not more than 0.4% AR1, not more than 0.3% AR1, not more than 0.2% AR1, not more than 0.1% AR1, or 0.0% AR1, Lt; / RTI >

In yet another embodiment, the DO concentration and / or pH is less than about 15% AR2, less than 14% AR2, less than 13% AR2, less than 12% AR2, less than 11% AR2, less than 10% AR2 less than 9%, AR2 less than 8%, AR2 less than 7%, AR2 less than 6%, AR2 less than 5%, AR2 less than 4.5%, AR2 less than 4%, AR2 less than 3.5% AR2 of not more than 3%, AR2 of not more than 2.5%, AR2 of not more than 2%, AR2 of not more than 1.9%, AR2 of not more than 1.8%, AR2 of not more than 1.7%, AR2 of not more than 1.6%, AR2 of not more than 1.5% AR2 of not more than 1.3%, AR2 of not more than 1.2%, AR2 of not more than 1.1%, AR2 of not more than 1%, AR2 of not more than 0.9%, AR2 of not more than 0.8%, AR2 of not more than 0.7%, AR2 of not more than 0.6% AR2, less than 0.5% AR2, less than 0.4% AR2, less than 0.3% AR2, less than 0.2% AR2, less than 0.1% AR2, or 0.0% AR2, AR < / RTI >

In certain embodiments, the pH and / or DO are selected such that the amount of acidic species in the protein or antibody sample is about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2% 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% , 75%, 80%, 85%, 90%, 95%, 100%, and ranges within at least one of the above.

For example, if five different pH conditions: 7.1, 7.0, 6.9, 6.8, and 6.7 were assessed during cell culture as described in Example 2 below, the percentage of acid species would be pH 7.1 Reduction from pH 2.7% at pH 6.7 to 21.5% at pH 6.7.

In addition, three different DO concentrations: 20% DO concentration, 30% DO concentration, and 50% DO concentration were evaluated during cell culture at 35 DEG C, as described in Example 2 below. The percentage of acid species as a whole was lower at higher DO concentrations. In particular, the percentage of acid species was reduced by a total of 3.6% reduction with an increase in DO concentration from 23.9% at 20% DO concentration sample to 20.3% at 50% DO concentration sample.

In certain embodiments, a low AR composition produced by cell culture can be obtained by culturing a cell culture that expresses the desired protein as described herein with a DO concentration and / or pH, with a suitable temperature or temperature shift strategy But are not limited to, for example, by maintaining a lower process operating temperature, a temperature transition to a lower temperature, or a selection of temperature transition at an initial incubation time. These culture conditions can be used in various culture methods including, but not limited to, batch, fed-batch, chemostat and perfusion, and in a shaking flask, a spinner flask, a sterilized bioreactor, But are not limited to, bioreactors, membrane bioreactors, reactors using cells immobilized / trapped on a solid support or as in microporous beads, and any other configuration suitable for optimal growth and productivity of the desired cell line It can be used with various cell culture equipment. In addition, these methods of controlling pH and / or DO and / or temperature may be used to maintain or achieve an AR of a target level, or to reduce the formation of AR during cell culture, , Niacinamide, and / or calcium, or a combination thereof.

Continuous / perfusion cell culture technology to regulate acidic species (AR)

In certain embodiments, the low AR composition is prepared by selection of a cell culture method. In certain embodiments, the use of continuous or perfusion techniques can be used to achieve the desired reduction of acid species in the combination. In some embodiments, this can be achieved by adjusting the medium exchange rate, wherein the exchange rate is the exchange rate of the inlet / outlet (in / out) of the reactor.

In certain non-limiting embodiments, the media exchange rate (working volume per day) of a cell culture run of from about 0 to about 20, or from about 0.5 to about 12, or from about 1 to about 8, or from about 1.5 to about 6, ) Can be used to achieve the desired reduction of acid species.

For example, as described in Example 4 below, when the medium exchange rate was selected to be 1.5, the acid species was 8.1%. With an additional increase in the exchange rate to 6, an additional reduction of the acid species to 6% was obtained.

In certain embodiments, the continuous or perfusion technique ( e.g., adjustment of the exchange rate) may include up to about 15% AR, up to 14% AR, up to 13% AR, up to 12% AR, up to 11% AR, less than 10% AR, less than 9% AR, less than 8% AR, less than 7% AR, less than 6% AR, less than 5% AR, less than 4.5% AR, less than 4% AR of less than 3.5%, AR of less than 3%, AR of less than 2.5%, AR of less than 2%, AR of less than 1.9%, AR of less than 1.8%, AR of less than 1.7%, AR of less than 1.6% AR, AR of less than 1.4%, AR of less than 1.3%, AR of less than 1.2%, AR of less than 1.1%, AR of less than 1%, AR of less than 0.9%, AR of less than or equal to 0.8%, less than or equal to 0.7% AR, AR of less than 0.6%, AR of less than 0.5%, AR of less than 0.4%, AR of less than 0.3%, AR of less than 0.2%, AR of less than or equal to 0.1%, or AR of less than or equal to 0.0% Lt; RTI ID = 0.0 > AR < / RTI >

In other embodiments, continuous or perfusion techniques (e.g., adjustment of the exchange rate) may include up to about 15% AR1, up to 14% AR1, up to 13% AR1, up to 12% AR1, up to 11% AR1 less than 10%, AR1 less than 9%, AR1 less than 8%, AR1 less than 7%, AR1 less than 6%, AR1 less than 5%, AR1 less than 4.5% AR1 less than 3.5%, AR1 less than 3%, AR1 less than 2.5%, AR1 less than 2%, AR1 less than 1.9%, AR1 less than 1.8%, AR1 less than 1.7%, AR1 less than 1.6% AR1 of not more than 1.4%, AR1 of not more than 1.3%, AR1 of not more than 1.2%, AR1 of not more than 1.1%, AR1 of not more than 1%, AR1 of not more than 0.9%, AR1 of not more than 0.8%, AR1 of not more than 0.7% AR1, up to 0.6% of AR1, up to 0.5% of AR1, up to 0.4% of AR1, up to 0.3% of AR1, up to 0.2% of AR1, up to 0.1% of AR1, or of up to 0.0% of AR1, Lt; RTI ID = 0.0 > AR < / RTI >

In yet another embodiment, the continuous or perfusion technique (e.g., adjustment of the exchange rate) comprises up to about 15% AR2, up to 14% AR2, up to 13% AR2, up to 12% AR2, up to 11% AR2 less than 10%, AR2 less than 9%, AR2 less than 8%, AR2 less than 7%, AR2 less than 6%, AR2 less than 5%, AR2 less than 4.5%, AR2 less than 4% , AR2 of not more than 3.5%, AR2 of not more than 3%, AR2 of not more than 2.5%, AR2 of not more than 2%, AR2 of not more than 1.9%, AR2 of not more than 1.8%, AR2 of not more than 1.7%, AR2 of not more than 1.6% AR2 of not more than 1.4%, AR2 of not more than 1.3%, AR2 of not more than 1.3%, AR2 of not more than 1.2%, AR2 of not more than 1.1%, AR2 of not more than 1%, AR2 of not more than 0.9%, AR2 of not more than 0.8% AR2 of not more than 0.6%, AR2 of not more than 0.5%, AR2 of not more than 0.4%, AR2 of not more than 0.3%, AR2 of not more than 0.2%, AR2 of not more than 0.1% or AR2 of not more than 0.0% ≪ RTI ID = 0.0 > AR < / RTI >

In certain embodiments, continuous or perfusion techniques (e. G., Adjustment of exchange rate) may be performed at a rate of about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2% %, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% AR compositions comprising ranges of at least 75%, 80%, 85%, 90%, 95%, 100%, and at least one of the foregoing.

In one embodiment, a medium containing additives such as, for example, one or more amino acids as described above, calcium and / or niacinamide, or combinations thereof, is used as the perfusion medium to maintain the target level of AR Or to reduce the formation of AR during cell culture.

In certain embodiments, low AR compositions are prepared, for example, by use of an intermittent collection strategy, or through the use of cell holding device technology.

IV. Downstream  Preparation of low AR compositions using process technology

In certain embodiments, the low AR composition of the present invention can be prepared using downstream process technology (e.g., purification) following cell culture of the protein. The downstream process technology may be used alone or in conjunction with the upstream process technology described in Sections III and 10 above.

The methods described herein for the preparation of compositions comprising low AR and / or low process-related impurities are, for example, multimodal (MM) media in which the ion exchange and hydrophobic interaction functional groups and E. G., An antibody or antigen-binding portion thereof, by chromatography such as multimodal (MM) chromatography involving both aqueous salt solutions. In one embodiment, the same or substantially the same aqueous salt solution is used as loading buffer and wash buffer.

In a further embodiment, the process described herein for the preparation of a composition comprising low AR and / or low process-related impurities comprises contacting the protein by chromatography comprising an anion exchange (AEX) resin and an aqueous salt solution , E. G., Purification of the antibody or antigen-binding portion thereof. In one embodiment, the same or substantially the same aqueous salt solution is used as loading buffer and wash buffer.

Also in a further embodiment, the process described herein for the preparation of a composition comprising low AR and / or low process-related impurities is carried out by chromatography comprising a cation exchange (CEX) adsorption resin and an aqueous salt solution Lt; / RTI > protein, e. G., An antibody or antigen-binding portion thereof. In one embodiment, the same or substantially the same aqueous salt solution is used as the loading buffer and wash buffer, and the target protein bound to the CEX adsorption resin is eluted with a buffer having a conductivity and / or pH higher than the loading / wash buffer .

Further, in a further embodiment, the methods described herein for the preparation of compositions comprising low AR and / or low process-related impurities can be carried out by using, for example, anion exchange (AEX) And purification of proteins, e. G., Antibodies or antigen-binding portions thereof, by chromatography with a suitable buffer, e. G., Cation exchange (CEX) adsorption resin in a Tris / formate buffer system. In one embodiment, the sample is a purified affinity chromatographic medium, e. G., Protein A, prior to ion chromatography. For example, in one embodiment, the method described herein for the manufacture of a composition comprising a low AR comprises an exemplary process as reflected in FIG.

In one embodiment, a method for preparing a low AR composition comprising an antibody or antigen-binding portion thereof comprises contacting a first sample comprising an antibody or antigen-binding portion thereof with a loading buffer (e.g., a low concentration of Tris / formate buffer ), Eluting the sample from the affinity chromatography medium as a first eluting sample, and eluting the first eluting sample with a first chromatographic medium in a loading buffer, for example, AEX chromatography Contacting the sample with a resin and eluting the sample from the AEX chromatography resin as a second eluting sample. The second eluted sample is then contacted with a second chromatographic medium in a loading buffer, e. G., A CEX chromatography resin, and the sample is eluted as a third eluting sample from the CEX chromatography resin. In one embodiment, the CEX chromatography resin is eluted once, twice, three times, or more. In one embodiment, the process optionally comprises at least one intermediate filtration step, a pH adjustment step and an inactivation step.

In one embodiment, the downstream process technology described herein may be used alone or in combination with other downstream process technologies, or in combination with one or more upstream process technologies, to produce up to 15% AR, 14% AR, 13% Or less AR, 12% or less AR, 11% or less AR, 10% or less AR, 9% or less AR, 8% or less AR, 7% or less AR, 6% or less AR, AR, AR of less than 4.5%, AR of less than 4%, AR of less than 3.5%, AR of less than 3%, AR of less than 2.5%, AR of less than 2%, AR of less than 1.9% AR of less than 1.7%, AR of less than 1.6%, AR of less than 1.5%, AR of less than 1.4%, AR of less than 1.3%, AR of less than 1.2%, AR of less than 1.1%, AR of less than or equal to 1% AR, AR of not more than 0.8%, AR of not more than 0.7%, AR of not more than 0.6%, AR of not more than 0.5%, AR of not more than 0.4%, AR of not more than 0.3%, AR of not more than 0.2%, AR of not more than 0.1% AR, or 0.0% AR, and ranges within one or more of the above. Lt; RTI ID = 0.0 > AR < / RTI > In one aspect of this embodiment, a low AR composition of the invention comprises from about 0.0% to about 10% AR, from about 0.0% to about 5% AR, from about 0.0% to about 4% AR, From about 3% to about 5% AR, from about 5% to about 8% AR, or from about 8% to about 10% AR, or from about 10% To about 15% AR, and ranges within one or more of the above.

In one embodiment, the downstream process technology described herein may be used alone or in combination with other downstream process technologies, or in combination with one or more upstream process technologies, to produce up to 15% AR1, up to 14% AR1, 13% AR1 less than 12%, AR1 less than 11%, AR1 less than 10%, AR1 less than 9%, AR1 less than 8%, AR1 less than 7%, AR1 less than 6%, AR1 less than 5% AR1, 4.5% or less AR1, 4% or less AR1, 3.5% or less AR1, 3% or less AR1, 2.5% or less AR1, 2% or less AR1, 1.9% or less AR1, AR1 of not more than 1.7%, AR1 of not more than 1.6%, AR1 of not more than 1.5%, AR1 of not more than 1.4%, AR1 of not more than 1.3%, AR1 of not more than 1.2%, AR1 of not more than 1.1%, AR1 of not more than 1% AR1 of not more than 0.8%, AR1 of not more than 0.7%, AR1 of not more than 0.6%, AR1 of not more than 0.5%, AR1 of not more than 0.4%, AR1 of not more than 0.3%, AR1 of not more than 0.2% AR1, or 0.0% AR1, and a range within one or more of the above ranges A low AR composition comprising the antibody or antigen-binding parts thereof prepared containing. In one aspect of this embodiment, a low AR composition of the invention comprises from about 0.0% to about 10% AR1, from about 0.0% to about 5% AR1, from about 0.0% to about 4% AR1, About 3% to about 5% AR1, about 5% to about 8% AR1, or about 8% to about 10% AR1, or about 10% To about 15% of AR1, and ranges within one or more of the above.

In one embodiment, the downstream process techniques described herein may be used alone or in combination with other downstream process technologies, or in combination with one or more upstream process technologies, to produce up to 15% AR2, up to 14% AR2, 13% AR2 less than 12%, AR2 less than 11%, AR2 less than 10%, AR2 less than 9%, AR2 less than 8%, AR2 less than 7%, AR2 less than 6%, AR2 less than 5% AR2 of not more than 4.5%, AR2 of not more than 4.5%, AR2 of not more than 3.5%, AR2 of not more than 3%, AR2 of not more than 2.5%, AR2 of not more than 2%, AR2 of not more than 1.9% AR2 of not more than 1.7%, AR2 of not more than 1.6%, AR2 of not more than 1.5%, AR2 of not more than 1.4%, AR2 of not more than 1.3%, AR2 of not more than 1.2%, AR2 of not more than 1.1%, AR2 of not more than 1% AR2 of not more than 0.8%, AR2 of not more than 0.7%, AR2 of not more than 0.6%, AR2 of not more than 0.5%, AR2 of not more than 0.4%, AR2 of not more than 0.3%, AR2 of not more than 0.2% AR2, or 0.0% AR2, and a range within one or more of the above ranges A low AR composition comprising the antibody or antigen-binding parts thereof prepared containing. In one aspect of this embodiment, a low AR composition of the present invention comprises from about 0.0 to about 10% AR2, from about 0.0% to about 5% AR2, from about 0.0% to about 4% AR2, About 3% to about 5% AR2, about 5% to about 8% AR2, or about 8% to about 10% AR2, or about 10% AR2, To about 15% AR2, and ranges within one or more of the above.

General protein purification

According to the upstream processing of the desired protein, the protein can be purified using downstream process technology. Once a purified solution or mixture containing, for example, a desired protein, e. G., An antibody or antigen-binding fragment thereof, is obtained, separation of the desired protein from the acidic species is achieved by affinity A combination of different purification techniques including, but not limited to, a separation step, an ion exchange separation step, a mixed mode separation step, and a hydrophobic interaction separation step, alone or in combination. The separation step separates the mixture of proteins based on their charge, degree of hydrophobicity, or size, or a combination thereof, depending on the particular type of separation, including chromatographic separation. In one aspect of the invention, the separation is carried out using chromatography involving cationic, anionic and hydrophobic interactions. Several different chromatographic resins can be used for each of these techniques to enable accurate tailoring of the purification scheme for the specific protein involved. Each separation method allows the protein to cross at a different rate through the column, thereby achieving a physical separation that increases as they pass further through the column or selectively attach to the separation medium. Proteins are then differentially eluted by different elution buffers. In some cases, the antibody is separated from the impurities, that is, the desired antibody variant is present in the Flow Through when the impurities are preferentially attached to the column and the antibody is less attached.

In certain embodiments, a low AR composition may be prepared using chromatographic separation to obtain the desired fractionation, e. G., Fractionation of acidic species and lysine variants of the sample comprising the desired protein and one or more process-related impurities Identify certain conditions sufficient to cause the profile, such as salt concentration, pH, DO concentration, temperature, loading and conditions, and wash conditions. In certain embodiments, the method further comprises pooling the obtained fractions containing the desired low AR composition.

The purification process can be initiated in the separation step using the upstream preparation method described above and / or after manufacturing the antibody by a conventional alternative manufacturing method in the art. Once a desired protein, e. G., A purified solution or mixture comprising an antibody is obtained, separation of the desired protein from the process-related impurities, e. G., Other proteins produced by the cell, and product- Separation of the material, e.g., acidic or basic variants, is carried out. In certain non-limiting embodiments, such separation is performed using CEX, AEX, and / or MM chromatography. In certain embodiments, a combination of one or more different purification techniques including affinity separation step (s), ion exchange separation step (s), mixing-type step (s), and / or hydrophobic interaction separation step Can also be used. These additional purification steps separate the mixture of proteins based on the charge, hydrophobicity, and / or size of the proteins. In one aspect of the invention, these additional separation steps are performed using chromatography comprising hydrophobic, anionic or cationic interactions (or combinations thereof). A number of chromatographic resins can be utilized for each of these techniques to enable accurate tailoring of the purification scheme for the particular protein involved. Each separation method allows the protein to cross at a different rate through the column to achieve physical separation that increases as they pass further through the column or selectively attach to the separation medium (or medium). Then, the proteins are differentially eluted by different elution eluents. In some cases, the desired protein is separated from the impurity when the impurity specifically adheres to the resin of the column and the target protein does not specifically and specifically adhere, that is, the desired protein is separated from the effluent , While in other cases the desired protein will attach to the resin of the column while the impurities and / or product-related substances are extracted from the resin of the column during the washing cycle.

Initial Recall and Virus Inactivation

In certain embodiments, the initial steps of the purification method of the present invention include purification and initial recovery of the antibody from the sample matrix. In certain embodiments, the initial recovery will include one or more centrifugation steps to separate the antibody product from the cells and cell debris. Centrifugation of the sample can be performed, for example, without limitation, from 7,000 x g to about 12,750 x g. With respect to large-scale purification, such centrifugation may occur on-line, for example, without limitation, at a flow rate set to achieve a turbidity level of 150 NTU in the resulting supernatant. These supernatants may then be collected for further purification or in-line filtered through one or more in-depth filters for further purification of the sample.

In certain embodiments, the initial recovery will include the use of one or more in-depth filtration steps to purify the sample matrix to assist in the purification of the subject antibodies of the invention. In another embodiment, the initial recovery will include the use of one or more in-depth filtration steps after centrifugation to further purify the sample matrix. Non-limiting examples of deep filters that can be used in connection with the present invention include Millistak + X0HC, F0HC, D0HC, A1HC, B1HC EMD Millipore, Cuno ™ Model 30 / 60ZA, 60/90 ZA, VR05, VR07, (delipid) deep filter (3M Corp.). A 0.2 μm filter, for example, a Sartorius 0.45 / 0.2 μm Sartopore ™ bi-layer or Millipore Express SHR or SHC filter cartridge is a typical deep filter.

In some embodiments, the initial recovery process may also be a time for reducing or inactivating viruses that may be present in the sample matrix. For example, any one or more of the various methods of virus reduction / inactivation can be performed by heat inactivation (pasteurization), pH inactivation, buffer / detergent treatment, UV and gamma irradiation, The same predetermined chemically inactivated agent, for example, US Pat. May be used during the initial recovery phase of the tablet, including the addition of copper phenanthroline as described in US Pat. No. 4,534,972. In certain embodiments of the invention, the sample matrix is exposed to detergent virus deactivation during the initial recovery phase. In another embodiment, the sample matrix is exposed to low pH deactivation during the initial recovery phase.

In these embodiments where virus reduction / inactivation is used, the sample mixture may be adjusted for further purification steps, as needed. For example, according to low pH virus inactivation, the pH of the sample mixture is typically adjusted to a more neutral pH, e. G., About 4.5 to about 8.5, before continuing with the purification process. In addition, the mixture is diluted with water for injection (WFI) to obtain the desired conductivity.

Additives to purified water

In certain embodiments, a low AR composition is prepared by supplementing a purified collection containing an antibody or antigen-binding portion thereof. The clarified collection can be removed from the cell culture, e. G., From a fermentation bioreactor after centrifugation to remove large solid particles and subsequent filtration to remove impurities from finer solid particles and materials have. Such purified collection may be supplemented (e.g., with calcium, niacinamide and / or basic amino acids, or combinations thereof) as described above, or may be supplemented to control, for example, to reduce AR formation the pH can be lowered (see Example 3).

Affinity chromatography

In certain embodiments, it may be advantageous to further purify the desired protein from the acid species by affinity chromatography on samples prepared by the techniques of the present invention. In certain embodiments, the chromatography material may selectively or specifically bind to the desired ("capture") protein. Non-limiting examples of such chromatographic materials include protein A, protein G, a chromatographic material comprising, for example, an antigen bound by the desired antibody, and a chromatographic material comprising an Fc binding protein. In certain embodiments, the affinity chromatography step comprises passing the original recovered sample through a column containing a suitable protein A resin. In certain embodiments, the protein A resin is useful for the isolation of affinity purification and various antibody isotypes, particularly IgG1, IgG2, and IgG4. Protein A is a bacterial cell wall protein that first binds to mammalian IgG through their Fc region. In its original state, protein A has five IgG binding domains and other domains of unknown function.

There are several commercial sources for protein A resins. One suitable resin is MabSelect ™ from GE Healthcare. Suitable resins include MabSelect SuRe (TM), MabSelect SuRe LX, MabSelect, MabSelect, MabSelect Xtra, rProtein A Sepharose from GE Healthcare, ProSep HC, ProSep Ultra, and ProSep Ultra Plus from EMD Millipore, MapCapture from Life Technologies, . A non-limiting example of a suitable column packed with MabSelect (TM) is a 1.0 cm diameter x approximately 21.6 cm long column (approximately 17 mL bed volume). Columns of this size are available for small scale purification and can be compared to other columns used for scale up. For example, a 20 cm x 21 cm column with a bed volume of about 6.6 L can be used for larger scale tablets. Regardless of the column, the column can be packed using a suitable resin such as MabSelect ™.

The protein A column can be equilibrated to the appropriate buffer prior to sample loading. After loading the column, a suitable set of buffers may be used to wash the column one or more times. Protein A column can then be eluted using a suitable elution buffer. For example, glycine-HCL or citric acid may be used as the elution buffer. The eluate can be monitored using techniques well known to those skilled in the art. The desired eluate fractions can be collected and then prepared for further processing.

The protein A eluate can be subjected to a virus inactivation step with a detergent or a low pH, but this step is not performed prior to the protein A capture operation. The appropriate detergent concentration or pH and time can be selected to obtain the desired virus inactivation result. After viral inactivation, the protein A eluate is generally pH and / or conductivity adjusted for subsequent purification steps.

The protein A eluate can be filtered through a deep filter to remove turbidity and / or various impurities from the desired antibody prior to further chromatographic polishing steps. Examples of in-depth filters include Miilistak + X0HC, F0HC, D0HC, A1HC, and B1HC Pod filters (EMD Millipore) or Zeta Plus 30ZA / 60ZA, 60ZA / 90ZA, degreasing, VR07, and VR05 filters But is not limited thereto. The protein A eluate pool may need to be conditioned to the proper pH and conductivity to obtain the desired impurity removal from the deep filtration step and product recovery.

The present invention is not limited to the capture of the desired protein using protein A chromatography. Non-protein A chromatography capture steps can also be performed. For example, cation exchange capture and non-chromatographic methods, such as aqueous two-phase extraction or precipitation, or other methods known in the art may be used.

Anion exchange chromatography

In certain embodiments, the low AR composition is prepared by subjecting the originally recovered sample to one or more anion exchange separation steps. In certain embodiments, the anion exchange step will occur after affinity chromatography, e. G., A protein A affinity step as described above.

The use of a cation exchange material, such as a cation exchange material, discussed in greater detail below with respect to anion exchange materials, is based on the local charge of the desired protein in a given solution. Therefore, it is within the scope of the present invention to use the anion exchange step before use of the cation exchange step, or the cation exchange step before the anion exchange step. It is also possible to use a combination of anion exchange step single, cation exchange step single, or any series of two steps (including a series of ion exchange steps of one or both of the other chromatographic separation techniques described herein) Is within the scope of the present invention.

In performing the separation, the initial protein composition may be contacted with an anion exchange material by using any of a variety of techniques, for example, by using a batch purification technique or a chromatography technique.

For example, in connection with batch purification, the anion exchange material is prepared in the desired starting buffer or equilibrated to the desired starting buffer. Upon manufacture or equilibration, a slurry of anion exchange material is obtained. The desired protein, e. G., Antibody solution, is contacted with the slurry to enable protein adsorption to the anion exchange material. Solutions containing acidic species that are not bound to the AEX material are separated from the slurry, for example, by allowing the slurry to settle and removing the supernatant. The slurry may be subjected to one or more washing steps and / or elution steps.

With respect to chromatographic separation, a chromatographic apparatus, which is typically cylindrical, is used to contain the chromatographic support material (e.g., AEX material) prepared in an appropriate buffer. If cylindrical, the chromatographic apparatus may have a diameter of about 5 mm to about 2 m, and a height of 5 cm to 50 cm, and in some embodiments use a height of 30 cm or less, particularly for large-scale processing. Once the chromatographic material is added to the chromatographic apparatus, the sample containing the desired protein, for example an antibody, is contacted with the chromatographic material to induce separation. Any portion of the solution that does not bind to the chromatographic material, which may include, for example, the desired protein, acidic species, depending on the AEX material used, can be removed by washing the material and collecting fractions from the column, . The chromatography material may be subjected to one or more washing steps. Then, if desired, the chromatographic material may be contacted with a solution designed to desorb any constituents in the solution bound to the chromatographic material.

In certain embodiments, the washing step may be performed in conjunction with AEX chromatography using conditions similar to loading conditions, or alternatively, by decreasing the pH of the wash liquor in a step wise or linear gradient fashion and / Can be carried out by increasing the conductivity. The resulting passing and washing fractions can be analyzed and appropriate fractions can be pooled to achieve the desired reduction of the charged variant species. In certain embodiments, the aqueous salt solution used as both the loading buffer and the washing buffer has an isoelectric point (pI) of the desired protein or a pH near the isoelectric point. In some embodiments, the pH is about 0-2 units higher or lower than the pI of the desired protein. In some embodiments, this may be in the range of 0 to 0.5 units higher or lower. In some embodiments, this will be the pI of the antibody.

In certain non-limiting embodiments, the anionic agent is selected from the group consisting of acetate, formate, or combinations thereof. In certain non-limiting embodiments, the cationic agent is selected from the group consisting of Tris, arginine, or combinations thereof. In one embodiment, the buffer is a Tris / formate buffer. In another embodiment, the buffer is selected from the group consisting of pyridine, piperazine, L-histidine, Bis-tris, Bis-tris propane, imidazole, N- ethylmorpholine, TEA (triethanolamine), Tris, morpholine, (2-amino-2-methyl-1,3-propanediol), diethanolamine, ethanolamine, AMP Diaminopropane, piperidine, and the like.

A packed anion-exchange chromatography column, anion-exchange membrane device, anion-exchange monolithic device, or a deep filter media can be used in a bind-elute, flow-through, Can operate in a hybrid manner indicative of binding to this chromatographic material and can also be washed from the column using a buffer that is the same as or substantially similar to the loading buffer. In the coupled-elution mode, the column or membrane device is conditioned with a buffer having appropriate ionic strength and pH, under the condition that the predetermined protein is immobilized on the resin-based matrix. For example, in some embodiments, during the feed liquid loading, the desired protein will be adsorbed to the resin due to electrostatic attraction. The column or membrane device is washed with an equilibration buffer or other buffer having a different pH and / or conductivity, and then the ionic strength (i.e., conductivity) of the elution buffer is increased to compete with the solute for the charged portion of the anion exchange matrix, Thereby achieving recovery. Changing the pH and thereby altering the charge of the solute is another way to achieve dissolution of the solute. The change in conductivity or pH may be gradual (concentration gradient elution) or stepwise (step elution). In the pass-through mode, the column or membrane device is maintained at a selected pH and conductivity such that the desired protein does not bind to the resin or membrane while the acidic species remains on the column or has a distinct elution profile relative to the desired protein . In connection with this hybrid strategy, the acidic species will bind to the chromatographic material (or the passage liquid) in a manner distinct from the desired protein, for example, while the desired protein and certain aggregates of the desired protein and / Or fragments may bind to the chromatography material and a wash may be applied to preferentially remove the desired protein. The column is then regenerated before the next use.

Non-limiting examples of anion exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and quaternary amine (Q) groups. Additional non-limiting examples include Poros 50PI and Poros 50HQ, which are rigid polymer beads having a backbone of cross-linked poly [styrene-divinylbenzene]; The high flow agarose beads Capto Q Impres and Capto DEAE; Polymeric basic beads Toyopearl QAE-550, Toyopearl DEAE-650, and Toyopearl GigaCap Q-650; Fractogel ^® EMD TMAE Hicap, a synthetic polymer resin with a tentacle ion exchanger; Sartobind ^® STIC nano, a salt-resistant chromatographic membrane with primary amine ligands; Sartobind Q nano, a strong anion exchange chromatography membrane; CUNO BioCap, an in-depth filter media prepared from inorganic filter aids, purified celluloses, and ion exchange resins; And XOHC, an in-depth filter media made from inorganic filter aids, cellulose, and mixed cellulose esters. Detailed information is listed in Table 1.

In certain embodiments, the protein loading of the mixture comprising the desired protein is adjusted to a total protein loading of about 50 to 500 g / L, or about 75 to 350 g / L, or about 200 to 300 g / L for the column. In certain embodiments, the protein concentration of the loaded protein mixture is adjusted to a protein concentration of the material for the column of about 37 g / L.

In some embodiments, additives may be added to improve the performance of separation, for example, polyethylene glycol, detergents, amino acids, sugars, chaotropic agents to achieve better recovery or product quality.

In certain embodiments, including, but not limited to, those associated with azalimumab, the methods of the present invention may be used to selectively remove, significantly reduce, or reduce the AR in the passing solution and wash fraction, while concentrating the AR in the flow- , Essentially all of which can be used to produce protein compositions with reduced AR or without AR. In certain embodiments of the tablet of Adalimumab, the method of the invention selectively removes, significantly reduces, or reduces AR1 charge variants in the passing and washing fractions, while concentrating AR1 charge variants in the flow eluting fraction , Essentially all of which can be used to produce protein compositions with reduced AR1 or without AR1 variants. In certain embodiments of adalimumab, the method of the present invention comprises selectively removing, significantly reducing, or reducing the AR2 charge variant in the passing and washing fractions, while concentrating AR2 charge variants in the flow- Lt; RTI ID = 0.0 > AR2 < / RTI > variants.

In certain embodiments, including, but not limited to, those associated with azalimumab, the methods of the present invention include selectively removing or significantly reducing MGO variants in the passing and washing fractions, while concentrating the MGO variants in the eluted fraction Or by essentially eliminating all of it, can be used to produce a protein composition with reduced MGO or no MGO variant (see, e. G., U. S. Patent Application Serial No. 61 / 777,883). In certain embodiments, including but not limited to, those associated with azalimumab, the method of the present invention is characterized by the concentration of glycated variants in the eluting fraction and the glycosylated variants (Schiff bases and permanent Glycosylated forms of glycosylated glycosaminoglycans) can be selectively removed, significantly reduced, or essentially eliminated altogether to produce protein compositions that do not contain reduced or glycosylated variants.

In certain embodiments, the load, pH, conductivity, and elution pH conductivity of the AEX chromatography step can be modified to achieve a desired distribution of the product-related material (AR or lysine variant). For example, but not by way of limitation, certain embodiments relate to the modulation of the lysine distribution of a purified sample of a desired protein, for example, an increase in Lys 0 and a decrease in Lys 1 and Lys 2. In certain embodiments, the method of the invention enables the production of a sample wherein the amount of Lys 0 is reduced while the amount of Lys 1 and / or Lys 2 is increased.

In certain embodiments, AEX chromatographic separation can be performed and, in addition, or instead of merely adjusting the charge mutant concentration, the combination of fractions can be pooled to provide desired process-related impurities and / or product- A combination can be achieved.

The level of AR species can be monitored on-line, at-line or in-line using spectroscopic methods such as UV, NIR, FTIR, fluorescence, and Raman, The spectroscopic method can be used to control the level of charge variants in the pooled material, e. G., Acidic species, collected from the AEX eluate.

In certain embodiments, certain signals resulting from chemical modification of proteins, such as glycosylation, MGO modification, deamidation, glycosylation, are those that result in the production of such in- Line, on-line, or by a spectroscopic method through an at-line method. In some embodiments, on-line, at-line, or in-line monitoring methods may be used for the effluent line of the chromatography step or in the collection vessel to enable achievement of desired product quality / recovery. In some embodiments, the UV signal can be used as a surrogate to achieve a suitable product quality / recovery, wherein the UV signal is selected from the group consisting of integration, differentiation, moving average, Such as, but not limited to, normal process variability and can be suitably processed such that target product quality can be achieved. In certain embodiments, such measurements can be combined with an in-line dilution method, whereby the ion concentration / conductivity of the load / wash can be controlled by feedback, thus product quality control can also be enabled.

In certain embodiments, the combination of AEX and CEX and the MM method can be used to produce product-related material-adjusted materials, including certain embodiments in which one technique is used in a complementary / complementary manner with other techniques . In some embodiments, such a combination may be performed such that a given technique removes most of the predetermined sub-species, so that the combination provides the desired final composition / product quality. In certain embodiments, such combination may be achieved by additional intervening chromatography, filtration, pH adjustment, and / or UF / DF steps to achieve desired AR, product quality, ion concentration, and / .

As described below and in Example 11, AEX chromatography can be used with recycle chromatography methods and continuous chromatography methods.

Cation exchange chromatography

The low AR composition of the present invention can be prepared by subjecting the composition, e.g., the initial recovery sample, to one or more cation exchange separation steps. In certain embodiments, the CEX step will occur after the above-described affinity chromatography, e. G., A protein A affinity step.

The use of anion exchange materials such as cation exchange materials versus the anion exchange materials discussed in detail above is based on the local charge of the desired protein in a given solution. Therefore, it is within the scope of the present invention to use the cation exchange step before use of the anion exchange step, or the anion exchange step before the cation exchange step. It is also possible to use a combination of a cation exchange step single, anion exchange step single, or any series of two steps (including a series of ion exchange steps of one or both of the other chromatographic separation techniques described herein) Is within the scope of the present invention.

In carrying out the separation, the initial protein mixture can be purified by using any of a variety of techniques, for example by using batch purification or chromatographic techniques in conjunction with protein A or AEX as described above, / RTI >

In certain embodiments, the aqueous salt solution used as both the loading buffer and the wash buffer has a pH lower than the isoelectric point (pI) of the desired protein. In certain embodiments, the pH is about 0-5 units lower than the pI of the desired protein. In some embodiments, this is in the range of one to two units lower. In some embodiments, this is in the range of 1 to 1.5 units lower.

In certain embodiments, the concentration of the anionic agent in the aqueous salt solution is from about 3.5 to 10.5, or from about 4 to 10, or from about 4.5 to 9.5, or from about 5 to 9, or from about 5.5 to 8.5, or from about 6 to 8 , Or a pH of about 6.5 to 7.5 is achieved. In certain embodiments, the concentration of the anionic agent in the aqueous salt solution is increased or decreased to achieve a pH of 5, or 5.5, or 6, or 6.5, or 6.8, or 7.5. Suitable buffer systems for use in the CEX method include, but are not limited to, trisformate, tris acetate, ammonium sulfate, sodium chloride, and sodium sulfate.

In certain embodiments, the conductivity and pH of the aqueous salt solution are adjusted by increasing or decreasing the concentration of the cationic agent. In certain embodiments, the cationic agent is maintained at a concentration in the range of about 20 mM to 500 mM, or about 50 to 350 mM, or about 100 to 300 mM, or about 100 to 200 mM. In certain non-limiting embodiments, the cationic agent is selected from the group consisting of sodium, Tris, tromethalmine, ammonium, arginine, or combinations thereof. In certain non-limiting embodiments, the anionic formulation is selected from the group consisting of formate, acetate, citrate, chloride anion, sulfate, phosphate, or combinations thereof.

Packed cation-exchange chromatography columns or cation-exchange membrane devices can operate in a coupling-release mode, in a pass-through mode, or in a hybrid manner in which the product exhibits binding to a chromatographic material, ≪ / RTI > can be washed from the column using a similar buffer. A detailed description of these schemes is summarized above.

Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S). Additional cationic materials include Capto SP ImpRes, a high flow agarose bead; CM Hyper D grade F, ceramic beads coated and infiltrated with functional hydrogels 250-400 ionic groups μeq / mL; Eshmuno S, a hydrophilic polyvinyl ether-based matrix with an ionic capacity of 50 to 100 μeq / mL; Nuvia C Prime, a hydrophobic cation exchange medium consisting of macroporous hyperbranched linked hydrophilic polymer matrix 55-75 μeq / mL; Nuvia S with UNOsphere base matrix with 90 to 150 μeq / mL ionic groups; Poros HS, a rigid polymeric bead having a backbone consisting of cross-linked poly [styrene-divinylbenzene]; Poros XS, a rigid polymeric bead having a backbone consisting of a cross-linked poly [styrene-divinylbenzene]; Toyo Pearl Giga Cap 650M, a polymeric base bead with an ionic capacity of 0.225 meq / mL; Polymer base bead, Toyo Pearl Giga Cap S 650M; But are not limited to, polymeric base beads, such as Toyo Pearl MX TRP. Detailed information on the above-mentioned materials is listed in Table 2. Note that CEX chromatography can be used with the MM resins described herein.

In certain embodiments, the protein loading of the mixture comprising the desired protein is about 5 to 150 g / L, or about 10 to 100 g / L, about 20 to 80 g / L, about 30 to 50 g / L, or about 40 to 50 g / L. In certain embodiments, the protein concentration of the loaded protein mixture is adjusted to a protein concentration of about 0.5 to 50 g / L, or about 1 to 20 g / L, of the material loaded into the column.

In certain embodiments, additives may be added to improve separation performance, for example, polyethylene glycol, detergents, amino acids, sugars, and chaotropic agents to achieve better recovery or product quality.

In certain embodiments, including but not limited to, those associated with azalimumab, the methods of the present invention may be used to selectively remove, significantly reduce, or reduce the AR in the passing and washing fractions, while concentrating the AR in the eluted fraction, By essentially eliminating all, it can be used to produce protein compositions with reduced AR or without AR. In certain embodiments of the tablet of Adalimumab, the method of the invention selectively removes, significantly reduces, or reduces AR1 charge variants in the passing and washing fractions, while concentrating AR1 charge variants in the flow eluting fraction , Essentially all of which can be used to produce protein compositions with reduced AR1 or without AR1 variants. In certain embodiments of adalimumab, the method of the present invention comprises selectively removing, significantly reducing, or reducing the AR2 charge variant in the passing and washing fractions, while concentrating AR2 charge variants in the flow- Lt; RTI ID = 0.0 > AR2 < / RTI > variants.

In certain embodiments including, but not limited to, those associated with azalimumab, the methods of the present invention selectively remove or significantly reduce the MGO variants in the eluted fraction, while concentrating the MGO variants in the passing and washing fractions Or by essentially eliminating it entirely, to produce protein preparations having reduced MGO or no MGO variants. In certain embodiments, including, but not limited to, those associated with azalimumab, the methods of the present invention may be used to concentrate glycated variants in the eluted fraction, including the Schiff base and the permanently glycosylated Type of glycoprotein is selectively removed, significantly reduced, or essentially entirely eliminated, so that the glycated variant can be used to produce a protein preparation that does not contain reduced or glycosylated variants.

In certain embodiments, the load, pH, conductivity, and elution pH conductivity of the CEX chromatography step can be modified to achieve a desired distribution of acidic species. For example, but not by way of limitation, certain embodiments relate to the modulation of the lysine distribution of a purified sample of a desired protein, for example, an increase in Lys 0 and a decrease in Lys 1 and Lys 2. In certain embodiments, the method of the invention enables the production of a sample wherein the amount of Lys 0 is reduced while the amount of Lys 1 and / or Lys 2 is increased.

In certain embodiments, CEX chromatographic separation can be performed and, in addition, or instead of merely adjusting the charge mutant concentration, the combination of the fractions can be pooled to provide the desired process-related impurities and / or product- A combination can be achieved.

In some embodiments, the level of product-related charge mutants, aggregates, low molecular weight variants (e. G., Fragments of the desired protein) are determined using spectroscopic methods such as UV, NIR, FTIR, fluorescence, and Raman Can be monitored on-line, at-line, or in-line, and then the spectroscopic method can be used to control the level of charge variants in the pooled material collected from the CEX eluate, for example, acid species . In certain embodiments, certain signals resulting from chemical modification of proteins, such as glycosylation, MGO modification, deamidation, glycosylation, may be achieved by such in-line, on-line, Lt; RTI ID = 0.0 > spectroscopic < / RTI > In some embodiments, on-line, at-line, or in-line monitoring methods may be used for the effluent line of the chromatography step or in the collection vessel to enable achievement of desired product quality / recovery. In some embodiments, the UV signal can be used as a surrogate to achieve a suitable product quality / recovery, wherein the UV signal includes, but is not limited to, such processing techniques as integration, differentiation, moving average, Process variability can be resolved and the target product quality can be properly processed. In certain embodiments, such measurements can be combined with an in-line dilution method, whereby the ion concentration / conductivity of the load / wash can be controlled by feedback, thus product quality control can also be enabled.

In certain embodiments, the combination of CEX and AEX and / or the MM method may be used to produce product-related material-adjusted materials, including certain embodiments in which one technique is used in a complementary / complementary manner . In certain embodiments, such a combination can be performed to remove most of the predetermined species by one technique, so that the combination provides the desired final composition / product quality. In certain embodiments, such combinations include the use of additional chromatography, filtration, pH adjustment, UF / DF steps to achieve desired product quality, AR, ion concentration, and / or virus reduction.

As described below and in Example 11, CEX chromatography can be used with recycle chromatography and continuous chromatography methods.

Mixed Chromatography

Mixed-mode ("MM") chromatography may also be used to prepare the low AR compositions of the present invention. MM chromatography, also referred to herein as "multimodal chromatography, " refers to two or more different moieties that interact with the material to be bound, and in some embodiments, a ligand capable of providing a co-operative moiety ≪ / RTI > In certain embodiments, one of these sites provides charge-charge interaction of the type of attraction between the ligand and the desired material, and the other site is capable of electron acceptor / donor interaction and / or hydrophobic and / or hydrophilic interaction . Electron donor-acceptor interactions include interactions such as hydrogen-bonding, π-π, cation -π, charge transfer, dipole-dipole, induced dipole,

In some embodiments, the resin used in the mixed mode separation is Capto Adhere. Capto Adhere is a strong anion exchanger with multimodal functionality. The base matrix thereof is a highly cross-linked agarose having a ligand (N-benzyl-N-methylethanolamine) exhibiting a number of functionalities for interactions such as ionic, hydrogen and hydrophobic interactions. In some embodiments, the resin used in the mixed mode separation is selected from PPA-HyperCel and HEA-HyperCel. The base matrix of PPA-HyperCel and HEA-HyperCel is a very porous, cross-linked cellulose. These ligands are phenylpropylamine and hexylamine, respectively. Phenylpropylamine and hexylamine provide different selectivity and hydrophobic options for protein separation. Further mixing method of the chromatographic support may include, but is Nuvia C Prime, Toyo Pearl 650M MX Trp, and Eshmuno ^® HCX, not limited to these.

In certain embodiments, the mixed chromatographic resin comprises ligands, either directly or through a spacer, sometimes coupled to an organic or inorganic support, referred to as a base matrix. The support may be in the form of particles, such as, for example, essentially spherical particles, monoliths, filters, membranes, surfaces, capillaries, and the like. In certain embodiments, the support is made from a natural polymer, such as a cross-linked carbohydrate material, such as agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate and the like. To obtain a high adsorption capacity, the support is porous, and then the ligand can be coupled to the pore surface as well as the outer surface. Such natural polymer scaffolds can be prepared according to standard methods, for example inverse suspension gelation (S Hjerten: Biochim Biophys Acta 79 (2), 393-398 (1964)). Alternatively, the support can be made from a synthetic polymer such as a cross-linked synthetic polymer, such as styrene or styrene derivatives, divinylbenzene, acrylamide, acrylate esters, methacrylate esters, vinyl esters, . This synthetic polymer can be prepared according to standard methods (see, for example, "Styrene based polymer supports developed by suspension polymerization" (R Arshady: Chimica e L'Industria 70 (9), 70-75 Porous natural or synthetic polymeric scaffolds are also available from commercial sources such as Amersham Biosciences (Uppsala, Sweden).

In certain embodiments, the protein loading of the mixture comprising the desired protein is adjusted to a total protein loading of about 50 to 750 g / L, or about 75 to 500 g / L, or about 100 to 300 g / L for the column. In certain embodiments, the protein concentration of the loaded protein mixture is adjusted to a protein concentration of about 1 to 50 g / L, or about 9 to 25 g / L of the material loaded into the column.

In certain embodiments, additives such as polyethylene glycols, detergents, amino acids, sugars, chaotropic agents may be added to improve separation performance to achieve better recovery or product quality.

In certain embodiments, including but not limited to, those associated with azalimumab, the MM method of the present invention selectively removes, or significantly reduces, the AR in the passing and washing fractions, while concentrating the AR in the flow- Or by essentially eliminating it entirely, to produce a protein composition that has reduced AR or does not contain AR. In certain embodiments of the tablet of Adalimumab, the method of the invention selectively removes, significantly reduces, or reduces AR1 charge variants in the passing and washing fractions, while concentrating AR1 charge variants in the flow eluting fraction , Essentially all of which can be used to produce protein compositions with reduced AR1 or without AR1 variants. In certain embodiments of adalimumab, the method of the present invention comprises selectively removing, significantly reducing, or reducing the AR2 charge variant in the passing and washing fractions, while concentrating AR2 charge variants in the flow- Lt; RTI ID = 0.0 > AR2 < / RTI > variants.

In certain embodiments including, but not limited to, those associated with azalimumab, the MM method of the present invention can be used to selectively remove MGO variants in the passing and washing fractions, while significantly reducing , Or by essentially eliminating all of them, to produce protein preparations having reduced MGO or no MGO variants. In certain embodiments, including, but not limited to, those associated with azalimumab, the methods of the present invention may be used to purify glycated variants (including Schiff base and permanently glycosylated Type of glycoprotein is selectively removed, significantly reduced, or essentially entirely eliminated, so that the glycated variant can be used to produce a protein preparation that does not contain reduced or glycosylated variants.

In certain embodiments, the loading, pH, conductivity, and elution pH conductivity, wash pH and conductivity, and elution pH, conductivity of the MM chromatography step can be modified to achieve a desirable distribution of acidic species. For example, but not by way of limitation, certain embodiments relate to the modulation of the lysine distribution of a purified sample of a desired protein, for example, an increase in Lys 0 and a decrease in Lys 1 and Lys 2. In certain embodiments, the method of the invention enables the production of a sample wherein the amount of Lys 0 is reduced while the amount of Lys 1 and / or Lys 2 is increased.

In some embodiments, the MM chromatographic separation can be performed and, in addition, or instead of merely adjusting the charge mutant concentration, the combination of fractions can be pooled to provide desired process-related impurities and / or product- A combination can be achieved.

In some embodiments, levels of AR species can be monitored on-line, at-line, or in-line using spectroscopic methods such as UV, NIR, FTIR, fluorescence, and Raman, The spectroscopic method can be used to control the level of charge variants in the pooled material, e. G., Acid species, collected from the MM eluate. In certain embodiments, certain signals resulting from chemical modification of proteins, such as glycosylation, MGO modification, deamidation, glycosylation, may be achieved by such in-line, on-line, Lt; RTI ID = 0.0 > spectroscopic < / RTI > In some embodiments, on-line, at-line, or in-line monitoring methods may be used for the effluent line of the chromatography step or in the collection vessel to enable achievement of desired product quality / recovery. In some embodiments, the UV signal can be used as a surrogate to achieve a suitable product quality / recovery, wherein the UV signal includes, but is not limited to, such processing techniques as integration, differentiation, moving average, Process variability can be resolved and the target product quality can be properly processed. In certain embodiments, such measurements can be combined with an in-line dilution method, whereby the ion concentration / conductivity of the load / wash can be controlled by feedback, thus product quality control can also be enabled.

In certain embodiments, the combination of mixing methods and AEX and CEX methods may be used to prepare the low AR compositions of the present invention, including certain embodiments in which one technique is used in a complementary / complementary manner with other techniques. In certain embodiments, such a combination can be performed to remove most of the predetermined species by one technique, so that the combination provides the desired final composition / product quality. In certain embodiments, such combinations include the use of additional chromatography, filtration, pH adjustment, UF / DF steps to achieve desired product quality, AR, ion concentration, and / or virus reduction.

As described below and in Example 11, MM chromatography can be used with recycle chromatography and continuous chromatography methods.

Continuous chromatography and recycle chromatography

Continuous chromatography and recycle chromatography methods can be used to prepare the low AR compositions of the present invention and are described below. These methods result in a significant improvement in the recovery of the desired protein, e. G., The antibody, while maintaining AR reduction levels. These continuous chromatographic and recycle chromatographic schemes can be used to separate the desired low acidic species components into (a) the desired low acidic constituent components are collected in the unbound fractions during chromatography (pass / wash liquid chromatography), or (b) The present invention is applicable to a chromatographic method in which components are first bound to a medium and subsequently recovered by washing the medium under conditions to elute the bound components.

Continuous chromatography and recycle chromatography-passing liquid / wash liquid chromatography

When the desired low acid species component is collected in the unbound fraction, the following approach is used to prevent the loss of the loaded material on the column.

In one embodiment, a recycle chromatography method is used in which the column is loaded and unbound fractions resulting in a target AR level are collected. Subsequently, instead of regenerating the column and losing the product, the column is washed under conditions that result in recovery of the product remaining bound to the column. These products recovered under these conditions contain significantly higher AR levels than the original feed material. Such a wash fraction can be adjusted to conditions suitable for achieving the desired separation for subsequent processing (typically similar conditions to the initial preparation) and combined with the original feed material, after the column has been properly prepared for the next cycle, Lt; / RTI > The amount of material that is prepared for the next cycle in combination with the wash fraction from the first cycle and the fresh material is determined as the target load capacity (typically similar to the target capacity for the first cycle) for the column to achieve the desired separation .

During the execution of the second cycle, a similar strategy is used to collect unbound fractions so that the target AR level is achieved, followed by washing the column under conditions to recover the product that remains on the column subsequently.

In one embodiment, this recycling chromatography scheme continues until all loading materials are used. The number of cycles can be controlled by appropriately designing the column size.

In the use of the recycle chromatography method, the recovery of the product loaded into the column is significantly improved while achieving the target AR level.

Several variations of the recirculation chromatography method can be used. In one embodiment, the collected fraction targeting a given AR level can be measured based on a predetermined criterion or on at-line, off-line, or on-line analysis of the elution or collected pool of the column have.

In another embodiment, the cleaning conditions used in the first cycle can be adjusted so that the desired amount of product is recovered to the desired product quality, and is limited only by the feasibility of producing an appropriate load mixture at the next step . In one aspect of this embodiment, the cleaning conditions may be similar to the loading conditions. In another aspect of this embodiment, the washing conditions can be stringent to recover all of the (desired or undesirable) product species remaining on the column.

In yet another embodiment, the load, the load condition, and the cleaning conditions used in the subsequent cycle may be modified to achieve the desired purity, provided that the load material for subsequent cycles contains a higher level of AR .

In another embodiment, the operation of the last cycle can be performed under different conditions to achieve target purity and target counts to optimize overall recovery and purity.

Further, the method for preparing the low AR composition of the present invention can be carried out by a continuous chromatography method. In this manner, two or more columns (denoted as "first" column and "second" column) are used. In one embodiment, the feed material is loaded onto the first column and the unbound fraction from the first column is collected such that the pool material contains the target AR level. The column is then washed under conditions under which the remaining product is recovered. This material is then dynamically diluted into a solution suitable for achieving the desired loading conditions, mixed with the fresh feed material, and sent to the second column. Unbound fractions from the second column are collected to achieve target AR levels. The second column is then washed under conditions to recover the product, diluted with a suitable solution, and dynamically mixed with fresh material to a first column (which is prepared to accommodate loading after regeneration / rinsing) . In one embodiment, such cycling continues until all load materials are used. The last cycle can be operated in a "typical" manner, where load and wash conditions are adjusted as needed, as needed.

In some embodiments, such a continuous chromatographic scheme can be performed such that a wash material containing a higher AR level can be sent back to the load tank after appropriate dilution. This material is then loaded into the second column either subsequently or concurrently, so that the operation of the two columns is not tandem, thereby reducing the complexity of the operation.

This continuous chromatography scheme is similar to the recycling chromatography scheme, but can be performed more effectively and therefore has a reduced processing time.

With respect to this continuous chromatographic scheme, several variations can be used. In one embodiment, the fraction collected targeting a given AR level is measured based on a predetermined criterion or on at-line, off-line, or on-line analysis of the elution or collected pool of the column .

In another embodiment, the cleaning conditions used in the first cycle can be adjusted so that the desired amount of product is recovered to the desired product quality, and is limited only by the feasibility of producing an appropriate load mixture at the subsequent step. In one aspect of this embodiment, the cleaning conditions may be similar to the loading conditions. In another aspect of this embodiment, the washing conditions can be stringent to recover all of the (desired or undesirable) product species remaining on the column.

In yet another embodiment, the load, the load condition, and the cleaning conditions used in the subsequent cycle may be modified to achieve the desired purity, provided that the load material for subsequent cycles contains a higher level of AR .

In other embodiments, the operation of the last cycle may be performed under different conditions to achieve target purity / recovery to optimize overall recovery and / or purity.

In one embodiment, the medium selection for recirculation or continuous mode may be one of a plurality of chromatographic resins having pendant hydrophobic and anion exchange functional groups, a monolith medium, a membrane adsorption medium or a deep filtration medium .

In some embodiments, a membrane or a deep filter-based medium ("convective media ") is used to separate the less enriched portion of the product, while taking into account the fact that the pure fraction is selectively pooled, , It can be used in a recycle or continuous chromatography method.

Continuous chromatography and recycle chromatography-elution chromatography

In chromatographic or separation elution schemes as exemplified by the CEX technique for AR reduction, the conditions for the loading and washing steps are such that the AR concentrated material is collected in the passing liquid and / or wash fraction, while the AR reduction Is selected to be collected in the eluted fraction. In a typical implementation of CEX technology, about 10 to 40% of the product (the desired charge mutant) may be lost in the passing liquid / wash fraction. Two modes of operation, i.e., recycle and continuous chromatography schemes, provide improved recovery while maintaining target AR levels.

In a recirculation chromatographic process, the loading material is typically processed over multiple cycles. In the implementation of the recirculation chromatography method, the loading material is prepared such that the eluate contains the target product purity or AR level. Under these conditions, the AR enriched material is collected in the passing liquid / wash fraction. These materials are pooled and additional fresh load material is added to achieve the appropriate loading capacity in the next cycle of chromatography on the same column. In particular, in one embodiment, the column is eluted under conditions under which the combined product (with low AR levels) is recovered, and subsequently regenerated and equilibrated to prepare for the next cycle.

In the next cycle, the combined load (pass / wash from cycle 1) is loaded into the target volume. Collect and pool the passing liquid / wash fraction. The column is re-eluted to obtain a second eluate containing the low AR composition. In one embodiment, this sequence continues until all load materials have been processed.

In another embodiment, by implementing the recycle chromatography scheme, the material that would not otherwise be discarded as an AR enrichment material is further purified to "recover" the pure protein product, thereby improving the overall recovery of the protein. In one embodiment, the level of recovery depends on the number of cycles used.

With respect to recirculation chromatography schemes, several variations can be used. In one embodiment, the entire pool of the passing liquid / wash fraction is typically combined with fresh material to maximize the total number of operations. However, to achieve higher purity or efficiency, some of the through-liquid washes may be discarded. For example, in one embodiment, certain fractions containing very high levels of AR species can be discarded. To enable such selective pooling, the level of AR can be measured directly or indirectly using an off-line, in-line, or at-line method.

In another embodiment, the conditions for loading and loading, washing and elution can be modified for the second and subsequent cycles to accommodate higher levels of AR that would be present in the load pool.

In yet another embodiment, the final cycle of the process can be performed under conditions that allow achievement of target purity and recovery to optimize overall recovery and purity.

Continuous chromatography schemes provide additional advantages in terms of time efficiency. In this manner of operation, two or more columns are used. Specifically, with respect to the recirculation method, conditions suitable for the load capacity, load, washing and elution conditions are selected for operation. The passing liquid and wash fraction (or portions thereof) are sent to a loading tank containing fresh material. After completion of the loading and washing steps, the first column is eluted and subsequently regenerated. On the other hand, the second column is loaded with fresh material and a mixture of washing and passing liquid from the previous cycle. The washing and passing liquid from the second column is returned to the loading tank again. The second column is then eluted and regenerated. The first column is then ready to be loaded, and the cycle continues. This continuous chromatographic approach is efficient because the product is processed continuously and the purified product is obtained in a semi-continuous manner.

Several variations of the continuous chromatographic scheme can be used. In one embodiment, the entire pool of the passing liquid / wash fraction is combined with fresh material to maximize the total number of operations. However, to achieve higher purity or efficiency, some of the through-liquid washes may be discarded. For example, certain fractions containing very high levels of AR species can be discarded. To enable such selective pooling, the level of acid species can be measured directly or indirectly using an off-line, in-line, or at-line method.

In another embodiment, the conditions for loading and loading, washing and elution can be modified for the second and subsequent cycles to accommodate higher levels of AR that would be present in the load pool.

In yet another embodiment, the operation of the last cycle can be performed under different conditions to optimize overall recovery and purity.

Recycle chromatography and continuous chromatography methods are not limited to use with any particular chromatography resin. The medium used in the recirculation or continuous mode may be one of a plurality of chromatographic resins having pendant hydrophobic and anion exchange functional groups, a monolith medium, a membrane adsorption medium or a deep filtration medium.

In some embodiments, the membrane deep layer filter-based medium ("convection medium") has a high selectivity for separation, considering the fact that the less concentrated portion of the product is "recirculated " while the pure fraction is selectively pooled. Is not required, it can be used in recycle or continuous chromatography methods.

Recycle chromatography and continuous chromatography methods can be used with the AEX, CEX, or MM chromatographic methods as described herein to produce the low AR compositions of the present invention. For example, Example 11 below describes recirculation chromatography for AR reduction using AEX, CEX and MM techniques.

Hydrophobic interaction chromatography

In addition, the low AR compositions of the present invention can be prepared using a hydrophobic interaction chromatography (HIC) step in addition to a displacement chromatography step.

In performing the separation, the sample mixture is contacted with the HIC material, for example, using batch purification techniques or using column or membrane chromatography. Prior to HIC purification, it may be desirable to adjust the concentration of the salt buffer to achieve the desired protein binding to the resin or membrane.

Ion exchange chromatography relies on the local charge of the desired protein for selective separation, while hydrophobic interaction chromatography uses the hydrophobic character of the protein to achieve selective separation. The hydrophobic group on the protein interacts with the hydrophobic group of the resin or membrane. The more hydrophobic, the stronger the protein, which will interact with the column or membrane. Thus, the HIC step removes process-related impurities (e.g., HCP) and product-related materials (e.g., aggregates and fragments).

Similar to ion exchange chromatography, the HIC column or membrane apparatus can also operate in a hybrid-mode manner in which the products exhibit binding to the chromatographic material, a coupling-elution mode, a passage mode, or the like, Can be washed from the column using a substantially similar buffer. A detailed description of these schemes is summarized above with respect to AEX purification.

Because hydrophobic interactions are strongest at high ionic strengths, this type of separation is conveniently performed according to salt elution steps such as those typically used in connection with ion exchange chromatography. Alternatively, the salt may be added to the low salt level feed stream prior to this step. Antibody adsorption to the HIC column is preferred for high salt concentration, but the actual concentration may vary over a wide range depending on the nature of the desired protein, the salt form and the particular HIC ligand selected. The various ions are so-called soluphobic series, depending on whether they cause hydrophobic interactions (salting-out effects) or destruction of water structures (chaotropic effects) The cation is ranked as Ba2 +, Ca2 +, Mg2 +, Li +, Cs +, Na +, K +, Rb + and NH4 + from the viewpoint of increasing the salting effect, while the anion is ranked as PO43-; -; CH3CO3-; Cl-; Br-; NO3-; ClO4-; I-; SCN-.

In general, Na +, K + or NH4 + sulfate effectively promotes ligand-protein interaction in HIC. Considering the following relationships, salts that affect the strength of the interaction can be formulated: (NH4) 2SO4> Na2SO4> NaCl> NH4C1> NaBr> NaSCN. In general, salt concentrations of about 0.75 M to about 2 M ammonium sulfate or about 1 M to 4 M NaCl are useful.

The HIC medium typically comprises a base matrix (e.g., a cross-linked agarose or synthetic co-polymer material) to which a hydrophobic ligand (e.g., an alkyl or aryl group) is coupled. Suitable HIC media include agarose resins or membranes functionalized with phenyl groups (e.g., Phenyl Sepharose from GE Healthcare or Phenyl Membrane from Sartorius). Many HIC resins are commercially available. Examples include Capto Phenyl, low substituted or highly substituted Phenyl Sepharose ™ 6 Fast Flow, Phenyl Sepharose ™ High Performance, Octyl Sepharose ™ High Performance (GE Healthcare); Fractogel 占 EMD Propyl or Fractogel 占 EMD Phenyl (E. Merck, Germany); Macro-Prep ™ Methyl or Macro-Prep ™ t-Butyl column (Bio-Rad, California); WP HI-Propyl (C3) ™ (J. T. Baker, New Jersey); And Toyopearl (TM) ether, phenyl or butyl (TosoHaas, PA).

Virus filtration

Virus filtration is a dedicated virus reduction step in the overall refining process. This step is generally performed after the chromatographic polishing step. Virus reduction includes, but is not limited to, Planova 20N ™, 50N or BioEx from Asahi Kasei Pharma, Viresolve ™ filters from EMD Millipore, ViroSart CPV from Sartorius, or Ultipor DV20 or DV50 ™ filters from Pall Corporation Can be achieved through the use of suitable filters. It will be clear to a person skilled in the art to select a filter suitable for obtaining the desired filtration performance.

Ultrafiltration / Dialysis filtration

Certain embodiments of the invention further concentrate and formulate the desired protein, e. G., Antibody product, using ultrafiltration and dialysis filtration steps. Ultrafiltration is described in Microfiltration and Ultrafiltration: Principles and Applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York, N.Y., 1996); And Ultrafiltration Handbook, Munir Cheryan (Technomic Publishing, 1986; ISBN No. 87762-456-9). One filtration process is described in Millipore catalog entitled "Pharmaceutical Process Filtration Catalog" 177-202 (Bedford, Mass., 1995/96). Ultrafiltration is generally considered to be an average filtration using a filter having a pore size of less than 0.1 [mu] m. By using filters with these small pore sizes, the volume of the sample can be reduced through the penetration of the sample buffer through the filter membrane pores while the protein, e.g., antibody, is retained on the membrane surface.

Dialysis filtration can be accomplished by removing and exchanging salts, sugars, and non-aqueous solvents to separate unbound species from bound species and / or removing low molecular weight species and / or rapid changes in ionic and / And a membrane filter is used to bring about the effect. The microsolute is most effectively removed by adding solvent to the dialysis filtered solution at a rate approximately equivalent to the permeation flux. This effectively cleanses the microspecies from the solution to purify the desired protein retained. In certain embodiments of the invention, the dialysis filtration step may be used prior to any additional chromatography or other purification steps, not only to exchange various buffers used in connection with the present invention, but also to remove impurities from the protein preparation do.

One of ordinary skill in the art can select a membrane filtration device suitable for UF / DF operation. Examples of membrane cassettes suitable for the present invention include Pellicon 2 or Pellicon 3 cassettes with 10 kD, 30 kD or 50 kD membranes from EMD Millipore, Kvick 10 kD, 30 kD or 50 kD membrane cassettes from GE Healthcare, Of Centramate or Centrasette 10kD, 30kD or 50kD cassettes.

Exemplary refining strategies

In certain embodiments, the initial recovery can be carried out by sequentially removing cells and cell debris (including HCP) from the product bioreactor collection using pH reduction, centrifugation, and filtration steps sequentially. In certain embodiments, the present invention relates to performing one or more AEX, CEX, and / or MM purification steps on the sample mixture from said initial recovery. Certain embodiments of the invention will include additional purification steps. Examples of further purification procedures that may be performed prior to, during, or after the ion exchange chromatography method include ethanol precipitation, isoelectric point electrophoresis, reverse phase HPLC, chromatography on silica, chromatography on heparin Sepharose ™ , Further anion exchange chromatography, and / or further cation exchange chromatography, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography For example, using protein G, an antibody, a specific substrate, a ligand or an antigen as a capture reagent).

Embodiments of this combination of strategies are set out below with specific data relating to the specific combinations useful in connection with the present invention included in Tables 80-87 and Tables 76-78.

In certain embodiments, the unbound passes and wash fractions may be further fractionated and the combination of fractions providing the target product fractions may be pooled.

In certain embodiments, the protein concentration is adjusted such that differential partitioning behavior between the antibody product and the product-related substrate is achieved such that the purity and / or yield can be further improved. In some embodiments, the load may be performed at different protein concentrations during load operation to improve product quality / yield during any particular purification step.

In certain embodiments, the column temperature can be independently varied to improve separation efficiency and / or yield of any particular purification step.

In certain embodiments, the loading and cleaning buffer matrix may be different or may comprise a mixture of chemicals to achieve the new " resin interaction "behavior while validating the new separation. For example, and not by way of limitation, the load and wash buffer may differ in terms of ionic strength or pH while maintaining substantially similar functionality in terms of the cleaner of the product achieved during the wash step. In certain embodiments, additives such as amino acids, sugars, PEG, etc. may be added to the loading or washing step to adjust the fractional behavior to achieve separation efficiency and / or yield.

In some embodiments, the loading & cleaning step may be controlled by in-line, at-line, or off-line measurements of column effluent or collected pool or product related impurity / And / or yield. In certain embodiments, the loading concentration can be controlled dynamically by in-line or batch or continuous dilution into buffers or other solutions to achieve the fractionation needed to improve separation efficiency and / or yield.

V. How to test sample purity

Assay of host cell protein

The present invention also provides a method for determining the residual level of host cell protein (HCP) concentration in a low AR composition of the invention. As described above, the HCP is preferably excluded from the final target material product. Exemplary HCPs include those derived from a source of antibody production Proteins. Failure to identify and sufficiently remove HCP from the target antibody can result in reduced efficiency and / or adverse reaction in the subject.

The term "HCP ELISA " as used herein refers to an ELISA specific for an HCP produced from a cell used for producing the desired antibody, for example, a CHO cell, wherein the second antibody used in the assay . The second antibody may be prepared according to conventional methods known to those skilled in the art. For example, a second antibody can be produced using HCP obtained by sham production and purification runs, i.e., using the same cell line used to produce the desired antibody, It is not transfected with DNA. In an exemplary embodiment, the second antibody is selected from a selected cell expression system, i. E., Expressed in a cell expression system used to produce the target antibody ≪ / RTI >

Generally, the HCP ELISA involves sandwiching antibodies, i.e., liquid samples containing HCP between two layers of a first antibody and a second antibody. The sample is incubated for a period of time during which the HCP in the sample is captured by the first antibody, but not limited to, for example, affinity purified chlorine anti-CHO (Cygnus). A second antibody, or a blend of antibodies, specific for the HCP prepared from the cells used to generate the antibody, e. G., Biotinylated anti-CHO HCP, is added and bound to the HCP in the sample do. In certain embodiments, the first and second antibodies are polyclonal antibodies. In certain aspects, the first and second antibodies are blends of polyclonal antibodies produced against HCP. The amount of HCP contained in the sample is determined using appropriate tests based on the labeling of the second antibody.

The HCP ELISA can be used to measure the level of HCP in an antibody composition, such as eluates or transdermal solutions, obtained using the processes described above. The present invention also provides a composition comprising an antibody that does not have a detectable level of HCP when measured by an HCP enzyme linked immunosorbent assay ("ELISA").

Assay of acidic species (AR)

The levels of acidic species in a chromatographic sample prepared using the techniques described herein can be analyzed as described in the Examples section. In certain embodiments, CEX-HPLC methods are used. For example, but not limited to, cation exchange chromatography can be performed on a Dionex Propac WCX-10, analytical column 4 mm x 250 mm (Dionex, Calif.). The Agilent 1200 HPLC system can then be used as the HPLC. In certain embodiments, mobile phases such as 10 mM dibasic pH 7.5 sodium phosphate (mobile phase A) and 10 mM dibasic sodium phosphate, 500 mM pH 5.5 sodium chloride (mobile phase B) may be used. In some embodiments, a binary gradient (94% A, 6% B: 0 to 20 min; 84% A, 16% B: 20 to 22 min; 0% A, 100% % A, 6% B: 28-34 min) can be used with detection at 280 nm. In some embodiments, the quantitation is based on the relative area percent of the detected peak. In certain embodiments, peaks eluting at a relative retention time less than a predetermined time are indicated together as acidic peaks.

size Mutant  black

In certain embodiments, levels of aggregates, monomers, and fragments in a chromatographic sample prepared using the techniques described herein are analyzed. In certain embodiments, aggregates, monomers, and fragments are measured using a size exclusion chromatography (SEC) method for each molecule. For example, but not limited to, TSK-gel G3000SWxL, 5 urn, 125 Å, 7.8 X 300 mm column (Tosoh Bioscience) can be used in connection with certain embodiments, while TSK- , 250 ANGSTROM, 4.6 X 300 mm column (Tosoh Bioscience) can be used in alternative embodiments. In certain embodiments, the above-mentioned columns are used with an Agilent or Shimazhu HPLC system. In certain embodiments, sample injection is performed under isocratic elution conditions using, for example, a mobile phase consisting of 100 mM sodium sulfate and 100 mM sodium phosphate at pH 6.8 and detected with UV absorbance at 214 nm. do. In some embodiments, the mobile phase will consist of 1X PBS at pH 7.4 and the elution profile is detected with UV absorbance at 280 nm. In certain embodiments, the quantification is based on the relative area of the detected peaks.

Any additional technique, e.g., mass spectroscopy, can be used to assay for size variants.

VI. Methods of treatment using the low AR composition of the present invention

The low AR compositions of the present invention can be used to treat any disorder in a subject suitable for treatment with the therapeutic protein contained in the composition.

A "disorder" is any condition that benefits from treatment with a protein. This includes chronic and acute disorders or diseases, including those pathological conditions that make the subject susceptible to suspicious disorders. In the case of an anti-TNF [alpha] antibody or antigen binding portion thereof, e.g., adalimumab, a therapeutically effective amount of a low AR composition may be administered to treat disorders where TNF [alpha] activity is detrimental.

Disorders in which TNFa activity is deleterious include disorders in which inhibition of TNFa activity is expected to alleviate the symptoms and / or progression of the disorder. This disorder can be evidenced, for example, by an increase in the concentration of TNF [alpha] in the biological fluid of a subject suffering from the disorder (e.g., an increase in TNF [alpha] concentration in the serum, plasma, synovial fluid, etc. of the subject) Lt; / RTI > antibody.

TNFa has been implicated in the pathophysiology of a wide variety of TNFa related disorders including sepsis, infections, autoimmune diseases, graft rejection and graft-versus-host disease ( see, for example , Moeller, A., et al. (1990) Cytokine 2 : 162-169; Moeller et al. , US Patent 5,231,024; Moeller, A., et al. Vasilli, P. (1992) Annu . Rev. Immunol . 10 : 411-452 See, for example, Patent Publication No. 260 610 B1; Tracey, KJ and Cerami, A. (1994) Annu . Rev. Med . 45 : 491-503). Thus, the low AR compositions or low process-related impurity compositions of the present invention may be used in the treatment of autoimmune diseases such as rheumatoid arthritis, pediatric idiopathic arthritis or psoriatic arthritis, bowel disorders such as Crohn's disease or ulcerative colitis , Spondyloarthropathies, e.g., ankylosing spondylitis, or skin disorders, such as psoriasis.

Disorders with deleterious effects of TNF [alpha] activity are well known in the art and are described in U.S. Pat. U.S. Patent No. 8,231,876 and U.S. Pat. No. 6,090,382, the entire disclosure of each of which is expressly incorporated herein by reference. In one embodiment, the term " harmful disorder of TNFa activity "includes, but is not limited to, sepsis (including septic shock, endotoxic shock, gram negative sepsis and toxic shock syndrome), autoimmune diseases (rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and gouty arthritis, (Including systemic lupus, Lupus nephritis and lupus encephalitis), Crohn's disease and autoimmune hearing loss), active-facial spondyloarthritis (including acute myeloid leukemia), autoimmune uveitis, nephrotic syndrome, multiple system autoimmune disease axSpA) and non-radiographic axillary arthritis (nr-axSpA), infectious diseases including malaria, meningitis, acquired immunodeficiency syndrome (AIDS), influenza and cachexia following infection, allograft rejection and graft- Malignant tumors, pulmonary disorders (adult respiratory distress syndrome (ARDS), lung shock, chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis, Inflammatory bowel disease, Crohn's disease and Crohn's disease-related disorders (such as bladder, vagina, and skin), including, but not limited to, chronic obstructive pulmonary disease and chronic obstructive airways disorder Including acute myelodysplastic syndromes, chronic obstructive pulmonary disease, intestinal obstruction, abscess, malnutrition, complications from the use of corticosteroids, joint inflammation, nodular erythema, Cardiovascular tissue damage caused by cardiac arrest, cardiovascular tissue injury caused by cardiac arrest, cardiogenic shock, and / or cardiac arrhythmia caused by cardiac ischemia, cardiac insufficiency, restenosis, congestive heart failure, coronary artery disease, angina pectoris, myocardial infarction, Including but not limited to hypertension, atherosclerosis, cardiomyopathy, coronary artery spasm, coronary artery disease, valve disease, arrhythmia, and cardiomyopathy), spondyloarthropathies (ankylosing spondylitis, psoriatic arthritis / Diabetic neuropathy, diabetic retinopathy, diabetic retinopathy, diabetic retinopathy, diabetic retinopathy, diabetic retinopathy, diabetic retinopathy, diabetic retinopathy, diabetic retinopathy, Anemia, pain (including acute and chronic pain such as neuropathic pain and post-surgical pain, chronic lower back pain, cluster headache (including diabetic ulcers including retinopathy ulcers and diabetic macular vasculopathy) Pain, pain during labor and childbirth, and sunburn, from a burn injury, a burn injury, a burn injury, a pain, (Including genitourinary-associated pain including pain resulting from postpartum pain, migraine, angina pain, and cystitis), liver disorders (including hepatitis, alcoholic hepatitis, viral hepatitis, Including hepatitis B and C, and fatty liver inflammation, cystic fibrosis, primary biliary cirrhosis, sclerosing cholangitis, and biliary obstruction), skin and nail disorders (psoriasis (Including, but not limited to, chronic plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis and other psoriatic disorders), pemphigus pemphigoid, scleroderma, atopic dermatitis (eczema), sarcoidosis, (Including Behcet's disease), and other disorders such as, for example, pediatric rheumatoid arthritis (JRA), endometriosis, prostatitis, choroid plexus Angiogenesis, sciatica, Sjogren's syndrome, uveitis, wet macular degeneration, osteoporosis and osteoarthritis.

The term "subject" as used herein is intended to include living organisms such as prokaryotes and eukaryotes. Examples of subjects include mammals such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments of the invention, the subject is a human.

The term "treating" or "treating" as used herein refers to both therapeutic treatment and prophylactic or preventative measures. Those requiring treatment include those that already have a disability, as well as those where disability should be prevented.

In one embodiment, the invention provides a method of administering to a subject a low AR composition comprising an anti-TNF [alpha] antibody or antigen-binding portion thereof, such that TNFa activity is inhibited or a disorder in which TNFa activity is detrimental. In one embodiment, TNF [alpha] is human TNF [alpha] and the subject is a human subject. And in one embodiment, wherein the antibody -TNFα Oh otherwise free MAB, also shown as a HUMIRA ^®.

The low AR composition can be administered by a variety of methods known in the art. Exemplary routes / routes of administration include subcutaneous injection, intravenous injection or infusion. In certain embodiments, the low AR composition can be administered orally. The route and / or manner of administration as will be understood by those skilled in the art will vary depending on the desired outcome.

Dosage regimens may be adjusted to provide the optimal desired response (e. G., Therapeutic or prophylactic response). For example, a single bolus may be administered, some sub-doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the therapeutic situation. In certain embodiments, it is particularly advantageous to formulate the parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as a single dose for the mammalian subject being treated, each unit being capable of producing the desired therapeutic effect in association with the required pharmaceutical carrier Calculated amount of the active compound. The description of the dosage unit form of the present invention is based on the assumption that (a) the particular characteristics of the active compound and the specific therapeutic or prophylactic effect achieved, and (b) the sensitivity of the combination of such active compound Quot; is dictated by the limitations inherent in the field, and is directly dependent on (a) and (b) above.

An exemplary non-limiting range for a therapeutically or prophylactically effective amount of a low AR composition of the invention is 0.01 to 20 mg / kg, or 1 to 10 mg / kg, or 0.3 to 1 mg / kg. For a low AR composition comprising an anti-TNF [alpha] antibody or antigen-binding portion thereof, e.g., adalimumab, an exemplary dose is 40 mg biweekly. In some embodiments, in particular, exemplary dosages for the treatment of ulcerative colitis or Crohn's disease include an initial dose of 160 mg (1 day) (e.g., 4 40 mg injections per day or 2 days 2 consecutive days) A second dose of 80 mg after two weeks, and a maintenance dose of 40 mg every other week starting two weeks later. Alternatively, in the case of psoriasis, for example, the dose may comprise 80 mg starting dose followed by 40 mg every other week starting one week after the starting dose.

It should be noted that the dose value may vary depending on the type and severity of the condition being alleviated. It should be understood that, for any particular subject, the particular dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering the composition or supervising the administration of the composition, and the dosage ranges set forth herein are exemplary only It is to be further understood that it is not intended to limit the scope or practice of the claimed composition.

VII. The pharmaceutical formulation containing the low AR composition of the present invention

The present invention further provides formulations and formulations comprising a low AR composition of the invention. Any of the antibodies and antibody fragments described herein, including antibodies and antibody fragments having any one or more of the structural and functional properties detailed throughout the application, It should be understood that it can be converted or manufactured. Where various formulations are described in this section as comprising an antibody, it is understood that such an antibody may be an antibody or antibody fragment having any one or more of the features of the antibody and antibody fragment described herein. In one embodiment, the antibody is an anti-TNFa antibody or antigen-binding portion thereof.

In certain embodiments, a low AR composition of the invention may be formulated as a pharmaceutical (therapeutic) composition with a pharmaceutically acceptable carrier and administered by any of the methods known in the art. As will be understood by those skilled in the art, the route and / or manner of administration will vary depending on the desired result. The term "pharmaceutically acceptable carrier" means one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient. Such formulations may routinely contain salts, buffers, preservatives, compatible carriers, and any other therapeutic agent. Such pharmaceutically acceptable preparations may also contain encapsulating materials which are routinely compatible with solid or liquid fillers, diluents, or for administration to humans. The term "carrier " refers to a natural or synthetic organic or inorganic ingredient in which the active ingredient is combined with it to be applicable. In addition, the components of the pharmaceutical composition may be co-mingled with the antibodies of the present invention and in a manner such that there is no interaction that substantially degrades the desired pharmaceutical efficacy.

The low AR compositions of the present invention are known in the art and exist in forms acceptable for therapeutic use. In one embodiment, the formulation of the low AR composition of the present invention is a liquid formulation. In another embodiment, the formulation of the low AR composition of the present invention is a reduced pressure lyophilized formulation. In a further embodiment, the formulation of the low AR composition of the present invention is a reconstituted liquid formulation. In one embodiment, the formulation of the low AR composition of the present invention is a suitable liquid formulation. In one embodiment, the liquid formulation of the low AR composition of the present invention is an aqueous formulation. In another embodiment, the liquid formulation is non-aqueous. In certain embodiments, a liquid formulation of a low AR composition of the invention is an aqueous formulation wherein the aqueous carrier is distilled water.

Formulations of the low AR compositions of the present invention comprise the antibody at a concentration resulting in an appropriate w / v for the desired dose. The antibody may be administered at a concentration of from about 1 mg / ml to about 500 mg / ml, such as greater than 1 mg / ml, greater than 5 mg / ml, greater than 10 mg / ml, greater than 15 mg / ml, greater than 20 mg / More than 30 mg / ml, more than 35 mg / ml, more than 40 mg / ml, more than 45 mg / ml, more than 50 mg / ml, more than 55 mg / ml, more than 60 mg / ml, more than 65 mg / ml, more than 70 mg / Or more, more than 80 mg / ml, more than 85 mg / ml, more than 90 mg / ml, more than 95 mg / ml, more than 100 mg / ml, more than 105 mg / ml, more than 110 mg / ml, more than 115 mg / ml, more than 120 mg / , At least 130 mg / ml, at least 135 mg / ml, at least 140 mg / ml, at least 150 mg / ml, at least 200 mg / ml, at least 250 mg / ml or at least 300 mg / ml.

In certain embodiments, formulations of a low AR composition of the invention comprise an antibody of the invention at least about 100 mg / ml, at least about mg / ml, at least about 130 mg / ml, or at least about 150 mg / ml.

In one embodiment, the antibody contained in the formulations of the present invention has a concentration of about 1 mg / ml to about 25 mg / ml, about 1 mg / ml to about 200 mg / ml, about 25 mg / ml to about 200 mg / ml to about 200 mg / ml, from about 75 mg / ml to about 200 mg / ml, from about 100 mg / ml to about 200 mg / ml, from about 125 mg / ml to about 150 mg / ml, from about 50 mg / ml to about 150 mg / ml, from about 75 mg / ml to about 150 mg / ml, from about 100 mg / ml to about 125 mg / ml, from about 50 mg / ml to about 125 mg / ml, from about 75 mg / ml to about 125 mg / ml, from about 100 mg / ml to about 100 mg / ml, from about 75 mg / ml to about 100 mg / ml, from about 25 mg / ml to about 75 mg / ml, from about 50 mg / ml to about 75 mg / ml, or from about 25 mg / ml to about 50 mg / ml.

In certain embodiments, a formulation of a low AR composition of the invention comprises between about 90 mg / ml and about 110 mg / ml or between about 100 mg / ml and about 210 mg / ml of an antibody.

Formulations of a low AR composition of the invention comprising an antibody may additionally comprise one or more active compounds required for a particular indication to be treated, which typically has complementary activity that does not adversely affect one another . These additional active compounds are present in suitable combinations in amounts effective for the intended purpose.

Formulations of the low AR compositions of the present invention can be prepared by mixing the antibody having the desired purity with a buffering agent, a saccharide, a salt, a surfactant, a solubilizer, a polyol, a diluent, a binder, a stabilizer, a salt, a lipophilic solution, or does not contain a preservative, such as, but limited to, by the carrier, is mixed with excipients, or stabilizers that are allowed in any physiological for storage (Goodman and Gilman's the Pharmacological Basis of Therapeutics, 12 ^th edition, L. Brunton, et al. and Remington ' s Pharmaceutical Sciences , 16th edition, Osol, A. Ed. (1999)) and may be prepared in the form of a reduced pressure lyophilized formulation or aqueous solution to the desired final concentration. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, such as buffers such as histidine, phosphate, citrate, glycine, acetate and other organic acids; Antioxidants including ascorbic acid and methionine; Preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol; Resorcinol, cyclohexanol, 3-pentanol, and m-cresol); Low molecular weight (less than about 10 residues) polypeptides; Proteins, such as serum albumin, gelatin, or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; Monosaccharides, disaccharides, and other carbohydrates including trehalose, glucose, mannose, or dextrin; Chelating agents such as EDTA; Sugars such as sucrose, mannitol, trehalose or sorbitol; Salt-forming counter-ions, such as sodium; Metal complexes (e. G., Zn-protein complexes); And / or non-ionic surfactants such as TWEEN, polysorbate 80, PLURONICS (TM) or polyethylene glycol (PEG).

The buffer may be histidine, citrate, phosphate, glycine or acetate. The saccharide vehicle may be trehalose, sucrose, mannitol, maltose or raffinose. The surfactant may be polysorbate 20, polysorbate 40, polysorbate 80, or Pluronic F68. Salt may be NaCl, KCl, MgCl _2, or CaCl _2.

Formulations of the low AR compositions of the present invention may include buffers or pH adjusting agents to provide improved pH control. The formulations of the present invention may be formulated to contain about 3.0 to about 9.0, about 4.0 to about 8.0, about 5.0 to about 8.0, about 5.0 to about 7.0, about 5.0 to about 6.5, about 5.5 to about 8.0, about 5.5 to about 7.0, ≪ / RTI > to about 6.5. In a further embodiment, the formulations of the present invention comprise about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, A pH of about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.5, about 8.0, about 8.5, . In certain embodiments, the formulations of the present invention have a pH of about 6.0. One of skill in the art will appreciate that the pH of the formulation should generally not be equal to the isoelectric point of the particular antibody used in the formulation.

Typically, the buffer is a salt prepared from an organic or inorganic acid or base. Representative buffers include salts of organic acids such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; But are not limited to, Tris, tromethamine hydrochloride, or phosphate buffer. In addition, the amino acid constituent may have a buffering ability. Representative amino acid components that may be used as buffers in the formulations of the present invention include, but are not limited to, glycine and histidine. In certain embodiments, the buffer is selected from histidine, citrate, phosphate, glycine, and acetate. In certain embodiments, the buffer is histidine. In another specific embodiment, the buffer is citrate. In another specific embodiment, the buffer is glycine. The purity of the buffer should be at least 98%, or at least 99%, or at least 99.5%. The term "purity" as used in connection with histidine and glycine is present in, for example, in. [The Merck Index, 13 ^th ed, O'Neil et al. ed. Quot; refers to the chemical purity of histidine or glycine as understood in the art as described in the literature (Merck & Co., 2001).

The buffer is typically used at a concentration or concentration in the range of about 1 mM to about 200 mM, or within the range, depending on the desired ionic strength and the buffering capacity required. Conventional concentrations of conventional buffering agents used in parenteral formulations are described in Pharmaceutical Dosage Forms: Parenteral Medications, Volume 1, 2 ^nd Edition, Chapter 5, p. 194, De Luca and Boylan, "Formulation of Small Volume Parenterals", Table 5: Commonly used additives in Parenteral Products. In one embodiment, the buffer is selected from the group consisting of about 1 mM, or about 5 mM, about 10 mM, or about 15 mM, or about 20 mM, or about 25 mM, or about 30 mM, or about 35 mM, or about 40 mM, or about 45 mM, About 60 mM, or about 70 mM, or about 80 mM, or about 90 mM, or about 100 mM. In one embodiment, the buffer is selected from the group consisting of 1 mM, or 5 mM, or 10 mM, or 15 mM, or 20 mM, or 25 mM, or 30 mM, or 35 mM, or 40 mM, or 45 mM, or 50 mM, or 60 mM, or 70 mM, , ≪ / RTI > or 100 mM. In certain embodiments, the buffer is present at a concentration of about 5 mM to about 50 mM. In another specific embodiment, the buffer is present at a concentration of about 5 mM to about 20 mM.

In certain embodiments, the formulation of the low AR composition of the present invention comprises histidine as a buffer. In one embodiment, the histidine is present in the formulations of the invention at a concentration of about 1 mM or more, about 5 mM or more, about 10 mM or more, about 20 mM or more, about 30 mM or more, about 40 mM or more, about 50 mM or more, about 75 mM or more, Or at a concentration of histidine of at least about 200 mM. In another embodiment, the formulations of the present invention are formulated for oral administration in an amount of from about 1 mM to about 200 mM, from about 1 mM to about 150 mM, from about 1 mM to about 100 mM, from about 1 mM to about 75 mM, from about 10 mM to about 200 mM, from about 10 mM to about 150 mM, About 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 20 mM to about 75 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, or about 20 mM to about 30 mM Of histidine. In a further embodiment, the formulations comprise about 1 mM to about 5 mM, about 10 mM to about 20 mM, to about 25 mM to about 30 mM to about 35 mM to about 40 mM to about 45 mM to about 50 mM to about 60 mM to about 70 mM to about 80 mM to about 90 mM, About 100 mM, about 150 mM, or about 200 mM histidine. In certain embodiments, the formulation may comprise about 10 mM, about 25 mM histidine, or no histidine.

Formulations of the low AR compositions of the present invention may include carbohydrate excipients. The carbohydrate excipient may serve as, for example, a viscosity improver, a stabilizer, a bulking agent, and / or a solubilizing agent. Carbohydrate excipients are generally present in an amount ranging from about 1% to about 99% by weight or volume, such as from about 0.1% to about 20%, from about 0.1% to about 15%, from about 0.1% to about 5% About 20%, about 5% to about 15%, about 8% to about 10%, about 10% to about 15%, about 15% to about 20% % To 10%, 10% to 15%, 15% to 20%, about 0.1% to about 5%, about 5% to about 10%, or about 15% to about 20%. In another specific embodiment, the carbohydrate excipient is present at 1%, or 1.5%, or 2%, or 2.5%, or 3%, or 4%, or 5%, or 10%, or 15%, or 20% do.

Suitable carbohydrate excipients for use in the formulations of the present invention include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; Disaccharides such as lactose, sucrose, trehalose, and cellobiose; Polysaccharides such as raffinose, melezitose, maltodextrin, dextran, and starch; And alditols such as mannitol, xylitol, maltitol, lactitol, and xylitol sorbitol (glucitol), but are not limited thereto. In one embodiment, the carbohydrate excipient for use in the present invention is selected from sucrose, trehalose, lactose, mannitol, and raffinose. In certain embodiments, the carbohydrate excipient is trehalose. In another specific embodiment, the carbohydrate excipient is mannitol. In another specific embodiment, the carbohydrate excipient is sucrose. In another specific embodiment, the carbohydrate excipient is raffinose. The purity of the carbohydrate excipient should be at least 98%, at least 99%, or at least 99.5%.

In certain embodiments, the formulation of the low AR composition of the present invention may comprise trehalose. In one embodiment, the formulations of the present invention comprise about 1% or more, about 2% or more, about 4% or more, about 8% or more, about 20% or more, about 30% or more or about 40% or more of trehalose. In another embodiment, the formulations of the present invention comprise from about 1% to about 40%, from about 1% to about 30%, from about 1% to about 20%, from about 2% to about 40% About 2% to about 20%, about 4% to about 40%, about 4% to about 30%, or about 4% to about 20% of trehalose. In a further embodiment, the formulations of the present invention comprise about 1%, about 2%, about 4%, about 6%, about 8%, about 15%, about 20%, about 30%, or about 40% . In certain embodiments, the formulations of the present invention comprise about 4%, about 6%, or about 15% of trehalose.

In certain embodiments, the formulation of a low AR composition of the invention comprises an excipient. In certain embodiments, the formulations of the present invention comprise one or more excipients selected from sugars, salts, surfactants, amino acids, polyols, chelating agents, emulsifying agents and preserving agents. In one embodiment, the formulations of the present invention comprise a salt selected from, for example, NaCl, KCl, CaCl ₂ , and MgCl ₂ . In certain embodiments, the formulation comprises NaCl.

Formulations of a low AR composition of the present invention may comprise at least about 10 mM, at least about 25 mM, at least about 50 mM, at least about 75 mM, at least about 80 mM, at least about 100 mM, at least about 125 mM, at least about 150 mM, at least about 175 mM, About 300 mM or more of sodium chloride (NaCl). In a further embodiment, the formulations are administered at a concentration of from about 10 mM to about 300 mM, from about 10 mM to about 200 mM, from about 10 mM to about 175 mM, from about 10 mM to about 150 mM, from about 25 mM to about 300 mM, from about 25 mM to about 200 mM, From about 50 mM to about 200 mM, from about 50 mM to about 200 mM, from about 50 mM to about 175 mM, from about 50 mM to about 150 mM, from about 75 mM to about 300 mM, from about 75 mM to about 200 mM, from about 75 mM to about 175 mM, from about 75 mM From about 100 mM to about 300 mM, from about 100 mM to about 200 mM, from about 100 mM to about 175 mM, or from about 100 mM to about 150 mM sodium chloride. In a further embodiment, the formulation may comprise about 10 mM, about 25 mM, about 50 mM, about 75 mM, about 80 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, or about 300 mM sodium chloride.

Formulations of the low AR compositions of the present invention may also include amino acids, such as lysine, arginine, glycine, histidine or amino acid salts. The formulation may comprise an amino acid greater than about 1 mM, greater than about 10 mM, greater than about 25 mM, greater than about 50 mM, greater than about 100 mM, greater than about 150 mM, greater than about 200 mM, greater than about 250 mM, greater than about 300 mM, greater than about 350 mM, . In other embodiments, the formulations comprise about 1 mM to about 100 mM, from about 10 mM to about 150 mM, from about 25 mM to about 250 mM, from about 25 mM to about 300 mM, from about 25 mM to about 350 mM, from about 25 mM to about 400 mM, from about 50 mM to about 250 mM, From about 100 mM to about 300 mM, from about 100 mM to about 400 mM, from about 150 mM to about 250 mM, from about 150 mM to about 300 mM, or from about 150 mM to about 300 mM, from about 50 mM to about 350 mM, from about 50 mM to about 400 mM, from about 100 mM to about 250 mM, To about 400 mM amino acid. In a further embodiment, the formulations of the invention comprise an amino acid of about 1 mM, about 1.6 mM, about 25 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM,

The formulation of the low AR composition of the present invention may further comprise a surfactant. The term "surfactant " as used herein refers to an organic material having an amphipathic structure; That is, they consist of groups of opposing solubility tendencies, typically oil-soluble hydrocarbon chains and water-soluble ionic groups. Surfactants can be classified as anionic, cationic, and nonionic surfactants depending on the charge of the surface-active moiety. Surfactants are often used as wetting, emulsifying, solubilizing, and dispersing agents for various pharmaceutical compositions and formulations of biological materials. Polysorbates (e.g., polysorbate 20 or 80); Polyoxamers (e. G., Polyoxamers 188); Triton; Sodium octyl glycoside; Lauryl-, myristyl-, linoleic-, or stearyl-sulfobetaine; Lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; Linoleyl-, myristyl-, or cetyl-betaine; Lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmitopropyl-, or isostearamidopropyl-betaine (for example lauroamidopropyl); Myristamidopropyl-, palmitopropyl-, or isostearamidopropyl-dimethylamine; Sodium methylcocoyl-, or disodium methyl oleyl-taurate; And copolymers of ethylene and propylene glycol (e.g., PLURONICS (TM), PF68, etc.), such as the MONAQUA ™ series (Mona Industries, Inc., Paterson, NJ), polyethylglycol, polypropylglycol, Surfactants may optionally be added to the formulation of the invention to reduce aggregation. In one embodiment, the formulation of the invention comprises polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. Surfactants are particularly useful when pumps or plastic containers are used to administer formulations. The presence of a pharmaceutically acceptable surfactant alleviates the tendency of the protein to aggregate. Formulations may include polysorbates present in a concentration ranging from about 0.001% to about 1%, or from about 0.001% to about 0.1%, or from about 0.01% to about 0.1%. In other specific embodiments, formulations of the present invention may contain 0.001%, or 0.002%, or 0.003%, or 0.004%, or 0.005%, or 0.006%, or 0.007%, or 0.008%, or 0.009%, or 0.01% , Or 0.015%, or 0.02%, by weight of the composition.

Formulations of the low AR compositions of the invention may optionally further comprise other conventional excipients and / or additives, including but not limited to diluents, binders, stabilizers, lipophilic solutions, preservatives, or adjuvants, . Pharmaceutically acceptable excipients and / or additives may be used in the formulations of the present invention. Commonly used excipients / additives, such as, for example, pharmaceutically acceptable chelating agents (such as, but not limited to, EDTA, DTPA, or EGTA) may optionally be added to the formulation of the present invention to reduce aggregation . These additives are particularly useful when a pump or plastic container is used to administer the formulation.

Preservatives such as phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenyl mercury nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride, Benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof, may be used in combination with the compounds of the present invention (such as, for example, hexahydrate), alkyl parabens The formulation may optionally be added at any suitable concentration, such as from about 0.001% to about 5%, or any range or value within the range. The concentration of the preservative used in the formulation of the present invention is sufficient to obtain a microbial effect. This concentration is dependent on the chosen preservative and is readily determined by those skilled in the art.

Other contemplated excipients / additives that may be used in the formulation of the present invention include, for example, flavoring agents, antibacterial agents, sweeteners, antioxidants, antistatic agents, lipids such as phospholipids or fatty acids, steroids such as cholesterol, Excipients such as serum albumin (human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and salt-forming counterions such as sodium. These and further known pharmaceutical excipients and / or additives suitable for use in the formulations of the present invention are described, for example, in Remington: The Science & Practice of Pharmacy, 21 ^st ed., Lippincott Williams & Wilkins, (2005), and, "Physician's Desk Reference" 60 th ed., are known in the art, as is listed in the Medical Economics, Montvale, NJ (2005 )]. Pharmaceutically acceptable carriers suitable for the mode of administration, solubility and / or stability of the antibody can be routinely selected as is well known in the art or as described herein.

In one embodiment, the low AR composition of the present invention, the literature [ "Highlights of Prescribing Information" for HUMIRA ® (adalimumab) Injection (Revised Jan. 2008), full text of the document is included by reference herein] described in Are formulated together with excipients and buffers identical or similar to those present in commercially available HUMIRA ( ^R ) formulations. For example, subcutaneous administration, each of the pre-filled syringe of HUMIRA ^® that is, the transmission to the subject of drug products of 0.8mL (40mg). Each 0.8 mL of HUMIRA ^® contains 40 mg of adalimumab, 4.93 mg of sodium chloride, 0.69 mg of monobasic sodium phosphate dihydrate, 1.22 mg of dibasic sodium phosphate dihydrate, 0.24 mg of sodium citrate, 1.04 mg of Citric acid monohydrate, 9.6 mg of mannitol, 0.8 mg of polysorbate 80, and water for injection, USP. Sodium hydroxide is added as needed to adjust the pH.

It will be understood by those skilled in the art that the formulations of the low AR compositions of the present invention may be isotonic with human blood, wherein the formulations of the present invention essentially have the same osmotic pressure as human blood. Such isotonic formulations will generally have an osmotic pressure of about 250 mOSm to 350 mOSm. Isotonicity can be measured, for example, by using a vapor pressure or an ice-freezing type osmometer. The tonicity of the formulation is adjusted by the use of a tension modifier. A "tension modifier" is a pharmaceutically acceptable inert material that can be added to a formulation to provide isotonicity of the formulation. Tension modifiers suitable for the present invention include, but are not limited to, saccharides, salts and amino acids.

In certain embodiments, formulations of a low AR composition of the invention have a viscosity of from about 100 mOSm to about 1200 mOSm, or from about 200 mOSm to about 1000 mOSm, or from about 200 mOSm to about 800 mOSm, or from about 200 mOSm to about 600 mOSm, From about 250 mOSm to about 400 mOSm, or from about 250 mOSm to about 350 mOSm.

The concentration of any one constituent or any combination of the various constituents of the formulation of the low AR composition of the present invention is adjusted so that the desired tension of the final formulation is achieved. For example, the ratio of carbohydrate excipient to antibody can be adjusted according to methods known in the art (e.g., U.S. Patent No. 6,685,940). In certain embodiments, the molar ratio of carbohydrate excipient to antibody is from about 100 to about 1000 moles of carbohydrate excipient for about 1 mole of antibody, or about 200 to about 6000 moles of carbohydrate excipient for about 1 mole of antibody, , Or about 100 to about 510 moles of carbohydrate excipient for about 1 mole of antibody, or about 100 to about 600 moles of carbohydrate excipient for about 1 mole of antibody.

In addition, the desired isotacticity of the final formulation can be achieved by adjusting the salt concentration of the formulation. Suitable pharmaceutically acceptable salts and tension modifiers for the present invention include, but are not limited to, sodium chloride, sodium sulphate, sodium sulphate, potassium chloride, magnesium chloride, magnesium sulphate, and calcium chloride. In certain embodiments, the formulations of the invention include NaCl, MgCl _2, and / or CaCl _2. In one embodiment, the concentration of NaCl is from about 75 mM to about 150 mM. In another embodiment, the concentration of MgCl ₂ is from about 1 mM to about 100 mM. Pharmaceutically acceptable amino acids including those suitable for the present invention as a tension modifier include, but are not limited to, proline, alanine, L-arginine, asparagine, L-aspartic acid, glycine, serine, lysine, no.

In one embodiment, the formulation of the low AR composition of the present invention is a pyrogen-free formulation that is substantially free of endotoxins and / or related exothermic materials. Endotoxins include toxins that are localized within the microorganism and released only when the microorganism is destroyed or killed. In addition, exothermic materials include thermo-labile materials (glycoproteins) from external membranes of bacteria and other microorganisms. Both of these materials can cause fever, hypertension and shock when administered to humans. Due to potentially harmful effects, even a low amount of endotoxin should be removed from the intravenously administered pharmaceutical drug solution. The United States Food and Drug Administration ("FDA") has set an upper limit of 5 endotoxin units per dose per kilogram of body weight in a single hour for intravenous drug applications (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1): 223 (2000)). If the therapeutic protein is administered in amounts of hundreds or thousands of milligrams per kilogram of body weight, as in the case of antibodies, trace amounts of harmful and dangerous endotoxins must be removed. In certain embodiments, the level of endotoxin and pyrogens in the composition is less than 10 EU / mg, or less than 5 EU / mg, or less than 1 EU / mg, or less than 0.1 EU / mg, or less than 0.01 EU / mg.

When used for in vivo administration, the formulations of the low AR compositions of the present invention must be sterile. The formulations of the present invention can be sterilized by various sterilization methods including sterile filtration, irradiation, and the like. In one embodiment, the antibody formulation is filter sterilized with a pre-sterilized 0.22-micron filter. The injectable sterile compositions may be formulated according to conventional pharmaceutical practice as described in "Remington: The Science & Practice of Pharmacy ", 21 ^st ed., Lippincott Williams & Wilkins, . Formulations comprising antibodies, such as those disclosed herein, will usually be stored in a reduced-pressure lyophilized form or in solution. Sterile compositions comprising an antibody can be used in a container having a sterile access port, for example, an intravenous solution bag or an adapter that enables retrieval of the formulation, for example, Is believed to be located in a vial having a stopper that can be pierced with a hypodermic needle. In one embodiment, the composition of the present invention is provided as a pre-filled syringe.

In one embodiment, the formulation of the low AR composition of the present invention is a reduced pressure lyophilized formulation. The term " reduced pressure lyophilized "or" freeze-dried "includes the state of a material subjected to a drying procedure such as reduced pressure freeze drying with at least 50% moisture removed.

The phrase "bulking agent" includes a compound that adds a bulk to a pharmaceutically acceptable and reduced pressure lyophilized cake. Bulking agents known in the art include, for example, carbohydrates including monosaccharides such as dextrose, ribose, and fructose, sugar alcohols such as mannitol, inositol and sorbitol, diols including trehalose, sucrose and lactose Natural polymers such as saccharides, starch, dextran, chitosan, hyaluronate, proteins (e.g., gelatin and serum albumin), glycogen, and synthetic monomers and polymers.

Lyoprotectant can be used to significantly prevent or reduce the chemical and / or physical instability of proteins upon reduced pressure freeze-drying and subsequent storage, when combined with a desired protein (e.g., an antibody of the invention) The lyophilized protective agents include sugars and their corresponding sugar alcohols, amino acids such as monosodium glutamate or histidine, methylamines such as betaine, lyotropic salts such as, for example, Magnesium sulfate, polyols such as trihydric or higher molecular weight sugar alcohols such as glycerin, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol; Polyethylene glycol, PLURONICS ™, and combinations thereof. Additional examples of lyophilized protective agents include glycerin and gelatin, sugars, melibiose Examples of reducing sugars include glucose, maltose, lactose, maltulose, iso-maltulose and lactulose, but they are not limited thereto. Examples of reducing sugars include glucose, maltose, lactose, Non-reducing sugar. Examples of non-reducing sugars include, but are not limited to, non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other linear polyhydric alcohols. Examples of sugar alcohols include monoglyco But are not limited to, compounds obtained by reduction of disaccharides such as carboxymethylcellulose, carboxymethylcellulose, carboxymethylcellulose, carboxymethylcellulose, carboxymethylcellulose, carboxymethylcellulose, Additional examples of sugar alcohols include, but are not limited to, glucitol, maltitol, lactitol, and iso-maltulose In certain embodiments, trehalose or sucrose is used as a lyophilized protective agent.

The lyophilized protective agent is characterized in that after lyophilization under reduced pressure of the protein in the presence of the lyophilized protective amount of the lyophilized protective agent, the protein has essentially the physical and chemical stability and integrity of the protein in vacuum lyophilization and storage Is added to the lyophilized pre-formulation as the "lyophilized protection dose ".

In one embodiment, the molar ratio of the lyophilized protective agent (e. G., Trehalose) and antibody molecules of the formulations of the present invention is at least about 10, at least about 50, at least about 100, at least about 200, In another embodiment, the molar ratio of the lyophilized protective agent (e. G., Trehalose) of the formulations of the invention to the antibody molecule is about 1, about 2, about 5, about 10, about 50, about 100, about 200, to be.

A "reconstituted" formulation is one that is prepared by dissolving the lyophilized antibody formulation in a diluent to disperse the antibody in the reconstituted formulation. The reconstituted formulation is suitable for administration to a patient to be treated with an antibody (e.g., parenteral administration) and may be suitable for intravenous administration in certain embodiments of the invention.

The intended "diluent " herein is useful for the preparation of a liquid formulation that is pharmaceutically acceptable (safe and non-toxic for administration to humans), for example, reconstituted formulation after reduced pressure freeze drying. In some embodiments, the diluent includes sterile water, injectable bacteriostatic water (BWFI), pH buffer (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution, or dextrose solution , But are not limited thereto. In alternative embodiments, the diluent may comprise an aqueous solution of a salt and / or buffer.

In certain embodiments, a formulation of a low AR composition of the invention is a reduced pressure lyophilized formulation comprising an antibody of the invention wherein at least about 90%, at least about 95%, at least about 97% At least 98%, or at least about 99%, of the volume of the vial can be recovered from the vial by shaking the vial filled with the formulation at a rate of 400 shakes per minute for 4 hours. In another embodiment, a formulation of the invention is a reduced pressure lyophilized formulation comprising an antibody of the invention wherein at least about 90%, at least about 95%, at least about 97%, at least about 98% Or about 99% or more of the volume of the vial can be recovered by performing three cycles of freezing / thawing from the vial filled with the formulation. In a further embodiment, a formulation of the invention is a reduced pressure lyophilized formulation comprising an antibody of the invention wherein at least about 90%, at least about 95%, at least about 97%, at least about 98% , Or about 99% or more of the composition can be recovered by reconstituting the reduced pressure lyophilized cake produced from the formulation.

In one embodiment, the reconstituted liquid formulation may comprise the antibody at the same concentration as the reduced pressure freeze-dried liquid formulation.

In another embodiment, the reconstituted liquid dosage form comprises the antibody in a concentration that is higher than the reduced-pressure lyophilization-pre-liquid formulation, such as about 2-fold, about 3-fold, about 4-fold , About 5 times, about 6 times, about 7 times, about 8 times, about 9 times, or about 10 times higher.

In another embodiment, the reconstituted liquid dosage forms comprise the antibody of the present invention at a lower concentration than the reduced pressure freeze-dried liquid formulation, for example, about 2-fold, about 3-fold, About 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, or about 10 times lower.

The pharmaceutical formulation of the low AR composition of the present invention is typically a stable, e.g., stable formulation at room temperature.

The terms "stability" and "stable ", as used herein in connection with formulations comprising an antibody of the invention, refer to the resistance of the antibody in the formulation to aggregation, degradation or fragmentation under given manufacturing, formulation, . A "stable" formulation of the invention retains biological activity under given manufacturing, formulation, delivery, and storage conditions. The stability of the antibody can be assessed by the HPSEC, static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular polarization dichroism (CD), urea unfolding technique, intrinsic tryptophan fluorescence, differential scanning calorimetry, Or by the degree of aggregation, degradation or fragmentation compared to the reference formulation, as measured by the ANS binding technique. For example, the reference formulation may be a reference standard frozen at -70 占 폚 consisting of 10 mg / ml of an antibody of the invention in PBS.

The therapeutic formulations of the low AR compositions of the present invention may be formulated for a particular dosage. Dosage regimens may be adjusted to provide the optimal desired response (e. G., Therapeutic response). For example, a single bolus may be administered, some sub-doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as a single dose for a subject to be treated; Each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The description of the dosage unit form of the present invention is based on the assumption that (a) the intrinsic characteristics of the antibody and the specific therapeutic effect achieved, and (b) the limitations inherent in the art of the formulation of such antibodies for the treatment of sensitivity in the individual , And is directly dependent on (a) and (b) above.

The therapeutic compositions of the low AR compositions of the present invention may be formulated for a particular route of administration such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. By way of example, in some embodiments, antibodies (including antibody fragments) are formulated for intravenous administration. In certain other embodiments, the antibodies (including antibody fragments) can be delivered to a subject via, for example, catheter, stent, wire, intracardiac delivery, intracardiac delivery, It is formulated for local delivery to cardiovascular.

Formulations of the low AR compositions of the present invention suitable for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound can be mixed with a pharmaceutically acceptable carrier under sterile conditions and with any preservative, buffer, or propellant that may be required (US Patents 7,378,110; 7,258,873; 7,135,180; 7,923,029; and US Publication No. 20040042972).

As used herein, the phrases "parenteral administration" and "parenterally administered " refer to a mode of administration by injection other than intestinal and topical administration, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, (Subcapsular), subarachnoid, intraspinal, epidural, and sternal (subcapsular) subcapsular, subarachnoid, subarachnoid, subepithelial, Injection and injection.

The actual dosage level of the active ingredient in the pharmaceutical composition of the low AR composition of the present invention will depend on the amount of active ingredient effective to achieve the desired therapeutic response to the particular patient, To be obtained. The selected dose level will depend upon a variety of factors including the activity of the particular composition of the present invention, or the ester, salt, or amide thereof used, the mode of administration, the time of administration, the rate of excretion of the particular compound employed, Depend on other pharmacokinetic factors, including other drugs, compounds and / or materials in combination with the age, sex, weight, condition, general health and prior history of the patient being treated, and similar factors well known in the medical arts something to do.

In certain embodiments, the antibodies of the invention can be formulated to be appropriately distributed in vivo . For example, the blood-brain barrier (BBB) excludes primarily highly hydrophilic compounds. In order to ensure that the therapeutic compounds of the present invention can cross the BBB (if desired), they can be formulated, for example, in liposomes. For a method of preparing liposomes, see, for example, US Pat. 4,522,811; 5,374,548; 5,399, 331]. Liposomes can include one or more moieties that enhance targeted drug delivery by being selectively delivered to a particular cell or organ (see, for example, VV Ranade (1989) J. Clin . Pharmacol . 29: 685 ]). Exemplary targeting moieties include folate or biotin (see, e. G., US Pat. No. 5,416,016); Manoside (Umezawa et al. , (1988) Biochem . Biophys . Res. Commun . 153: 1038); Antibodies (PG Bloeman et al. (1995) FEBS Lett . 357: 140; M. Owais et al. (1995) Antimicrob . Agents Chemother. 39: 180); Surfactants, protein A receptors (Briscoe et al. (1995) Am. J. Physiol. 1233: 134), formulations of the invention, and various components of the molecules of the invention; p120 (Schreier et al. (1994) J. Biol . Chem . 269: 9090); Also, see K. Keinanen; ML Laukkanen (1994) FEBS Lett . 346: 123; JJ Killion; IJ Fidler (1994) Immunomethods 4: 273]. In one embodiment of the invention, the therapeutic compounds of the invention can be formulated in liposomes; In another embodiment, the liposome comprises a targeting moiety. In another embodiment, the therapeutic compound in the liposome is delivered to a site close to the desired region by a bolus injection. When administered in this manner, the composition must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. Additionally or alternatively, the antibodies of the invention may be delivered locally to the brain to alleviate the risk that the blood-brain barrier will slow effective delivery.

In certain embodiments, the low AR compositions of the invention may be administered in a medical device known in the art. For example, in certain embodiments, the antibody or antibody fragment is administered topically via a catheter, stent, wire, or the like. For example, in one embodiment, the therapeutic compositions of the present invention may be used in needleless subcutaneous injection devices, such as those described in U.S. Pat. Pat. 5,399, 163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; 4,596,556. ≪ / RTI > Examples of well-known implants and modules useful in the present invention include, but are not limited to, U.S. Pat. Pat. 4,487, 603; ≪ / RTI > which discloses a therapeutic device for administering medicament through the skin. Pat. 4,486,194; ≪ / RTI > which discloses a medication infusion pump for delivering medicament at a precise infusion rate. Pat. 4,447,233; ≪ / RTI > discloses a variable flow implantable infusion device for continuous drug delivery. Pat. 4,447,224; U.S. Pat. No. 5,204,507 to Omega-3 discloses an osmotic drug delivery system having multiple-chamber compartments. Pat. 4,439,196; ≪ / RTI > which discloses an osmotic drug delivery system. Pat. 4,475,196. Many other such implants, delivery systems, and molecules are known to those skilled in the art.

The effective dose and dosage regimen for a low AR composition of the invention will depend on the disease or condition being treated and can be determined by one of skill in the art. One of ordinary skill in the art can determine this amount based on such factors as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

VIII. Alternative formulations containing a low AR composition of the invention

Alternative aqueous formulation

In addition, the present invention is described in U.S. Pat. As described in U.S. Patent Nos. 8,420,081 and WO2012 / 065072, the entire contents of which are incorporated herein by reference, provides a low AR composition formulated as an aqueous formulation comprising protein and water. In these aqueous formulations, the protein is stable without requiring additional formulations. Such aqueous formulations are more stable than conventional formulations in the art without the need for additional excipients and without the need for additional excipients to maintain the stability of the protein in water, Lt; RTI ID = 0.0 > concentration. ≪ / RTI > They are also advantageous because the proteins in the formulation remain stable during storage, for example, at 7 DEG C or in freezing / thawing conditions, even in high protein concentrations and repeated frozen / thaw processing steps in liquid form Storage characteristics. In one embodiment, the formulations described herein comprise a high concentration of protein such that the aqueous formulation does not exhibit significant opalescence, aggregation, or precipitation.

In one embodiment, there is provided an aqueous low AR composition comprising a protein, e. G., An antibody, such as an anti-TNFa antibody or antigen-binding portion thereof, and water, wherein the formulation is not limited thereto But have, for example, low conductivity, such as conductivity less than about 2.5 mS / cm, protein concentration greater than about 10 μg / mL, osmotic concentration less than about 30 mOsmol / kg, The protein has a molecular weight (Mw) of greater than about 47 kDa. In one embodiment, the formulations include, but are not limited to, stability in a liquid formulation over an extended period of time (e.g., greater than about 3 months, or greater than about 12 months), or stability over one or more freeze / thaw cycles And no further refrigeration / thaw cycles are present). In one embodiment, the formulations are stable for at least about three months in a form selected from the group consisting of frozen, reduced pressure lyophilized, or spray-dried forms.

In one embodiment, the formulations may include, for example, less than about 2.5 mS / cm conductivity, less than about 2 mS / cm conductivity, less than about 1.5 mS / cm conductivity, less than about 1 mS / cm conductivity, / cm, < / RTI >

In other embodiments, the low AR composition contained in the formulation may be, for example, at least about 1 mg / mL, at least about 10 mg / mL, at least about 50 mg / mL, at least about 100 mg / mL, at least about 150 mg / mL, / mL, or greater than about 200 mg / mL. < / RTI > In another embodiment, the formulation of the present invention has an osmolality concentration of about 15 mOsmol / kg or less.

The aqueous formulations described herein may be formulated with standard excipients such as, for example, a tension modifier, a stabilizer, a surfactant, an antioxidant, a cryoprotectant, a bulking agent, a lyophilized protective agent, a basic component, Do not depend on it. In another embodiment of the invention, the formulations contain water, one or more proteins, and nonionic excipients (e. G., Salts, free amino acids).

In certain embodiments, the aqueous formulations described herein comprise a low AR composition comprising a protein concentration of at least 50 mg / mL and water, wherein the formulation has an osmotic concentration of 30 mOsmol / kg or less. The lower limit of the osmolality of the aqueous formulation is also encompassed by the present invention. In one embodiment, the osmolality concentration of the aqueous formulation is 15 mOsmol / kg or less. The aqueous formulations of the present invention may have an osmotic concentration of less than 30 mOsmol / kg and also have a high protein concentration, for example, a protein concentration of at least 100 mg / ml and may be present at least 200 mg / ml. The ranges cited above and in the middle of the osmotic concentration units are also intended to be part of the present invention. It is also intended that ranges of values using any combination of the values listed above as upper and / or lower limits are also intended to be included.

The concentrations of the aqueous formulations described herein are not limited by the protein size, and the formulations may include proteins of any size range. Such as 40 mg / mL, 65 mg / mL, 130 mg / mL, or 195 mg / mL, which may be in the size range from 5 kDa to 150 kDa, Formulations are included within the scope of the present invention. In one embodiment, the protein in the formulation of the invention is

Size greater than about 15 kD, size greater than about 20 kD; A size of about 47 kD or more; A size of about 60 kD or more; A size of about 80 kD or more; A size of about 100 kD or more; A size of about 120 kD or more; A size of about 140 kD or more; A size of about 160 kD or more; Or greater than about 160 kD. It is also contemplated that the intermediate range of the recited sizes is part of the present invention. It is also intended that ranges of values using any combination of the values listed above as upper and / or lower limits are also intended to be included.

The aqueous formulations described herein are characterized by the hydrodynamic diameter (D _h ) of the protein in solution. The hydrodynamic diameter of the protein in solution can be measured using dynamic light scattering (DLS), an established analytical method for measuring the protein D _h . A typical value for a monoclonal antibody, e. G. IgG, is about 10 nm. Low-ionic formulations can be characterized in that the D _h of the protein is significantly lower than that of a protein formulation comprising an ionic excipient. The D _h value of the antibody in the aqueous formulation made using the dialysis filtration / ultrafiltration (DF / UF) process as described in US Patent No. 8,420,081, using purified water as the exchange medium, Independently, significantly lower than the D _h of the antibody in conventional formulations. In one embodiment, the antibodies in the aqueous formulations described herein have a D _h of less than 4 nm, or less than 3 nm.

In one embodiment, the D _h of the protein in the aqueous formulation is less than the D _h of the same protein in the buffered solution, regardless of protein concentration. Thus, in some embodiments, a protein in an aqueous formulation produced according to the methods described herein will have a D _h of 25% or less less than the D _h of the protein in the buffered solution at the same defined concentration. Examples of buffered solutions include, but are not limited to, phosphate buffered saline (PBS). In certain embodiments, the protein in the aqueous formulation of the invention is at least 50% less than the D _h of the protein in PBS at a defined concentration; At least 60% less than the D _h of the protein in PBS at a given concentration; At least 70% less than the D _h of the protein in PBS at a given concentration; At a given concentration has a lower D _h D _h than in greater than 70% of the protein in PBS. It is contemplated that ranges that are midway between the percentages recited above, such as 55%, 56%, 57%, 64%, and 68%, are also part of the present invention. Also, it is intended that the range of values that use a combination of any of the recited values as an upper limit and / or lower limit, for example, from about 50% to about 80%.

In one aspect, the aqueous formulation comprises a protein at a dose of from about 0.01 mg / kg to about 10 mg / kg. In another aspect, the protein dose comprises about 1 mg / kg administered every other week, or about 0.3 mg / kg administered weekly. Those skilled in the art will know the appropriate dosage and dosage for administration to the subject.

Alternative solid unit formulations

The present invention is further described in the Attorney Docket No. < RTI ID = 0.0 > 117813-31001), a low AR composition of the present invention formulated as a stable solid composition of a protein (preferably a therapeutic protein) and a stabilizer, represented herein as a solid unit. Specifically, despite having a higher proportion of sugar relative to the protein, the solid unit of the present invention maintains structural rigidity when stored for extended periods of time under ambient conditions, such as room temperature and room humidity And resisting changes in shape and / or volume. The solid unit of the present invention maintains free-flowing and can maintain long-term physical and chemical stability of the protein without significant degradation and / or aggregation formation. The solid units of the present invention have a number of advantages over the art, including that they can be formulated for oral delivery and are easily reconstituted in a diluent such as water. Since solid units are readily soluble, they can be used in dual chamber delivery devices and can be manufactured directly as devices for patient use.

The term "solid unit" as used herein refers to a composition suitable for pharmaceutical administration and comprising a protein, such as an antibody or peptide, and a stabilizing agent, for example, a sugar. The solid units have structural rigidity and resistance to changes in shape and / or volume. In a preferred embodiment, the solid unit is obtained by vacuum lyophilization of the pharmaceutical formulation of the therapeutic protein. The solid unit may be in any geometric form including, but not limited to, spheres, cubes, pyramids, hemispheres, cylinders, and teardrops, including any form including irregularly shaped units. In one embodiment, the solid unit has a volume ranging from about 1 ml to about 20 ml. In one embodiment, the solid unit is not obtained using a spray drying technique, for example, the solid unit is not a powder or granules.

As used herein, the phrase "a plurality of solid units" refers to a collection or group of solid units, wherein the collection comprises two or more solid units having substantially uniform morphology, e.g., spherical and / . In one embodiment, the plurality of solid units is free-flowing.

IX. Kits comprising the low AR compositions of the invention and articles of manufacture

Also, kits comprising the low AR compositions of the present invention and instructions for use are within the scope of the present invention. The term "kit " as used herein refers to a packaged product comprising components for administering an antibody or antigen-binding portion thereof of the invention to treat a disease or disorder. The kit may include a box or container holding its components. Boxes or containers are labeled with labeling or KFDA approval protocols. The box or container holds components of the present invention that can be contained in plastic, polyethylene, polypropylene, ethylene, or propylene containers. The container may be a capped-tube or bottle. The kit may also include instructions for administering an antibody of the invention.

The kit may comprise one or more additional reagents, such as an immunosuppressive agent, a cytotoxic agent or a radioactive toxic agent, or one or more additional antibodies of the invention (e. G., An epitope in a TNFa antigen distinct from the anti- Lt; / RTI > antibody with complementary activity that binds to the antibody). A kit typically includes a label that indicates its intended use for the kit's contents. The term label includes any article, or recording material, that is supplied on or otherwise supplied with or on the kit.

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with a liquid formulation or a reduced-pressure lyophilized formulation of the antibody or antibody fragment thereof of the present invention. In one embodiment, the container filled with the liquid formulation of the present invention is a pre-filled syringe. In certain embodiments, the formulations of the present invention are formulated into a single dose vial as a sterile liquid. For example, the formulation may be supplied in a 3 cc USP Type I borosilicate amber vial (West Pharmaceutical Services - Part No. 6800-0675) with a target volume of 1.2 ml. Optionally, a warning reflecting the agency's approval for manufacture, use or sale for human administration in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products may be combined with such container (s) .

In one embodiment, the container filled with the liquid formulation of the present invention is a pre-filled syringe. Any pre-filled syringe known to those skilled in the art may be used with the liquid formulations of the present invention. Pre-filled syringes that may be used include, but are not limited to, those disclosed in PCT Publications WO05032627, WO08094984, WO9945985, WO03077976, US Patents US6792743, US5607400, US5893842, US7081107, US7041087, US5989227, US6807797, US6142976, US5899889, US7699811, US7540382, US7998120, US7645267, and U.S. Patent Publication No. US20050075611. The pre-filled syringe can be made of a variety of materials. In one embodiment, the pre-filled syringe is a glass syringe. In another embodiment, the pre-filled syringe is a plastic syringe. One of skill in the art will appreciate that the nature and / or quality of the materials used to make the syringe may affect the stability of the protein formulation stored in the syringe. For example, it is understood that a silicone based lubricant attached to the inner surface of the syringe chamber may affect particle formation in the protein formulation. In one embodiment, the pre-filled syringe includes a silicone-based lubricant. In one embodiment, the pre-filled syringe comprises baked on silicone. In another embodiment, the pre-filled syringe does not include a silicone-based lubricant. Those skilled in the art will also appreciate that small amounts of contaminants that leak into the formulation from syringe barrels, syringe tip caps, plungers or stoppers may also affect the stability of the formulation. . For example, it is understood that the tungsten introduced during the manufacturing process may have deleterious effects on the formulation stability. In one embodiment, the pre-filled syringe may contain tungsten at a level of at least 500 ppb. In another embodiment, the pre-filled syringe is a low tungsten syringe. In another embodiment, the pre-filled syringe comprises tungsten from about 500 ppb to about 10 ppb, from about 400 ppb to about 10 ppb, from about 300 ppb to about 10 ppb, from about 200 ppb to about 10 ppb, from about 100 ppb to about 10 ppb, 25 ppb to about 10 ppb.

In certain embodiments, also, purification or immunoprecipitation of the protein of interest from the cells, the examination of the protein of interest in vitro Or in vivo A kit comprising an antibody of the invention useful for a variety of purposes, including detection, for example, in research and diagnosis, is provided. For the isolation and purification of the desired protein, the kit may contain an antibody coupled to a bead (e.g., sepharose bead). Kits may be provided comprising an antibody for detecting and quantifying the desired protein in vitro, for example, by ELISA or Western blotting. With respect to article of manufacture, the kit includes a container and a label or package insert on or in combination with the container. The container holds a composition comprising one or more antibodies of the invention. For example, additional containers containing diluents and buffers, control antibodies may be included. The label or package insert may provide guidance for intentional in vitro or diagnostic use as well as a detailed description of the composition.

The present invention also includes a finished packaged and labeled pharmaceutical product. Such articles of manufacture include any suitable unit dosage form in a suitable container or container, for example, a glass vial, a pre-filled syringe or other container that has been drained. In one embodiment, the unit dosage form is provided as a sterile, particulate free solution comprising an antibody suitable for parenteral administration. In another embodiment, the unit dosage form is provided as a sterile pressure-reduced lyophilized powder comprising an antibody suitable for reconstitution.

In one embodiment, the unit dosage form is suitable for intravenous, intramuscular, intranasal, oral, topical or subcutaneous delivery. Thus, the present invention comprises a sterile solution suitable for each delivery route. The present invention further comprises a sterile vacuum lyophilized powder suitable for reconstitution.

With respect to any pharmaceutical product, packaging materials and containers are designed to protect the stability of the product during storage and transportation. The product of the present invention also includes instructions for administering the medicament as well as instructions for appropriately preventing or treating the suspicious disease or disorder and instructions for use or other information to inform the physician, technician or patient about the frequency of administration. In other words, the article of manufacture includes instructional means that direct or suggest dosing regimens, including, but not limited to, actual doses, monitoring procedures and other monitoring information.

More particularly, the present invention relates to a method of making a packaging material, such as a box, bottle, tube, vial, container, pre-filled syringe, spray, insufflator, intravenous (iv) bag, Etc; And one or more unit dosage forms of a pharmaceutical formulation comprising a liquid formulation containing an antibody contained within the packaging material. The packaging material includes instructions that indicate how the antibody may be used to prevent and / or treat and / or manage one or more symptoms associated with the disease or disorder.

The present invention is further illustrated by the following examples, which should not be construed as limiting in any way.

X. Example

Example  1: Method for reducing the degree of acidic species in cell culture by addition of medium components

Production of recombinant proteins by host cells may result in product-related charge heterogeneity present in a population of proteins produced by the cells. The presence of acidic species in a population of proteins is an example of product-related charge heterogeneity. Control of the amount of acidic species present in a population of proteins produced by the host cell can be accomplished by modifying the culture conditions of the host cell.

Experiments in these examples demonstrate that supplementing the cell culture medium with supplemental amounts of amino acids, calcium chloride and niacinamide improves product quality by reducing the amount of acidic species in the culture harvest. The amino acids included in the study were arginine, lysine, ornithine and histidine belonging to the group of basic amino acids. Studies include examples from multiple cell lines and antibodies, in shake flasks and bioreactors, and in a batch and fed-batch culture format. A dose-dependent effect of the degree of reduction of acid species by increasing the concentration of the supplements was observed. It has also been demonstrated that these media additives can be supplemented individually or in combination in an appropriate amount for acidic species reduction.

Materials and methods

Cell source and adaptation culture

In the studies discussed below, three adalimumab productivity cell lines (cell line 1, cell line 2, and cell line 3), one mAb1 producing cell line and one mAb2 producing cell line were used. For Adalimumab-producing cell lines, the cells were cultured in a vented non-baffled shaking flask (Corning) on a shaker platform with 110 RPM (cell line 1), 180 RPM (cell line 2), 140 RPM (cell line 3) 10L or 20L wave bags (GE) were combined and cultured in their respective growth medium (chemically defined medium (medium 1) or hydrolyzate-based medium (medium 2 or medium 3)). For cell-based experiments in the hydrolyzate-based medium (medium 3), cells were thawed in medium 1 and then adapted to medium 3 over several passages. To obtain the required number of cells to be able to initiate production phase cultures, cell lines 1 and 2 were incubated in a 5% CO ₂ incubator at 35 ° C and 36 ° C for cell line 3 in a 5% CO ₂ incubator Cultures were grown.

For mAb1 productivity cell lines, cells were cultured in a chemically defined growth medium (Medium 1) in combination with a ventilated non-baffled shaking flask (Corning) on a shaker platform at 130 RPM and a 20 L wavebag (GE). To obtain the required number of cells to initiate production stage cultures, the cultures were grown in a 5% CO ₂ incubator at 36 ° C.

For mAb2 productive cell lines, cells were cultured in a chemically defined growth medium (Medium 1) in combination with a ventilated non-baffled shaking flask (Corning) on a shaker platform at 140 RPM and a 20 L wavebag (GE). Cultures were grown in a 5% CO ₂ incubator at 35 占 폚 in order to obtain the required number of cells to be able to initiate production stage cultivation.

Cell culture medium

Growth and production medium were prepared from a chemically defined medium formulation (medium 1) or hydrolyzate-based medium (medium 2 and medium 3). For the preparation of medium 1, L-glutamine, sodium bicarbonate, sodium chloride, and methotrexate solution were supplemented to the medium (IVGN GIA-1, the registered trademark base medium from Invitrogen). The production medium consisted of all components in the growth medium except methotrexate. In addition, for cell line 1, insulin was supplemented in both growth and production medium. For the mAb1 and mAb2 productivity cell lines, insulin was supplemented to the growth medium.

For the hydrolyzate-based formulation (medium 2), the growth medium was PFCHO (a chemically defined formulation of a registered trademark from SAFC), dextrose, L-glutamine, L-asparagine, HEPES, Poloxamer 188, Recombinant human insulin, yeastolate (BD), python peptone (BD), mono- and di-basic sodium phosphate, sodium bicarbonate, sodium chloride and methotrexate. The production medium consisted of all components listed in the growth medium except methotrexate.

For the hydrolyzate-based formulation (Medium 3), growth media consisted of OptiCHO (Invitrogen), L-Glutamine, Isotolate (BD), Python Peptone (BD) and Methotrexate. The production medium consisted of all components listed in the growth medium except methotrexate.

The amino acids used in the experiments were reconstituted in Milli-Q water to produce a stock solution of 100 g / L, which was subsequently supplemented in both growth and production basal medium. After the addition of the amino acids, the medium was transferred to a pH similar to that of the medium not supplemented (control) with 5N hydrochloric acid / 5N NaOH and adjusted to the osmotic concentration similar to that of the uncompensated (control) medium by adjusting the sodium chloride concentration .

Calcium chloride dihydrate (Sigma or Fluka) used in the experiments was reconstituted in Milli-Q water to prepare a stock solution, which was subsequently supplemented to the production basal medium. After the addition of calcium chloride, the medium was transferred to a pH similar to that of the medium not supplemented (control) with 6N hydrochloric acid / 5N NaOH, and the concentration of osmotic agent similar to that of the medium (control) .

The niacinamide (Sigma or Calbiochem) used in the experiments was reconstituted in Milli-Q water to prepare a stock solution, which was subsequently supplemented to the production basal medium. After addition of the niacinamide, the medium was transferred to a pH similar to that of the medium not supplemented (control) using 6N hydrochloric acid / 5N NaOH and the osmotic concentration similar to that of the medium not supplemented (control) by adjusting the concentration of sodium chloride .

All media was filtered through a Corning 1 L filter system (0.22 μm PES) and stored at 4 ° C. until use.

Production culture

The production culture was started in a 500 ml shaking flask (Corning) or in a 3 L bioreactor (Applikon). For shake flask experiments, a double 500 mL Corning aeration-baffle shake flask (200 mL working volume) was used for each condition. The shake flasks were maintained at 110 rpm for adalimumab-producing cell line 1, 180 rpm for adalimumab-producing cell line 2, 140 rpm for adalimumab-producing cell line 3, 130 rpm for mAb1-producing cell line, Maintained at 35 ° C or 36 ° C and 5% CO ₂ on a shaker platform set at 140 rpm. For a bioreactor experiment, a 3 L bioreactor (1.5 L working volume) was operated at 35 ° C, 30% dissolved oxygen (DO), 200 rpm, a pH profile of 7.1 to 6.9 within 3 days, Lt; / RTI > pH 6.9. In all experiments, cells were transferred from the seed train to the production phase at a 1: 5 split ratio.

Cultivation was performed in a batch or fed-batch manner. In batch mode, cells were cultured in each production medium. When the medium glucose concentration is reduced to less than 3 g / L 1.25% (v / v) of a 40% glucose stock solution was fed. In the oil-price expression, Using IVGN feed (chemically defined feed formulations from Invitrogen) according to the feed schedule - (4% (v / v) - 6 days, 7 days, and 8 days respectively) -Cell PFCHO feed (chemically defined formulation of registered trademark) -3% (v / v). In addition, when the glucose concentration was less than 3.0 g / L, 1.25% (v / v) 40% glucose stock solution was supplied to the culture.

A retention sample for 2 x 1.5 mL of titer analysis was prepared on day 8 Collected daily for bioreactor experiments and frozen at -80 ° C. After each Samples were assayed for potency.

The collection procedure of the shake flask and the reactor included centrifugation of the culture sample for 30 minutes at 3,000 RPM and storage of the supernatant in the PETG bottle at -80 DEG C before performing Protein A purification and WCX-10 analysis Respectively.

WCX -10 black

This method is used for quantification of acidic species and other variants present in cell culture collection samples. Cation exchange chromatography was performed on a Dionex ProPac WCX-10 analytical column (Dionex, CA).

For the adalimumab and mAb1 samples, the mobile phase used was 10 mM dibasic sodium phosphate pH 7.5 (mobile phase A) and 10 mM dibasic sodium phosphate, 500 mM sodium chloride pH 5.5 (mobile phase B). Binary concentration gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% : 28-34 min) was used with detection at 280 nm.

For the mAb2 sample, the mobile phase used was 20 mM (4-morpholino) ethanesulfonic acid monohydrate (MES) pH 6.5 (mobile phase A) and 20 mM MES, 500 mM sodium chloride pH 6.5 (mobile phase B). Optimized concentration gradient (minutes /% B): 0/3, 1/3, 46/21, 47/100, 52/100, 53/3, 58/3 were used with detection at 280 nm.

The quantification is based on the relative area percent of the detected peaks. Peaks eluting at a relative retention time faster than the major peak corresponding to the drug product are indicated together as the acid peak (Figure 1).

Methylglyoxal ( MGO ) Lysine-C for quantification Peptides Mapping

A typical trypsin digestion, which is almost universally used for peptide mapping, cleaves denatured and reduced and alkylated proteins at the carboxyl side chains of two basic amino acids, lysine and arginine. Methylglyoxal (MGO) is a small molecule metabolite that is induced as a corresponding byproduct that can modify arginine residues. Modification of arginine prevents the trypsin from cutting these sites, resulting in mis-cleavage. The challenge to quantify the amount of MGO-modified peptides is that they are not compared against equivalent non-modified peptides but rather against two peptides that tend to have different ionization potentials than modified peptides . In order to measure an actually accurate direct measurement of the MGO-modified peptide, it should be expressed as a percentage as compared to its non-modified counterpart. When endoproteinase lysine-C is used as an alternative enzyme, cleavage occurs only at the lysine residue. The result is a direct comparison of the same peptides in the presence and absence of MGO variants, which also provides high accuracy in the determination of trace levels of modified species.

Procedure: Samples were diluted to a nominal concentration of 4 mg / mL. 8M of guanidine-HCl was added to the sample in a 3: 1 ratio resulting in a concentration of 1 mg / mL in 6 M guanidine-HCl. The sample was reduced with 10 mM final concentration DTT for 30 minutes at 37 DEG C and then alkylated at 37 DEG C for 30 minutes in a dark place with 25 mM final concentration of iodoacetic acid. The sample was then buffer exchanged to 10 mM Tris pH 8.0 using a NAP-5 column. The samples were then digested at 37 ° C for 4 hours using 1: 20 enzyme-protein ratio endoproteinase Lys-C. The digestion was quenched by adding 5 [mu] L of formic acid to each sample. Samples were analyzed by LC / MS peptide mapping. Briefly, 50 μL of sample was loaded onto a Waters BEH C18 1.7 μ 1.0 x 150 mm UPLC column with 98% 0.08% formic acid, 0.02% TFA in water and 0.08% formic acid in 0.02% TFA in acetonitrile. The composition was changed to 65% 0.08% formic acid in water, 0.02% TFA in water and 0.08% formic acid in 35% 0.02% TFA in acetonitrile using a Waters Acquity UPLC system. Elution peaks were monitored using a Thermo Scientific LTQ-Orbitrap mass spectrometer. Specific mass traces were obtained for both modified and non-modified peptides in order to accurately quantify the total amount of MGO deformation at each site. Mass spectra were also analyzed for specific regions of the chromatogram to confirm peptide identity. An example data set is shown in Fig.

result

Effect of arginine supplementation on cell culture medium

The addition of arginine was tested in several experimental systems across multiple cell lines, media and monoclonal antibodies. The following is a detailed description of two representative experiments in which two different mulberry barley productivity cell lines (cell line 2 and cell line 3) were cultured in a chemically defined medium (medium 1).

Cell line 2 was cultured in medium 1 with various total amounts of arginine (1 (control), 1.25, 1.5, 2, 3, 5, 9 g / L) Culture was performed in a shake flask in a batch format with only glucose supply as described in the materials and methods above. Cells were grown to the maximum viable cell density (VCD) in the range of 18 to 22 x ¹⁰⁶ cells / ml for the various conditions tested. Growth and viability profiles were similar between the various test conditions (Figs. 1 and 2), although a slight decrease in the live cell density profile was observed in the samples with arginine test conditions of 9 g / l. The collection condition was similar between conditions (FIG. 3). On day 10 and day 12 of culture, double shaker flasks were harvested for each condition and subsequently analyzed using WCX-10 after protein A purification and the total peak (s) area corresponding to the acid species Percentages were quantified (Figures 4 and 5). The percentage of acid species in the control sample was as high as 19.7% at 10 days. In the sample with a total maximum concentration of arginine (9 g / L) in this experiment, the percentage of acid species was reduced to 12.2%. A dose-dependent reduction of acid species was observed under test conditions with an arginine concentration of greater than 2 g / L (Figure 4). A similar trend was also observed in the reduction of acid species with arginine increase in the collection sample on day 12 (Figure 5). In addition, the degree of acidic species in the 1 g / L arginine sample increased from 19.7% (collected on day 10) to 25.5% (collected on day 12), but this increase in the 9 g / L arginine test condition was 12.2% Collection) to 13.9% (collected on day 12). Thus, the increase in total arginine resulted in a reduction in the rate of increase of acidic species with the degree of total acidic species at the specific time of culture and with the incubation time.

Cell line 3 was cultured in medium 1 with various total amounts of arginine (1 (control), 3, 5, 7, 9 g / L). Culture was performed in a shake flask in a batch format with only glucose supply as described in the materials and methods above. The cells grew to a maximum VCD within the range of 7-10 x ¹⁰⁶ cells / ml for the various conditions tested. Growth and viability profiles were similar between the various test conditions, although slight reductions in viable cell density and viability profiles were observed in samples at 9 g / L arginine conditions (FIGS. 6 and 7). In addition, product yields were similar among all conditions (Figure 8). On the 10th day of culture, double shake flasks were collected for each condition and subsequently analyzed using WCX-10 after protein A purification and the percentage of the total peak (s) corresponding to the acid species was quantitated (Fig. 9). The percentage of acid species in the control sample was as high as 23.3% at 10 days. In the sample with the highest total concentration of arginine (9 g / L) in this experiment, the percentage of acid species was reduced to 17.0%. A dose-dependent reduction of acidic species was observed under conditions with higher arginine concentrations.

Additional experiments were performed using multiple cell lines in chemically defined media or hydrolyzate-based media to demonstrate broad applicability of this method. The experimental setup for each of these experiments was similar to that described above. A summary of the results of the various experiments performed on Adil Mammab is summarized in Figures 10, 11, and 12. Also in each case, a decrease in acid species with increased arginine concentration was observed.

In addition to adalimumab, the use of this method for acid species reduction has also been demonstrated for processes involving other mAb productive cell lines (cell lines producing mAb1 and mAb2). The experimental setup for each of these experiments was similar to that described in the section above and in the materials and methods. The reduction of acid species with increased arginine concentrations for experiments corresponding to each mAb is summarized in Figures 13 and 14. As for mAb2, a significant reduction of acid species was observed at an arginine concentration of 9 g / L.

Attorney Docket No. 117813-74101 (the disclosure of which is incorporated herein by reference), the inventors describe the use of arginine supplementation in culture media for the adjustment of lysine variant distribution. In addition to the fractionation of acidic species that are completely removed from the entire protein population, the fractionation of acidic species is also possible to migrate (from Lys 0 to Lys 1 and Lys 2) as the lysine mutant moves. Prior to WCX-10, samples of protein A eluates from a representative set of arginine supplementation experiments were pretreated with enzyme carboxypeptidase to estimate acid species reduction independent of this redistribution of lysine variants. One set of samples from the Avidan Mabab experiment and another set of samples from the mAb2 experiment were used for this analysis. The carboxy peptidase treatment of the sample resulted in cleavage of the C-terminal lysine residue as evidenced by complete conversion of Lys1 / Lys2 to Lys0 in each of these samples (data not shown herein). As a result of this conversion, the acidic species quantified in these samples had previously been displaced corresponding to lysine mutant movements, possibly including those species that may not have been calculated for samples not treated with carboxypeptidase prior to WCX-10 Which corresponded to the sum of the aggregates of the acidic species expected to be. A dose-dependent reduction of acidic species was observed in the carboxy peptidase treated samples with increasing concentrations of arginine (Figures 15 and 16). This suggests that the acid species reduction described herein does not contribute fully to the possible migration of acidic species corresponding to lysine variant redistribution.

Effect of lysine supplementation on cell culture medium

The addition of lysine was tested in several experimental systems across multiple cell lines, media and monoclonal antibodies. The following is a detailed description of two representative experiments in which two different cell lines (cell line 2 and cell line 3) were cultured in a chemically defined medium (medium 1) for the production of adalimumab.

Cell line 2 was cultured in medium 1 with various concentrations of lysine (1 (control), 5, 7, 9, 11 g / L). Culture was performed in a shake flask in a batch format with only glucose supply as described in the materials and methods above. The cells grew to a maximum viable cell density (VCD) in the range of 17 to 23 x ¹⁰⁶ cells / ml for the various conditions tested. A slight dose-dependent reduction in the viable cell density profile was observed for all samples for the control samples (Figure 17). Viability profiles were similar between conditions (Figure 18). On days 10 and 11 of incubation, samples were collected for potency analysis (FIG. 19). The reverse for all conditions was similar. On the eleventh day of culture, double shake flasks were collected for each condition and subsequently analyzed using WCX-10 after protein A purification and the percentage of the total peak (s) corresponding to acid species was quantitated (Fig. 20). The percentage of acid species in the control group was as high as 26.5%. In this experiment, in samples with the highest test concentration of lysine (11 g / L), the percentage of acid species was reduced to 15.0%. A dose-dependent reduction of acid species was observed under test conditions with higher total lysine concentration.

Cell line 3 was cultured in medium 1 with various total concentrations of lysine (1 (control), 3, 5, 7, 9, 11 g / L). Culture was performed in a shake flask in a batch format with only glucose supply as described in the materials and methods above. The cells grew to a maximum VCD within the range of 9.5 to 11.5 x 10 ⁶ cells / ml for the various conditions tested. Growth and viability profiles were similar between the various test conditions (Figs. 21 and 22), although a slight reduction in viable cell density and viability profile was observed in samples with lysine concentrations higher than lysine concentrations in control samples. Samples were collected for potency analysis on day 10, day 11 and day 12 of culture (FIG. 23). The reverse for all conditions was similar. On the twelfth day of culture, double shake flasks were collected for each condition, and subsequently analyzed using WCX-10 after protein A purification, and the percentage of the total peak (s) corresponding to the acid species was quantified (Fig. 24). The percentage of acid species in the control sample was as high as 26.6%. In this experiment, in samples with the highest test concentration of lysine (11 g / L), the percentage of acid species was reduced to 18.1%. A dose-dependent reduction of acid species was observed under test conditions with higher total lysine concentration.

Additional experiments were performed using multiple cell lines in chemically defined media or hydrolyzate-based media to demonstrate broad applicability of this method. The experimental setup for each of these experiments was similar to that described above and in the Materials and Methods section above. A summary of the results of various experiments performed on Adil Mammab is summarized in Figures 25, 26, and 27. Also, in each case, a decrease in acid species with increased lysine concentration was observed.

The use of this method for acid species reduction, in addition to the hydronephrine, has also been demonstrated for processes involving two different mAbs. The experimental setup for each of these experiments was similar to that described above and in the Materials and Methods section above. The reduction of acid species with lysine addition to the experiment corresponding to each mAb is summarized in Figures 28 and 29. [ With respect to mAb2, a significant decrease in acidic species was observed at a lysine concentration of 11 g / L.

Attorney Docket No. 117813-74101, the disclosure of which is incorporated herein by reference, describes the use of lysine replenishment in culture media for the modulation of lysine variant distribution. Prior to WCX-10, samples of protein A eluates from a representative set of lysine replicate experiments were pretreated with enzyme carboxypeptidase to estimate acid species reduction independent of this redistribution of lysine variants. One set of samples from the Avidan Mabab experiment and another set of samples from the mAb2 experiment were used for this analysis. The carboxypeptidase treatment of the sample resulted in cleavage of the C-terminal lysine residue as evidenced by the conversion of Lys1 / Lys2 to Lys0 in each of these samples. As a result of this conversion, the acid species quantified in these samples had previously been displaced in response to lysine mutagenesis, possibly including those species that may not have been calculated for samples not treated with carboxypeptidase prior to WCX-10 Which corresponds to the sum of aggregates of acidic species expected to be. A dose-dependent reduction of acid species in the carboxy peptidase-treated sample with increasing concentrations of lysine versus 26.1% of the non-supplemented samples versus the adalimumab sample to 21.1% of the 10 g / L lysine supplemented samples And total acid species showed a decrease of 5.7% (Fig. 30). Similar results were also observed for mA2 samples (Figure 31). This suggests that the acid species reduction described herein does not contribute fully to the possible migration of acidic species corresponding to lysine redistribution.

Effect of histidine supplementation on cell culture medium

The addition of histidine was tested in several experimental systems across multiple cell lines, media and monoclonal antibodies. The following is a detailed description of two representative experiments in which two different cell lines (cell line 2 and cell line 3) were cultured in a chemically defined medium (medium 1) for the production of adalimumab.

Cell line 2 was cultured in medium 1 with various total concentrations of histidine (0 (control), 4, 6, 8, 10 g / L). Culture was performed in a shake flask in a batch format with only glucose supply as described in the materials and methods above. The cells grew to a maximum VCD within the range of 12 to 22 x ¹⁰⁶ cells / ml for various conditions tested. A dose dependent decrease in the viral cell density profile was observed with a 10 g / L histidine condition with significant reduction in growth (Figure 32). In addition, a corresponding effect on viability was observed (Figure 33). On day 10, day 11 and day 12 of culture, samples were collected for potency analysis and the collection days for each sample were reported (FIG. 34). There was a small dose-dependent reduction in potency against conditions with histidine replacement. On days 11-12, the double shake flask was harvested and subsequently analyzed using WCX-10 after protein A purification and the percentage of total peak (s) area corresponding to the acid species was quantified (Figure 35 ). The percentage of acid species in the control group was as high as 26.5%. In the sample with the highest test concentration of histidine (10 g / L) in this experiment, the percentage of acid species was reduced to 15.6%. A dose-dependent reduction of acidic species was observed under test conditions with increased histidine concentration.

Cell line 3 was cultured in medium 1 with various total concentrations of histidine (0 (control), 2, 4, 6, 8 g / L). Culture was performed in a shake flask in a batch format with only glucose supply as described in the materials and methods above. Cells were grown to a maximum viable cell density (VCD) in the range of 6-10 x ¹⁰⁶ cells / ml for various conditions tested. A dose-dependent reduction of the viable cell density profile was observed under all conditions with histidine concentrations higher than the histidine concentrations in the control samples (Figure 36). The viability profile was more similar between conditions using these cell lines (Figure 37). On day 12 of incubation, samples were collected for potency analysis (FIG. 38). The reverse for all conditions was similar. On the twelfth day of culture, double shake flasks were collected for each condition, and subsequently analyzed using WCX-10 after protein A purification, and the percentage of the total peak (s) corresponding to the acid species was quantified (Fig. 39). The percentage of acid species in the control sample was as high as 26.2%. In the sample with the highest test concentration of histidine (11 g / L) in this experiment, the percentage of acid species was reduced to 20.0%. A dose-dependent reduction of acidic species was observed under test conditions with increased histidine concentration.

Additional experiments were performed using multiple cell lines in chemically defined media or hydrolyzate-based media to assess the broad applicability of this method. The experimental setup for each of these experiments was similar to that described above and in the Materials and Methods section above. A summary of the results of various experiments performed on Adil Mammab is shown in Figures 40, 41, and 42. Reduction of acidic species with increased histidine concentration was observed with cell line 1 (FIG. 40) in medium 1 and with cell line 2 (FIG. 42) in medium 3. A dose-dependent reduction of acidic species was observed (Figure 42), with respect to cell line 2 in medium 3, to histidine up to 4 g / L with no significant decrease in histidine at higher concentrations. With respect to cell line 1 in medium 2, no significant decrease in acidic species was observed within the histidine concentration range (0 to 4 g / L) (FIG. 41). The use of this method for acid species reduction, in addition to the hydronephrine, has also been demonstrated for processes involving two different mAbs. The experimental setup for each of these experiments was similar to that described above and in the Materials and Methods section above. The reduction of acidic species with increased histidine concentration for experiments corresponding to each mAb is summarized in Figures 43 and 44. Concerning mAb 2, in contrast to the results reported with arginine and lysine replenishment previously shown, a significant significant dose-dependent reduction in total acidic species from 28.1% in the control group to 21.5% in the 4 g / L histidine sample was observed .

Attorney Docket No. 117813-74101, the disclosure of which is incorporated herein by reference, describes the use of increased histidine in the culture medium for the modulation of lysine variant distribution. In addition, prior to WCX-10, protein A eluate samples from a representative set of histidine supplement experiments were pretreated with enzyme carboxypeptidase to estimate acid species reduction independent of this redistribution of lysine variants. One set of samples from the Avidan Mabab experiment and another set of samples from the mAb2 experiment were used for this analysis. The carboxy peptidase treatment of the sample resulted in cleavage of the C-terminal lysine residue as evidenced by complete conversion of Lys1 / Lys2 to Lys0 in each of these samples (data not shown herein). A dose-dependent reduction of acidic species was observed in the carboxy peptidase treated samples with increasing concentrations of histidine (Figures 45 and 46). This suggests that the acid species reduction described herein does not contribute fully to the possible migration of acidic species corresponding to lysine redistribution.

Effect of ornithine supplementation on cell culture medium

The addition of ornithine was tested in several experimental systems across multiple cell lines, media and monoclonal antibodies. The following is a detailed description of two representative experiments in which two different cell lines (cell line 2 and cell line 3) were used in a chemically defined medium (medium 1) for the production of adalimumab.

Cell line 2 was cultured in medium 1 with various total concentrations of histidine (0 (control), 4, 6, 8, 10 g / L). Culture was performed in a shake flask in a batch format with only glucose supply as described in the materials and methods above. The cells grew to a maximum VCD within the range of 15 to 22 x ¹⁰⁶ cells / ml for the various conditions tested. A slight decrease in viable cell density with ornithine supplementation was observed (Figure 47). In addition, corresponding differences in viability profiles were observed (Figure 48). On day 11 of culture, samples were collected for potency analysis (Figure 49). The titers for all conditions were similar. On day 11, double shaker flasks were collected for each condition and subsequently analyzed using WCX-10 after protein A purification and the percentage of total peak (s) area corresponding to acid species was quantified (Fig. 50). The percentage of acid species in the control sample was 26.5%. In the sample with the highest test concentration of ornithine (10 g / L) in this experiment, the percentage of acid species was reduced to 16.1%. A dose-dependent reduction of acid species was observed under test conditions with increased ornithine concentrations.

Cell line 3 was cultured in medium 1 supplemented with various concentrations of ornithine (0 (control), 2, 4, 6, 8 g / L). Culture was performed in a shake flask in a batch format with only glucose supply as described in the materials and methods above. The cells grew to a maximum viable cell density (VCD) in the range of 9.5 to 11.5 x ¹⁰⁶ cells / ml for the various conditions tested. The viable cell density and viability profiles were similar (Figures 51 and 52). On day 12 of incubation, samples were collected for potency analysis (Figure 53). The reverse for all conditions was similar. On the twelfth day of culture, double shake flasks were collected for each condition, and subsequently analyzed using WCX-10 after protein A purification, and the percentage of the total peak (s) corresponding to the acid species was quantified (Fig. 54). The percentage of acid species in the control sample was as high as 24.8%. In this experiment, in the sample with the highest test concentration of ornithine (8 g / L), the percentage of acid species was reduced to 20.5%. A dose-dependent reduction of acid species was observed under test conditions with increased ornithine concentrations.

Additional experiments were performed using multiple cell lines in chemically defined media or hydrolyzate-based media to assess the broad applicability of this method. The experimental setup for each of these experiments was similar to that described above and in the Materials and Methods section above. A summary of the results of the various experiments performed on Adil Mammab is summarized in Figures 55, 56, and 57. A dose-dependent decrease was observed for cell line 1 in medium 1 (FIG. 55). However, no significant reduction of acidic species was observed with respect to cell line 1 in medium 2, the hydrolyzate medium (Fig. 56). With respect to cell line 2 in medium 3, a reduction of acid species from 22.1% to 18.7% in the control sample was observed in the 2 g / L ornithine sample without further reduction at higher ornithine concentrations (FIG. 57).

In addition to adalimumab, the use of this method for acid species reduction has also been demonstrated for processes involving two different mAbs. The experimental setup for each of these experiments was similar to that described in the section above and in the Materials and Methods section. The reduction of acidic species with ornithine addition to the experiment corresponding to each mAb is summarized in Figures 58 and 59. In the case of mAb1, a dose-dependent reduction of 7.3% of total acid species was observed within the tested concentration range. In the case of mAb2, a 2% reduction was observed in the 1 g / L ornithine concentration sample with a minimal further decrease at higher ornithine concentrations.

Similar to the analysis performed with other amino acids, a protein A eluate sample from a representative set of ornithine experiments was also pre-treated with WCX-10 enzyme-carboxy peptidase. One set of samples from the Avidan Mabab experiment and another set of samples from the mAb2 experiment were used for this analysis. A dose-dependent reduction of acidic species was observed in carboxy peptidase treated samples with increasing concentrations of ornithine (Figs. 60 and 61). In addition, the percentage of acidic species was similar between the untreated sample and the carboxypeptidase treated sample for ornithine at a certain concentration. This indicates that acid species reduction is independent of possible migration of acidic species which may correspond to any lysine redistribution.

Increased effect of arginine, lysine, histidine, and ornithine supplementation on cell culture medium

In this experiment, the combination of four amino acids arginine, lysine, histidine and ornithine for acid species reduction is demonstrated. The experiments described herein were carried out using Adalimumab-producing cell line 2 in a chemically defined medium (medium 1). In this experiment, the concentration range of arginine and lysine was 1 to 3 g / L, while the concentration range for histidine and ornithine in this experiment was 0 to 2 g / L. An additional decrease in total acid species was observed under conditions in which the combination of amino acids in the medium was increased, compared to a lower concentration, or a condition in which the single amino acid concentration was increased (FIG. 62). A gradual decrease in total acid species was observed when more amino acids were increased in the blend. The percentage of acidic species was reduced from 21.9% in the lowest concentration sample to 12.3% in the sample with all four amino acids at high concentration.

Increased  Arginine and Lysine  Control of acidic species by selection of cell culture and collection criteria and / or adjustment of pH

The increase in the concentration of amino acids (arginine, lysine) in the base medium can also be combined with the selection when collecting the cultures to achieve an optimal reduction of the total acid species. In this example, the study was carried out in a 3 L bioreactor with cell line 1 (producing adalimumab) in medium 1. Two sets of conditions were tested: control conditions (arginine 1 g / L, lysine 1 g / L); Test condition 1 (arginine 3 g / L, lysine 5 g / L). Cell growth, viability, and potency profiles were similar between conditions (Figures 63, 64, and 65). A small amount of cell culture collection was collected every day from day 4 to day 10 from each reactor, and Protein A purification and WCX-10 analysis were performed. The percentage of acidic species in the control condition increased from 12.1% (day 4) to 24.6% (day 10) (FIG. 66). The percentage of acid species in Test Condition 1 was lower than that observed in the control condition at each corresponding culture day. In addition, the percentage of acid species in the test conditions also increased from 8.7% (from day 4) to 18.8% (day 10). In addition, the rate of growth of acidic species over the duration of incubation correlated with the lowering of viability for both conditions, and increased rapidly on day 8. Thus, a decrease in the desired acid species can be achieved using the selection of collection / collection survival rates at the collection day, together with increasing arginine and lysine concentrations in the culture medium.

Increases in the amino acid (arginine, lysine) concentration in the base medium can be combined with process pH adjustment to achieve further reduction of total acid species. In this example, the study was carried out in a 3 L bioreactor with cell line 1 (producing adalimumab) in medium 1. Three sets of conditions were tested in duplicate: control conditions (arginine (1 g / L), lysine (1 g / L), pH 7.1 → 6.9 within 3 days, then pH 6.9); Test conditions 1 (arginine (3 g / L), lysine (3 g / L), pH 7.1 → 6.9 within 3 days, then pH 6.9); Test conditions 2 (arginine (3 g / L), lysine (3 g / L), pH 7.1 → 6.8 within 3 days, then pH 6.8). In comparison with the control, there was a slight decrease in the VCD profile and collection titer for Condition 2 (Figs. 67, 68 and 69). Cultures were harvested when viability was less than 50%, and protein A and WCX-10 assays were performed on the culture supernatant. The percentage of acid species in the control sample was 19.1%. The percentage of acidic species was reduced to 14.3% in test condition 1 and to 12.8% in test condition 2 (FIG. 70). Thus, it proves that the increase in the concentration of amino acids with the selection of a lower final process pH can be used in combination to further reduce the degree of acidic species.

To cell culture medium CaCl ₂ Supplementary effect

The addition of calcium chloride was tested in several experimental systems across multiple cell lines, media and monoclonal antibodies. The following is a detailed description of two representative experiments in which two different cell lines (cell line 2 and cell line 3) were cultured in a chemically defined medium (medium 1) for the production of adalimumab.

Cell line 2 was cultured in medium 1 with various concentrations of calcium (0.14, 0.84 and 1.54 mM). Culture was performed in a shake flask in a batch format with only glucose supply as described in the materials and methods above. Cells were grown to the maximum viable cell density (VCD) in the range of 22-24.5 x ¹⁰⁶ cells / ml for the various conditions tested. The viable cell density and viability profiles for all test conditions were similar (FIGS. 71 and 72). On day 10 of incubation, samples were collected for potency analysis (Figure 73). The titers for all conditions were similar. On day 10, double shaker flasks were collected for each condition and subsequently analyzed using WCX-10 after protein A purification and the percentage of total peak (s) area corresponding to the acid species was quantified (Fig. 74). The percentage of acid species in the 0.14 mM calcium condition was 23.8%. In the sample with the highest test concentration of calcium (1.54 mM) in this experiment, the percentage of acid species was reduced to 21.6%. A dose-dependent reduction of acidic species was observed under test conditions with increased calcium concentration.

Cell line 3 was cultured in medium 1 with various total concentrations of calcium (0.14, 0.49, 0.84, 1.19, 1.54, 1.89 mM). Culture was performed in a shake flask in a batch format with only glucose supply as described in the materials and methods above. The cells grew to a maximum viable cell density (VCD) in the range of 9.5 to 10.5 x 10 ⁶ cells / ml for the various conditions tested. The viable cell density and viability profiles for all test conditions were similar (Figures 75 and 76). On day 11 of culture, samples were collected for potency analysis. Collecting titers for all conditions were similar (Figure 77). On day 11, double shaker flasks were collected for each condition and subsequently analyzed using WCX-10 after protein A purification and the percentage of total peak (s) area corresponding to acid species was quantified (Fig. 78). The percentage of acid species at 0.14 mM calcium condition was 23.7%. In the sample with the highest test concentration of calcium (1.89 mM) in this experiment, the percentage of acid species was reduced to 20.7%. A dose-dependent reduction of acidic species was observed under test conditions with increased calcium concentration.

Additional experiments were performed using multiple cell lines in chemically defined media or hydrolyzate-based media to assess the broad applicability of this method. The experimental setup for each of these experiments was similar to that described above and in the Materials and Methods section above. A summary of the results of various experiments performed on Adil Mammab is summarized in Figures 79, 80, and 81. In addition, a decrease in acid species with increased calcium concentration was observed in each case.

In addition to adalimumab, the use of this method for acid species reduction has also been demonstrated for processes involving two different mAbs. The experimental setup for each of these experiments was similar to that described above. The reduction of acidic species with ornithine addition to the experiment corresponding to each mAb is summarized in Figures 82 and 83. In the case of mAb1, a small but significant acid species reduction was observed from 15.4% (0.14 mM calcium sample) to 11.8% (1.54 mM calcium chloride supplemented sample). In the case of mAb2, a greater acid dose-dependent reduction from 28.9% (0.14 mM calcium sample) to 23.1% (1.40 mM calcium chloride supplemented sample) was observed.

Effects of increasing concentrations of arginine, lysine, calcium chloride, and niacinamide in combination

In this experiment, the combined effects of amino acid arginine, lysine, inorganic salts of calcium chloride and vitamin niacinamide for acid species reduction were evaluated. The experiments described herein were conducted in a chemically defined medium (medium 1) supplemented with 3% (v / v) PFCHO (a suitably chemically defined medium formulation from SAFC) Cell line 2 was used. In this experiment, a central composite DOE experimental design was used. The basal medium for each condition was supplemented with four replenishments at various concentrations. Cell culture was performed in duplicate for each condition. Upon collection, WCX-10 analysis was performed after protein A purification. In Table 4 below, the experimental conditions from the DOE design are summarized, including the concentration of each component supplemented and the% of total acid species (or AR) obtained for each condition. Reductions in acid species have been observed over increasing concentrations in combination with these constituents. For example, in a condition (# 24) in which all four components were present at their maximum concentrations, the total AR% was reported to be reduced to 9.7%. Using the data from the above experiment, a model (R ² : 0.92, P <0.0001) for predicting the effect of addition of these constituents on the medium for AR reduction is described in FIG. The model predicted contribution from each of the four components to acid species reduction. It may also be possible to use this model to predict the selection of the concentration of these various constituents in the medium to achieve target reduction of the total AR.

Use of niacinamide supplementation in cell culture medium for acidic species reduction

In addition to the use of niacinamide in combination with other supplements as described in the previous section, it is also possible to add niacinamide independently of other supplements, as evidenced by the following experiments on two mAbs: Can be used.

For the experiments corresponding to adalimumab, cell line 1 was cultured in Medium 1 supplemented with various amounts of niacinamide (0, 0.2, 0.4, 0.8 and 1.6 mM). Culture was performed in a shake flask in a batch format with only glucose supply as described in the materials and methods above. The cells grew to a maximum VCD within the range of 8.5 to 11 x ¹⁰⁶ cells / ml for the various conditions tested. A slight decrease in the viable cell density profile was observed with maximal niacin supplementation (1.6 mM in this experiment) (Figure 85). The viability profiles for the test conditions were similar (Figure 86). On day 12 of culture, samples were collected for potency analysis. The titers for all conditions were similar (Figure 87). On day 11 and day 12, double shaker flasks were harvested for each condition and subsequently analyzed using WCX-10 after protein A purification and the percentage of total peak (s) corresponding to acid species (Figs. 88 and 89). The percentage of acidic species in the control sample (niacinamide supplement) was 19.6% on day 10. In the 10 day sample with the highest test concentration (1.6 mM) of niacinamide in this experiment, the percentage of acidic species was reduced to 15.9%. A dose-dependent reduction of acidic species was observed under conditions with higher arginine concentrations. At day 12, a similar acid species reduction was observed with niacinamide supplementation.

For experiments corresponding to mAb2, mAb2 productive cell lines were cultured in Medium 1 supplemented with various amounts of niacinamide (0, 0.1, 0.5, 1.0, 3.0 and 6.0 mM). Culture was performed in a shake flask in a batch format with only glucose supply as described in the materials and methods above. The cells grew to a maximum viable cell density (VCD) in the range of 14-21.5 x ¹⁰⁶ cells / ml for the various conditions tested. A slight decrease in the viable cell density profile was observed for the conditions at concentrations of 3.0 mM and 6.0 mM niacinamide (FIG. 90). The viability profiles for all test conditions were similar (Figure 91). On day 12 of culture, samples were collected for potency analysis (FIG. 92). The titers for all conditions were similar. On day 12, double shaker flasks were collected for each condition and subsequently analyzed using WCX-10 after protein A purification and the percentage of total peak (s) area corresponding to the acid species was quantified (Fig. 93). The percentage of acid species in the control sample (niacinamide supplement) was 27.0%. In the sample with the highest test concentration (6.0 mM) of niacinamide in this experiment, the percentage of acid species was reduced to 19.8%. A dose-dependent reduction of acidic species was observed under test conditions with niacinamide supplementation.

The antibody, methylglyoxal ( MGO ) Basic amino acids in cell culture medium for reduction of depletion Supplementation of arginine and lysine

In this experiment, the effect of MGO modification on acid species reduction was investigated. Adalimumab-producing cell line 1 was cultured in a chemically defined medium (medium 1) supplemented with amino acids as described below.

Materials and methods

Cell source and adaptation culture

Cells were cultured in their respective growth medium (chemically defined medium (medium 1)) in combination with a 10 L or 20 L wavebag (GE) and aeration non-baffled shaking flask (Corning) on a shaker platform at 110 RPM . To obtain the required number of cells to initiate production phase cultures, the cultures were grown in a 5% CO ₂ incubator at 36 占 폚.

Cell culture medium

For the preparation of medium 1, L-glutamine, sodium bicarbonate, sodium chloride, and methotrexate solution were supplemented to the medium (IVGN GIA-1, a registered trademark base medium formulation from Invitrogen). The production medium consisted of all components in the growth medium except methotrexate. In addition, insulin was supplemented in both growth and production medium.

The amino acids used in the experiments (Arginine (Sigma, A8094) and lysine (Calbiochem, 4400)) were reconstituted in Milli-Q water to produce a stock solution of 100 g / L, . After addition of the amino acids, the medium was transferred to a pH similar to that of the medium not supplemented (control group) with 6N hydrochloric acid / 5N NaOH and adjusted to the osmotic concentration similar to that of the non-supplemented (control) medium by adjusting the sodium chloride concentration .

All media was filtered through a Corning 1 L filter system (0.22 μm PES) and stored at 4 ° C. until use.

Production culture was initiated in a 3L bioreactor (Applikon). For bioreactor experiments, a 3 L bioreactor (1.5 L working volume) was run at a pH set point of 35 ° C, 30% DO, 200 rpm, 7.1. Cells were transferred from the seed train to the production phase at a 1: 5 split ratio.

Cultivation was performed in a batch or fed-batch manner and cultured in medium 1 supplemented with each production medium (arginine (4 g / L) or lysine (4 g / L). When the medium glucose concentration is reduced to less than 3 g / L 1.25% (v / v) of a 40% glucose stock solution was fed.

Retention samples for 2 x 1.5 mL of titer analysis were collected daily on day 8 and frozen at -80 ° C. After each Samples were assayed for potency.

The collection procedure of the shake flask and the reactor included centrifugation of the culture sample for 30 minutes at 3,000 RPM and storage of the supernatant in the PETG bottle at -80 DEG C before performing Protein A purification and WCX-10 analysis Respectively.

WCX -10 black

The WCX-10 assay method as described above was used in the Materials and Methods section.

MGO  Lysine-C for quantification Peptides Mapping

Procedures for lysine-C peptide mapping for MGO quantitation were performed as described above in the Materials and Methods section above.

Results and discussion

The majority of the cultures grew with similar peak VCDs in the range of 9-10 x ¹⁰⁶ cells / mL (Figure 94A). In addition, the viability profile of the cultures was similar to the harvest viability of 10-25%. The incubation duration (10 days) was similar between conditions (Figure 94B).

The percent of total peak (s) area corresponding to the acidic species was quantified using WCX-10 for the collected sample after protein A purification. The percentage of acid species in the control sample was 36.5%. The percentage of total acid species in samples from cultures supplemented with arginine and lysine was reduced to 20.1% and 28.0%, respectively (Figure 94C). In addition, a significant decrease in AR1% was observed in these cultures: 16.8% to 7.3% (arginine supplemented culture) and 12.8% (lysine supplemented culture) in the control sample (FIG. 94C). In addition, the degree of MGO deformation was quantified using Lys-C peptide mapping and reported as a percentage of MGO modified peptides among those more sensitive to MGO variants. From these results, it was found that the% MGO strain was also significantly reduced in cultures supplemented with amino acids (FIG. 94D).

Example  2: Methods of reducing the extent of acidic species in cell cultures by adjusting process parameters

Experiments described below in this example demonstrate that on-line modification of cell culture process parameters can be used to adjust the acidic species of the desired protein, e. G., Antibody dalidamabe or mAb2 / RTI &gt; and / or &lt; / RTI &gt; For example, an increase in dissolved oxygen concentration and / or a decrease in final pH can result in a decrease in AR.

Materials and methods

Cell source and adaptation culture

In the study covered in this example, two Adalimumab-producing CHO cell lines (cell line 1 and cell line 3) and one mAb2-producing cell line were used. At thawing, the Adalimumab Productive Cell Line 3 was cultured in a chemically defined growth medium (Medium 1) in combination with a ventilated shake flask on a shaker platform at 140 RPM and a 20 L wavebag. To obtain the required number of cells to initiate production stage cultures, the cultures were grown in a 5% CO ₂ incubator at 36 ° C.

At thawing, Adalimumab productivity cell line 1 was cultured in hydrolyzate-based growth medium (Medium 2) in combination with a 20 L wavebag on a shaker platform at 35 RPM on a shaker platform at 35 RPM in a 5% CO ₂ incubator . In some cases, the cultures can be transferred to a seed reactor at pH 7.1, 35 ° C and 30% DO. Cultures can be adapted for Medium 1 or Medium 2 by growing in a 10 L or 20 L wavebag for 7 to 13 days in one or two passages prior to commencement of the production stage culture.

Upon thawing, mAb2 productivity cells were cultured in Medium 1 in combination with a ventilated non-baffled shaking flask (Corning) on a shaker platform at 140 RPM and a 20 L wavebag (GE). Cultures were grown in a 5% CO ₂ incubator at 35 占 폚 in order to obtain the required number of cells to be able to initiate production stage cultivation.

Cell culture medium

Medium 1, chemically defined growth or production medium was prepared from primary IVGN CD medium (trademark formulations). For the preparation of IVGN CD medium formulations, the trademarked media was supplemented with L-glutamine, sodium bicarbonate, sodium chloride, and methotrexate solution. The production medium consisted of all components in the growth medium except methotrexate. In addition, for cell line 1 and mAb 2, the medium was supplemented with insulin. In addition, for the cell line 3 or 1, 10 mM or 5 mM of galactose (Sigma, G5388) and 0.2 μM or 10 μM of manganese (Sigma, M1787) were supplemented to the production medium, respectively. The osmotic concentration was adjusted by the concentration of sodium chloride. All media was filtered through a filter system (0.22 μm PES) and stored at 4 ° C. until use.

Medium 2 is a hydrolyzate-based medium containing the basic registered trademark medium, Bacto TC Yeastolate, and Phytone Peptone.

Production culture

Production culture was initiated in a 3L bioreactor (Applikon). The 3L bioreactor (1.5 to 2.0 L working volume) contained a pH profile of 7.1 to 6.9 within 3 days at 35 ° C, 30% DO (dissolved oxygen), 200 rpm, Lt; / RTI &gt; pH 6.9. In all experiments, the cells were transferred from the wavebag to the production phase with a split ratio of 1: 5.6 (except for mAb2 at a ratio of 1: 5). When the medium glucose concentration is reduced to less than 3 g / L Approximately 1.25% (v / v) of a 40% glucose stock solution was fed.

The collection procedure of the reactor included centrifugation of the culture sample for 30 minutes at 3,000 RPM and storage of the supernatant in PETG bottle at -80 占 폚 before performing protein A purification and WCX-10 analysis.

WCX-10 Black

The acidic species and other variants present in the cell culture collected samples were quantitated. Cation exchange chromatography was performed on a Dionex ProPac WCX-10 analytical column (Dionex, CA). For the Adalimumab productivity cell line, a Shimadzu LC10A HPLC system was used as HPLC. The mobile phase used was 10 mM dibasic sodium phosphate pH 7.5 (mobile phase A) and 10 mM dibasic sodium phosphate, 500 mM sodium chloride pH 5.5 (mobile phase B). Binary concentration gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% : 28-34 min) was used with detection at 280 nm. The WCX-10 method used for mAb B used various buffers. The mobile phase used was 20 mM (4-morpholino) ethanesulfonic acid monohydrate (MES) pH 6.5 (mobile phase A) and 20 mM MES, 500 mM sodium chloride pH 6.5 (mobile phase B). Optimized concentration gradient (minutes /% B): 0/3, 1/3, 46/21, 47/100, 52/100, 53/3, 58/3 were used with detection at 280 nm.

The quantification is based on the relative area percent of the detected peaks. Peaks eluting at a relative retention time faster than the major peak corresponding to the drug product are indicated together as the acid peak.

result

Effect of Process pH on Medium 1 using Cell Line 1

Five different pH conditions were evaluated in this study: 7.1, 7.0, 6.9, 6.8, and 6.7. Culture was started at a pH set point of 7.1 and then reduced to the target pH set point within 4 days. All cultures except for cultures at pH 6.7 conditions, where the maximum cell density was much lower than other cultures, reached the same maximum viable cell density on day 8 (Figure 95). In addition, the viability of cultures at pH 7.1 and pH 7.0 dropped much faster than other cultures. The viability of cultures at pH 7.1 and pH 7.0 was 38% and 54% at 10 days, respectively; On the other hand, the viability of cultures at lower pH (including pH 6.9, 6.8 and 6.7) exceeded 70% on the same day (FIG. 96). Samples collected on the last day of culture were measured for IgG concentration. The reversal of each test condition increased from 1.2 g / L at pH 7.1 to 1.8 g / L at pH 6.8, corresponding to a decrease in pH, but the product potency did not continue to increase at pH 6.7 (1.6 g / L) (Fig. 97). Cultures were collected on day 10 or day 12. The collected material was purified with protein A and then analyzed using WCX-10. The peak area obtained from the WCX-10 assay was quantified (Figure 98). The percentage of acid species decreased from 56.0% at pH 7.1 to 14.0% at pH 6.7, corresponding to a decrease in pH. Since cultures at pH 6.9, 6.8 and 6.7 were present at 70% viability on day 10, additional samples were collected on day 12 for these cultures when viability reached approximately 50%. WCX-10 analysis was also performed on these samples. Percentage of acidic species at day 12 increased relative to day 10 for these three conditions (i.e., pH 6.9, 6.8, and 6.7); The increase in the percentage of acid species was smaller at lower pH. The percentage of acid species increased to 18.8% (pH 6.9), 8.1% (pH 6.8), and 3.5% (pH 6.7) on day 10 (70% viability) to day 12 (50% viability). Thus, the percentage of acid species was lower at lower pH at day 12. The percentage of acid species decreased from 39.1% at pH 6.9 to 17.5% at pH 6.7 with a decrease of 21.6% on day 12 with a decrease in pH.

In addition, the effect of process pH on the specific reduction of specific acidic variants in larger fractions of total acid species was evaluated. Table 5 summarizes the extent of some species of acidic species fractions. With the reduction of total acid species, the methods presented in this section can also be used for the reduction of subspecies, including but not limited to AR1, AR2 and MGO modified product variants.

Effect of Process pH on Medium 2 using Cell Line 1

Three different pH conditions were evaluated in this study: 7.0, 6.9, and 6.8. The cultures were started at a pH of 7.1 and then reduced to the target pH set point within 3 days of incubation. By day 8, viable cell density and viability over different pH setpoints were similar. After day 8, viable cell density and viability were slightly higher at lower pH set points (FIG. 99 and FIG. 100). Cultures were harvested at about 50% viability. The product reversal was slightly higher at pH 6.8 compared to pH 6.9 and 7.0 (Figure 101). The peak area obtained from the WCX-10 assay was quantified (Fig. 102). The percentage of acid species decreased from 20.7% at pH 7.0 to 18.1% at pH 6.8, a total reduction of 2.6%.

Effect of Process pH on Medium 1 using Cell Line 3

Five different pH conditions were evaluated in this study: 7.1, 7.0, 6.9, 6.8, and 6.7. Cultivation was started at a pH set point of 7.1; It was then reduced to the target pH set point within 4 days of incubation. The pH set points using these cell lines and media showed significant effects on cell growth and survival. Cell density was lower at higher pH, and viability also dropped faster at higher pH (FIGS. 103 and 104). Cells were harvested at day 10 or when viability dropped to 50% or less. The potency slightly increased with decreasing pH and reached the highest titers at pH 6.8 (Figure 105). The peak area obtained from the WCX-10 assay was quantified (Figure 106). The percentage of acid species decreased from 29.7% at pH 7.1 to 21.5% at pH 6.7, a total reduction of 8.2%.

Lt; RTI ID = 0.0 &gt; 35 C &lt; / RTI &gt; Dissolved  Effect of oxygen (DO)

Three different dissolved oxygen (DO) conditions were evaluated in this study: 20%, 30%, and 50%. The culture was set at 35 캜. Cell density and viability were very similar in different DO conditions (Figures 107 and 108). Cultures were collected at a target viability of 50% for each condition. The harvesting station was higher at 50% DO than at 20% DO (Fig. 109). In addition, the collected material was subjected to Protein A purification before analysis of WCX-10. The percentages of acidic species in each of the test conditions were 20.6% (20% DO), 19.0% (30% DO), and 17.7% (50% DO), respectively (FIG. The percentage of acid species was generally lower at higher dissolved oxygen concentrations. The percentage of acid species decreased by 20.6% at 20% DO to 17.7% at 50% DO with a decrease of 2.9% with increasing DO.

Lt; RTI ID = 0.0 &gt; 33 C &lt; / RTI &gt; Dissolved  Effect of oxygen (DO)

Three different DO conditions were evaluated in this study: 20%, 30%, and 60%. Cell density, viability, and product turnover were very similar in different DO conditions (Figures 111, 112, and 113). The percentages of acidic species in each of the test conditions were 20.1% (20% DO), 17.8% (30% DO), and 17.7% (60% DO), respectively (FIG. The percentage of acid species was generally lower at higher dissolved oxygen concentrations. The percentage of acid species decreased with a decrease of 2.4%, from 20.1% at 20% DO to 17.7% at 60% DO with increasing DO.

Lt; RTI ID = 0.0 &gt; 35 C &lt; / RTI &gt; Dissolved  Effect of oxygen (DO)

Three different dissolved oxygen (DO) conditions were evaluated in this study: 20%, 30%, and 50%. The culture was set at 35 캜. Cell density and viability were very similar under different DO conditions (Figures 115 and 116). The cultures were harvested at 40% of the target viability for each condition. Collection was higher at 30% and 50% DO compared to 20% DO (Figure 117). In addition, the collected material was subjected to Protein A purification before analysis of WCX-10. The percentages of acid species in each of the test conditions were 23.9% (20% DO), 22.4% (30% DO), and 20.3% (50% DO), respectively (FIG. The percentage of acid species was generally lower at higher dissolved oxygen concentrations. The percentage of acid species was reduced from 23.9% at 20% DO to 20.3% at 50% DO with a decrease of 3.6% with increasing DO.

In culture medium 1 using cell line 3 Dissolved  Effect of oxygen (DO)

The study was conducted at four different temperature levels (33 ° C, 34 ° C, 35 ° C and 36 ° C) with two different DO conditions (20% DO and 50% DO). Generally, cell growth at different levels of dissolved oxygen was similar except for growth at 35 DEG C, where cell density was lower at 50% DO (FIG. 119). The culture was harvested at day 10 or at about 50% viability (FIG. 120). The reversal at about 50% viability was similar in the different DO conditions (Figure 121). The percentage of acid species was generally lower at higher dissolved oxygen than at each test temperature condition (Figure 122). On day 10, the percentage of acid species decreased from 25.2% at 20% DO to 22.7% at 50% DO, with a decrease of 2.5% with increasing DO; The percentage of acid species was reduced from 23.2% at 20% DO to 19.4% at 50% DO with a total reduction of 3.8% with increasing DO at 35 ° C; The percentage of acid species was reduced by a total of 1.1% reduction from 18.2% at 20% DO to 17.1% at 50% DO under 34% DO at 34 ° C, , From 14.3% at 20% DO to 12.9% at 50% DO, with a total reduction of 1.4%. On day 12, when the viability was about 50% for the 34 ° C test conditions, the percentage of acid species increased from 21.5% at 20% DO to 20.6% at 20% DO, % Of the total. Finally, on day 14, when the viability was about 50% for the 33 ° C test conditions, the percentage of acid species increased from 19.7% at 20% DO to 17.9% at 50% DO, Which was reduced by 1.8%. In summary, at all test temperature conditions for different collection days, the percentage of acid species was lower at higher dissolved oxygen concentrations.

mAb2 Lt; RTI ID = 0.0 &gt; Dissolved  Effect of oxygen (DO)

Six different DO conditions were evaluated: 10%, 20%, 30%, 50%, 60% and 80%. The culture was set at 35 캜. In general, cell density, viability, and potency were similar at different dissolved oxygen conditions (FIG. 123, FIG. 124, and FIG. 125). The percentage of acid species in each test condition was 26.5% (10% DO), 27.3% (20% DO), 27.3% (30% DO), 25.8% (50% DO), 24.4% ) And 24.5% (80% DO) (Fig. 126). The percentage of acid species was generally lower at higher dissolved oxygen concentrations. The percentage of acid species was reduced from 27.3% at 20% DO to 24.5% at 80% DO with a decrease of 2.8% with increasing DO.

Example  3: Methods for reducing acidic species by adding amino acids to purified cell culture collections and by changing the pH of the clarified collection

This example describes a process for reducing and controlling the level of acidic species in an antibody preparation. Specifically, this example provides a method of reducing the acidic variant content in the clarified collection, and a method of reducing the formation rate of acidic species in the purified collection. The method includes adding an additive such as various amino acids to the purified collection, or adjusting the pH of the purified collection using an acidic substance.

As shown below, the antibody acidic species in the clarified collection can be reduced by adding to the clarified collection more than 100 mM and 50 mM, respectively, of an additive such as arginine or histidine. In addition, the AR reduction can be achieved by adjusting the pH of the clarified collection to pH 6 or pH 5. In addition, acidic variant formation rates can be reduced through the use of arginine or histidine in a concentration-dependent manner, or by low pH treatment of the clarified extract.

Materials and methods

Purified collection material

Different batches of the dodabimab purified collection material were used in the following experiments described below. The clarified collection can be prepared by subjecting the desired composition, for example, centrifugation to remove large solid particles, and subsequent filtration to remove finer solid particles and impurities from the material, Is a liquid material containing the desired monoclonal antibody. The clarified collection was used in the low pH treatment studies described herein. The clarified collection was also used in experiments to study the effect of the amino acid concentration on the presence of acidic species in the clarified collection and in the acid type-pH treatment studies described herein. Different batches of mAb-B and mAb-C purified collection material were used for studying the effect of amino acids on the presence of acidic species described herein and for low pH treatment studies.

Manufacturing of materials

First, the purified collected material was adjusted to pH 4 using 3M citric acid. The material at pH 4 was then stirred for 60 minutes before pH adjustment to 3, 5, 6, or 7 target pH with 3M sodium hydroxide. The material was then stirred for an additional 60 minutes. The samples were then centrifuged at 7300xg for 15 minutes on a Sorvall Evolution RC with a SLA-3000 centrifuge bowl. The supernatant from the centrifuged material was then deeply filtered using a B1HC deep-well filter (Millipore) followed by a 0.22 mu m sterile filter. Filters at different pHs were then held for different times to assess the rate of formation of acidic variants. After the retention, the material was purified with a protein A affinity column, the eluate was sampled and analyzed using the WCX-10 method. The manufacturing scheme is shown in FIG. 127 below.

Materials for studying the effects of arginine on acidic species were prepared in two ways. For lower target arginine concentrations of 5 mM, 10 mM, 30 mM and 100 mM, they were prepared by adding the appropriate amount of 0.5 M arginine stock buffer at pH 7 (pH adjusted with acetic acid) to obtain the desired target arginine concentration . For higher target arginine concentrations of 50 mM, 100 mM, 300 mM, 500 mM, 760 mM, 1 M and 2 M, they were supplemented with the appropriate amount of arginine (solids) to the sample to obtain the target arginine concentration and subsequently with glacial acetic acid 7 &lt; / RTI &gt; Arginine was adjusted to a final concentration of 100 mM using two methods to determine if the manufacturing method could lead to different effects. For all experiments, the clarified collection treated after arginine addition was held at room temperature for the indicated duration, then purified with Protein A column and analyzed for acidic variants. This study provided two results: (1) the data of the samples from Day 0 provided the effect of arginine on acid species reduction in the clarified collection, (2) the data of samples with different retention days Provided the effect of arginine on the reduction of the formation rate of acidic species. The manufacturing scheme is shown in FIG.

Materials for studying the effects of histidine were prepared at target concentrations of 5 mM, 10 mM, 30 mM, 50 mM, 100 mM, 200 mM and 250 mM. Samples were prepared by adding an appropriate amount of histidine (solids) to the sample to obtain a target histidine concentration and subsequently titrating to a final pH of 7 using glacial acetic acid. The sample preparation scheme is shown in FIG.

Materials for studying the effects of lysine were prepared at target concentrations of 5 mM, 10 mM, 30 mM, 50 mM, 100 mM, 200 mM, 300 mM, 500 mM and 1000 mM. Samples were prepared by adding the appropriate amount of lysine hydrochloride (solids) to the sample to obtain the target lysine concentration and subsequently titrating to a final pH of 7 using hydrochloric acid. The sample preparation scheme is shown in FIG.

Materials for studying the effects of methionine were prepared at target concentrations of 5 mM, 10 mM, 30 mM, 50 mM, 100 mM, 200 mM and 300 mM. Samples were prepared by adding the appropriate amount of methionine (solids) to the sample to obtain the target methionine concentration and subsequently titrating to a final pH of 7 using glacial acetic acid. The sample preparation scheme is shown in FIG.

The materials for studying the effect of different amino acids were evaluated by using two 20 amino acids each to be evaluated as well as two controls using sodium acetate instead of the amino acid and another control using the glacial acetic acid, &Lt; / RTI &gt; were prepared at different target concentrations. The target concentrations for the amino acids are shown in Table 6 below.

Samples were prepared by adding the appropriate amount of amino acid (solids) to the sample to obtain the target amino acid concentration shown in Table 6, and subsequently purifying it to a final pH of 7 using glacial acetic acid. The sample preparation scheme is shown in FIG.

Materials for studying the effects of additives other than amino acids were prepared by adding the respective additives to be evaluated as well as sodium hydroxide instead of arginine to the pH of the material to pH 10 and then neutralizing it to pH 7 with glacial acetic acid With different target concentrations. The target concentrations for the additives are shown in Table 7 below.

Samples were prepared by adding appropriate amounts of additives to the samples to obtain the target amino acid concentrations shown in Table 6 or Table 7 and subsequently titrating to a final pH of 7 using glacial acetic acid.

Using the following scheme shown in Figure 133, materials were studied to study the effects of the above-mentioned methods on CDM purified collection.

Purified collections of mAb B hydrolyzate were used to study the effects of the above mentioned methods.

Purified collections of mAb C hydrolyzate were used to study the effects of the above mentioned methods.

Retention studies on the treated purified effluent

After preparation of the above-mentioned samples, the samples were placed in separate sterile stainless steel containers for the purpose of holding at 4 ° C or at room temperature. For each material, a different container was used for each day of the evaluated retention. For the acidified samples, the acidic mutant compositions of the samples were evaluated on days 0, 3, 7 and 14 of retention at any of the above temperatures. For the arginine containing material, the acidic mutant compositions of the samples were evaluated on days 0, 5 and 8 of retention at room temperature. For the histidine-containing material, the acidic mutant compositions of the samples were evaluated on days 0, 3 and 7 of retention at room temperature.

Mountain type  And the pH effect on the clarified collection

In this study, the effects of acid type, purified water pH and arginine content on acid mutant reduction were evaluated. Samples were prepared in triplicate over a series of three consecutive days at a target arginine concentration of 0 mM (not arginine added) or 500 mM and then incubated with either a glacial acetic acid, phosphoric acid, 3M citric acid or 6M hydrochloric acid at a target pH of 5, 6 or 7 Lt; / RTI &gt; One other sample was prepared by adding 2M arginine acetate pH 7 stock buffer to the clarified collection to obtain a target arginine concentration of 500 mM. The sample preparation scheme is shown in FIG.

Protein A Purification

Purification of the protein A of the sample was performed using a 5 mL rProtein A FF Hitrp column (GE Healthcare) of 10 g adalimumab per kilogram of loading resin and operating at a flow rate of 3.4 mL / min. Following the equilibration of 5 column volumes (CV) (1X PBS pH 7.4), the sample was loaded and then the column was washed with equilibration buffer to remove non-specifically bound impurities, followed by the addition of 0.1 M acetic acid, The protein is eluted with 0.15M sodium chloride.

Samples of eluate were collected and neutralized to pH 6.9 to 7.2 using 1 M Tris pH 9.5 from 45 min to 75 min after collection. The sample was then frozen at -80 占 폚 for more than one day and then thawed to perform WCX-10 analysis.

Purification method for purified effluent, effect of acid concentration and neutralization

In this study, the effects of purification method, acid concentration and pH neutralization using different types of chromatographic resins for acidic mutant reduction were evaluated. The following samples were prepared as shown in Table 8 below.

Subsequently, each of the prepared materials was subjected to double capture of Mabselect Sure or Fractogel S. Samples of eluate were collected and neutralized to pH 6.9 to 7.2 using 1 M Tris pH 9.5 from 45 min to 75 min after collection. The sample was then frozen at -80 占 폚 for more than one day and then thawed to perform WCX-10 analysis.

acid Mutant  analysis( WCX -10 black)

Cation exchange chromatography was performed on a 5 mm x 250 mm Dionex ProPac WCX-10 analytical column (Dionex, Calif.). HPLC assays were performed using a Shimadzu LC10A HPLC system. The mobile phase used was 10 mM dibasic sodium phosphate pH 7.5 (mobile phase A) and 10 mM dibasic sodium phosphate, 500 mM sodium chloride pH 5.5 (mobile phase B). Binary concentration gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% : 28-34 min) was used with detection at 280 nm.

The quantification is based on the relative area percent of the detected peaks. Peaks eluting at a relative retention time less than the residence time of the predominant lysine 0 peak are indicated together as the acidic variant peak (AR).

result

Subsequent  Effect of low pH treatment using neutralization

The results of the low pH treatment and subsequent neutralization are shown in Figures 135 and 136 below. Figure 136 shows that low pH treatment and subsequent neutralization to pH 5 or 6 reduces the rate of acidic variant formation over time. However, as shown in Figure 135, there is no significant decrease in the initial acidic variant content.

Effect of arginine treatment

The results of the arginine treatment are shown in Figures 137 and 138. Figures 137 and 138 show that the sample preparation method resulted in different levels of acidic species in the purified effluent. Addition of 0.5 M arginine pH 7 stock buffer tended to increase acid species whereas addition of pure arginine and subsequent acetic acid titration to pH 7 reduced acidic variants to arginine concentrations greater than 100 mM. In addition, the effect of the treatment method is demonstrated when comparing two 100 mM arginine samples, which represents an absolute difference of 1% of the acidic variants between the two methods.

Figure 139 shows that the acidic variant formation rate decreases with increasing arginine concentration in the clarified collection and is plateauing at or above the ambient concentration of 500 mM arginine. However, the two methods of sample preparation do not result in significantly different rates of formation of acidic variants.

Effect of histidine treatment

The results of the histidine treatment are shown in FIG. 140 and FIG. Similar to the arginine treatment effect, when histidine was added to the purified collection and subsequently pH neutralized with acetic acid, the acidic mutants were reduced to a histidine concentration higher than 50 mM, as shown in Fig. Figure 141 shows that the acidic variant formation rate decreases with increasing concentration of histidine in the clarified collection and is stable at or above the ambient concentration of 200 mM histidine.

Effect of ricin treatment

The results of lysing treatment are shown in Fig. 142 and Fig. Similar to the arginine treatment effect, acidic mutants were significantly reduced by about 1% or more when lysine was added to the clarified collection and subsequently pH neutralized with acetic acid, as shown in Figure 149. Figure 153 shows that the acidic variant formation rate decreases with increasing lysine concentration in the clarified collection.

Effect of methionine treatment

The results of the methionine treatment are summarized in Figures 154 and 165. [ Similar to the arginine treatment effect, when methionine is added to the clarified collection, and subsequently pH neutralized with acetic acid, the acidic mutant has a concentration of about 1% or more at a concentration of more than 10 mM Respectively. Figure 145 shows that the acidic variant formation rate is not significantly affected by the presence of methionine in the purified collection.

Effect of other amino acid treatment

The results of treatment with various amino acids are summarized in Figs. 146 and 147 below. As shown in Figure 146, the addition of 14 amino acids, including arginine, histidine, lysine and methionine, resulted in lower amounts of acidic variant content in the clarified collection. In addition, the addition of sodium acetate or the use of acetic acid also caused a decrease in acidic mutant content. Figure 147 shows that the acidic variant formation rate is reduced by some amino acids, including arginine, histidine, lysine, aspartic acid, glutamic acid, and leucine.

Effect of alternative additives treatment

The results of treatment with other additives are summarized in Figures 148 and 149 below. As shown in Figure 148, the addition of any additives did not result in lower acid variant content in the clarified collection of adalimumab hydrolyzate. However, Figure 149 shows that the acidic variant formation rate is reduced by most additives.

Adalmumab CDM  Effect of Low pH / Arginine Treatment on Purified Collection

The results of the CDM purified collection study are summarized in Figures 150 and 151 below. As shown in Figure 150, the low pH / arginine treatment did not result in lower acid variant content in the agarimammud CDM purified water. However, Figure 151 shows that the acid mutant formation rate is significantly reduced by all treatments.

mAb  B Hydrolyzate  Effect of Low pH / Arginine Treatment on Purified Collection

The results of the purified collections of mAb B hydrolyzate are summarized in Figures 152 and 153 below. As shown in Figures 152 and 153, low pH / arginine treatment results in both lower acid variant content and slower acidic variant formation rate in purified collections of mAb B hydrolyzate.

mAb  C Hydrolyzate  Effect of Low pH / Arginine Treatment on Purified Collection

The results of the mAb C hydrolyzate purified collection study are summarized in Figures 154 and 155 below. As shown in Figures 154 and 155, low pH / arginine treatment results in both lower acid variant content and slower acid variant formation rate in purified mAb C hydrolyzate digests.

Mountain type  And the effect of pH

The results obtained from the acid-pH study are summarized in FIG. Greater acid species reduction is obtained at lower pH. In addition, the addition of arginine further reduces the acid species content, but not to a significant extent when considering the high concentration used (500 Mm). The results also show that the use of arginine acetate stock buffer can achieve an acid species reduction of about 1%, although the use of pure arginine powder as a subsequent acid titration is performed somewhat better. With respect to the acid form used for pH adjustment, there were no significant differences between the different acids observed.

Purification method, effect of acid concentration and neutralization

The results obtained from this study are summarized in Figures 157, 158, 159, and 160. Figures 157 and 158 show that when the acid used is present at a higher concentration, there is a reduction in the acidic variant content in the hydrolyzate-purified collection compared to the lower concentration of acid used. Figures 159 and 160 show that there is an increase in acidic variant content when the clarified collection is base neutralized to pH 7 after treatment with low pH. In addition, these figures show that Fractogel resin can remove acidic variants better than Mabselect Sure.

Example 4: Reduction of AR in cell culture using continuous media perfusion technique

As demonstrated in Example 3 above, the production or formation of acidic species in a population of proteins can occur during the retention of the antibody in the clarified collection or spent media. Thus, the possibility of improving the stability of the product antibody and reducing the production of acid species has been explored using continuous / perfusion based cell culture techniques. Control or reduction of the amount of acidic species present in the population of proteins obtained at the end of cell culture can be achieved by altering the rate of exchange of the fresh medium to the bioreactor (removal of the medium consumed by the product antibody from the bioreactor) have.

Materials and methods

Cell source

In one study dealt with here, a single dalmamov productivity CHO cell line (cell line 1) was used. Upon thawing, the vials were incubated in a chemically defined growth medium (Medium 1) in a series of air-agitating shaking flasks on a shaker platform at 35 rpm in a 5% CO ₂ incubator at 110 rpm. The culture was propagated to obtain a sufficient number of cells for inoculation of perfusion cultibag.

Cell culture medium

Chemically defined growth or production media were used in this study. For the preparation of media formulations, L-glutamine, sodium bicarbonate, sodium chloride, recombinant human insulin and methotrexate solution were supplemented in a suitable medium (Invitrogen). The perfusion stage medium consisted of all the constituents in the growth medium, excluding the methotrexate solution, except for higher concentrations of recombinant human insulin.

Perfusion culture

Sartorius Cultibag RM 10L perfusion pro 1.2my (lot 1205-014) perfusion culture was performed with a Sartorius BIOSTAT RM 20 optical perfusion system (SN # 00582 | 12) in a perfusion bag. A perfusion bag was run with a working culture volume of 1.5 L and operating conditions of pH 7.00, 30% dissolved oxygen, 25 rpm, 35 캜, 0.3 slpm air overlay and 15 sccm CO ₂ overlay. PH control was initiated on the third day of culture. The pH was controlled by the addition of 0.5 M sodium hydroxide and CO ₂ .

Perfusion was performed by 'collecting' the spent cultures through an integrated 1.2 μm filter integrated in a perfumed tea bag. Fresh media was added to the culture via feed line at the same rate as collection. Perfusion started at 1.0 exchange / day (ex / day) on the fourth day of the process. The perfusion rate was adjusted to the glucose requirement, lactate accumulation and sampling schedule. On days 5 to 6 of perfusion, perfusion cell-free collection samples were collected at 1.5, 3.0, and 6.0 exchange volumes / day. Fresh collection bags were used for each collection sample. Samples were then purified using Protein A and analyzed using WCX-10 assays.

The perfusion culture was terminated on the eighth day of the process.

WCX -10 black

The acidic species and other charge variants present in the cell culture collected samples were quantified. Cation exchange chromatography was performed on a Dionex ProPac WCX-10 analytical column (Dionex, CA).

The mobile phase used was 10 mM dibasic sodium phosphate pH 7.5 (mobile phase A) and 10 mM dibasic sodium phosphate, 500 mM sodium chloride pH 5.5 (mobile phase B). Binary concentration gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% : 28-34 min) was used with detection at 280 nm. The WCX-10 method used in mAb2 samples used different buffers. The mobile phase used was 20 mM (4-morpholino) ethanesulfonic acid monohydrate (MES) pH 6.5 (mobile phase A) and 20 mM MES, 500 mM sodium chloride pH 6.5 (mobile phase B). Optimized concentration gradient (minutes /% B): 0/3, 1/3, 46/21, 47/100, 52/100, 53/3, 58/3 were used with detection at 280 nm. The quantification is based on the relative area percent of the detected peaks as described above.

result

The use of perfusion technology and the effect of selection of medium exchange rate on acid species

Adalimumab-producing cell line 1 was cultured in medium 1, and culturing was performed as described in the above materials and methods. As described in Table 8, the exchange rate was varied over a period of 24 hours between 5 and 6 days to investigate the effect of medium exchange rate on the degree of acidic species. The product antibodies in the consumed medium were collected in a collection bag at a continuous rate of 1.5 times the volume of culture medium / day for a period of 17 hrs. The collection bag was then replaced with a new bag, and the previous bag was transferred to 4 ° C. Subsequently and subsequently, the rate of medium exchange was increased to 3 to 6 volumes / day, and the product collected was collected over a period of 5 and 2 hrs, respectively. After holding overnight at 4 [deg.] C, three collection samples were processed through protein A and analyzed for acidic species using WCX-10. The percentage of acid species in the sample was 8.1% with a medium exchange rate of 1.5 volumes / day. In the samples with the highest exchange rates tested in these experiments, the percentage of acid species was reduced to 6%. Exchange rate-dependent decreases in acid species were observed in three samples (Table 9). Reduction of different sub-species within acidic variants (AR1 and AR2) is also known. An increase in volumetric productivity with exchange rate was also observed.

Example  5: Use of continuous perfusion techniques and reduction of acid species by addition of amino acids to the culture medium

As shown in Example 4 above, the reduction in the amount of acidic species present in the population of proteins obtained at the end of cell culture is achieved by modifying the rate of exchange of fresh media into the bioreactor (or by using product antibodies from the bioreactor) Thereby removing the spent culture medium). In this example, the ability to further reduce acidic species through the use of a high rate of media exchange in combination with supplementation of basic amino acids (arginine and lysine) in the culture medium is described.

Materials and methods

Cell source

Adalimumab-producing CHO cell line (cell line 1) was used. Upon thawing, the vials were incubated in a chemically defined growth medium (Medium 1) in a series of air-agitating shaking flasks on a shaker platform at 35 rpm in a 5% CO ₂ incubator at 110 rpm. The culture was propagated to obtain a sufficient number of cells for the inoculation of the peristaltic tea bag.

Cell culture medium

Chemically defined growth or production media were used in this study. For the preparation of media formulations, L-glutamine, sodium bicarbonate, sodium chloride, recombinant human insulin and methotrexate solution were supplemented in a suitable medium (Invitrogen). The perfusion stage medium consisted of all the constituents in the growth medium, excluding the methotrexate solution, except for higher concentrations of recombinant human insulin. Arginine and lysine were added directly to the media solution as a powder. After the addition of the amino acid, the pH was adjusted to the pH of the non-supplemented medium using 5N NaOH and 5N HCl as needed, and the osmolarity was adjusted to the osmotic concentration of the uncompensated medium by varying the concentration of sodium chloride.

Perfusion culture

Sartorius Cultibag RM 10L perfusion pro 1.2my (lot 1205-014) perfusion culture was performed with a Sartorius BIOSTAT RM 20 optical perfusion system (SN # 00582 | 12) in a perfusion bag. A perfusion bag was run with a working culture volume of 1.5 L and driving conditions of pH 7.00, 30% dissolved oxygen, 25 rpm, 35 캜, 0.3 slpm air overlay and 15 sccm CO ₂ overlay. PH control was initiated on the third day of culture. The pH was controlled by the addition of 0.5 M sodium hydroxide and CO ₂ .

Perfusion was carried out by 'collecting' the spent culture through an integrated 1.2 μm filter integrated in a perfumed tea bag. Fresh media was added to the culture via feed line at the same rate as collection. The perfusion rate was adjusted to the glucose requirement, lactate accumulation and sampling schedule during run. On days 6 to 8 of perfusion, perfusion cell-free collection samples were collected at 1.5, 3.0, 4.0, 6.0 and 8.0 exchange volumes / day. Fresh collection bags were used for each collection sample. Samples were then purified using Protein A and analyzed using WCX-10 assays.

WCX-10 Black

The acidic species and other charge variants present in the cell culture collected samples were quantified. Cation exchange chromatography was performed on a Dionex ProPac WCX-10 analytical column (Dionex, CA).

The mobile phase used was 10 mM dibasic sodium phosphate pH 7.5 (mobile phase A) and 10 mM dibasic sodium phosphate, 500 mM sodium chloride pH 5.5 (mobile phase B). Binary concentration gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% : 28-34 min) was used with detection at 280 nm. The WCX-10 method used in mAb2 samples used different buffers. The mobile phase used was 20 mM (4-morpholino) ethanesulfonic acid monohydrate (MES) pH 6.5 (mobile phase A) and 20 mM MES, 500 mM sodium chloride pH 6.5 (mobile phase B). Optimized concentration gradient (minutes /% B): 0/3, 1/3, 46/21, 47/100, 52/100, 53/3, 58/3 were used with detection at 280 nm. The quantification is based on the relative area percent of the detected peaks as described above.

Results: The use of perfusion technology and the effect of selection of medium exchange rate on acid species

Adalimumab-producing cell line 1 was cultured in medium 1, and culturing was performed as described in the above materials and methods. The exchange rate was varied over a period of 2 days between 6 days and 8 days to investigate the effect of medium exchange rate on the degree of acidic species. The product antibodies in the spent medium were collected in a collection bag at a continuous rate of 1.5 times the volume of culture medium / day for a period of 22 hrs. The collection bag was then replaced with a new bag, and the previous bag was transferred to 4 ° C. Subsequently and subsequently, the rate of media exchange was increased from 3 to 6 volumes / day at day 7 and from 4 to 8 volumes / day at day 8, and the resulting collection was collected and transferred to 4 ° C. Collection samples were processed through protein A and analyzed for acidic species using WCX-10. The percentage of acid species in the control sample was 7.7% with a medium exchange rate of 1.5 volumes per day. The percentage of acid species in arginine and lysine supplemented cell cultures with a medium exchange rate of 1.5 volumes per day was 4.3%. In the samples according to the highest exchange rates (8 volumes / day) tested in these experiments, the percentage of acid species was reduced to 5.4% in the control sample and to 3.0% in the arginine and lysine supplemented cell culture samples. An exchange rate-dependent decrease in acid species was observed in both cultures (Figure 161). Thus, a combination of arginine / lysine replenishment with exchange rate modulation in the culture medium can be used to further reduce AR.

Example  6: to achieve target AR% or AR reduction Upstream  And Downstream  Process combination

Upstream and downstream process technology, such as cell culture and chromatographic separation of the presently disclosed subject matter, may be combined together or combined with methods in the art to provide a final target AR value or achieve an AR% reduction can do. Upstream methods for AR reduction include, but are not limited to, those described in this application. Downstream methods for AR reduction are also described herein. Examples of upstream and downstream process technologies include cell culture additives and conditions; Clarified collection additive and pH / salt conditions; Mixed type medium separation; Anion exchange medium separation; And cation exchange medium separation, but are not limited thereto.

This embodiment demonstrates the combined effect of one or more of these techniques in achieving target AR values or AR reduction, thereby enabling the production of antibody materials with specific charge heterogeneity. Additional examples of combinations of downstream technologies and upstream techniques are provided herein.

In this example, the combination of the upstream and downstream methods is based on the reduction of acid species in a 3L bioreactor cell culture supplemented with arginine (2 g / L) and lysine (4 g / L), as previously proved in the present application I am involved. The results of this strategy are summarized in Table 10. The total acid species decreased from 20.5% of the control sample to 10.2% of the sample from the culture supplemented with the additive. In this study, adalimumab-producing cell line 1 was cultured in Medium 1 (chemically defined medium) supplemented with amino acid arginine (2 g / L) and lysine (4 g / L) in a 300 L bioreactor. On day 12 of culture, the cultures were harvested and subsequently analyzed using WCX-10 after protein A purification and the percentage of total peak (s) area corresponding to acid species was quantified. The percentage of acid species was estimated to be 9.1% in the 300 L collection sample.

Materials produced by the 300L bioreactor using arginine and lysine addition, which effectively reduced the AR level to 9.1%, were purified using a downstream process using mixed-mode chromatography as the primary AR reduction method.

Adalimumab was purified by protein A chromatography step followed by low pH virus inactivation step. The filtered virus-inactivated material was buffer exchanged and loaded onto a Capto Adhere column. Subsequently, the passing liquid of the Capto Adhere material was purified using a HIC column in a bind / elute system as well as a passing liquid system. As shown in Table 11, the AR reduction was first achieved with the MM step with a predetermined contribution from the other steps. The table also shows that, using these combined techniques, additional product-related materials, such as aggregates and process-related impurities, such as HCP, can be effectively reduced.

As is apparent from the above example, the MM method further reduced the AR level to 2.26%. Thus, an upstream technique for reduction can achieve AR level / AR reduction in combination with downstream technology.

Example  7: Anion exchange ( AEX ) Chromatography Example

Materials and methods

Chromatographic method

The materials and methods described in connection with this example were used in Examples 8 and 9 below, except as noted herein.

Except as noted, pre-packed resin columns were used in the following experiments. The column was equilibrated in a buffer system with appropriate pH and conductivity. The column load is buffer exchanged from the protein A affinity chromatography eluate or the concentrated CEX chromatography elution (when the eluate is present as a different buffer component from the mixed-mode target buffer system) Addition of a stock solution and / or water to obtain a target pH and conductivity, when the eluate was present in the same buffer component as the mixing type target buffer system, was prepared. The prepared load material was filtered, loaded onto the column according to the target loading (protein g / resin L) as specified, and then washed with a buffer similar to the equilibration buffer or equilibration buffer of the indicated volume. The column passing liquid / washing liquid was collected as a fraction or as a pool. Mixed columns were regenerated using 0.1 M acetic acid, 0.15 M NaCl pH 3, or 0.1 M acetic acid solution, pH 3, or as indicated. A 1 M NaOH solution was used for column washing.

Buffer  Manufacturing method

The buffer for AEX was prepared by fixing the anion concentration (acid) to the target value and targeting the specific ion concentration to the anion by adjusting the solution having the cation component (base) to achieve the proper pH. For example, to prepare 10 mM acetate-Tris buffer, pH 8.7, glacial acetic acid was dissolved in water to a target concentration of 10 mM and adjusted to pH 8.7 with concentrated Tris-base. Also, for example, to prepare 10 mM formate-Tris buffer, pH 8.7, formic acid was dissolved in water to a target concentration of 10 mM and adjusted to pH 8.7 with concentrated Tris-base.

The buffer solution for CEX was prepared by fixing the cation concentration (base) to the target value and targeting a specific ion concentration to the cation by adjusting the solution having the anion component (acid) to achieve an appropriate pH. For example, to prepare a 140 mM Tris-formate buffer, pH 7.5, the Tris base was dissolved in water to a target concentration of 140 mM and adjusted to pH 7.5 using formic acid.

AR reduction and recovery rate calculation

Generally, the passage / wash fluid fractions were collected and analyzed for AR levels using the WCX-10 method. Recovery and corresponding AR levels were calculated by actual or calculated pooling of the fractions.

Adalmumab  About WCX -10

The acidic species and other charge mutants present in the Adalimumab process sample were quantified according to the following method. Analytical column Dionex ProPac WCX-10 4 x 250 mm (Dionex, CA) was subjected to cation exchange chromatography. An Agilent 1200 HPLC system was used as the HPLC. The mobile phase used was 10 mM dibasic sodium phosphate pH 7.5 (mobile phase A) and 10 mM dibasic sodium phosphate, 500 mM sodium chloride pH 5.5 (mobile phase B). Binary concentration gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% : 28-34 min) was used with detection at 280 nm.

The quantification is based on the relative area percent of the detected peaks. Peaks eluting at a relative retention time less than a predetermined time are displayed together as acidic peaks.

mAb For -B WCX -10

Acid species and other charge variants present in mAb-B process samples were quantified according to the following method. Analytical column Dionex ProPac WCX-10 4 x 250 mm (Dionex, CA) was subjected to cation exchange chromatography. An Agilent 1200 HPLC system was used as the HPLC. The mobile phase used was 20 mM 4-morpholineethanesulfonic acid (MES), pH 6.5 (mobile phase A) and 20 mM 4-morpholineethanesulfonic acid (MES), 500 mM sodium chloride pH 6.5 (mobile phase B). B: 35-40 min; 0% A, 100% B (0% A, 13% B: 0-5 min; 87% A; 13% B; 5-35 min; 75% : 40 to 43 minutes; 87% A, 13% B: 43 to 46 minutes; 87% A, 13% B: 46 to 55 minutes) 280 nm, bw 8 nm; ref 360 nm, bw 100 nm.

The quantification is based on the relative area percent of the detected peaks. All peaks eluting prior to major isoform peaks were summed as acidic areas and all peaks eluted after the LYS-2 peak would be summed as basic areas.

mAb For -C WCX -10

The mAb-C method was used for the quantification of acidic species and other charge variants present in mAb-C process samples. Analytical column Dionex ProPac WCX-10 4 x 250 mm (Dionex, CA) was subjected to cation exchange chromatography. An Agilent 1200 HPLC system was used as the HPLC. The mobile phase used was 20 mM 4-morpholineethanesulfonic acid (MES), pH 6.5 (mobile phase A) and 20 mM 4-morpholineethanesulfonic acid (MES), 250 mM sodium chloride pH 6.0 (mobile phase B). 100% B: 46-47 minutes; 0% A, 100% B (0% A, 3% B: 0-1 min; 79% : 47 to 52 minutes; 97% A, 3% B: 52 to 53 minutes; 97% A, 3% B: 53 to 60 minutes) 280 nm, bw 8 nm; ref 360 nm, bw 100 nm.

The quantification is based on the relative area percent of the detected peaks. All peaks eluting prior to the major isoform peak will be summed as the acid region and all peaks eluted after the major isoform peak will be summed as the basic region.

Size exclusion chromatography

The molecular weight distribution of the collected samples was quantified according to the following method. Size exclusion chromatography (SEC) was performed using a TSK-gel G3000SWxL, 5 탆, 125 Å, 7.8 X 300 mm column (Tosoh Bioscience) for the HP Agilent HPLC system. The injection is carried out under isocratic elution conditions using 200 mM sodium sulfate, 100 mM sodium phosphate, pH 6.8 mobile phase, and is detected with absorbance at 214 nm. The quantification is based on the relative area of the detected peaks.

The host cell protein ( HCP ) ELISA

HCP assays are based on process specific antigen-based ELISAs. Sample dilution was applied to achieve reading within the calibration range. The assay has a limit of 0.625 ng / mL.

UV spectroscopy A ₂₈₀

After protein A elution with UV A280, the protein concentration of the sample was measured. Assays were performed on an Agilent UV spectrophotometer. The protein concentration is determined using the Beer-Lambert's Law, A =? Lc (where A is the absorbance,? Is the extinction coefficient, I is the path length, and c is the concentration) Respectively. The absorbance was taken at 280 nm, the path length was 1 cm, and the extinction coefficients were 1.39, 0.38, and 1.43 for mAb and mAb B, respectively.

Example AEX  7.1: Mab :badge: Buffer  Determination of the appropriate operating conditions for the combination

A demonstration of the invention for a particular antibody &lt; / RTI &gt; resin is provided in this example, which is as follows.

One. Selection of the anion concentration to allow products and impurities to bind at a given pH above the pI of the product.

2. Performing pH gradient elution over a range of pI of the product, of the pI of the product, and less than the pI of the product.

3. Determination of the pH range over which proteins are eluted from anion exchange medium.

In this embodiment, Adalimumab and Poros 50PI are selected. Experiments were performed at a concentration of 5 mM acetate (anion). The column was equilibrated with 5 mM acetate / Tris at a pH of 9.0. Adalimumab was prepared in 5 mM acetate / Tris pH 9.0 and loaded onto the column with 20 g protein / resin L. [ The column was washed with 10 CV of equilibration buffer. Subsequently, a pH concentration gradient of 9.0 to 7.0 was performed at an anion concentration of 5 mM acetate / Tris. The process chromatogram is shown in Figure 165.

A demonstration of the invention for a particular antibody &lt; / RTI &gt; resin is provided in this example, which is as follows.

One. For a given pH, the choice of the starting anion concentration allows the products and impurities to bind to the AEX adsorbent.

2. Performing a linear concentration gradient elution by increasing the loading of a small amount of protein to the column followed by increasing the anion concentration while maintaining the pH constant.

3. Determination of the range of anion concentration in which proteins are eluted from an anion exchange medium.

In this embodiment, Adalimumab and Poros 50HQ are selected. Experiments were performed at pH 8.7. The column was equilibrated with 10 mM acetate / Tris at a pH of 8.7. Adalimumab in 10 mM acetate / Tris pH 8.7 was prepared and loaded onto the column with 20 g-protein / resin L. The column was washed with 10 CV of equilibration buffer. A linear concentration gradient from 10 to 100 mM acetate / Tris at pH 8.7 was performed. The process chromatogram is shown in Figure 166.

Using this general approach, suitable operating conditions for any resin / mAb combination are determined as shown in Table 12 to implement the present invention.

In practicing the present invention, preferred acid species reduction can be achieved by proper pooling of the load and wash fraction. The cumulative AR reduction and cumulative yield can be calculated using the weighed mean up to a given fraction, by collecting each fraction over the load and washout and subsequently measuring the product quality thereof. In addition, the instantaneous yield can be estimated by comparing the recovered protein to the total protein loaded into the column in a given fraction. The sample calculation is shown below.

Sample Calculation A: cumulative yield up to a given fraction

Sample calculation B: Cumulative AR reduction to a given fraction

Sample Calculation C: Instantaneous yield for a given fraction

A demonstration of the invention for a particular antibody &lt; / RTI &gt; resin is provided in this example, which is as follows.

One. Anion concentration and anion exchange medium for a given pH.

2. The load of anion exchange medium over the dynamic binding capacity of the product for a given condition.

3. Column washing with buffers containing similar pH and anion concentrations used in the equilibration and loading steps

4. Collection of fractions over the loading and washing stages and subsequent measurement of product quality profiles (e.g., AR, aggregates, etc.).

In this embodiment, Adalimumab and Poros 50PI are selected. Experiments were performed at 5 mM acetate / arginine pH 8.8. The column was equilibrated with 5 mM acetate / arginine at pH 8.8. Adalimumab in 5mM acetate / arginine pH 8.8 was prepared and loaded onto the column with 300g-protein / resin L. The column was washed with 20 CV of equilibration buffer. Fractions were collected in a volume representing 30 g-protein / resin L, as shown in Figure 167. Each fraction was then analyzed for product quality and cumulative yield, and the AR reduction was calculated and is shown in Table 13. From this example, it will be apparent to those skilled in the art to determine the performance conditions that yield the target product quality and / or step yield.

Using this general approach, the performance against a given operating condition for any resin / mAb / buffer combination is evaluated.

Example AEX  7.2: AEX  Demonstration of AR reduction with adsorbent

This data set demonstrates that AR reduction was achieved with three different AEX adsorbents. Each of the resins was evaluated using the Acidimabbe at acetate concentrations measured from the process outlined in Example 7.1 and at pH values below pI, near pI, and above pI (e.g., pH 8.5 to 9.0) Respectively. Table 14 summarizes the results from these experiments.

It is evident that AR reduction with eight different AEX adsorbents was achieved with this data set. Each resin was tested using an advanced screening method using the process outlined in Example 7.1 and was tested at two different pHs (e.g., pH 8.7 and 9.0) and two different acetate concentrations (e.g., 0.0 &gt; 20mM) &lt; / RTI &gt; In these experiments, instantaneous (e.g., not cumulative) AR reduction was determined by analyzing the load fraction at 150 g / L and subsequently compared for all resins. Table 15 summarizes the results from these experiments.

It is demonstrated that AR reduction with two different AEX chromatography membranes has been achieved according to this data set. Each membrane was tested using the conditions outlined in Table 15. &lt; tb &gt; &lt; TABLE &gt; The results from these experiments are shown in Table 16.

It is evident that AR reduction is achieved with two different charged deep filters according to this data set. The results from these experiments are shown in Table 17.

Example AEX  7.3: Other antibodies, Mab  B and Mab  Demonstration of AR reduction with C

The AR reduction technique of the present invention has been demonstrated with multiple antibodies using AEX adsorbents. Antibodies have different amounts of charged residues at different positions, resulting in charge interaction behavior for AEX columns that classify different antibodies from one antibody. Thus, the effect of anionic type, anion concentration is different for each antibody.

The following Table 18 and Table 19 show data for mAb B and mAb C, respectively. The data clearly demonstrate that AR reduction techniques work very effectively against other antibodies.

Example AEX  7.4: Demonstration of AR reduction with different pH conditions - Adalmumab

The AR species of the present invention are incorporated during the loading step; Therefore, binding pH is a key parameter. The anion concentration providing the desired performance will vary with the operational pH.

In this example, data according to different experiments are found to demonstrate the effect on the AR reduction of pH selection for pi of protein. This data set provides a person skilled in the art with a basis for determining the pH range for carrying out the experiments to carry out the present invention. It also emphasizes the fact that pH selection depends on several factors and that the relationship between pH and AR reduction is also mAb dependent.

In this example, Adalimumab and Poros 50PI were selected. Experiments were performed at concentrations of acetate / arginine at 5 mM at each of the specified pHs. Adalimumab was prepared with 5 mM acetate / arginine at each specified pH and loaded onto the column with 300 g protein / resin L. The column was washed with 20 CV of equilibration buffer. The results showing the pH effect on AR reduction are shown in Figure 168.

It is also evident that AR reduction can be achieved using the present invention with a pH selection range in the range of +/- 0.5 pH units from the pI of multiple mAbs listed in Table 20. Each of these experiments was carried out with a 300 g / L loaded Poros 50HQ resin in an acetate / Tris buffer system.

Example AEX  7.5: Demonstration of AR reduction with different ion concentrations - Adalmumab

The anion concentration is a key parameter in the performance of anion exchange chromatography. For all combinations of antibody / resin / pH, there is a range of anionic concentrations that provide AR reduction; The AR reduction and the corresponding number of times for each anion concentration can be measured according to the strategy outlined in Example 7.1.

Table 21 below shows the effect of anion concentration on AR reduction. The table also includes the effect of anion concentration on different pH values. The data demonstrate that AR reduction can be effectively achieved over a range of anion concentrations at each pH, and that the concentration range depends on the pH.

Example AEX  7.6: Ahlally Muhammad  Different Buffer  Demonstration of AR reduction with system

Anion type and concentration are key parameters in anion exchange chromatography. The present invention has been demonstrated using acetate and formate as the anionic type, and Tris and arginine as the counter cation type. Optimal pH and cation concentrations were different for each cation type / mixture and were derived using the outlined strategy in Example 7.1. Table 22 shows the AR reduction and corresponding collection data for different anion / cation types.

Example AEX  7.7: Demonstration of AR reduction with different loads

In addition, the strategy outlined in Example 7.1 for reducing acidic species through careful control of buffer anion type, anion concentration, AEX adsorbent, and pH can be applied to any range of protein loading. The range of relevant protein loading (e.g., 100-350 g / L) for Poros 50HQ at pH 8.7 with acetate as an anion is shown in Table 23, indicating a strong AR reduction over the irradiated load range.

Example AEX  7.8: Demonstration of AR reduction with different load concentrations

In addition, the strategy outlined in Example 7.1 for reducing acidic species through careful control of buffer anion type, anion concentration, AEX adsorbent, and pH can be applied to any range of column feed streams of varying protein concentrations have. The range of varying protein loading concentrations (e. G., 100-350 g / L) for 300 g / L load of Adalimumab in Poros 50HQ at 15 mM acetate / Tris pH 8.7 is shown in Table 24.

In practice AEX  7.9: Alternative cleaning methods

In this example, Adalimumab and Poros50HQ resin were selected. In each experiment, changes were made to the equilibrium, load and wash pH values at a given acetate concentration (as specified). Table 25 and Table 26 show the effect of pH change at step yield and AR reduction.

As discussed in the previous section, the relationship between the working pH and its product pi is important for AR species reduction in AEX. Similarly, the working pH for the pKa of the AEX adsorbent is also important because the majority of the mAbs are similar to the pKa of the AEX adsorbent. This effect is demonstrated for mAb B using several different AEX adsorbents, using several different pKa values, performed with acetate / Tris buffer at pH 9.1.

As discussed in the previous section, the AR for Adalamumab is further grouped into two areas named AR1 and AR2 based on the predetermined retention time of the peaks shown in the WCX-10 method. The characteristics of mutants in these two regions are predicted to be different, and for this reason, a method for reducing mutants belonging to these groups can be specifically described.

Additionally, in addition to achieving the desired AR reduction, it may be desirable to achieve a certain absolute level of AR level in view of reducing or eliminating certain variants. The ability of the present invention in attaining the predetermined absolute levels AR, AR1, and AR2 is demonstrated in Table 27. &lt; tb &gt; &lt; TABLE &gt; The method of the present invention can effectively reduce AR2 levels, thus achieving a total reduction in AR levels. The method can be used to achieve a target absolute level as illustrated by the data presented in Table 27. [ There are many species under the group of AR2, and the method of the present invention can be used to reduce these species. The method of the present invention can effectively achieve target absolute levels of acidic species as well as AR reduction, as exemplified by the data presented in Table 27. [

Example AEX  7.10: In addition to AR reduction HCP  And demonstration of decreased aggregate

AEX chromatography is effective in reducing aggregate and HCP levels. In the present invention it has been demonstrated that HCP and aggregate levels can be effectively reduced under operating conditions selected for AR reduction. Tables 28 and 29 show aggregates and HCP removal achieved with AR reduction. The data clearly demonstrate that other processes and product-related substances / impurities involved can be achieved using the present invention for AEX adsorbents and, for this reason, serve as effective polishing steps in the large-scale purification of monoclonal antibodies.

Example AEX  7.11: Proof of means to control AR reduction

Controlling the final product quality by modifying the process based on the quality of the intermediate material is an approach that has been proposed as an effective method of securing product quality in terms of ensuring safety and efficacy.

Considering that the AR levels generated during cell culture and other upstream steps may change, it is desirable to design a downstream process step that implements the means to control product quality; It is desirable to have specific means of controlling process parameters that affect the quality of the product.

In the present invention, this control is possible since the pH and load (i.e., g / L) are parameters that can be modified to achieve the desired separation of AR species. For example, to achieve a higher level of AR reduction at a given anion concentration and pH, the load on the column can be reduced. In addition, for a given anion concentration and load, the pH can be increased to achieve a reduction in higher AR species.

By way of example and not limitation in any way, it has been demonstrated in this embodiment that the AR level can be controlled by varying the loading liquid and the pH of the wash liquid and the total load on the column. For this study, a pilot scale Poros HQ column (10 cm diameter x 22.5 cm height, 1.8 L) was used.

Both the loading material and the stock buffer are prepared with 18 mM acetate / Tris at the specified pH by titrating the affinity trapped material into the stock Tris solution. The AR level of the load material was the same for both runs. These experiments demonstrate how the final AR level can be adjusted by adjusting the pH and the protein loading on the column as shown in Table 30 while maintaining acceptable yields.

Example AEX  7.12: Tris / Formate Buffer  System-based AEX : Formic acid Wan In the infusion system Poros  50 HQ For Adalmumab  Reduction of Acid Species

This example provides a demonstration of the use of a Tris / formate buffer system for AR reduction with AEX. In the practice of this embodiment, And by appropriate pooling of the wash fraction. Cumulative AR reduction and cumulative yield can be calculated using the weighed averages up to a given fraction by collecting each fraction over load and wash and subsequently measuring the product quality of these fractions. In addition, the instantaneous yield can be estimated by comparing the recovered protein to the total protein loaded into the column in a given fraction.

AEX  absorbent

Poros 50HQ (Applied Biosciences, part # 1-2459-11), a rigid 50 탆 polymeric bead with a backbone consisting of cross-linked poly [styrene-divinylbenzene], was used in this experiment.

AEX chromatography method

Poros 50HQ was packed into a 1.0 cm x 10.0 cm (OmniFit) column. The column was equilibrated in a buffer system with appropriate pH and conductivity. Loads were made in the equilibration buffer by addition of the stock solution to obtain the target ion concentration, loaded onto the column, and then washed with equilibration buffer for 20 CV. Antibody products were collected in the passage and wash fractions during loading and washing steps. The column / housing was then regenerated with 100 mM formate and a 1M NaOH solution was used for column washing.

The sample calculation is shown below:

Sample Calculation A: cumulative yield up to a given fraction

Sample calculation B: Cumulative AR reduction to a given fraction

Sample Calculation C: Instantaneous yield to a given fraction

In this example, Adalimumab and Poros 50HQ were selected. Experiments were performed at 10 mM, 15 mM, 20 mM, 30 mM, and 40 mM formate / Tris pH 8.8. Columns were equilibrated to pH 8.8 using each formate / Tris for each run. Adalimumab was prepared with 10 mM, 15 mM, 20 mM, 30 mM, and 40 mM formate / Tris pH 8.8 and loaded into the column with 300 to 500 g protein / L resin. Fractions were collected in volumes representing approximately 25 g-protein / L-resin. These fractions were analyzed for product quality, cumulative yield, and cumulative AR reduction during execution (shown in Figure 189). Instantaneous yields and AR reductions at 100, 200, 300, 400, and 500 g / L loading are shown in Table 31. This example demonstrates the effectiveness of a Tris / formate buffer system in general and, in particular, the effectiveness of formate ions on AEX columns for AR reduction. In addition, this embodiment confirms that the AEX AR reduction method is applied to various buffer systems.

Example 8: Cation Exchange Chromatography Example

Example CEX 8.1: Mab: Resin: Determination of operating conditions appropriate for buffer combination

A demonstration of the invention for a particular antibody &lt; / RTI &gt; resin is provided in this example, which is as follows.

One. Selection of pH below pI of protein.

2. Select NaCl concentration in the range of 100 to 150 mM and perform experiments at, for example, 115, 125, 135 concentrations.

3. Determination of the distribution of acidic species in the effluent / eluent of the wash fraction.

4. The choice of NaCl concentration to provide the desired acid species level and recovery.

In this example, Adalmumab was selected and Poros XS was selected. Experiments were performed at pH 6.0. The process chromatogram is shown in Figure 169. The recovery versus AR reduction curve for each experiment is shown in Figure 170 and Table 32. [ From this set of experiments, a sodium concentration of 125 mM can be selected to give a recovery of 74% of eluate, which provides an AR reduction of 5.4%. Alternatively, you can choose an AR reduction value of 5.4%, which will provide a recovery of about 75%.

Using this general approach, the present invention is implemented by determining appropriate operating conditions for any resin / mAb combination.

In the practice of certain embodiments of the invention, the preferred acid species reduction can be achieved by an absolute pooling of the wash fraction and the elution fraction. In the embodiment described in the previous section, the eluted fraction can achieve an AR reduction of about 1% to about 7%, depending on the pooled and pooled fractions with the wash fraction, as shown in Table 32. [ This approach can be implemented to achieve target yield and AR reduction as illustrated in Figure 170. [

Example CEX 8.2: Demonstration of AR Reduction with CEX Adsorbent

This data set is compiled to demonstrate the AR reduction achieved using eight different CEX sorbents. The conditions for each resin were derived based on the strategy outlined in Example 8.1 above. Table 33 summarizes the conditions used and the AR reduction achieved and the corresponding number of times achieved.

The data show that the technique is robust in calculating the AR reduction in all 10 resins. As described in Example 8.1 above, AR reduction can be balanced with recovery and optimal conditions can be selected. The experiment was performed at pH 7.5. 29 mM Tris-acetate was used for pH control.

Example CEX 8.3: Other antibodies: Demonstration of AR reduction with mAb B and mAb C

The AR reduction technique of the present invention has been demonstrated with multiple antibodies using CEX adsorbents. Antibodies have different amounts of charged residues at different positions, which leads to charge interaction behavior on a CEX column where one antibody is different from the other antibody. Thus, the effect of cation type, cation concentration is different for each antibody.

For each antibody / resin combination, the cation concentration for each cation type provided AR reduction was determined using the experimental strategy outlined in Example 8.1 above.

Tables 34 and 35 below show data for mAb B and mAb C, respectively. The data clearly demonstrate that AR reduction techniques apply very effectively to other antibodies. It is also evident that the concentration range differs between the different antibodies. The selected pH range was related to the isoelectric point of the antibody and was chosen to be approximately 1-2 units less than the pI of the molecule.

Example CEX  8.4: Demonstration of AR reduction with different pH conditions - Adalmumab

The AR species of the present invention are removed with a passing liquid / wash fraction. Thus, binding pH is a key parameter. The cation concentration providing the desired performance will vary with the binding pH. Thus, for each binding pH, the experimental strategy outlined in Example 8.1 above is performed to determine the range of ion concentrations that result in AR reduction.

The results of the experiment using different pH for Adalimumab are shown in Table 36. [ As shown, at lower pH, the cation concentration required to achieve AR reduction in the wash fraction is higher. At low pH, the AR reduction at pH higher (closer to pi) is significantly more potent and not optimal. It is not obvious to those skilled in the art to operate the cation exchange chromatography at a pH closer to the pi as shown in Table 37. The literature data suggests an optimal pH that is at least 3 units less than the pI of the molecule.

Example CEX  8.5: Demonstration of AR reduction with different ion concentrations - Adalmumab

The cation concentration is a key parameter in the performance of cation exchange chromatography. There is a range of cation concentrations that provide AR reduction for all combinations of antibody / resin / pH; The AR reduction and the corresponding number of times for each cation concentration can be measured according to the strategy outlined in Example 8.1 above.

Table 38 below shows the effect of cation concentration on AR reduction. The table also includes the effect of the cation concentration on the different pH values. The data demonstrate that AR reduction over a range of cation concentrations for each pH can be effectively achieved and that the concentration range is pH dependent. The table also includes examples of concentration ranges for different cation types.

Example CEX  8.6: Ahlally Muhammad  Different Buffer  Demonstration of AR reduction with system

Cationic types and concentrations are key parameters in cation exchange chromatography. The present invention has been demonstrated to have an effective reduction of AR using Tris, sodium / Tris, ammonium / Tris and arginine / Tris as cationic types / mixtures in each case. As will be understood by those skilled in the art, the optimum pH and cation concentration are different for each cation type / mixture, which is derived by using the strategy outlined in Example 8.1 above. The experiment was performed at pH 7.5. 29 mM Tris-acetate was used for pH control. Table 39 shows data of the AR reduction and the corresponding number of times for different cation types / mixtures.

Example CEX  8.7: Demonstration of AR reduction with different loads

In addition, the strategy outlined above in Example 8.1 for reducing acid species through careful control of buffer cation type, concentration, and pH, can be used to determine the mode of operation of the coupling following elution, That is, it can be applied to any range of protein load that is not overloaded. The range of relevant protein loading for Poros XS at pH 7.5 with Tris as the cation is shown in Table 40 to indicate strong AR reduction.

Example CEX  8.8: Demonstration of AR reduction with different load concentrations

In addition, the strategy outlined above in Example 8.1 for reducing acidic species through careful control of buffer cation type, concentration, and pH can be applied to any range of column feed streams of varying protein concentrations. The range of varying protein loading concentrations for Poros XS at pH 7.5 with Tris as the cation is shown in Table 41 to indicate a strong AR reduction.

As described above, the AR for Adalimumab is further grouped into two areas named AR1 and AR2 based on the predetermined retention time of the peaks shown in the WCX-10 method. The characteristics of mutants in these two regions are predicted to be different, and for this reason, a method for reducing mutants belonging to these groups can be specifically described.

Additionally, in addition to achieving the desired AR reduction, it may be desirable to achieve a certain absolute level of AR level in view of reducing or eliminating certain variants. The ability of the present invention in achieving the predetermined absolute levels AR, AR1, and AR2 is demonstrated in Table 42. [

Identification and quantification of specific species, including AR1 species, can demonstrate the reduction of this species by the method of the present invention. Two of these species, glycated mAbs and MGO modified mAbs, have been identified and found to be reduced by the method of the present invention. These species are among the acidic species of the charge mutants, but the acidic species typically described in the literature are apparently different deamidated mAbs.

The method of the present invention can effectively reduce AR2 levels, thus achieving a total reduction in AR levels. The method can be used to achieve a target absolute level as exemplified by the data presented in Table 42. [

The method of the present invention can effectively achieve target absolute levels of acidic species as well as AR reduction, as exemplified by the data presented in Table 42. [

Example CEX  8.9: Glycosylated  Species and Methylglyoxylated  Proof of species decline

The strategy outlined above in Example 8.1 for reducing acidic species through careful control of buffer cation type, concentration and pH can be further extended to specific post-translational modifications. Acidic species are defined herein as impurities that are less retained than the main peaks for analytical weak cation exchange (WCX) HPLC columns in the present application, but are not limited to specific metabolic modifications such as glycosylation and methylglyoxal (MGO) The product-related material of the invention can be specifically identified as being part of an acidic species. Figures 171 and 172 show the results of an in vitro labeling experiment demonstrating that the glycated antibody and the MGO modified antibody are unique species that can be resolved by the WCX method in the AR1 region of the chromatogram and concentrated in vitro. In addition, the present invention described herein provides that the glycated antibody and the MGO modified antibody can be effectively removed through careful control of the buffer cation type, concentration and pH using CEX as described in Example 8.1 above . The quantitative reduction of AR1 and consequently the glycosylated species and MGO species by CEX and CEX-mixed resins are shown in Tables 43 and 44.

Example CEX  8.10: Proof of lysine distribution transformation

The strategy outlined in Example 8.1 above for reducing acidic species can also be used to adjust the distribution of C-terminal Lys variants, which are known post-translational modifications leading to charge heterogeneity. Since the WCX analysis is a compositional analysis, a small change in the distribution of Lys isoforms is predicted through the reduction of acid species. However, through careful control of the buffer cation type, concentration and pH, in addition to reducing acidic species, the elution pool can be enriched for the more basic isoforms (Lys 1 and Lys 2). Tables 45 and 173 show non-limiting examples of the effect of pH and cation (Tris) concentration on basic isoform concentration.

Example CEX  8.11: In addition to AR reduction HCP  And demonstration of decreased aggregate

In the present invention it has been demonstrated that, after washing away the AR enriched species in the passing liquid / wash fraction, the HCP and aggregate levels can be effectively reduced by proper adjustment of the elution conditions.

Tables 46 and 47 show HCP and aggregate removal achieved with AR reduction. The data clearly demonstrate that other process-related impurities and product-related materials can be achieved for the CEX adsorbent using the present invention, thus functioning as an effective polishing step in the large-scale purification of monoclonal antibodies.

Example CEX  8.12: Proof of AR reduction control measures

Controlling the final product quality by modifying the process based on the quality of the intermediate is an approach that has been proposed as an effective way to ensure product quality in terms of ensuring safety and efficacy.

Considering that the AR levels generated during cell culture and other upstream steps may change, it may be desirable to perform a method of controlling the product quality and to control the process parameters, It is desirable to design process steps.

In the present invention, this control is possible because the cation concentration is a single parameter that can be modified to achieve the desired separation of the AR species. For example, to achieve a higher level of AR reduction, the Tris concentration of the loading material and wash buffer may be reduced, and thus the AR concentrated paper may be collected in the passing fraction.

By way of example and not limitation in any way, it has been demonstrated in this example that the AR level can be controlled by varying the Tris concentration in the loading liquid and wash liquor. A pilot scale Poros XS column (10 cm diameter x 22 cm height, 1.7 L) was used in this study.

Both the loading material and the stock buffer are prepared at 300 mM Tri concentration at the same pH. The AR level of the load material was measured as X%. The loading material and the equilibration / wash buffer dilute in-line with the target Tris concentration based on a predetermined correlation between the AR level and the Tris concentration. As demonstrated in this example, a final AR reduction of 4.1 was achieved when the Tris concentration was adjusted to 156 mM, whereas a final AR level of 3.1 was achieved when the Tris concentration was adjusted to 150 mM (Table 48) . This enables highly predictable control of the AR level ensuring attainment of the desired product quality.

In addition to the acid species reduction demonstrated in Example CEX 8.1 through careful control of the loading (process stream) and the pH cation type and concentration in the equilibration / wash buffer, the composition of the elution buffer further improves the product quality profile Can be used. The effect of various cation types, concentrations and pH to elute the product was tested. As shown in Table 49, there is a wide selection of elution buffers. The experiment was carried out using Poros XS resin.

Example CEX 8.13: Demonstration of AR Reduction with Cation-HIC Mixed Resin

The strategy outlined above in Example 8.1 for reducing acidic species through careful control of buffer cation type, concentration and pH involves other chromatographic adsorbents such as mixing systems or multi-mode adsorbents including cation exchange mechanisms . &Lt; / RTI &gt; Table 50 summarizes the conditions used and the AR reduction achieved for the two cation-hydrophobic interaction mixed resins. The data clearly show that the technique outlined in Example 8.1 is robust in producing AR reductions for these types of resins. As described in Example 8.1 above, AR reduction can be balanced with recovery and optimal conditions can be selected. As a further demonstration, mAb 2 was also evaluated to have the same result indicating a relationship between cation concentration, recovery and AR reduction. As indicated previously in Example 8.1, the optimal conditions for the different molecules vary. In addition, when applied to CEX-HIC mixed resin, this technique also shows the reduction of impurities as described above.

As described in the previous section, the AR for Adalimumab is further grouped into two regions named AR1 and AR2 based on the predetermined retention time of the peaks shown in the WCX-10 method. The characteristics of mutants in these two regions are predicted to be different, and for this reason, a method for reducing mutants belonging to these groups can be specifically described.

Additionally, in addition to achieving the desired AR reduction, it may be desirable to achieve a certain absolute level of AR level in view of reducing or eliminating certain variants. The ability of the present invention in achieving certain absolute levels of AR, AR1, and AR2 is demonstrated in Tables 52a and 52b and Table 52c, which illustrate additional process-related impurities or levels of acid species.

Identification and quantification of specific species, including AR1 species, can demonstrate the reduction of this species by the method of the present invention. These species are among the acidic species of the charge mutants, but the acidic species typically described in the literature are apparently different deamidated mAbs. These results indicate that cation exchange resins with additional pendant hydrophobic interaction functionality can effectively provide AR reduction similar to CEX adsorbents.

[Table 52a]

[Table 52b]

[Table 52c]

Example  8.14: Tris / Formate  Buffer system and CEX : Different Tris / Formate  AR reduction with concentration - Adalmumab

This example provides demonstration of AR reduction with Tris / formate buffer system and CEX. The cation (e.g., Tris) concentration is a key parameter in the performance of cation exchange chromatography.

CEX  absorbent:

Poros XS (Applied Biosciences, part # 4404338), a 50 탆 polymeric bead having a backbone consisting of cross-linked poly [styrene-divinylbenzene], was used in this experiment.

CEX  Chromatographic method

Poros XS was packed into a 1.0 cm x 10.0 cm (OmniFit) column. The column was equilibrated in a buffer system with appropriate pH and conductivity. Column loadings were made in equilibration buffer by buffer exchange or addition of stock solution to achieve a target ion concentration as specified, loaded into the column with approximately 40 g protein / L resin (or as specified), then 20 CV And washed with equilibration buffer (as indicated). The antibody product was then eluted and the column was regenerated.

In this example, Adalimumab and Poros XS were selected. The experiment was carried out with formic acid at a concentration of 120-150 mM Tris buffered to pH 7.5. Columns were equilibrated to pH 7.5 using each Tris / formate for each run. Adalimumab was prepared with each Tris / formate pH 7.5 and loaded into the column with 35 to 45 g protein / L resin. The column was then washed with 20 CV using equilibration buffer and then eluted at pH 5.2 with 140 mM Tris / formate + 140 mM sodium sulfate buffer. The eluate was analyzed for product quality and yield.

Table 53 below shows the effect of Tris concentration on AR reduction and aggregate reduction on adalimumab on Poros XS in a Tris / formate buffer system at a pH of 7.5. The data demonstrate that AR and aggregate reduction can be effectively achieved over a range of Tris concentrations and column loading. This example demonstrates the effectiveness of Tris / formate buffer systems in general, and specifically the effectiveness of Tris cations, in connection with formates for CEX columns for AR reduction. In addition, the CEX AR reduction method confirms that it is applied to various buffer systems.

CEX  Wash volume for chromatography

Experiments were performed using Protein A eluate as CEX loading material. Run 1 was performed under load / wash buffer conditions of a 128 mM Tris-formate buffer system, pH 7.5, 40 g / L load. Washing was carried out using 20 CV of wash buffer. Run 2 was performed under load / wash buffer conditions of a 160 mM Tris-formate buffer system, pH 7.5, 40 g / L load. Washing was carried out using 6CV of washing buffer.

As shown in Table 54, Run 1 and Run 2 provided similar yields and AR reductions. Thus, these results demonstrate that process performance can be achieved by varying the cleaning volume in accordance with the corresponding load conditions.

Example  9: Mixture chromatography Example

Example  MM 9.1: resin and pH combination

In this embodiment, operating conditions for implementing the present invention were determined using the approaches outlined in the general description. Specifically, the response surface design DOE was applied to evaluate mAb AR reduction and recovery yield.

A demonstration of the invention for a particular antibody &lt; / RTI &gt; resin is provided in this example, which is as follows.

One. Selection of pH in the range of 6.8 to 8.4.

2. Selection of the conductivity in the range of 2.3 to 13.7 mS / cm.

3. Determination of acid species distribution in the passing liquid / wash fraction.

4. Selection of optimum pH and conductivity to provide desired acid species levels and recovery.

In this example, Adalimumab and Resin Capto Adhere were selected. Experiments were performed using a Tris / acetate buffer system at the target pH and conductivity listed in Table 55. [ The loading material was from protein A affinity capture and the pH was adjusted. These studies have demonstrated the effect of load pH and conductivity on acid species reduction. Acidic species reduction can be significantly affected by manipulating the pH. The AR decrease was increased with increasing pH and / or decreasing conductivity (Table 55, Table 56 and Figure 174).

Example  MM 9.2: Fraction Pooling

In this example, Adalimumab and Resin Capto Adhere were selected. Experiments were conducted using a Tris / acetate buffer system at pH 7.85 and 2.5 mS / cm conductivity. The loading material was from protein A affinity capture and the pH was adjusted. The column passed liquid was fractionated over the full load and wash phases. Each fraction was analyzed for acidic species and protein recovery. Figures 175, 176 and 57 demonstrate the AR reduction achieved with the corresponding number of times. These AR reductions and recoveries correspond to a cumulative pool of fractions from initiation to various points in time during load / wash. This is shown in Table 57, where the AR reduction corresponds to each of these pools. This data is plotted in Figure 175.

Example  MM 9.3: Demonstration of AR reduction with mixed adsorbent

In this example, Adalimumab was selected. Experiments were performed using a Tris / acetate buffer system at pH 7.85 and 2.5, 3.5, and 4.5 mS / cm conductivity. The same loading materials were applied to the different mixed type resin columns. The loading material was from protein A affinity capture and the pH was adjusted. Table 58 shows that all three mixed resins can reduce the mAb acid species. Due to differences in resin ligands, the level of AR reduction may vary slightly under certain conditions.

Example  MM 9.4: Other Antibodies: mAb  B and mAb  Demonstration of AR reduction with C

In this example, two different monoclonal antibodies (mAb B and mAb C) and resin Capto Adhere were selected in addition to adalimumab. Experiments were performed using a Tris / acetate buffer system at multiple pH and conductivity conditions. The loading material of all mAbs was from protein A affinity capture and the pH was adjusted. mAb C was also applied to two other MM resins besides Capto Adhere under the same operating conditions. Table 59 summarizes the operating conditions and the AR reduction achieved and the corresponding number achieved. The results demonstrate that the technique can also reduce acid species for other monoclonal antibodies with optimal pH and conductivity conditions. Experiments were performed using a Tris-acetate buffer system.

Figure 177 shows mAb B accumulated pool AR, which passed through a Capto Adhere column operated at a conductivity of 7.0 and 3.0 mS / cm using Tris-acetate buffer. Figure 178 shows mAb C accumulated pool AR through a Capto Adhere column operated at a conductivity of pH 8.5 and 3.0 mS / cm using Tris-acetate buffer. Both graphs demonstrate a similar AR pass curve with different AR values compared to Azali Mumab (FIG. 176). Figure 179 shows the AR pass curves of Mab C according to three different blended resins with Tris-acetate buffer operated at a conductivity of pH 8.5 and 3.0 mS / cm. The data clearly demonstrate that the AR reduction technique with mixed resin works very effectively against other antibodies.

Example  MM 9.5: Adalmumab  Proof of Relative pH for AR Reduction with Different Resins with Antibody Material

In this example, data edited from different experiments are found to demonstrate the effect of pH selection on pi of protein versus AR reduction. These data sets provide a basis for determining the pH range for carrying out the present invention to those skilled in the art. Additionally, this underscores the fact that pH selection depends on several factors and the relationship between pH and AR reduction is also mAb dependent. Figure 180 demonstrates the effect of pH-pI and conductivity on AR reduction with data compiled from experiments performed using Capto Adhere under the conditions listed in Table 60. &lt; tb &gt; &lt; TABLE &gt; Figure 181 shows the effect of pH-pI and conductivity on mAb B AR reduction, including experiments run with Tris / acetate buffer system and multi-mix resin under the conditions listed in Table 61. Figure 182 shows the effect of pH-pI and conductivity on mAb C AR reduction, including experiments run with the Tris / acetate buffer system and multiple mix resin under the conditions listed in Table 62. All loading materials were from protein A affinity capture and pH adjusted. It is also clear that the AR reduction can be achieved with the present invention using a range of pH selections within the range of + 0.5 to -2.5 pH units from pI for adalimumab. One skilled in the art can select a pH that is appropriate to achieve a target AR reduction.

Example MM 9.6: Effect of pH on AR reduction

Response surface design DOE was applied to assess the effect of pH and conductivity on mAb AR reduction. In this example, Adalmumab and Capto Adhere were selected. This experiment was performed using a Tris / acetate buffer system. The loading material was from protein A affinity capture and the pH was adjusted. In addition to the ranges of pH and conductivity tested in Table 63 and Table 64, higher pH ranges were also studied (Figure 183).

The results demonstrate that the mAb acid species can be reduced over a wide pH range of 6.8 to 9.5 in Figures 183 and 184.

Example MM 9.7: Demonstration of AR reduction with different ion concentration / ionic strength -

In this example, Adalimumab was selected. In addition to the previously tested conductivity ranges, lower conductivity and higher conductivity ranges have also been studied as Capto Adhere. Tables 65 and 66 show DOE study conditions using a Tris / acetate buffer system and Capto Adhere. The loading material was from protein A affinity capture and the pH was adjusted. Column run-through pools were collected from 50 mAU of UV A280 for the rising side of the peak at each run and 150mAU for the falling side of the peak. Figure 185 demonstrates the effect of pH (6.8 to 8.4), conductivity (2.3 to 13.7 mS / cm), and protein loading (116 to 354 g / L). Figure 186 demonstrates AR reduction at a conductivity as low as about 1 mS / cm. Table 67 demonstrates AR reduction at a conductivity of 86 mS / cm using an ammonium sulphate-Tris-acetate buffer system.

The results demonstrate that mAb acid species can be reduced over a wide range of conductivities from 1 to 86 mS / cm.

Example  MM 9.8: Ahlally Muhammad  Different Buffer  Demonstration of AR reduction with system

In this example, Adalimumab and Resin Capto Adhere were selected. Experiments were performed using different buffer systems listed in the following table at multiple pH and conductivity conditions. The pH of the loading material was adjusted from the protein A eluate or the CEX eluate. The results demonstrate that mAb acid species can be reduced using various buffer systems in Tables 68 and 69.

Example  MM 9.9: Demonstration of AR reduction with different loads

Experiments were conducted using a Tris / acetate buffer system under the conditions in Table 66. The loading material was adalimumab from protein A affinity capture and the pH was adjusted. Column run-through pools were collected from 50 mAU of UV A280 for the rising side of the peak at each run and 150mAU for the falling side of the peak. As seen from the profile (Figure 186), the load capability has an effect on the AR reduction, but the AR reduction can be achieved over a wide range of load capabilities, and is merely a trade-off between the AR reduction and the number of times.

Example  MM 9.10: Demonstration of AR reduction with different load concentrations

In this example, Capto Adhere was selected. Experiments were performed with a Tris / acetate buffer system at pH 7.8 ± 0.1 and conductivity 3.0 ± 0.05 mS / cm. The loading material was the agar medium from the concentrated CEX trap and the pH was adjusted. Then, the prepared load material was divided into two parts. One was directly loaded onto the Capto adhere column and the other was diluted 2 fold with equilibration buffer to produce different protein concentrations. Table 70 demonstrates that the loading protein concentration had no significant effect on mAb acid species reduction.

Example  MM 9.11: Alternative cleaning form

In this example, mAb Adalmumab and Resin Capto Adhere were selected. Experiments were performed using a Tris / acetate buffer system and the loading material pH was adjusted from the protein A eluate. The equilibration buffer for both runs was Tris / Acetic acid pH 7.8 ± 0.1 and Conductivity 3.0 ± 0.1 mS / cm. In a concentration gradient conductivity wash study, the second buffer had a Tris / acetic acid pH of 7.8 ± 0.1 and a conductivity of 6.0 mS / cm.

The results demonstrate that pH and conductivity after loading can vary with the minimum AR reduction effected (see Table 71).

Example  MM 9.12: Demonstration of Absolute Value of AR Level in Antibody Preparations Using Mixed Chromatography

In this example, mAb dival also was selected. Experiments were performed using multiple buffer systems and multi-MM adsorbents under the conditions listed in Table 72. The loading material pH was adjusted from the protein A eluate.

As described above, the AR for adalimumab is further grouped into two regions labeled AR1 and AR2 based on the predetermined retention time of the peaks shown in the WCX-10 method. The characteristics of mutants in these two regions are predicted to be different, and for this reason, a method for reducing mutants belonging to these groups can be specifically described. Additionally, in addition to achieving the desired AR reduction, it may be desirable to achieve a certain absolute level of AR level in view of reducing or eliminating certain variants. The ability of the present invention in achieving the predetermined absolute levels AR, AR1, and AR2 is demonstrated in Table 72. [

Example  MM 9.13: In addition to AR reduction HCP  And demonstration of decreased aggregate

In addition to acid species reduction, the MM adsorbent can effectively reduce other product / process related substances / impurities. In the practice of the present invention, AR reduction is achieved and other impurities / materials are expected to be significantly removed, since they must bind more strongly than acidic species. The data presented in Tables 73 and 74 demonstrate significant HCP and aggregate reduction using different resins, buffer system, pH, conductivity and molecules.

Example  MM 9.14: Alternative Separation Strategy and MM Combination

Acid species reduction by the MM adsorbent is expected to be carried out after capture of the antibody by other means, or after one or more intermediate steps following the capture step. In the following example, the MM adsorbent step was performed after the cation exchange capture step or the protein A affinity capture step. As shown in Table 75, AR reduction was achieved at two different conductivities after protein A chromatography and CEX chromatography.

Adalimumab was purified by a CEX chromatography step followed by a low pH virus inactivation step. The filtered virus-inactivated material was buffer exchanged and loaded onto a Capto Adhere column. Subsequently, the passing solution of the Capto Adhere material was purified by a HIC column using a coupling / elution method. As shown in Table 76, AR reduction was achieved primarily by contributions from MM steps and other steps.

Adalimumab purified the protein A chromatography step followed by a low pH virus inactivation step. The filtered virus-inactivating material was buffer exchanged and loaded onto a Capto Adhere column. Subsequently, the passing solution of the Capto Adhere material was purified by a HIC column using a coupling / elution system and a passage system. As shown in Table 77, AR reduction was mainly achieved by contributions from MM steps and other steps.

Example  10: Target AR %  or AR  To achieve reduction Upstream  And Downst Rim process combination

This embodiment demonstrates the combined effect of one or more upstream and downstream process techniques in achieving target AR values or AR reduction, thereby enabling the production of antibody compositions with specific charge heterogeneity.

Example  10.1: MM  Used Upstream  And Downstream  A combination of technologies

In this example, the combination of the upstream and downstream methods involves the reduction of acid species in a 3L bioreactor cell culture supplemented with arginine (2 g / L) and lysine (4 g / L). The results of this strategy are summarized in Table 78. Total acid species decreased from 20.5% in the control sample to 10.2% in the culture-derived sample supplemented with the additive.

In this study, adalimumab productivity cell line 1 was cultured in medium 1 (chemically defined medium) supplemented with amino acid arginine (2 g / L) and lysine (4 g / L) in a 300 L bioreactor. On day 12 of culture, the culture was harvested and subsequently analyzed using Protein A purification after WCX-10 and the percentage of total peak (s) area corresponding to acid species was quantified. The percentage of acid species is estimated to be 9.1% in the 300 L collection sample.

The material produced by the 300L bioreactor using arginine and lysine addition, which effectively reduced the AR level to 9.1%, was purified using a downstream process using mixed-mode chromatography as the primary AR reduction method.

Adalimumab was purified by a protein A chromatography step followed by a low pH virus inactivation step. The filtered virus-inactivated material was buffer exchanged and loaded onto a Capto Adhere column. Subsequently, the passing solution of the Capto Adhere material was purified by a HIC column using a coupling / elution system and a passage system. As shown in Table 79, AR reduction was mainly achieved by contributions from MM steps and other steps. In addition, the table shows that these combined techniques can be used to effectively reduce additional product-related materials, such as aggregates, and process-related impurities, such as HCP.

As is apparent from the above examples, the MM method further reduced the AR level to 2.26%. Thus, upstream techniques for reduction may be combined with downstream techniques to achieve a desirable AR level / AR reduction.

Example CEX  10.2: In the process combination AR  Proof of reduction

The methods described above for reducing acid species using cation exchange can be used as independent processes or in other process steps that provide additional acid species reduction or other process steps that provide additional complementary and complementary purification (See Tables 80 to 87). Here, the following process combinations are provided as non-limiting examples.

One. Affinity → MM → CEX

2. Affinity → AEX → CEX

3. Affinity → CEX

4. CEX capture → CEX

Example  10.3: Process combination: Tris / Formate  Protein A using a buffer system, AEX , CEX  Combination

In Example 10.3, AR reduction through a combination of protein A affinity capture processes followed by microfabrication using AEX and CEX chromatography in a Tris / formate buffer system was tested.

Materials and methods

material

Antibody

The affinity capture of the clarified collection resulted in an adalimumab monoclonal antibody preparation. The eluate from the capture step was buffer exchanged as needed.

AEX  absorbent:

In this experiment, Poros 50HQ (Applied Biosciences, part # 1-2459-11), a 50 μm polymeric bead with a backbone consisting of cross-linked poly [styrene-divinylbenzene] was used.

CEX  absorbent:

In this experiment, Poros XS (Applied Biosciences, part # 4404338), a 50 μm polymeric bead with a backbone consisting of cross-linked poly [styrene-divinylbenzene] was used.

Way

AEX  Chromatographic method

Poros 50HQ was packed onto a 1.0 cm x 10.0 cm (OmniFit) column. The column was equilibrated in a buffer system with appropriate pH and conductivity. To obtain the target ion concentration as specified, a loading was made in equilibration buffer by addition of stock solution, loaded into the column as specified, and then washed with equilibration buffer for 20 CV. Antibody products were collected in the passage and wash fractions during loading and washing steps. The column / housing was then regenerated with 100 mM formate and a 1M NaOH solution was used for column washing.

CEX  Chromatographic method

A 1.0 cm x 10.0 cm (OmniFit) column was packed with Poros XS. The column was equilibrated in a buffer system with appropriate pH and conductivity. A load was made in the equilibration buffer by buffer exchange or addition of stock solution to achieve the target ion concentration as specified, and approximately 40 g (or as specified) of protein / L resin was loaded onto the column, followed by 20 CV (Or as specified) with equilibration buffer. The antibody product was then eluted and the column was regenerated.

Method of making buffer

A buffer for AEX was prepared to target specific ion concentrations for anions by fixing the anion concentration (acid) to the target value and adjusting the solution to achieve the proper pH using a cationic component (base). For example, to prepare 10 mM formate-Tris buffer, pH 8.7 formic acid was dissolved in water to a target concentration of 10 mM and adjusted to pH 8.7 with concentrated Tris-base.

A buffer for CEX was prepared to target a specific ion concentration to the cation by fixing the cation concentration (base) to the target value and adjusting the solution to achieve the proper pH using the anion component (base). For example, to prepare a 140 mM Tris-formate buffer, a pH 7.5 Tris base was dissolved in water to a target concentration of 140 mM and adjusted to pH 7.5 using formic acid.

AR  Reduction and counting

Generally, the eluate fractions and the passage liquid (FT) / wash fractions were collected and analyzed for AR levels using the WCX-10 method. Recovery and corresponding AR levels were calculated by actual or calculated pooling of fractions.

Analysis method

Adalmumab  About WCX -10

The acidic species and other charge mutants present in the Adalimumab process sample were quantified according to the following method. Cation exchange chromatography was performed on Dionex ProPac WCX-10, an analytical column 4 mm x 250 mm (Dionex, Calif.). An Agilent 1200 HPLC system was used as the HPLC. The mobile phase used was 10 mM dibasic sodium phosphate pH 7.5 (mobile phase A) and 10 mM dibasic sodium phosphate, 500 mM sodium chloride pH 5.5 (mobile phase B). Binary concentration gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% : 28-34 min) was used with detection at 280 nm.

The quantification was based on the relative area percent of the detected peaks. Peaks eluting at a relative retention time less than a predetermined time are also indicated as acidic peaks.

Size exclusion chromatography

The molecular weight distribution of the collected samples was quantified according to the following method. Size exclusion chromatography (SEC) was performed using a TSK-gel G3000SWxL, 5 urn, 125 Å, 7.8 X 300 mm column (Tosoh Bioscience) on an HP Agilent HPLC system. Under the conditions of isocratic elution, using 200 mM sodium sulfate, 100 mM sodium phosphate, pH 6.8, and detected by absorbance at 214 nm. The quantification was based on the relative area of the detected peaks.

UV  Spectroscopy A ₂₈₀

After protein A elution with UV A280, the protein concentration of the sample was measured. Assays were performed on an Agilent UV spectrophotometer. The protein concentration was measured using Beer-Lambert's law, A =? Lc, where A is the absorbance,? Is the extinction coefficient, I is the path length, and c is the concentration. Absorbance was measured at 280 nm, the path length was 1 cm, and the extinction coefficient was 1.39, 1.38 for mAb B and 1.43 for mAb C, respectively.

result

AR reduction through a process combination of protein A affinity capture following microfabrication with Poros 50HQ and Poros XS in a Tris / formate buffer system was performed as follows, resulting in 1.4% final AR. This exemplary low AR process according to the flow path is shown in FIG.

Protein A

For protein A affinity capture, a 2.2 x 20 cm MabSelect SuRe (GE Healthcare) column was packed and qualification confirmed by HETP / Asymmetry analysis. Chromatography was performed using a coupling-elution method with a retention time of 4 minutes. 37 g of protein per liter of resin were loaded onto the column.

The column was washed with high concentration Tris / formate buffer, washed with low concentration Tris / formate buffer and subsequently eluted with low pH Tris / formate buffer. The column was then regenerated and washed with the appropriate hydroxide solution in resin.

MabSelect Sure eluate pool was titrated to pH 3.7 with formic acid and left for 1 hour. The acidified material was mixed for 1 hour at ambient temperature. The VI pool was neutralized to a pH of 8.7 (i.e., AEX load). The AEX load was filtered before loading.

AEX chromatography

All AEX chromatographic experiments were performed on the AKTAavant25 system using a 1.0 cm diameter x 9.5 cm long column packed with Poros 50HQ resin and qualification confirmed by HETP / Asymmetry analysis. Each experiment was performed at ambient temperature. The AEX step was carried out with a resin load of 225 g / L. Equilibration and loading were carried out at a pH of 8.7 using a low concentration of formate / Tris buffer, for example 15 mM formate / Tris buffer. Washing was performed at the same pH using acetate and Tris. Each run was performed at ambient temperature with a load concentration of about 10 g / L at a retention time of 3 minutes. The column was regenerated and washed with a solution appropriate for the resin.

Passages were collected from the following fractions: 100mAu-175g / L, 175g / L to 200g / L, 200g / L to 225g / L + 1CV. The fractions were then measured by A280 mass spectrometry and analyzed by WCX-10 and SEC assay.

The stock solution of Tris and formic acid was used to adjust the Poros 50HQ FTW pool to 135 mM Tris / formate pH 7.5.

CEX Chromatography

All CEX chromatographic experiments were performed on an AKTAavant150 system using a 1.0 cm diameter x 11 cm long column packed with Poros XS resin and qualification confirmed by HETP / Asymmetry analysis. Each experiment was carried out at ambient temperature using a load of 5.8 mg / mL, a load of 40 g / L resin, and a residence time of 6 minutes. Equilibration, loading, and washing were performed at a pH of 7.5 using a high concentration of Tris / formate buffer. Sodium sulfate and Tris / formate buffer. The eluate was collected in one fraction of 400 mAU to 100 mAU. Three cycles were performed. The column was regenerated and washed with a solution appropriate for the resin.

Virus filtration was performed on the Poros XS eluate prior to UFDF processing using the Virosart CPV virus filter.

An Ultracel 3 Biomax 30 kDa filter was used for dialysis filtration (in water) and concentration of the CEX eluate.

The cumulative AR of the Poros50HQ fraction was less than 6%, allowing pooling together and was adjusted to CEX loading conditions. Three cycles of CEX were performed. Three CEX elution cycles all had less than 3% AR, and less than 0.2% HMW and were pooled together.

The step yield for each unit operation is listed in Table 88 along with the final overall yield achieved at 38%. The process was able to achieve an ordinal marmer composition with 1.4% final AR (0.0% AR1 and 1.4% AR2) and 0.10% final HMW.

Example 11 "Recirculation" AR reduction with AEX and CEX techniques

This embodiment describes a "recycle" scheme of chromatography for AR reduction using AEX, CEX, and MM techniques.

Materials and methods

material

Antibody

The adalimumab monoclonal antibody preparation was a material obtained after the affinity capture of the purified collection. The eluate from the capture step was buffer exchanged as needed.

AEX adsorbent:

Poros 50HQ (Applied Biosciences, part # 1-2459-11), a 50 탆 polymeric bead having a backbone consisting of crosslinked-linked poly [styrene-divinylbenzene], was used in this experiment.

CEX adsorbent:

Poros XS (Applied Biosciences, part # 4404338), a 50 탆 polymeric bead having a backbone consisting of cross-linked poly [styrene-divinylbenzene], was used in this experiment.

Way

AEX chromatography method

Poros 50HQ was packed into a 1.0 cm x 10.0 cm (OmniFit) column. The column was equilibrated to the appropriate pH and conductivity in a buffer system. A loading was made in the equilibration buffer by addition of the stock solution to obtain the target ion concentration, which was loaded onto the column and then washed with 20 CV of equilibration buffer. Antibody products were collected in the passage and wash fractions during loading and washing steps. The column / housing was then regenerated using 100 mM formate and a 1 M NaOH solution was used for column washing.

CEX Chromatography Method

Poros XS was packed into a 1.0 cm x 10.0 cm (OmniFit) column. The column was equilibrated to the appropriate pH and conductivity in a buffer system. Column loadings were made in equilibration buffer by buffer exchange or addition of a stock solution to achieve a target ion concentration as specified, which was loaded into the column with approximately 40 g protein / L resin (or as specified) And washed with equilibration buffer at 20 CV (or as indicated). The antibody product was then eluted and the column was regenerated.

Method of making buffer

The buffer for AEX was prepared by fixing the anion concentration (acid) to the target value and targeting the specific ion concentration to the anion by adjusting the solution having the cation component (base) to achieve the proper pH. For example, to prepare 10 mM formate-Tris buffer, pH 8.7, formic acid was dissolved in water to a target concentration of 10 mM and adjusted to pH 8.7 with concentrated Tris-base.

The buffer solution for CEX was prepared by fixing the cation concentration (base) to the target value and targeting a specific ion concentration to the cation by adjusting the solution having the anion component (acid) to achieve an appropriate pH. For example, to prepare a 140 mM Tris-formate buffer, pH 7.5, the Tris base was dissolved in water to a target concentration of 140 mM and adjusted to pH 7.5 using formic acid.

AR reduction and recovery rate calculation

In general, the eluate fractions and the pass-through / wash fractions were collected and analyzed for AR levels using the WCX-10 method. Recovery and corresponding AR levels were calculated by actual or calculated pooling of the fractions.

Analysis method

WCX-10 for Adalmumab

The acidic species and other charge mutants present in the Adalimumab process sample were quantified according to the following method. Cation exchange chromatography was performed on Dionex ProPac WCX-10, an analytical column 4 mm x 250 mm (Dionex, Calif.). The mobile phase used was 10 mM dibasic sodium phosphate pH 7.5 (mobile phase A) and 10 mM dibasic sodium phosphate, 500 mM sodium chloride pH 5.5 (mobile phase B). Binary concentration gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% : 28-34 min) was used with detection at 280 nm.

The quantification was based on the relative area percent of the detected peaks. Peaks eluting at a relative retention time less than a predetermined time are also indicated as acidic peaks.

Size exclusion chromatography

The molecular weight distribution of the collected samples was quantified according to the following method. Size exclusion chromatography (SEC) was performed using a TSK-gel G3000SWxL, 5 urn, 125 Å, 7.8 X 300 mm column (Tosoh Bioscience) on an HP Agilent HPLC system. Under the conditions of isocratic elution, using a mobile phase of 200 mM sodium sulfate, 100 mM sodium phosphate, pH 6.8, and detected by absorbance at 214 nm. The quantification was based on the relative area of the detected peaks.

UV spectroscopy A ₂₈₀

The protein concentration of the sample after protein A elution was measured using UV A280 spectroscopy. Assays were performed on an Agilent UV spectrophotometer. The protein concentration was measured using Beer-Lambert's law, A =? Lc, where A is the absorbance,? Is the extinction coefficient, I is the path length, and c is the concentration. Absorbance was measured at 280 nm, the path length was 1 cm, and the extinction coefficient was 1.39, 1.38 for mAb B and 1.43 for mAb C, respectively.

Example 11.1: Recycling AEX chromatography

In this example, the recycling strategy was used to increase the recovery yield for a given target product quality contribution. The AR reduction for a given formate concentration and pH can be adjusted by adjusting the load. In addition, the recovery yield is fixed for a given load. In this strategy, the load is selected to achieve a target AR level in the passing liquid fraction. The column is then eluted with a slightly higher formate concentration than the load. The eluate is collected and then diluted with water to match the load formate concentration and added back to the loading tank. This column circulation is repeated several times (and is referred to as "recirculation" chromatography).

For this experiment, the target AR level in the FT pool was set at 5%. First, a Poros 50HQ column was loaded onto 200 g / L of resin and the passage was collected from the 20 g / L protein loaded onto the resin using equilibration / wash buffer and loading conditions 15 mM acetate / Tris pH 8.7. WCX-10 assay was performed on the passage fraction and the cumulative AR wave rate was calculated. A cumulative AR wave rate of 5% occurred at 150 g / L of protein loaded on the resin, and all subsequent experiments were carried out at a load of 150 g / L.

The cyclic phase included the schemes described in Table 89. &lt; tb &gt; &lt; TABLE &gt; The AEX load was prepared by adjusting the MabSelect SuRe eluate to the appropriate pH using 3M Tris, diluted with 15 mM acetate, and then filtered. Load and wash through solutions were collected in two separate containers. The wash was mixed with sufficient MabSelect SuRe eluate to perform another circulation at 150 g / L and the conditions were adjusted to 15 mM acetate / Tris pH 8.7. A total of four cycles were performed using the sequential steps described above. Each run was performed with a residence time of 3 minutes at ambient temperature according to the chromatographic conditions listed in Table 89. The passage liquid from 100 mAu to the end of the step was collected in one fraction and was collected from the beginning of the step to 50 mAu of wash solution. The FT washes were then measured by A280 and analyzed by WCX-10 and SEC assays.

In this study, circulation of the wash fraction to the AEX column was performed as a means of controlling the level of process impurities. The wash fractions were collected in each cycle (C _n ), adjusted to the appropriate loading conditions and loaded in the subsequent cycle (C _{n + 1} ). The load was adjusted to provide an AR wave rate of 5%. A total of four cycles were performed.

Step yield and product quality are listed in Table 90. &lt; tb &gt; &lt; TABLE &gt; Lysine% (Lys 0, Lys 1 and Ly2, which are total lysine variants, i.e. mAbs containing 0, 1 or 2 terminal lysine) are quantities of the preferred (non-AR containing) fraction of the product, To &lt; / RTI &gt; recover more than 93% of the desired product. The product containing higher levels of AR is recovered in the wash fraction of each cycle, which is then recycled back to the subsequent AEX cycle. Recirculation of the wash fraction improves the cumulative yield while maintaining product quality as shown in Table 91. The cumulative yield increased from 65% to 81% in four cycles while maintaining the AR level at about 6% and the monomer level at about 99.4%.

Example 11.2: Recycling CEX Chromatography

These experiments were performed using protein A eluates as CEX loading material. Cycle 1 (control) was performed under load / wash buffer conditions of 160 mM Tris-acetate, pH 7.5, 40 g protein / L resin. Cycle 2 was performed by combining the wash liquor portion from cycle 1 and fresh protein A eluate as loading material. Earlier wash solutions containing higher AR (before reaching the peak) were removed and the remainder of the wash solution was included in the load. The loading and washing conditions were the same as in cycle 1. Cycle 3 and Cycle 4 were carried out in the same manner as Cycle 2.

The results shown in Table 92 indicate that the recycle chromatography with four runs increases the yield from 53.4% to 65.1%. The AR decrease for cycle 1 is 8.84%, while the 4 cycle recycle chromatography is 7.79%. While achieving similar product quality, a recycle chromatography approach can significantly improve yield.

Example 11.3: Reduction of AR with MM Recirculation Chromatography

The following materials and methods were used for Example 11.3.

Materials and methods

The material captured by protein A affinity chromatography

The crude dalmobil purified collection material obtained from the 300L bioreactor (SUL101912) was loaded onto protein A affinity column chromatography (e.g. MabSelect SuRe) and only the buffer components used in the downstream process product train Lt; / RTI &gt; buffer system. In the case of this study, Adalimumab coupled to MabSelect SuRe resin was eluted with 20 mM acetic acid.

Suzy

Multimodal media have ligand and / or base matrices with multifunctional groups that provide different selectivity compared to traditional ion exchange media. In these examples, multimodal media with anion exchange and hydrophobic interaction functional groups are found to remove acidic species and other impurities from the antibody preparation.

In this study, a strong anion exchange chain with multimodality functionality, Capto adhere (GE Healthcare, HiScreen ™ prepacked column, Cat # 28-9269-81) was evaluated. The base matrix thereof is a highly crosslinked-linked agarose having a ligand (N-benzyl-N-methylethanolamine) exhibiting a number of functionalities for interaction, for example, ionic, hydrogen and hydrophobic interactions to be. These ligands provide different selectivity and hydrophobic options for protein separation.

Capto Adhere ligand structure

Way

Chromatographic method

In the following experiment, a pre-packed resin column was used. The column was equilibrated in a buffer system with appropriate pH and conductivity. The process is described as Figure 191. Column loadings were prepared from protein A affinity chromatography. The prepared load material was filtered and loaded onto the column according to the target loading (g protein / L resin) as indicated and then washed with wash buffer equal to the equilibration buffer and volume of equilibration buffer as indicated. The column passing solution during the load was collected as a pool and the column passing solution during the wash was collected separately. The column was then regenerated with 0.1 M acetic acid (pH 3) solution for next cycle use. Cycle A The wash pool was mixed with the protein A eluate to prepare antibody material for loading with the target capacity for the following cycles. The pH and conductivity of the combined pools (wash pool plus protein A eluate) were adjusted using 2M Tris and Milli Q water to achieve the designed pH and conductivity. This material was then filtered through a 0.45 mu m filter (Corning polystyrene).

Method of making buffer

By starting the anionic component solution (acid) with the target value, adjusting the solution to the cationic component (base) to achieve the proper pH, and subsequently adding water to achieve target conductivity, Was prepared. For example, to prepare a Tris-acetate buffer having a conductivity of pH 7.85 and 2.5 mS / cm, the pH of the 250 mM acetic acid solution was adjusted to 7.85 ± 0.05 with 3 M Tris solution, The conductivity was adjusted to 2.5 ± 0.5 mS / cm, and then the pH of the final solution was confirmed or adjusted to 7.85 ± 0.05 by the addition of 3 M acetic acid solution or 3 M or 2 M Tris solution as needed.

In this study, Tris / acetate buffer with pH 7.9 and conductivity 2.5 mS / cm was used for column equilibration; A Tris / acetate buffer having a pH of 7.9 and a conductivity of 5.0 mS / cm was used as post-loading wash buffer.

Capto Adhere

Cycle A:

The protein A eluate was titrated to pH 7.9 with 2M Tris and diluted with Milli Q water to a conductivity of 2.5 mS / cm. The prepared material was then filtered through a 0.22 mu m filter before being loaded onto the column.

Cycle B:

A full cycle A wash pool was mixed with the protein A eluate to produce sufficient loading for the following cycles. After mixing with 2M Tris and Milli Q water, pH and conductivity were adjusted to achieve a conductivity of pH 7.9 and 2.5 mS / cm. This material was then filtered through a 0.45 占 퐉 filter (Corning polystyrene filter) before being loaded onto the column.

Cycle C:

A full cycle B wash pool was mixed with the protein A eluate to produce sufficient loading for the following cycles. After mixing with 2M Tris and Milli Q water, pH and conductivity were adjusted to achieve a conductivity of pH 7.9 and 2.5 mS / cm. This material was then filtered through a 0.45 占 퐉 filter (Corning polystyrene filter) before being loaded onto the column.

Cycle D:

A full cycle C wash pool was mixed with the protein A eluate to produce sufficient loading for the following cycles. After mixing with 2M Tris and Milli Q water, pH and conductivity were adjusted to achieve a conductivity of pH 7.9 and 2.5 mS / cm. This material was then filtered through a 0.45 占 퐉 filter (Corning polystyrene filter) before being loaded onto the column.

AR reduction and recovery rate calculation

Generally, the passage liquid / wash fraction was collected and analyzed for AR levels using the WCX-10 method. Recovery and corresponding AR levels were calculated by actual or calculated pooling of the fractions.

Analysis method

WCX-10 for Adalmumab

The acidic species and other charge mutants present in the Adalimumab process sample were quantified according to the following method. Cation exchange chromatography was performed on Dionex ProPac WCX-10, an analytical column 4 mm x 250 mm (Dionex, Calif.). An Agilent 1200 HPLC system was used as the HPLC. The mobile phase used was 10 mM dibasic sodium phosphate pH 7.5 (mobile phase A) and 10 mM dibasic sodium phosphate, 500 mM sodium chloride pH 5.5 (mobile phase B). Binary concentration gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% : 28-34 min) was used with detection at 280 nm.

The quantification was based on the relative area percent of the detected peaks. Peaks eluting at a relative retention time less than a predetermined time are also indicated as acidic peaks.

UV spectroscopy A ₂₈₀

After protein A elution with UV A ₂₈₀ , the protein concentration of the sample was measured. Assays were performed on an Agilent UV spectrophotometer. The protein concentration was measured using Beer-Lambert's law, A =? Lc, where A is the absorbance,? Is the extinction coefficient, I is the path length, and c is the concentration. Absorbance was measured at 280 nm, the path length was 1 cm, and the extinction coefficient was 1.39 for Adil Mammab.

Proof of Recirculation and Continuous Chromatography

In this example, Adalimumab and Resin Capto Adhere were selected. In each cycle, 75 grams of hydronephrine per liter resin was loaded onto the Capto Adhere column and a total of four cycles were performed. A single run with 100 g / L loading material loaded on the Capto Adhere column was run as a reference to compare AR reduction and mAb recovery. Figure 192 illustrates the cumulative AR% in the load, the through-flow pool (FT), the cleanup pool (clean) AR percentages, and the total FT for each cycle of the MM process. As shown in Figure 192, the load AR% in each cycle increased due to the higher AR% in the wash pool obtained from the previous cycle was re-processed, resulting in a slight increase in the AR% in the passing liquid pool .

From Figure 192 it becomes apparent that the AR level in the collected pool achieving a total reduction of approximately 5% is maintained in all four cycles. Thus, it is clear that the recycle mode can maintain the product AR level. Table 93 shows the recovery rates and overall recovery rates obtained for each step. It is clear that the recirculation scheme results in a significant improvement in recovery (10% increase) in recovery compared to a single run that achieves similar product quality when four cycles are run. As shown in Table 93, the cumulative recovery rate increases with each additional cycle. Thus, further improvement can be achieved by increasing the number of cycles. It is also clear that a 20% increase in recovery can be achieved by using a chromatographic approach when comparing performance (cycles) in cycle 1 versus performance (cycles) in cycles 1-4.

Example 12: Storage of AR reduction

The present invention provides a method for reducing acidic species for a given protein of interest. In this example, adalimumab was prepared using a combination of arginine and lysine replenishment to cell cultures as shown in the present invention, together with AEX and CEX purification techniques as described herein, to yield 2.5% And a high-AR sample with a final AR of 6.9%. Both of the samples were incubated for 10 weeks in a controlled environment at 25 ° C and 65% relative humidity and AR was measured every 2 weeks. Figure 164 shows the growth of AR for each sample over a 10 week incubation period. From Figure 164 it is clear that the growth rate of AR is linear and similar between both low-AR and high-AR samples. Based on these results, the reduced AR material can be stored three times longer before reaching the same AR level as the high-AR sample. This is significantly useful because it can be very beneficial for the storage and handling of other proteins for therapeutic use. Further, as indicated above, the formation of the storage-induced AR can be suppressed when the preparation is stored under certain conditions. For example, aqueous formulations can be partially or completely inhibited from forming AR by storage at certain temperatures. In addition, formed or storage-induced ARs can be partially inhibited in aqueous formulations stored between about 2 ° C and 8 ° C, and can be completely inhibited when stored at -80 ° C. In addition, the low AR composition can be freeze dried to partially or completely inhibit the formation of the storage-induced AR.

Example 13: Increased biological activity of low AR compositions

This example describes the increased efficacy of an exemplary low AR composition comprising in vivo dalimab in vivo. The low AR composition used in this example was prepared using the CEX reduction method as described in Example 8.14 above. In particular, the low AR composition used in this example was prepared using a Poros XS column in a Tris / formate buffer system at a pH of 7.5. The low AR composition has 3.1% AR, wherein the composition comprises 0.1% AR1 and 3.0% AR2. In this example, this composition is referred to as "low AR composition ".

Animal models for arthritis

In order to study the efficacy of such low AR-dicalimab compositions, experiments were performed in vivo using human TNF-Tg197 mice. The TNF-Tg197 mouse model is a mouse model of well-recognized arthritis used to test anti-human TNFa treatment regimens. A TNF-Tg197 mouse model is described in Keffer, J. et al ., (1991) EMBO J 10: 4025-4031, the entire contents of which are incorporated herein by reference. We have developed a transgenic mouse carrying the human TNF gene to study the effect of excessive TNF production in vivo.

Tg197 mice develop swelling and impaired movement in the ankle joints of both hind feet, which is very similar to human rheumatoid arthritis. Clinical manifestations of the disease in Tg197 mice begin at 4 weeks of age and include slower weight gain, joint sprains and swelling, joint deformity and ankylosis, and impaired movements. Histopathologic analysis was performed to assess the degree of hypertrophy of synovial membrane, leukocyte infiltration at approximately 3 weeks of age, followed by pannus formation, articular cartilage destruction at the advanced stage of disease at age 9-11, Explain the mass production. These models have been used in the development of anti-TNFa therapeutics including adalimumab.

Way

Groups of mice (6 males and 6 females) were dosed with one of the following adalimumab formulations: low AR composition (Group 5), low host cell protein (HCP) composition (Group 7), AR1 composition (Group 8), and Lys-1/2 composition (containing only Lys 1 and Lys 2 variants) (Group 9). These compositions (fractions) are shown in the chromatograph of Figure 193. Another group of mice was administered a control composition, also referred to as a "control AR composition" or "normal" composition, containing unaltered AR levels and adalimumab with unmodified Lys variants. A placebo group containing 6 mice was also included.

Each composition containing the control AR composition was started with a tolerizing dose of Adalimumab at 1 week of age and then injected into each group of mice for 10 weeks with an additional weekly dose of 1 mg / . Weekly weight and arthritis score measurements were performed from 2.5 to 13.5 weeks and serum was collected weekly. At the end of the study, tissue samples from perfused mice were also obtained and analyzed. The following tissues were collected for testing drug levels, anti-drug antibodies (ADA), and complex and free TNF levels: paw, inguinal, dwarf and mesenteric lymph nodes, spleen, tail (for skin samples), knee. Femoral and spinal tissues were collected for micro-CT scanning.

result

As shown in Figure 194A, mice receiving a low AR composition had a lowest arthritic score among all the compositions tested, including the control AR composition, indicating increased efficacy in the treatment of arthritis. In addition, as shown in Figure 194B, the mice to which the low AR composition was administered exhibited an average weight gain similar to that of the control composition, indicating that the low AR composition, which affects the safety of the low AR composition and the weight gain and growth of the mouse The absence of side effects.

As shown in Figure 195, during the 12 to 13 week treatment period of the mice, the low AR composition provided the highest protection against the onset of arthritis in mice as measured by an arthritic score as compared to the other compositions tested. The Lys-1/2 composition was the next most effective. The AR1 composition provided the least protection against the onset of the arthritis score, which was less protective than the control AR composition.

Serum and drug levels of ADA were measured at ages of 3 to 14 weeks. As shown in Figure 196B, the animals to which the low AR composition was administered exhibited a low mean level of ADA over the measured time frame. In addition, the animals to which the low AR composition was administered exhibited drug serum levels similar to the control (Figure 196A), indicating that the absence of drug in serum did not involve low levels of serum ADA.

As shown in Figure 197, cumulative serum concentration values (PK) over a 10 week treatment period were highest for the animals receiving the low AR composition and lowest for the animals receiving the AR1 composition. The Lys-1/2 composition was next the highest low AR composition and higher than the AR control composition. In addition, as shown in Figure 197, the lowest ADA titer was observed for the animals to which the AR1 composition was administered, and for the animals to which the low AR composition was administered, the highest ADA titer.

Complex TNF levels also showed that the cumulative serum concentration values during the 10 week treatment period were the highest for the animals to which the control AR composition was administered and the lowest for the animals to which the AR1 composition was administered (Figure 198). The cumulative serum concentration values for the low AR composition were slightly lower than the levels of the control AR composition.

Histopathological evaluation of the joints of mice showed that the highest protection was provided by the low AR composition and the Lys-1/2 composition, indicating that the low AR composition and the Lys-1/2 composition inhibited arthritis &Lt; / RTI &gt; As shown in Figure 199, the low AR composition protected cell infiltration, synovial hyperplasia, proteoglycan loss, cartilage destruction and bone erosion more effectively than other compositions comprising the control AR composition. Protection by the AR1 composition was lower than that of the control AR composition, indicating a deleterious effect of AR1 on joint damage.

Figures 200A-200D show the average drug (PK) levels for various tissues (foot, lymph node, spleen, skin, knee and serum) for the low AR composition, the control AR composition, the AR1 composition, and the Lys- . As shown here, the animals to which the low AR composition was administered had higher drug levels than the animals to which the other compositions tested were administered or higher than the animals.

201A-201D illustrate the mean ADA levels in the same tissue for the same composition (low AR composition, control AR composition, AR1 composition, and Lys-1/2 composition). As shown in Figures 201A-201D, for low AR compositions, the highest ADA concentration is present in the paw (which corresponds to the highest level of inflammation site in the animal), and in the serum.

202A-202D and 203A-203D show that at the end of the study where low AR, control AR, AR1, Lys-1/2 and naive, The results of micro CT analysis of spine and femur obtained from mouse are shown. Samples were analyzed for L5 vertebral bone volume, L5 vertebra trabecular number, L5 vertebral column thickness, and L5 vertebral column space. As shown in Figs. 202A to 202D and Figs. 203A to 203D, the low AR composition and the Lys-1/2 composition exhibited higher bone volume, soju water, soju thickness and soju space Respectively.

FIGS. 204A-204D show micro-CT images of vertebrae and femurs obtained from transgenic mice at the end of the study in which a low AR composition, a control AR composition, an AR1 composition, a Lys-1/2 composition, The results of the addition of the analysis are shown. Samples were analyzed for the presence of soju in the femoral bone, in the femoral bone, in the femoral bone, and in the femoral bone. As shown in Figs. 204A-204D, the low AR composition was found to have a significant effect on the soju bone volume in the larger femoral bone simplification, soju number in the femoral osseous simplification, and soju in the femoral osseous simplification Thickness.

Figures 205 and 206 also show actual micro-CT images of the spine and femur from each of the six groups of mice to which the following compositions were administered: Naive, vehicle (control), low AR composition (group 5) (Composition containing only Lys 1 and Lys 2 variants) (Group 9), a low-cost cell protein (HCP) composition (Group 7), an AR1 composition (containing only AR1 acid mutants) . As seen in both the vertebrae and femur, the low AR composition (Group 5) provided protection against bone erosion compared to the vehicle because it had less visible bone erosion in the Group 5 image compared to the vehicle to be.

The results of these experiments show that the weekly dose of 1 mg / kg of adalimumab in TNF-Tg197 mice was significantly lower than that in the TNF-Tg197 mouse model when measured by arthritis score and histopathology score in the TNF-Tg197 mouse model, &Lt; / RTI &gt; injury and local inflammation). Thus, control AR compositions with normal levels of AR variants were effective at certain levels.

Formulations containing either low AR formulations or Lys-1/2 compositions provided maximum protection against arthritis onset compared to control AR groups when measured by arthritic score and histopathology score, Increased efficacy compared to control AR groups in both tested parameters including proliferation, proteoglycan loss, cartilage destruction, and bone erosion. Thus, the low AR and Lys-1/2 compositions increased the efficacy in the treatment and prevention of arthritis compared to the control AR composition.

The adalimumab AR1 composition was found to be superior in all aspects examined in this study: less weight gain, higher arthritic score indicating a deleterious effect exerted by AR1, higher histopathology scores in higher joints, Were less effective than normal AR containing the MAb control group.

Significant differences were observed at serum levels of various formulations including the following: Animals treated with the AR1 composition had the lowest concentration of adalimumab in comparison with the other groups, and animals treated with a low &lt; RTI ID = 0.0 &gt; The group had the highest concentration of dalavimab compared to the group. In addition, the AR1 composition had the highest titer of ADA in serum.

* * *

The invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the detailed description and the accompanying drawings, in order to practice the invention. Such modifications are intended to fall within the scope of the appended claims.

The contents of all cited references, including literature references, granted patents, and published patent applications cited throughout this application, are expressly incorporated herein by reference. It is further to be understood that the drawings and the sequence listing and the full text of the tables attached hereto are expressly incorporated herein by reference. The entirety of the following applications are also expressly incorporated herein by reference: US Provisional Patent Application 61 / XXX, XXX, Attorney Docket Number 117813-31001, entitled " STABLE SOLID PROTEIN COMPOSITIONS AND METHODS OF MAKING SAME "; U.S. Provisional Patent Application 61 / XXX, XXX, Attorney Docket Number 117813-73602, entitled "LOW ACIDIC SPECIES COMPOSITIONS AND METHODS FOR PRODUCING THE SAME USING DISPLACEMENT CHROMATOGRAPHY" U.S. Provisional Patent Application 61 / XXX, XXX, Attorney Docket Number 117813-73802, entitled " MUTATED ANTI-ANTIBODIES AND METHODS OF THEIR USE "filed on such date; U.S. Provisional Patent Application 61 / XXX, XXX, Attorney Docket Number 117813-74101, entitled " MODULATED LYSINE VARIANT SPECIES AND METHODS FOR PRODUCING AND USING THE SAME " And U. S. Patent Application 61 / XXX, XXX, Attorney Docket Number 117813-74301, entitled " PURIFICATION OF PROTEINS USING HYDROPHOBIC INTERACTION CHROMATOGRAPHY. &Quot;

                               SEQUENCE LISTING <110> ABBVIE INC. <120> LOW ACIDIC SPECIES COMPOSITIONS AND METHODS FOR PRODUCING AND USING THE SAME <130> 117813-73920 &Lt; 150 > PCT / US2013 / 031485 <151> 2013-03-14 &Lt; 150 > PCT / US2013 / 031681 <151> 2013-03-14 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> adalimumab light chain variable region <400> 1 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly  1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Ser Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 2 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> adalimumab heavy chain variable region <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg  1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr             20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val     50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> adalimumab light chain variable region CDR3 <220> <221> VARIANT <222> 9 <223> Xaa = Thr or Ala <400> 3 Gln Arg Tyr Asn Arg Ala Pro Tyr Xaa  1 5 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> adalimumab heavy chain variable region CDR3 <220> <221> VARIANT <222> 12 &Lt; 223 > Xaa = Tyr or Asn <400> 4 Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Xaa  1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> adalimumab light chain variable region CDR2 <400> 5 Ala Ala Ser Thr Leu Gln Ser  1 5 <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> adalimumab heavy chain variable region CDR2 <400> 6 Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu  1 5 10 15 Gly      <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> adalimumab light chain variable region CDR1 <400> 7 Arg Ala Ser Gln Gly Ile Arg Asn Tyr Leu Ala  1 5 10 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> adalimumab heavy chain variable region CDR1 <400> 8 Asp Tyr Ala Met His  1 5 <210> 9 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> adalimumab light chain variable region <400> 9 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtagggga cagagtcacc 60 atcacttgtc gggcaagtca gggcatcaga aattacttag cctggtatca gcaaaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaatcagg ggtcccatct 180 cggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag cctacagcct 240 gaagatgttg caacttatta ctgtcaaagg tataaccgtg caccgtatac ttttggccag 300 gggaccaagg tggaaatcaa a 321 <210> 10 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> adalimumab heavy chain variable region <400> 10 gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ccggcaggtc cctgagactc 60 tcctgtgcgg cctctggatt cacctttgat gattatgcca tgcactgggt ccggcaagct 120 ccagggaagg gcctggaatg ggtctcagct atcacttgga atagtggtca catagactat 180 gcggactctg tggagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240 acggcgtat taccttagca ccgcgtcctc ccttgactat tggggccaag gtaccctggt caccgtctcg 360 agt 363 <210> 11 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> adalimumab light chain <400> 11 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly  1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Ser Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 12 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> adalimumab heavy chain <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr             20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val     50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys     210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met                 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His             260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val         275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr     290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile                 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val             340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser         355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu     370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val                 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met             420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser         435 440 445 Pro Gly Lys     450

Claims (133)

A low acid species composition comprising an antibody or antigen-binding portion thereof, wherein the composition comprises less than about 15% of an acidic species, wherein the composition comprises an antigen or antigen-binding portion thereof. 2. The composition of claim 1, wherein the acidic species comprises a first acidic region (AR1) and a second acidic region (AR2). The composition of claim 1, wherein the composition comprises up to about 5% acidic species (AR). The composition of claim 1, wherein the composition comprises up to about 4% acidic species (AR). The composition of claim 1, wherein the composition comprises up to about 3% acidic species (AR). 3. The composition of claim 2, wherein the composition comprises up to about 0.1% AR1 and up to about 3% AR2. 3. The composition of claim 2, wherein the composition comprises up to about 1% AR1. 3. The composition of claim 2, wherein the composition comprises up to about 0.1% AR1. 7. The composition of claim 6, wherein the composition comprises up to about 3% AR2. The composition of claim 1, wherein the composition comprises up to about 2% acidic species (AR). 11. The composition of claim 10, wherein the composition comprises up to about 1.4% acidic species (AR). 3. The composition of claim 2, wherein the composition comprises about 1.4% AR2 and about 0.0% AR1. 13. The composition of any one of claims 1 to 12, wherein the acidic species (AR) comprises at least one of a charge mutant, a structural mutant, or a fragmented mutant. 14. The composition of claim 13, wherein said acidic species is AR1 and said charge mutant comprises a deamidated variant, a glycation variant, an afucosylation variant, an MGO variant or a citric acid variant. 14. The composition of claim 13, wherein the acidic species is AR1, and wherein the structural variant comprises a glycosylation variant or an acetone variant. 14. The composition of claim 13, wherein the fragment is an acidic species AR1, wherein the fragment variant comprises a Fab fragment variant, a C-terminal truncation mutant, or a variant in which the heavy chain variable domain is lost. 14. The composition of claim 13, wherein said acidic species is AR2 and said charge mutant comprises a deamidated or glycosylated variant. Wherein the composition is substantially free of host cell protein (HCP), a host nucleic acid, a chromatography material, and / or a media component. 19. The composition according to any one of claims 1 to 12 or 14 to 18, wherein said antibody or antigen-binding portion thereof is an anti-TNFa antibody or antigen-binding portion thereof. 20. The method of claim 19 wherein the antibody or an antigen-binding portion, which is dissociated from about 1 x 10 ^-8 M or less and a K _d of 1 x 10 ^-3 S human TNFα with a K _off rate constant of ^-1 or less, the composition . 20. The method of claim 19, wherein the anti-TNFa antibody or antigen-binding portion thereof comprises a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and an amino acid sequence of SEQ ID NO: A light chain variable region (LCVR) having a CDR3 domain; And a heavy chain variable region (HCVR) comprising a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 20. The composition of claim 19, wherein said anti-TNFa antibody or antigen-binding portion thereof comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: . 23. The composition of any one of claims 19-22, wherein the anti-TNFa antibody or antigen-binding portion thereof is adalimumab or an antigen-binding portion thereof. The composition of claim 1, wherein the composition exhibits increased cartilage tissue penetration compared to a non-low acid species composition. The composition of claim 1, wherein the composition exhibits increased TNF affinity as compared to a non-hypoallergenic seed composition. 2. The composition of claim 1, wherein the composition exhibits reduced cartilage destruction compared to a non-low acid species composition. 2. The composition of claim 1, wherein the composition exhibits reduced bone erosion compared to a non-low acid species composition. 2. The composition of claim 1, wherein the composition exhibits reduced synovial growth as compared to a non-low acid species composition. 2. The composition of claim 1, wherein the composition exhibits reduced cellular infiltration as compared to a non-low acid species composition. 2. The composition of claim 1, wherein the composition exhibits reduced chondrocyte cell death compared to a non-hypoallergenic seed composition. 2. The composition of claim 1, wherein the composition exhibits reduced proteoglycan loss as compared to a non-low acid species composition. 3. The composition of claim 1, wherein said composition, when administered to an animal model of arthritis, exhibits increased protection against the onset of an arthritic score as compared to a non-hypoallergenic species composition. 3. The composition of claim 1, wherein said composition, when administered to an animal model of arthritis, exhibits increased protection against the onset of a histopathology score, as compared to a non-hypoallergenic species composition. A low acid species composition comprising an anti-TNF [alpha] antibody or antigen-binding portion thereof,
A light chain variable region (LCVR) having a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3; And a heavy chain variable region (HCVR) having a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, The composition comprises less than about 10% acidic species (AR)
Lt; RTI ID = 0.0 &gt; anti-TNFa &lt; / RTI &gt; antibody or antigen-binding portion thereof. 36. The antibody of claim 34, wherein said anti-TNFa antibody or antigen-binding portion thereof comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: Composition. 35. The composition of claim 34, wherein the anti-TNFa antibody or antigen-binding portion thereof is adalimumab or an antigen-binding portion thereof. 35. The composition of claim 34, wherein the acidic species comprises a first acidic region (AR1) and a second acidic region (AR2). 38. The composition of claim 37, wherein the composition comprises up to about 0.1% AR1 and up to about 3% AR2. 39. The composition of any one of claims 34-38, wherein the acidic species (AR) comprises at least one of a charge mutant, a structural mutant, or a fragmented mutant. 40. The composition of claim 39, wherein said acidic species is AR1 and said charge mutant comprises a deamidated variant, a glycosyl variant, an afacosyl variant, an MGO variant or a citric acid variant. 41. The composition of claim 40, wherein the deamidated variant is from an asparagine residue comprising Asn393 and Asn329 and from a deamidation occurring at a glutamine residue comprising Gln3 and Gln6. 41. The composition of claim 40, wherein the glycated variant is from a glycation occurring in Lys98 and Lys151. 40. The composition of claim 39, wherein the acidic species is AR1, and wherein the structural variant comprises a glycosylation variant or an acetone variant. 40. The composition of claim 39, wherein the acidic species is AR1 and the fragmented variant comprises a Fab fragment variant, a C-terminal truncation variant, or a variant in which the heavy chain variable domain is lost. 40. The composition of claim 39, wherein said acidic species is AR2, and said charge mutant comprises a deamidated or glycosylated variant. 46. The composition of claim 45, wherein the deamidated variant is derived from an asparagine residue comprising Asn393 and Asn329 and from a deamidation occurring at a glutamine residue comprising Gln3 and Gln6. 46. The composition of claim 45, wherein the glycated variant is from a glycation occurring in Lys98 and Lys151. 35. The composition of claim 34, wherein the composition exhibits increased cartilage tissue penetration compared to a non-acidic species composition. 35. The composition of claim 34, wherein the composition exhibits increased TNF affinity as compared to a non-hypoallergenic seed composition. 35. The composition of claim 34, wherein the composition exhibits reduced cartilage destruction compared to a non-low acid species composition. 35. The composition of claim 34, wherein the composition exhibits reduced bone erosion compared to a non-low acid species composition. 35. The composition of claim 34, wherein the composition exhibits reduced synovial growth as compared to a non-hypoallergenic seed composition. 35. The composition of claim 34, wherein the composition exhibits reduced cellular infiltration as compared to a non-low acid species composition. 35. The composition of claim 34, wherein the composition exhibits reduced chondrocyte cell death compared to a non-hypoxic species composition. 35. The composition of claim 34, wherein the composition exhibits reduced proteoglycan loss as compared to a non-low acid species composition. 35. The composition of claim 34, wherein said composition, when administered to an animal model of arthritis, exhibits increased protection against the onset of an arthritic score as compared to a non-hypoallergenic species composition. 35. The composition of claim 34, wherein said composition, when administered to an animal model of arthritis, exhibits increased protection against the onset of a histopathology score, as compared to a non-hypoallergenic species composition. The pharmaceutical composition according to any one of claims 1 to 12, 14 to 18, 25 to 38 or 40 to 57, wherein the composition further comprises a pharmaceutically acceptable carrier &Lt; / RTI &gt; The method according to any one of claims 1 to 12, 14 to 18, 25 to 38 or 40 to 57, wherein the acidic species percentage is determined by using WCX-10 HPLC &Lt; / RTI &gt; 57. The method according to any one of claims 1 to 12, 14 to 18, 25 to 38 or 40 to 57, wherein said acid species percentage is at least one of isoelectric focusing (IEF) . &Lt; / RTI &gt; The method of any one of claims 1 to 12, 14 to 18, 25 to 38 or 40 to 57, wherein said acidic paper is a product- Composition. 63. The composition of claim 62, wherein said acidic species is a cell culture-derived acidic species. The composition of any one of claims 1 to 12, 14 to 18, 25 to 38 or 40 to 57, wherein the acidic paper is a storage-derived acidic species, . There is provided a method of treating a subject having a deleterious disorder, wherein the TNFa comprises administering to the subject having a deleterious disorder a composition according to any one of claims 1 or 34, thereby treating the subject having a deleterious disorder of TNFa Methods for treatment. 65. The method of claim 64, wherein said disorder wherein TNF [alpha] is harmful is rheumatoid arthritis (RA). 65. The method of claim 64, wherein said disorder wherein TNF [alpha] is harmful is psoriasis. 65. The method of claim 64, wherein said disorder wherein TNF [alpha] is detrimental is psoriatic arthritis. 65. The method of claim 64, wherein said disorder wherein TNF [alpha] is detrimental is ankylosing spondylitis. 65. The method of claim 64, wherein the disorder is a Crohn's disease wherein the TNFa is detrimental. 65. The method of claim 64, wherein the disorder is TNF-a deleterious ulcerative colitis. 65. The method of claim 64, wherein said disorder wherein TNFa is detrimental is childhood idiopathic arthritis. A method for producing a low acid species composition comprising an antibody or antigen-binding portion thereof, said method comprising the step of determining the concentration of an amino acid in a cell culture medium used to produce a non-acidic species composition comprising an antibody or antigen- The step of culturing cells expressing the antibody or antigen-binding portion thereof in a cell culture medium containing an amino acid selected from the group consisting of arginine, lysine, ornithine and histidine, or a combination thereof at an increased concentration &Lt; / RTI &gt; wherein the antibody or antigen-binding portion thereof comprises an antigen binding portion. 73. The method of claim 72, wherein the low acid species composition comprises less than about 10% acid species. 73. The method of claim 72, wherein the amino acid concentration in the culture medium is about 0.025 to 20 g / L. A method for producing a low acid species composition comprising an antibody or antigen-binding portion thereof, the method comprising contacting a cell with a calcium concentration in a cell culture medium used to produce a non-acidic species composition comprising an antibody or antigen- A method for producing a low acidic species composition comprising an antibody or an antigen-binding portion thereof, comprising culturing cells expressing an antibody or an antigen-binding portion thereof in a cell culture medium containing an increased concentration of calcium . 77. The method of claim 75, wherein the calcium concentration is from about 0.005 to about 5 mM. 77. The method of claim 75, wherein the cell culture medium further comprises an increased concentration of arginine, lysine &lt; RTI ID = 0.0 &gt; , Ornithine, and histidine, or a combination thereof. 78. The method of claim 75 or claim 77, wherein the low acid species composition comprises less than about 10% acid species. A method for producing a low acid species composition comprising an antibody or antigen binding portion thereof,
Calcium and amino acids in the cell culture medium used to produce the non-acidic species composition comprising the antibody or antigen-binding portion thereof, as compared to the concentrations of niacinamide, calcium, Comprising culturing a cell expressing an antibody or an antigen-binding portion thereof in a cell culture medium containing the antibody or an antigen-binding portion thereof. 80. The method of claim 79, wherein said at least one amino acid is selected from the group consisting of arginine, lysine, ornithine and histidine, and combinations thereof. 80. The method of claim 79 or 80 wherein the low acid species composition comprises less than about 10% acid species. A method for producing a low acid species composition comprising an antibody or antigen-binding portion thereof, comprising culturing a cell expressing the antibody or antigen-binding portion thereof in a cell culture medium having a pH of about 7.1 to 6.8, Or an antigen-binding portion thereof. 83. The method of claim 82, wherein the low acid species composition comprises less than about 10% acid species. A method for producing a low acid species composition comprising an antibody or antigen-binding portion thereof,
Cells expressing the antibody or its antigen-binding portion thereof in a cell culture medium having a modified exchange rate as compared to the exchange rate of the cell culture medium used for preparing the non-low acid species composition comprising the antibody or antigen-binding portion thereof, Wherein the antibody or antigen-binding portion thereof comprises an antibody or antigen-binding portion thereof. 85. The method of claim 84, wherein the low acid species composition comprises less than about 10% acid species. A method for producing a low acid species composition comprising an antibody or antigen-binding portion thereof,
Comprising the step of culturing cells expressing the antibody or antigen-binding portion thereof, extracting the purified collection from the cell culture, and adding one or more amino acids to the purified collection, A method for making a low acid species composition. 90. The method of claim 86, wherein said at least one amino acid is selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid and leucine, and combinations thereof. 87. The method of claim 86 or 87, wherein the low acid species composition comprises less than about 10% acid species. A method for producing a low acidic species composition comprising an antibody or antigen-binding portion thereof, comprising culturing cells expressing an antibody or antigen-binding portion thereof, extracting the purified collection from the cell culture, To 4.5 to 6.5. &Lt; Desc / Clms Page number 24 &gt; 87. The method of claim 87, wherein the low acid species composition comprises less than about 10% acid species. (a) contacting a chromatographic medium with a first sample comprising an antibody or antigen-binding portion thereof, wherein said contacting occurs in connection with a loading buffer;
(b) washing the chromatographic medium with wash buffer substantially equal to the loading buffer; And
(c) collecting a chromatographic sample
A method for producing a low acid species composition comprising an antibody or antigen-binding portion thereof, comprising preparing a low acid species composition comprising an antibody or antigen-binding portion thereof,
Wherein the chromatographic sample comprises a composition of an antibody or antigen-binding portion thereof that contains less than about 10% acidic species. 92. The method of claim 91, wherein the bound antibody material is eluted with a buffer having a different composition than the wash buffer. 92. The method of claim 91, wherein the chromatographic medium is selected from the group consisting of an anion exchange adsorbent material, a cation exchange adsorbent material, a mixed mode medium, a cation exchange mixed mode medium, and an anion exchange mixed mode medium. 92. The method of claim 91, wherein the chromatographic medium is a mixed-mode medium comprising cation exchange (CEX) and hydrophobic interaction functional groups. 92. The method of claim 91, wherein the chromatographic medium is a mixed media comprising anion exchange (AEX) and hydrophobic interaction functional groups. 93. The method of claim 93, wherein the mixed mode medium is a Capto MMC resin. 93. The method of claim 93, wherein the cation exchange (CEX) adsorbent material is selected from the group consisting of CEX resin and CEX membrane adsorbent. 98. The method of claim 97, wherein the CEX resin is a Poros XS resin. 93. The method of claim 93, wherein the anion exchange (AEX) adsorbent material is selected from the group consisting of AEX resin and AEX membrane adsorbent. 102. The method of claim 99, wherein the AEX resin is a Poros 50HQ resin. 92. The method of claim 91 wherein the chromatographic medium is a CEX adsorbent material or a mixed media and the pH of the loading and washing buffers is less than the isoelectric point of the antibody. 92. The method of claim 91, wherein the chromatographic sample contains a reduced level of antibody fragments as compared to the first sample. 92. The method of claim 91, wherein the chromatographic sample contains a reduced level of host cell protein as compared to the first sample. 92. The method of claim 91, wherein the chromatographic sample contains at least one of a reduced level of a charge mutant, a structural mutant, or a fragment variant as compared to the first sample. 105. The method of claim 104, wherein the chromatographic sample comprises a reduced level of an acidic species AR1, wherein the charge mutant comprises a deamidated variant, a glycosyl variant, an afacosyl variant, an MGO variant, or a citrate variant . 105. The method of claim 104, wherein the chromatographic sample contains a reduced level of an acidic species AR1, wherein the structural variant comprises a glycosylation variant or an acetonyl variant. 105. The method of claim 104, wherein the chromatographic sample comprises a reduced level of an acidic species AR1, wherein the fragmented variant comprises a Fab fragment variant, a C-terminal truncation variant, or a variant in which the heavy chain variable domain has been lost. . 105. The method of claim 104, wherein the chromatographic sample contains a reduced level of an acidic species AR2, wherein the charge mutant comprises a deamidated mutant or a glycosylated mutant. (a) contacting a first sample comprising an antibody or antigen-binding portion thereof with an affinity chromatography medium in a loading buffer, and eluting the sample from the affinity chromatography medium as a first eluted sample;
(b) contacting the first eluted sample with an anion exchange (AEX) chromatographic adsorbent material in a loading buffer and eluting the sample from the AEX chromatographic adsorbent material as a second eluted sample; And
(c) contacting said second eluted sample with a cation exchange (CEX) chromatography adsorbent material in a loading buffer and eluting said sample from said CEX chromatographic adsorbent material as a third eluted sample, A method for producing a low acid species composition comprising an antibody or antigen-binding portion thereof, comprising preparing a low acid species composition comprising an antigen-binding portion thereof,
Wherein said third eluted sample comprises a composition of an antibody or antigen-binding portion thereof that contains less than 3% of an acidic species. 109. The method of claim 109, wherein the affinity chromatography medium is a protein A resin. 109. The method of claim 109, wherein the affinity chromatography medium is washed with a high concentration of Tris / formate buffer after contacting the affinity chromatography medium with the first sample. 109. The method of claim 109, further comprising washing the first sample on the affinity chromatography medium with a low concentration of Tris / formate buffer. 109. The method of claim 109, wherein the first sample is eluted from the affinity chromatography medium using a low pH Tris / formate buffer. 110. The method of claim 109, wherein the loading buffer for the AEX chromatographic adsorbent material and the CEX adsorbent material is a low concentration Tris / formate buffer. 114. The method of claim 114, wherein the loading buffer has a pH of about 8.7. 109. The method of claim 109, wherein the AEX chromatographic adsorbent material is washed with a low concentration Tris / formate buffer after contacting the first eluted sample with an AEX chromatographic adsorbent material. 109. The method of claim 109, wherein the low concentration buffer has a pH of about 8.7. 109. The method of claim 109, wherein the second eluted sample is contacted with a CEX chromatographic adsorbent material and the CEX chromatographic adsorbent material is then washed with a high concentration of Tris / formate buffer. 110. The method of claim 109, wherein step (c) is repeated at least one additional number of times. 109. The method of claim 109, wherein step (c) is repeated three times. 109. The method of claim 109, wherein the cation exchange (CEX) adsorbent material is selected from the group consisting of CEX resin and CEX membrane adsorbent. 121. The method of claim 121, wherein the CEX resin is a Poros Xs resin. 109. The method of claim 109, wherein the anion exchange (AEX) adsorbent material is selected from the group consisting of AEX resin and AEX membrane adsorbent. 124. The method of claim 123, wherein the AEX resin is a Poros 50HQ resin. 110. The method of claim 109, further comprising performing virus filtration on the third eluted sample to obtain a filtered sample. 126. The method of claim 125, further comprising filtering the filtered sample using ultrafiltration / dialysis filtration (UF / DF). (a) an antibody or antigen-binding portion thereof is reacted with an anion exchange (AEX) chromatography adsorbate, a cation exchange (CEX) chromatography adsorbate, a mixed mode medium, a cation exchange mixed medium, and an anion Exchange mixed culture medium, and
(b) eluting the sample from the AEX chromatography adsorbent material, the CEX chromatography adsorbent material, the mixed mode medium, the cation exchange mixed mode medium, or the anion exchange mixed mode medium, A method for preparing a low acid species composition comprising an antibody or antigen-binding portion thereof, comprising preparing a low acid species composition,
Wherein the eluted sample comprises a composition of an antibody or antigen-binding portion thereof that contains less than about 3% of an acidic species. 127. The method of claim 127, further comprising contacting the eluted sample with a hydrophobic interaction chromatography (HIC) medium. 72. The method according to any one of claims 72, 75, 79, 82, 84, 89, 91, 109, or 127, wherein the antibody or antigen- Lt; RTI ID = 0.0 &gt; anti-TNFa &lt; / RTI &gt; antibody or antigen-binding portion thereof. The method of claim 129, wherein said antibody or an antigen-binding portion, which is dissociated from about 1 x 10 ^-8 M or less and a K _d of 1 x 10 ^-3 S human TNFα with a K _off rate constant of ^-1 or less, wherein . 127. The method of claim 129, wherein said anti-TNFa antibody or antigen-binding portion thereof comprises a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and an amino acid sequence of SEQ ID NO: A light chain variable region (LCVR) having a CDR3 domain; And a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4. 129. The method of claim 129, wherein said anti-TNF [alpha] antibody or antigen-binding portion thereof comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: . 132. The method of any one of claims 130,131, or 132, wherein said anti-TNFa antibody or antigen-binding portion thereof is adalimumab or an antigen-binding portion thereof.

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