WO2013158275A9 - Procédés de culture cellulaire pour réduire des espèces acides - Google Patents

Procédés de culture cellulaire pour réduire des espèces acides Download PDF

Info

Publication number
WO2013158275A9
WO2013158275A9 PCT/US2013/031485 US2013031485W WO2013158275A9 WO 2013158275 A9 WO2013158275 A9 WO 2013158275A9 US 2013031485 W US2013031485 W US 2013031485W WO 2013158275 A9 WO2013158275 A9 WO 2013158275A9
Authority
WO
WIPO (PCT)
Prior art keywords
media
acidic species
protein
cell culture
acidic
Prior art date
Application number
PCT/US2013/031485
Other languages
English (en)
Other versions
WO2013158275A1 (fr
Inventor
Kartik Subramanian
Xiaobei ZENG
Diane D. DONG
Wen Chen LIM
Kathreen A. GIFFORD
Original Assignee
Abbvie Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Abbvie Inc. filed Critical Abbvie Inc.
Priority to EP13786072.2A priority Critical patent/EP2836515A1/fr
Priority to CA2926384A priority patent/CA2926384A1/fr
Priority to SG11201504260UA priority patent/SG11201504260UA/en
Priority to KR1020157029562A priority patent/KR20150129033A/ko
Priority to CA2899308A priority patent/CA2899308C/fr
Priority to BR112015017307A priority patent/BR112015017307A2/pt
Priority to CA2926301A priority patent/CA2926301A1/fr
Priority to PCT/US2013/065749 priority patent/WO2014158231A1/fr
Priority to AU2013384204A priority patent/AU2013384204B2/en
Publication of WO2013158275A1 publication Critical patent/WO2013158275A1/fr
Publication of WO2013158275A9 publication Critical patent/WO2013158275A9/fr
Priority to HK15107319.2A priority patent/HK1206755A1/xx

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature

Definitions

  • the instant invention relates to the field of protein production, and in particular to compositions and processes for controlling the amount of acidic species generated during expression of a protein of interest by host cells, as well as the reduction of acidic species present in the clarified cell culture broth.
  • controlling the amount of acidic species generated during expression of a protein of interest is achieved by modifying the culture media of the cells.
  • controlling the amount of acidic species generated during expression of a protein of interest is achieved by modifying the culture process parameters.
  • controlling the amount of acidic species of a protein of interest is achieved by modifying a cell culture clarified harvest comprising the protein of interest.
  • the production of proteins for biopharmaceutical applications typically involves the use of cell cultures that are known to produce proteins exhibiting varying levels of product-related substance heterogeneity.
  • heterogeneity includes, but is not limited to, the presence of acidic species.
  • acidic species heterogeneities can be detected by various methods, such as WCX- 10 HPLC (a weak cation exchange chromatography) or IEF (isoelectric focusing).
  • the acidic species identified using such techniques comprise a range of product-related impurities such as antibody product fragments (e.g., Fc and Fab fragments), and/or post-translation modifications of the antibody product, such as, deamidated and/or glycoslyated antibodies.
  • the present invention is directed to compositions and methods that control (modulate or limit) acidic species heterogeneity in a population of proteins.
  • the presence of such acidic species corresponds to heterogeneity of the distribution of charged impurities, e.g., a mixture of protein fragments (e.g., Fc and Fab fragments of antibodies), and/or post-translation modifications of the proteins, such as, deamidated and/or glycoslyated proteins, in the population of proteins, and such heterogeneity particularly of interest when it arises in the context of recombinant protein production.
  • the acidic species heterogeneity arises from differences in the amount and/or type of acidic species in a population of proteins.
  • the acidic species heterogeneity is present in a population of proteins produced by cell culture.
  • control is exerted over the amount of acidic species of protein produced by cell culture.
  • the control is exerted over the amount of acidic species formed while the protein is present in a cell culture broth, while the culture is actively maintained or while the cells are removed.
  • the protein is an antibody.
  • control over the amount of acidic species produced by cell culture is exerted by employing certain media components during production of a protein, for example, an antibody, of interest.
  • control over the amount of acidic species of protein produced by cell culture is exerted by supplementing the media of cells expressing the protein of interest with one or more amino acids.
  • the one or more amino acids are arginine, lysine, ornithine, histidine or combinations thereof.
  • control over the amount of acidic species of protein produced by cell culture is exerted by supplementing the media of cells expressing the protein of interest with calcium, for example, by supplementing the media with calcium chloride dihydrate.
  • control over the amount of acidic species of protein produced by cell culture is exerted by supplementing the media of cells expressing the protein of interest with vitamin niacinamide.
  • control over the amount of acidic species of protein produced by cell culture is exerted by supplementing the media of cells expressing the protein of interest with suitable combinations of arginine, lysine, calcium chloride and niacinamide.
  • control over the amount of acidic species produced by cell culture is exerted by ensuring that the production of a protein, for example, an antibody, of interest occurs under specific conditions, including specific pH.
  • control over the amount of acidic species of protein produced by cell culture is exerted by supplementing the media of cells expressing the protein of interest with arginine and lysine and by controlling the pH of the cell culture.
  • the pH of the cell culture is adjusted to a pH of about 6.9. In certain embodiments, the pH of the cell culture is adjusted to a lower pH of about 6.8.
  • control over the amount of acidic species of protein produced by cell culture is exerted by supplementing the media of cells expressing the protein of interest with arginine and lysine and by choice of cell culture harvest criteria.
  • the harvest criterion is a particular culture day. In certain embodiments, the harvest criterion is based on harvest viability.
  • control over the amount of acidic species produced by cell culture is exerted by supplementing a cell culture clarified harvest comprising a protein or antibody of interest with one or more amino acids.
  • the one or more amino acids is arginine, histidine, or combinations thereof.
  • control over the amount of acidic species produced by cell culture is exerted by adjusting the pH of a cell culture clarified harvest comprising a protein or antibody of interest.
  • the pH of the cell culture clarified harvest is adjusted to a pH of about 5.
  • the pH of the cell culture clarified harvest is adjusted to a pH of about 6.
  • control over the amount of acidic species produced by cell culture is exerted by the use of a continuous or perfusion technology. In certain embodiments, this may be attained through choice of medium exchange rate. In certain, non-limiting, embodiments, maintenance of the medium exchange rates (working volumes/day) of a cell culture run between 0 and 20, or between 0.5 and 12 or between 1 and 8 or between 1.5 and 6 can be used to achieve the desired reduction in acidic species. In certain embodiments, the choice of cell culture methodology that allows for control of acidic species heterogeneity can also include, for example, but not by way of limitation, employment of an intermittent harvest strategy or through use of cell retention device technology.
  • the methods of culturing cells expressing a protein of interest reduces the amount of acidic species present in the resulting composition.
  • the resulting composition is substantially free of acidic species.
  • the sample comprises a cell culture harvest wherein the cell culture is employed to produce specific proteins of the present invention.
  • the sample matrix is prepared from a cell line used to produce anti-TNF-a antibodies.
  • the purity of the proteins of interest in the resultant sample product can be analyzed using methods well known to those skilled in the art, e.g., weak cation exchange chromatography (WCX), capillary isoelectric focusing (cIEF), size- exclusion chromatography, PorosTM A HPLC Assay, HCP ELISA, Protein A ELISA, and western blot analysis.
  • the invention is directed to one or more pharmaceutical compositions comprising an isolated protein, such as an antibody or antigen-binding portion thereof, and an acceptable carrier.
  • the compositions further comprise one or more pharmaceutical agents. 4. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 50 depicts the effect of total ornithine concentration in adalimumab producing cell line 2, media 1 on WCX 10 profile total acidic regions.
  • Figure 103 depicts the effect of pH modulation adalimumab producing cell line 3, media 1 on viability (n-2).
  • Figure 106 depicts an acidification sample preparation scheme.
  • Figure 107 depicts an arginine sample preparation scheme.
  • Figure 108 depicts a histidine sample preparation scheme.
  • Figure 109 depicts a lysine sample preparation scheme.
  • Figure 110 depicts a methionine sample preparation scheme.
  • Figure 111 depicts an amino acid sample preparation scheme.
  • Figure 112 depicts a CDM clarified harvest sample preparation scheme.
  • Figure 113 depicts an acid-type pH study sample preparation scheme.
  • Figure 114 depicts the effect of low pH treatment with subsequent neutralization on initial acidic variant content.
  • Figure 115 depicts the effect of low pH treatment with subsequent neutralization on acidic variant formation rate.
  • Figure 116 depicts the effect of sample preparation method on initial acidic variant content.
  • Figure 117 depicts the effect of sample preparation method on initial acidic variant content.
  • Figure 118 depicts the dose dependent effect of arginine on reduction of acidic variant formation rate
  • Figure 119 depicts the effect of histidine concentration on initial acidic variant content.
  • Figure 120 depicts the effect of histidine concentration on acidic variant formation rate.
  • Figure 121 depicts the effect of lysine on initial acid variant content.
  • Figure 122 depicts the effect of lysine on acidic variant formation rate.
  • Figure 123 depicts the effect of methionine on initial acid variant content.
  • Figure 124 depicts the effect of methionine on acidic variant formation rate.
  • Figure 125 depicts the effect of amino acids on initial acid variant content.
  • Figure 126 depicts the effect of amino acids on acidic variant formation rate.
  • Figure 127 depicts the effect of alternative additives on initial acid variant content.
  • Figure 128 depicts the effect of alternative additives on acidic variant formation rate.
  • Figure 129 depicts the effect of low pH/arginine treatment on D2E7
  • CDM initial acid variant content
  • Figure 130 depicts the effect of low pH/arginine treatment on D2E7 CDM acidic variant formation rate.
  • Figure 131 depicts the effect of low pH/arginine treatment on mAb B hydrolysate initial acid variant content.
  • Figure 132 depicts the effect of low pH/arginine treatment on mAb B hydrolysate acidic variant formation rate.
  • Figure 133 depicts the effect of low pH/arginine treatment on mAb C hydrolysate initial acid variant content.
  • Figure 134 depicts the effect of low pH/arginine treatment on mAb C hydrolysate acidic variant formation rate.
  • Figure 135 depicts the effect of acid type/pH on acid variant content.
  • Figure 136 depicts the effect of acid concentration on acid variant content.
  • Figure 137 depicts the effect of acid concentration on acid variant content.
  • Figure 138 depicts the effect of neutralization on acid variant content.
  • Figure 139 depicts the effect of neutralization on acid variant content.
  • Figure 140 depicts LC MS peptide mapping analysis of exemplary antibodies expressed in the context of the cell culture conditions of the instant invention, including preparation of specific mass traces for both modified and non- modified peptides in order to accurately quantify the total amount of MGO modification. Mass spectra are also analyzed for the specific region of the chromatogram to confirm the peptide identity.
  • Figure 141 depicts a chromatogram wherein the total acidic species associated with the expression of Adalimiumab is divided into a first acidic species region (AR1) and a second acidic species region (AR2).
  • Figure 142 depicts the AR Growth at 25°C of low and high AR containing samples.
  • the instant invention relates to the field of protein production.
  • the instant invention relates to compositions and processes for controlling the amount of acidic species expressed by host cells when used to produce a protein of interest.
  • Certain embodiments of the invention relate to culturing said cells to express said proteins under conditions that limit the amount of acidic species that are expressed by the cells.
  • the methods described herein employ culturing said cells in media supplemented with one or more amino acids and/or calcium (e.g., as calcium chloride dihydrate) and/or niacinamide.
  • the methods described herein employ culturing said cells in a culture with appropriate control of process parameters, such as pH.
  • methods described herein employ culturing cells at a lower process pH.
  • control of acidic species heterogeneity can be attained by the choice of cell culture methodology.
  • use of a continuous or perfusion technology may be utilized to achieve the desired control over acidic species heterogeneity. In certain embodiments, this may be attained through choice of medium exchange rate.
  • the present invention is directed toward pharmaceutical compositions comprising one or more proteins, such as, but not limited to an antibody or antigen-binding portion thereof, purified by a method described herein.
  • the terms “acidic species” and “acidic species heterogeneity” refer to a characteristic of a population of proteins wherein the population includes a distribution of product-related impurities identifiable by the presence of charge heterogeneities.
  • acidic species heterogeneities can be detected by various methods, such as, for example, WCX-10 HPLC (a weak cation exchange chromatography), or IEF (isoelectric focusing).
  • the acidic species identified using such techniques comprise a mixture of product-related impurities containing antibody product fragments (e.g., Fc and Fab fragments), chemical modifications (e.g., methylglyoxal modified species (as described in the U.S. patent application having attorney reference no. ABV1 1886USL1), glycated species) and/or post- translation modifications of the antibody product, such as, deamidated and/or glycoslyated antibodies.
  • product-related impurities containing antibody product fragments e.g., Fc and Fab fragments
  • chemical modifications e.g., methylglyoxal modified species (as described in the U.S. patent application having attorney reference no. ABV1 1886USL1), glycated species
  • post- translation modifications of the antibody product such as, deamidated and/or glycoslyated antibodies.
  • the acidic species heterogeneity comprises a difference in the type of acidic species present in the population of proteins.
  • the population of proteins may comprise more than one acidic species variant.
  • the total acidic species can be divided based on chromatographic residence time.
  • Figure 141 depicts a non-limiting example of such a division wherein the total acidic species associated with the expression of Adali iumab is divided into a first acidic species region (AR1) and a second acidic species region (AR2).
  • the compositions of particular acidic species regions may differ depending on the particular antibody of interest, as well as the particular cell culture, purification, and/or chromatographic conditions employed.
  • the heterogeneity of the distribution of acidic species comprises a difference in the amount of acidic species in the population of proteins.
  • the population of proteins may comprise more than one acidic species variant, and each of the variants may be present in different amounts.
  • the term "antibody” includes an immunoglobulin molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH).
  • the heavy chain constant region is comprised of three domains, CHI , CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino -terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
  • antigen-binding portion of an antibody includes fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., in the case of Adalimumab, hTNFa). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a fuH- length antibody.
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment comprising the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment comprising the VH and CHI domains; (iv) a Fv fragment comprising the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546, the entire teaching of which is incorporated herein by reference), which comprises a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • CDR complementarity determining region
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883, the entire teachings of which are incorporated herein by reference).
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, e.g., Holliger, P., et al. (1993) Proc, Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1 121-1123, the entire teachings of which are incorporated herein by reference).
  • an 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.
  • immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al.
  • Antibody portions such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies.
  • antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.
  • the antigen binding portions are complete domains or pairs of complete domains.
  • the phrase "clarified harvest” refers to a liquid material containing a protein of interest, for example, an antibody of interest such as a monoclonal or polyclonal antibody of interest, that has been extracted from cell culture, for example, a fermentation bioreactor, after undergoing centrifugation to remove large solid particles and subsequent filtration to remove finer solid particles and impurities from the material.
  • human antibody includes antibodies having variable and constant regions corresponding to human gennline immunoglobulin sequences as described by Kabat et al. (See Kabat, et al. (1991) Sequences of proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
  • the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs and in particular CDR3.
  • the mutations can be introduced using the "selective mutagenesis approach.”
  • the human antibody can have at least one position replaced with an amino acid residue, e.g., an activity enhancing amino acid residue which is not encoded by the human germline immunoglobulin sequence.
  • the human antibody can have up to twenty positions replaced with amino acid residues which are not part of the human gennline immunoglobulin sequence. In other embodiments, up to ten, up to five, up to three or up to two positions are replaced. In one embodiment, these replacements are within the CDR regions.
  • the term "human antibody", as used herein is not intended to include antibodies in which CDR sequences derived from the gennline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • recombinant human antibody includes human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295, the entire teaching of which is incorporated herein by reference) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
  • recombinant means such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobul
  • Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242).
  • such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • such recombinant antibodies are the result of selective mutagenesis approach or back-mutation or both.
  • an “isolated antibody” includes an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds hTNFa is substantially free of antibodies that specifically bind antigens other than hTNFa).
  • An isolated antibody that specifically binds hTNFa may bind TNFa molecules from other species.
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • Adalimumab (Abbott Laboratories). As used herein, the term "adalimumab”, also known by its trade name
  • Humira ® refers to a human IgG antibody that binds the human form of tumor necrosis factor alpha.
  • the heavy chain constant domain 2 (CH2) of the adalimumab IgG-Fc region is glycosylated through covalent attachment of oligosaccharide at asparagine 297 (Asn-297).
  • Weak cation-exchange chromatography (WCX) analysis of the antibody has shown that it has three main charged-variants (i.e. Lys 0, Lys 1 , and Lys 2). These variants, or charged isomers, are the result of incomplete posttranslational cleavage of the C-terminal lysine residues.
  • the WCX- 10 analysis measures the presence acidic species. These acidic regions (i.e., acidic species) are classified as product-related impurities that are relatively acidic when compared to the lysine variants and elute before the Lys 0 peak in the chromatogram ( Figure 1).
  • activity includes activities such as the binding specificity/affinity of an antibody for an antigen, and includes activities such as the binding specificity/affinity of an anti-TNFa antibody for its antigen, e.g., an anti- TNFa antibody that binds to a TNFa antigen and/or the neutralizing potency of an antibody, e.g., an anti-TNFa antibody whose binding to hTNFa inhibits the biological activity of hTNFa.
  • nucleic acid molecule includes DNA molecules and RNA molecules.
  • a nucleic acid molecule may be single-stranded or double- stranded, but in one aspect is double- stranded DNA.
  • isolated nucleic acid molecule as used herein in reference to nucleic acids encoding antibodies or antibody portions (e.g., VH, VL, CDR3), e.g. those that bind hTNFa, and includes a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than hTNFa, which other sequences may naturally flank the nucleic acid in human genomic DNA.
  • nucleic acid molecule includes a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than hTNFa, which other sequences may naturally flank the nucleic acid in human genomic DNA.
  • an isolated nucleic acid of the invention encoding a VH region of an anti-TNFa antibody contains no other sequences encoding other VH regions that bind antigens other than, for example, hTNFa.
  • isolated nucleic acid molecule is also intended to include sequences encoding bivalent, bispecific antibodies, such as diabodies in which VH and VL regions contain no other sequences other than the sequences of the diabody.
  • recombinant host cell includes a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell” as used herein.
  • the term "recombinant protein” refers to a protein produced as the result of the transcription and translation of a gene carried on a recombinant expression vector that has been introduced into a host cell.
  • the recombinant protein is an antibody, preferably a chimeric, humanized, or fully human antibody.
  • the recombinant protein is an antibody of an isotype selected from group consisting of: IgG (e.g., IgGI, IgG2, IgG3, IgG4), IgM, IgAl, IgA2, IgD, or IgE.
  • the antibody molecule is a full-length antibody (e.g., an IgGI or IgG4 immunoglobulin) or alternatively the antibody can be a fragment (e.g., an Fc fragment or a Fab fragment).
  • the term "cell culture” refers to methods and techniques employed to generate and maintain a population of host cells capable of producing a recombinant protein of interest, as well as the methods and techniques for optimizing the production and collection of the protein of interest. For example, once an expression vector has been incorporated into an appropriate host, the host can be maintained under conditions suitable for high level expression of the relevant nucleotide coding sequences, and the collection and purification of the desired recombinant protein. Mammalian cells are preferred for expression and production of the recombinant protein of the present invention, however other eukaryotic cell types can also be employed in the context of the instant invention.
  • Suitable mammalian host cells for expressing recombinant proteins according to the invention include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) PNAS USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621, the entire teachings of which are incorporated herein by reference), NSO myeloma cells, COS cells and SP2 cells.
  • monkey kidney cells (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDC , 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 N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), the entire teachings of which are incorporated herein by reference.
  • the protein of interest can be produced intracellularly, in the periplasmic space, or directly secreted into the medium.
  • the particulate debris either host cells or lysed cells (e.g., resulting from homogenization)
  • supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, e.g., an AmiconTM or Millipore PelliconTM ultrafiltration unit, which can then be subjected to one or more additional purification techniques, including but not limited to affinity chromatography, including protein A affinity chromatography, ion exchange chromatography, such as anion or cation exchange chromatography, and hydrophobic interaction chromatography.
  • a commercially available protein concentration filter e.g., an AmiconTM or Millipore PelliconTM ultrafiltration unit
  • additional purification techniques including but not limited to affinity chromatography, including protein A affinity chromatography, ion exchange chromatography, such as anion or cation exchange chromatography, and hydrophobic interaction chromatography.
  • on-line refers to processes that are accomplished in the context of an on-going cell culture run. For example, the administration of a particular nutrient or changes in temperature, pH, or dissolved oxygen level occur on-line when such administrations or changes are implemented in an existing cell culture run. Similarly, measurements of certain data are considered on-line if that data is being collected in the context of a particular cell culture run. For example, on-line gas analysis refers to the measurement of gases introduced into or released from a particular cell culture run.
  • off-line refers to actions taken outside the context of a particular cell culture run. For example, the production of cell culture media comprising specific concentrations of particular components is an example of an off-line activity.
  • modifying is intended to refer to changing one or more amino acids in the antibodies or antigen-binding portions thereof.
  • the change can be produced by adding, substituting or deleting an amino acid at one or more positions.
  • the change can be produced using known techniques, such as PCR mutagenesis.
  • control is intended to refer to both limitation as well as to modulation.
  • the instant invention provides methods for controlling diversity that decrease the diversity of certain characteristics of protein populations, including, but not limited to, the presence of acidic species. Such decreases in diversity can occur by: (1) promotion of a desired characteristic; (2) inhibition of an unwanted characteristic; or (3) a combination of the foregoing.
  • control also embraces contexts where heterogeneity is modulated, i.e., shifted, from one diverse population to a second population of equal, or even greater diversity, where the second population exhibits a distinct profile of the characteristic of interest.
  • antibody refers to an intact antibody or an antigen binding fragment thereof.
  • the antibodies of the present disclosure can be generated by a variety of techniques, including immunization of an animal with the antigen of interest followed by conventional monoclonal antibody methodologies e.g., the standard somatic cell hybridization technique of ohler and Milstein (1975) Nature 256: 495. Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed e.g., viral or oncogenic transformation of B lymphocytes.
  • hybridomas One preferred animal system for preparing 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.
  • An antibody preferably can be a human, a chimeric, or a humanized antibody.
  • Chimeric or humanized antibodies of the present disclosure can be prepared based on the sequence of a non-human monoclonal antibody prepared as described above.
  • DNA encoding the heavy and light chain immunoglobulins can be obtained from the non-human hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques.
  • murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Patent No. 4,816,567 to Cabilly et al.).
  • murine CDR regions can be inserted into a human framework using methods known in the art (see e.g., U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101 ; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).
  • the antibodies of this disclosure are human monoclonal antibodies.
  • Such human monoclonal antibodies can be generated using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system.
  • HuMAb Mouse® Medarex, Inc.
  • KM Mouse® Medarex, Inc.
  • XenoMouse® Amgen
  • alternative transchromosomic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise antibodies of the disclosure.
  • mice carrying both a human heavy chain transchromosome and a human light chain transchromosome referred to as "TC mice” can be used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727.
  • cows carrying human heavy and light chain transchromosomes have been described in the art (e.g., Kuroiwa et al. (2002) Nature Biotechnology 20:889-894 and PCT application No. WO 2002/092812) and can be used to raise antibodies of this disclosure.
  • Recombinant human antibodies of the invention can be isolated by screening of a recombinant combinatorial antibody library, e.g., a scFv phage display library, prepared using human VL and VH cDNAs prepared from mRNA derived from human lymphocytes. Methodologies for preparing and screening such libraries are known in the art.
  • kits for generating phage display libraries e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01 ; and the Stratagene SurfZAPTM phage display kit, catalog no. 240612, the entire teachings of which are incorporated herein
  • methods and reagents particularly amenable for use in generating and screening antibody display libraries can be found in, e.g., Ladner et al. U.S. Patent No. 5,223,409; ang et al. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al. PCT Publication No.
  • Human monoclonal antibodies of this disclosure can also be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. Such mice are described in, for example, U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.
  • the methods of the invention include anti-
  • the invention provides treatment with an isolated human antibody, or an antigen-binding portion thereof, that dissociates from hTNFa with a Kd of about 1 x 10 ⁇ 8 M or less and a off rate constant of 1 x 10 "3 s "1 or less, both determined by surface plasmon resonance.
  • an anti-TNFot antibody purified according to the invention competitively inhibits binding of Adalimumab to TNFa under physiological conditions.
  • Antibodies or fragments thereof can be altered wherein the constant region of the antibody is modified to reduce at least one constant region-mediated biological effector function relative to an unmodified antibody.
  • the immunoglobulin constant region segment of the antibody can be mutated at particular regions necessary for Fc receptor (FcR) interactions (see, e.g., Canfield and Morrison (1991) J. Exp. Med. 173: 14834491 ; and Lund et al. (1991) J. of Immunol. 147:2657- 2662, the entire teachings of which are incorporated herein).
  • Reduction in FcR binding ability of the antibody may also reduce other effector functions which rely on FcR interactions, such as opsonization and phagocytosis and antigen- dependent cellular cytotoxicity.
  • DNAs encoding the protein are inserted into one or more expression vector such that the genes are operatively linked to transcriptional and translational control sequences.
  • operatively linked is intended to mean that a gene encoding the protein of interest is li gated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the gene.
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • the protein of interest will comprising multiple polypeptides, such as the heavy and light chains of an antibody.
  • genes encoding multiple polypeptides, such as antibody light chain genes and antibody heavy chain genes can be inserted into a separate vector or, more typically, the genes are inserted into the same expression vector.
  • Genes are inserted into expression vectors by standard methods (e.g., ligation of complementary restriction sites on the gene fragment and vector, or blunt end ligation if no restriction sites are present).
  • the expression vector may already carry additional polypeptide sequences, such as, but no limited to, antibody constant region sequences.
  • one approach to converting the anti-TNFa antibody or anti-TNFa antibody- related VH and VL sequences to full-length antibody genes is to insert them into expression vectors already encoding heavy chain constant and light chain constant regions, respectively, such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector.
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the protein from a host cell.
  • the gene can be cloned into the vector such that the signal peptide is linked in- frame to the amino terminus of the gene.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
  • a recombinant expression vector of the invention can carry one or more regulatory sequence that controls the expression of the protein coding genes in a host cell.
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the protein coding genes.
  • Such regulatory sequences are described, e.g., in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990), the entire teaching of which is incorporated herein by reference.
  • Suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • a recombinant expression vector of the invention may carry one or more additional sequences, such as a sequence that regulates replication of the vector in host cells (e.g., origins of replication) and/or a selectable marker gene.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Patents Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al., the entire teachings of which are incorporated herein by reference),
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • An antibody, or antibody portion, of the invention can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell.
  • a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and secreted into the medium in which the host cells are cultured, from which medium the antibodies can be recovered.
  • Standard recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (3989), Ausubel et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Patent Nos. 4,816,397 & 6,914,128, the entire teachings of which are incorporated herein.
  • the expression vector(s) encoding the protein is (are) transfected into a host cell by standard techniques.
  • the various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram- positive organisms, e.g., Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B.
  • Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus
  • Salmonella e.g., Salmonella typhimurium
  • Serratia e.
  • E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coH XI 776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide encoding vectors.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), . bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.
  • waltii ATCC 56,500
  • . drosophilarum ATCC 36,906
  • K. thermotolerans K. marxianus
  • yarrowia EP 402,226
  • Pichia pastoris EP 183,070
  • Candida Trichoderma reesia
  • Neurospora crassa Neurospora crassa
  • Schwanniomyces such as Schwanniomyces occidentalis
  • filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • Suitable host cells for the expression of glycosylated proteins are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified.
  • a variety of viral strains for transfection are publicly available, e.g., the L- 1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
  • Suitable mammalian host cells for expressing the recombinant proteins of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) PNAS USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (3982) Mol. Biol. 159:601-621, the entire teachings of which are incorporated herein by reference), NS0 myeloma cells, COS cells and SP2 cells.
  • Chinese Hamster Ovary CHO cells
  • dhfr- CHO cells described in Urlaub and Chasin, (1980) PNAS USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (3982) Mol. Biol. 159:601-621, the entire teachings of which are incorporated herein by reference
  • NS0 myeloma cells COS cells and SP2
  • the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS- 7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc.
  • mice 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 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary rumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), the entire teachings of which are incorporated herein by reference.
  • Host cells are transformed with the above-described expression or cloning vectors for protein production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the host cells used to produce a protein may be cultured in a variety of media.
  • Commercially available media such as Ham's F10TM (Sigma), Minimal Essential MediumTM (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's MediumTM (DMEM), (Sigma) are suitable for culturing the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamycin drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, H, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • Host cells can also be used to produce portions of intact proteins, for example, antibodies, including Fab fragments or scFv molecules. It is understood that variations on the above procedure 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 either the light chain or the heavy chain (but not both) of an antibody. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to an antigen. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention.
  • bifunctional antibodies may be produced in which one heavy and one light chain are an antibody of the invention and the other heavy and light chain are specific for an antigen other than the target antibody, depending on the specificity of the antibody of the invention, by crosslinking an antibody of the invention to a second antibody by standard chemical crosslinking methods.
  • a recombinant expression vector encoding the protein for example, both an antibody heavy chain and an antibody light chain, is introduced into dhfr-CHO cells by calcium phosphate- mediated transfection.
  • the protein gene(s) are each operatively linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of transcription of the gene(s).
  • the recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification.
  • the selected transformant host cells are cultured to allow for expression of the protein, for example, the antibody heavy and light chains, and intact protein, for example, an antibody, is recovered from the culture medium.
  • Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the protein from the culture medium.
  • the protein for example, antibodies or antigen binding fragments thereof, can be produced intracellularly, in the periplasmic space, or directly secreted into the medium.
  • the particulate debris either host cells or lysed cells (e.g., resulting from homogenization)
  • supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, e.g., an AmiconTM or Millipore PelliconTM ultrafiltration unit.
  • Numerous populations of proteins expressed by host cells may comprise a number of acidic species, and are therefore amenable to the instant invention's methods for control of acidic species heterogeneity.
  • adalimumab may comprise a number of acidic species, and are therefore amenable to the instant invention's methods for control of acidic species heterogeneity.
  • weak cation-exchange chromatography (WCX) analysis of adalimumab has shown the presence of acidic regions. These acidic species are classified as product-related impurities that are relatively acidic when compared to the adalimumab protein population. The presence of these acidic species provides an exemplary system to identify those cell culture conditions that allow for control over acidic species heterogeneity. 5.3.1 Adjusting Amino Acid Concentration to Control
  • the variation in raw materials used in cell culture, particularly in the context of media preparation, can vary product quality significantly.
  • control of acidic species heterogeneity can be attained by adjustment of the media composition of the cell culture run.
  • such adjustment will be to increase the amount of one or more amino acids in the media
  • the necessary adjustment to achieve the desired control over acidic species heterogeneity will involve a decrease in the amount of one or more amino acids in the media.
  • Such increases or decreases in the amount of the one or more amino acids can be of a magnitude of 1%, 5%, 10%, 15%, 20%, 25%, 30%», 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%», 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the preceding, of the original amount.
  • a cell culture media will include one or more of the amino acids, or other compositions, described herein as facilitating a reduction in acidic species.
  • the amount of the amino acid, or other composition, that is necessary to be supplemented may be adjusted to account for the amount present in the media prior to supplementation.
  • the cell culture media is supplemented with one or more amino acids wherein each of the one or more amino acids is supplemented in an amount of between about 0.025 and 20 g/L, or between about 0.05 and 15 g/L, or between about 0.1 and 14 g/L, or between about 0.2 and 13 g/L, or between about 0.25 and 12 g/L, or between about 0.5 and 11 g/L, or between about 1 and 10 g/L, or between about 1.5 and 9.5 g/L, or between about 2 and 9 g/L, or between about 2.5 and 8.5 g/L, or between about 3 and 8 g/L, or between about 3.5 and 7.5 g/L, or between about 4 and 7 g/L, or between about 4.5 and 6.5 g/L, or between about 5 and 6 g/L.
  • the cell culture media is supplemented with one or more amino acids wherein each of the one or more amino acids is supplemented 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/L, or about 8 g/L, In certain embodiments, the cell culture media is supplemented with one or more amino acids wherein each of the one or more amino acids is supplemented in an amount effective to reduce the amount of acidic species heterogeneity in a protein or antibody sample by 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%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the preceding.
  • the one or more amino acids used to supplement the cell culture media is a basic amino acid.
  • the one or more amino acids is arginine, lysine, histidine, ornithine, or certain combinations of arginine or lysine with ornithine or of all four amino acids.
  • the amino acids are provided as single peptides, as dipeptides, as tripeptides or as longer oligopeptides.
  • 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 composed of two or more particular amino acids.
  • the amount of amino acid supplemented to the cell culture to achieve concentrations of about 0 to about 9 g 1 for arginine, about 0 to about 1 1 g/1 for lysine, about 0 to about 11 g 1 histidine, and about 0 to about 1 1 g/I ornithine.
  • concentrations of about 0 to about 9 g 1 for arginine, about 0 to about 1 1 g/1 for lysine, about 0 to about 11 g 1 histidine, and about 0 to about 1 1 g/I ornithine.
  • wider ranges are also within the scope of the instant invention, including, but not limited to: about 0 to about 30 g/1 for arginine, about 0 to about 30 g 1 for lysine, about 0 to about 30 g/1 histidine, and about 0 to about 30 g/1 ornithine.
  • Example 6.1 when the production medium employed in the example was supplemented with arginine to achieve a total concentration of 9 g/L arginine, the total amount of acidic species of adalimumab present in a cell culture sample after purification was reduced from 19.7% of a control sample to 12.2% of the sample purified from the cells cultured with the arginine supplemented media.
  • the production medium employed in the example was supplemented with lysine,, or histidine, or ornithine to achieve total concentrations of 11 g/L lysine, 10 g/L ornithine or 10 g/L histidine, respectively, the total amount of acidic species of adalimumab present in a cell culture sample after purification was reduced by 1 1.5%, 10.4% and 10.9%, respectively, compared to a control sample.
  • control over the amount of acidic species of protein produced by cell culture is exerted by supplementing the media of cells expressing the protein of interest medium supplements described herein such that they can be included in the medium at the start of culture, or can be added in a fed-batch or in a continuous manner.
  • the feed amounts may be calculated to achieve a certain concentration based on offline or online measurements.
  • the supplements may be added as multimers, e.g., arg ⁇ arg, his-his, arg-his-orn, etc., and/or as chemical variants, e.g., of amino acids or analogs of amino acids, salt forms of amino acids, controlled release of amino acids by immobilizing in gels, etc, and/or in fully or partially dissolved form.
  • the addition of one or more supplement may be based on measured amount of acidic species.
  • the resulting media can be used in various cultivation methods including, but not limited to, batch, fed-batch, chemostat and perfusion, and with various cell culture equipment including, but not limited to, shake flasks with or without suitable agitation, spinner flasks, stirred bioreactors, airlift bioreactors, membrane bioreactors, reactors with cells retained on a solid support or immobilized/entrapped as in microporous beads, and any other configuration appropriate for optimal growth and productivity of the desired cell line.
  • the harvest criterion for these cultures may be chosen, for example based on choice of harvest viability or culture duration, to further optimize a certain targeted acidic species profile. 5.3.1 Adjusting CaCl 2 and/or Niacinamide Concentration to Control Acidic Species
  • the cell culture media is supplemented with calcium (e.g., as calcium chloride dihydrate), wherein the calcium is supplemented to achieve a calcium concentration of between about 0.05 and 2.5 niM, or between about 0.05 and 1 mM, or between about 0.1 and 0.8 mM, or between about 0.15 and 0.7 mM, or between about 0.2 and 0.6 mM, or between about 0.25 and 0.5 mM, or between about 0.3 and 0.4 mM.
  • calcium e.g., as calcium chloride dihydrate
  • the cell culture media is supplemented with calcium (e.g., as calcium chloride dihydrate) wherein the calcium is supplemented in an amount effective to reduce the amount of acidic species heterogeneity in a protein or antibody sample by 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%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the preceding.
  • calcium e.g., as calcium chloride dihydrate
  • Example 6.3 when the production medium employed in the example was supplemented with calcium (e.g., as calcium chloride dihydrate) at a concentration of 1.05 mM, the total amount of acidic species of adalimumab present in a cell culture sample after purification was reduced from 23.2% of a control sample to 16.5% of the sample purified from the cells cultured with the calcium supplemented media.
  • the cell culture can be supplemented with a combination of calcium, e.g., CaCl 2 , and one or more a basic amino acids.
  • the one or more basic amino acids is arginine, lysine, histidine, ornithine, or combinations of arginine or lysine with ornithine or of all four amino acids.
  • the amino acids are provided as single peptides, as dipeptides, as tripeptides or as longer oligopeptides.
  • 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 composed of two or more particular amino acids.
  • the amount of basic amino acid concentrations in combination with calcium in the cell culture is between about 0 to about 9 g/1 for arginine, about 0 to about 11 g 1 for lysine, about 0 to about 1 1 g/1 histidine, and about 0 to about 1 1 g 1 ornithine.
  • wider ranges are also within the scope of the instant invention, including, but not limited to: about 0 to about 30 g/1 for arginine, about 0 to about 30 g 1 for lysine, about 0 to about 30 g/1 histidine, and about 0 to about 30 g/1 ornithine.
  • the cell culture media is supplemented with niacinamide, wherein the niacinamide is supplemented to achieve a niacinamide concentration of between about 0.2 and 3.0 mM, or between about 0.4 and 3.0 mM, or between about 0.8 and 3.0 mM.
  • the cell culture media is supplemented with niacinamide wherein the niacinamide is supplemented in an amount effective to reduce the amount of acidic species heterogeneity in a protein or antibody sample by 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%, 75%, 80%, 85%o, 90%, 95%, 100%i, and ranges within one or more of the preceding.
  • Example 6.3 when the production medium employed in the example was supplemented with niacinamide at a concentration of 1.6 mM, the total amount of acidic species of adalimumab present in a cell culture sample after purification was reduced from 19.9% of a control sample to 15.9% of the sample purified from the cells cultured with the niacinamide supplemented media.
  • the total amount of acidic species of adalimumab present in a cell culture sample after purification was reduced from 27.0% of a control sample to 19.8% of the sample purified from the cells cultured with the niacinamide supplemented media.
  • the cell culture can be supplemented with a combination of niacinamide, calcium, e.g., CaC , and/or one or more a basic amino acids.
  • the one or more basic amino acids is arginine, lysine, histidine, ornithine, or combinations of arginine or lysine with ornithine or of all four amino acids.
  • the amino acids are provided as single peptides, as dipeptides, as tripeptides or as longer oligopeptides.
  • 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 composed of two or more particular amino acids.
  • the amount of basic amino acid concentrations (after supplementation) in combination with calcium in the cell culture is between about 0 to about 9 g/1 for arginine, about 0 to about 1 1 g 1 for lysine, about 0 to about 1 1 g 1 histidine, and about 0 to about 1 1 g/1 ornithine.
  • control over the amount of acidic species of protein produced by cell culture is exerted by supplementing the media of cells expressing the protein of interest medium supplements described herein such that they can be included in the medium at the start of culture, or can be added in a fed-batch or in a continuous manner.
  • the feed amounts may be calculated to achieve a certain concentration based on offline or online measurements.
  • the addition of the supplement may be based on measured amount of acidic species.
  • Other salts of particular supplements, e.g., calcium may also be used, for example Calcium Nitrate.
  • the resulting media can be used in various cultivation methods including, but not limited to, batch, fed-batch, chemostat and perfusion, and with various cell culture equipment including, but not limited to, shake flasks with or without suitable agitation, spinner flasks, stirred bioreactors, airlift bioreactors, membrane bioreactors, reactors with cells retained on a solid support or immobilized/entrapped as in microporous beads, and any other configuration appropriate for optimal growth and productivity of the desired cell line.
  • various cell culture equipment including, but not limited to, shake flasks with or without suitable agitation, spinner flasks, stirred bioreactors, airlift bioreactors, membrane bioreactors, reactors with cells retained on a solid support or immobilized/entrapped as in microporous beads, and any other configuration appropriate for optimal growth and productivity of the desired cell line.
  • control over amount and/or rate of formation of acidic species is achieved by supplementing a clarified harvest.
  • a clarified harvest can be supplemented as described above (e.g., with calcium, niacinamide, and/or basic amino acids) to achieve a reduction the amount of acidic species and/or a reduction in the rate such acidic species form.
  • control of acidic species heterogeneity can be attained by adjustment of pH of the cell culture run.
  • such adjustment will be to decrease in the pH of the cell culture.
  • Such decreases in the pH can be of a magnitude of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the preceding, of the original amount.
  • pH is either increased or decreased in order to increase or decrease the amount of acidic species and/or the rate at which such acidic species form.
  • a reduction in pH to 6.7 from a control pH of 7.1 can be employed to decrease the acidic species during cell culture and the rate of acidic species formation in the context of a clarified harvest.
  • the pH is maintained in such a manner as to reduce the amount of acidic species in a protein or antibody sample by 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%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the preceding.
  • control over the amount of acidic species of protein produced by cell culture can be exerted by maintaining the pH of the cell culture expressing the protein of interest as described herein along with choice of suitable temperature or temperature shift strategies, for example, but not limited to, lower process temperature of operation, temperature shift to a lower temperature or a temperature shift at an earlier culture time point.
  • culture conditions can be used in various cultivation methods including, but not limited to, batch, fed-batch, chemostat and perfusion, and with various cell culture equipment including, but not limited to, shake flasks with or without suitable agitation, spinner flasks, stirred bioreactors, airlift bioreactors, membrane bioreactors, reactors with cells retained on a solid support or immobilized/entrapped as in microporous beads, and any other configuration appropriate for optimal growth and productivity of the desired cell line.
  • These may also be used in combination with supplementation of culture media with amino acids, niacinamide, and/or calcium salt, as described above. 5.3.4 Continuous/Perfusion cell culture technology to
  • control of acidic species heterogeneity can be attained by the choice of cell culture methodology.
  • use of a continuous or perfusion technology may be utilized to achieve the desired control over acidic species heterogeneity.
  • this may be attained through choice of medium exchange rate (where the exchange rate is the rate of exchange of medium in/out of a reactor).
  • medium exchange rate where the exchange rate is the rate of exchange of medium in/out of a reactor.
  • Such increases or decreases in medium exchange rates may be of magnitude of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%), and ranges within one or more of the preceding, of the original amount.
  • maintenance of the medium exchange rates ⁇ working volumes/day) of a cell culture run between 0 and 20, or between 0.5 and 12 or between 1 and 8 or between 1.5 and 6 can be used to achieve the desired reduction in acidic species.
  • the medium exchange rate was chosen to be 1.5
  • the acidic species was 8,1%.
  • the choice of cell culture methodology that allows for control of acidic species heterogeneity can also include, for example, but not by way of limitation, employment of an intermittent harvest strategy or through use of cell retention device technology.
  • the methods of the present invention can be used in combination with techniques for protein purification to provide for the production of a purified protein preparation, for example, a preparation comprising an antibody or an antigen binding fragment thereof, from a mixture comprising a protein and at least one process-related impurity or product-related substance.
  • separation of the protein of interest from the process-related impurities and/or product-related substances can be performed using a combination of different purification techniques, including, but not limited to, affinity separation steps, ion exchange separation steps, mixed mode separation steps, and hydrophobic interaction separation steps.
  • the separation steps separate mixtures of proteins on the basis of their charge, degree of hydrophobicity, or size.
  • separation is performed using chromatography, including cationic, anionic, and hydrophobic interaction.
  • each of the separation methods is that proteins can be caused either to traverse at different rates down a column, achieving a physical separation that increases as they pass further down the column, or to adhere selectively to the separation medium, being then differentially eluted by different solvents.
  • the antibody is separated from impurities when the impurities specifically adhere to the column and the antibody does not, i.e., the antibody is present in the flow through.
  • the separation steps of employed in connection with the cell culture methods of the instant invention facilitate the separation of an antibody from one or more process-related impurity and/or product-related substance.
  • Antibodies that can be successfully purified using the methods described herein include, but are not limited to, human IgAl, IgA2, IgD, IgE, IgGl , IgG2, IgG3, IgG4, and IgM antibodies.
  • Protein A affinity chromatography can be useful, however, in certain embodiments, the use of Protein A affinity chromatography would prove useful, for example in the context of the purification of IgG3 antibodies, as IgG3 antibodies bind to Protein A inefficiently.
  • Other factors that allow for specific tailoring of a purification scheme include, but are not limited to: the presence or absence of an Fc region (e.g., in the context of full length antibody as compared to an Fab fragment thereof) because Protein A binds to the Fc region; the particular germline sequences employed in generating to antibody of interest; and the amino acid composition of the antibody (e.g., the primary sequence of the antibody as well as the overall charge hydrophobicity of the molecule).
  • Antibodies sharing one or more characteristic can be purified using purification strategies tailored to take advantage of that characteristic.
  • the primary recovery process can also be a point at which to reduce or inactivate viruses that can be present in the sample mixture.
  • any one or more of a variety of methods of viral reduction/inactivation can be used during the primary recovery phase of purification including heat inactivation (pasteurization), pH inactivation, solvent/detergent treatment, UV and ⁇ -ray irradiation and the addition of certain chemical inactivating agents such as ⁇ -propiolactone or e.g., copper phenanthroline as in U.S. Pat. No. 4,534,972, the entire teaching of which is incorporated herein by reference..
  • the primary recovery may also include one or more centrifugation steps to further clarify the sample mixture and thereby aid in purifying the protein of interest.
  • Centrifugation of the sample can be run at, for example, but not by way of limitation, 7,000 x g to approximately 12,750 x g.
  • centrifugation can occur on-line with a flow rate set to achieve, for example, but not by way of limitation, a turbidity level of 150 NTU in the resulting supernatant.
  • Such supernatant can then be collected for further purification.
  • the primary recovery may also include the use of one or more depth filtration steps to further clarify the sample matrix and thereby aid in purifying the antibodies produced using the cell culture techniques of the present invention.
  • Depth filters contain filtration media having a graded density. Such graded density allows larger particles to be trapped near the surface of the filter while smaller particles penetrate the larger open areas at the surface of the filter, only to be trapped in the smaller openings nearer to the center of the filter.
  • the depth filtration step can be a delipid depth filtration step.
  • certain embodiments employ depth filtration steps only during the primary recovery phase, other embodiments employ depth filters, including delipid depth filters, during one or more additional phases of purification.
  • Non-limiting examples of depth filters that can be used in the context of the instant invention include the CunoTM model 30/60ZA depth filters (3M Corp.), and 0.45/0.2 ⁇ SartoporeTM bi-layer filter cartridges.
  • the chromatographic material is capable of selectively or specifically binding to the protein of interest.
  • Non-limiting examples of such chromatographic material include: Protein A, Protein G, chromatographic material comprising, for example, an antigen bound by an antibody of interest, and chromatographic material comprising an Fc binding protein.
  • the affinity chromatography step involves subjecting the primary recovery sample to a column comprising a suitable Protein A resin.
  • Protein A resin is useful for affinity purification and isolation of a variety of antibody isotypes, particularly IgGl, IgG2, and IgG4.
  • Protein A is a bacterial cell wall protein that binds to mammalian IgGs primarily through their Fc regions. In its native state, Protein A has five IgG binding domains as well as other domains of unknown function.
  • Protein A resin there are several commercial sources for Protein A resin.
  • One suitable resin is MabSeiectTM from GE Healthcare.
  • a non-limiting example of a suitable column packed with MabSeiectTM is an about 1.0 cm diameter x about 21.6 cm long column (-17 mL bed volume). This size column can be used for small scale purifications and can be compared with other columns used for scale ups. For example, a 20 cm x 21 cm column whose bed volume is about 6.6 L can be used for larger purifications. Regardless of the column, the column can be packed using a suitable resin such as MabSeiectTM.
  • Ion exchange separation includes any method by which two substances are separated based on the difference in their respective ionic charges, and can employ either cationic exchange material or anionic exchange material.
  • a cationic exchange material versus an anionic exchange material is based on the localized charges of the protein. Therefore, it is within the scope of this invention to employ an anionic exchange step prior to the use of a cationic exchange step, or a cationic exchange step prior to the use of an anionic exchange step.
  • the initial protein mixture can be contacted with the ion exchange material by using any of a variety of techniques, e.g., using a batch purification technique or a chromatographic technique.
  • Anionic or cationic substituents may be attached to matrices in order to form anionic or cationic supports for chromatography.
  • anionic exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine(Q) groups.
  • Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S).
  • Cellulose ion exchange resins such as DE23TM, DE32TM, DE52TM, CM-23TM, CM-32TM, and CM-52TM are available from Whatman Ltd. Maidstone, Kent, U.K.
  • SEPHADEX®-based and -locross-linked ion exchangers are also known.
  • DEAE-, QAE-, CM-, and SP- SEPHADEX® and DEAE-, Q-, CM-and S- SEPHAROSE® and SEPHAROSE® Fast Flow are all available from Pharmacia AB.
  • DEAE and CM derivitized ethylene glycol-methacrylate copolymer such as TOYOPEARLTM DEAE-650S or M and TOYOPEARLTM CM-650S or M are available from Toso Haas Co., Philadelphia, Pa.
  • Ultrafiltration is described in detail in: Microfiltration and Ultrafiltration: Principles and Applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York, N.Y., 1996); and in: Ultrafiltration Handbook, Munir Cheryan (Technomic Publishing, 1986; ISBN No. 87762-456-9).
  • a preferred filtration process is Tangential Flow Filtration as described in the Millipore catalogue entitled “Pharmaceutical Process Filtration Catalogue” pp. 177-202 (Bedford, Mass., 1995/96).
  • Ultrafiltration is generally considered to mean filtration using filters with a pore size of smaller than 0.1 ⁇ .
  • Diafiltration is a method of using ultrafilters to remove and exchange salts, sugars, and non-aqueous solvents, to separate free from bound species, to remove low molecular-weight material, and/or to cause the rapid change of ionic and/or pH environments.
  • Microsolutes are removed most efficiently by adding solvent to the solution being ultrafiltered at a rate approximately equal to the ultratfiltration rate. This washes micro species from the solution at a constant volume, effectively purifying the retained protein, in certain embodiments of the present invention, a diafiltration step is employed to exchange the various buffers used in connection with the instant invention, optionally prior to further chromatography or other purification steps, as well as to remove impurities from the protein preparations.
  • hydrophobic interaction chromatography it will be advantageous to subject a sample produced by the techniques of the instant invention to hydrophobic interaction chromatography in order to purify the protein of interest away from process-related impurities and/or product-related substances.
  • a first eluate obtained from an ion exchange column can be subjected to a hydrophobic interaction material such that a second eluate having a reduced level of impurity is obtained.
  • Hydrophobic interaction chromatography (HIC) steps such as those disclosed herein, are generally performed to remove protein aggregates, such as antibody aggregates, and process- related impurities.
  • the sample mixture is contacted with the HIC material, e.g., using a batch purification technique or using a column.
  • HIC purification it may be desirable to remove any chaotropic agents or very hydrophobic substances, e.g., by passing the mixture through a pre-column.
  • hydrophobic interaction chromatography uses the hydrophobic properties of the protein. Hydrophobic groups on the protein interact with hydrophobic groups on the column. The more hydrophobic a protein is the stronger it will interact with the column. Thus the HIC step removes host cell derived impurities (e.g., DNA and other high and low molecular weight product-related species).
  • Hydrophobic interactions are strongest at high ionic strength, therefore, this form of separation is conveniently performed following salt precipitations or ion exchange procedures.
  • Adsorption of the protein of interest to a HIC column is favored by high salt concentrations, but the actual concentrations can vary over a wide range depending on the nature of the protein and the particular HIC ligand chosen.
  • Various ions can be arranged in a so-called soluphobic series depending on whether they promote hydrophobic interactions (salting-out effects) or disrupt the structure of water (chaotropic effect) and lead to the weakening of the hydrophobic interaction.
  • Cations are ranked in terms of increasing salting out effect as Ba ; Ca ; Mg ; Li ; Cs ; Na ; K + ; Rb ; NH4 + , while anions may be ranked in terms of increasing chaotropic effect as P0 “"” ; S0 4 “” ; CH3CO3 “ ; CI “ ; Br “ ; N0 3 “ ; CIO4 “ ; I “ ; SCN “ .
  • Salts may be formulated that influence the strength of the interaction as given by the following relationship: ( H 4 ) 2 S0 4 > Na 2 S0 4 > NaCI > NH4CI > NaBr > NaSCN.
  • salt concentrations of between about 0.75 and about 2 M ammonium sulfate or between about 1 and 4 M NaCI are useful.
  • HIC columns normally comprise a base matrix (e.g., cross-linked agarose or synthetic copolymer material) to which hydrophobic ligands (e.g., alkyl or aryl groups) are coupled.
  • a suitable HIC column comprises an agarose resin substituted with phenyl groups (e.g., a Phenyl SepharoseTM column).
  • phenyl groups e.g., a Phenyl SepharoseTM column.
  • Many HIC columns are available commercially.
  • Examples include, but are not limited to, Phenyl SepharoseTM 6 Fast Flow column with low or high substitution (Pharmacia LKB Biotechnology, AB, Sweden); Phenyl SepharoseTM High Performance column (Pharmacia LKB Biotechnology, AB, Sweden); Octyl SepharoseTM High Performance column (Pharmacia LKB Biotechnology, AB, Sweden); FractogelTM EMD Propyl or FractogelTM EMD Phenyl columns (E. Merck, Germany); Macro-PrepTM Mehyl or Macro-PrepTM t-Butyl Supports (Bio-Rad, California); WP HI-Propyl (C3)TM column (J. T. Baker, New Jersey); and ToyopearlTM ether, phenyl or butyl columns (TosoHaas, PA).
  • Phenyl SepharoseTM 6 Fast Flow column with low or high substitution Pharmacia LKB Biotechnology, AB, Sweden
  • Multimodal chromatography is chromatography that utilizes a multimodal media resin.
  • a resin comprises a multimodal chromatography ligand.
  • a ligand refers to a ligand that is capable of providing at least two different, but co-operative, sites which interact with the substance to be bound. One of these sites gives an attractive type of charge- charge interaction between the ligand and the substance of interest. The other site typically gives electron acceptor-donor interaction and/or hydrophobic and/or hydrophilic interactions.
  • Electron donor- acceptor interactions include interactions such as hydrogen-bonding, ⁇ - ⁇ , cation- ⁇ , charge transfer, dipole-dipole, induced dipole etc.
  • Multimodal chromatography ligands are also known as "mixed mode" chromatography ligands.
  • the multimodal chromatography resin is comprised of multimodal ligands coupled to an organic or inorganic support, sometimes denoted a base matrix, directly or via a spacer.
  • the support may be in the form of particles, such as essentially spherical particles, a monolith, filter, membrane, surface, capillaries, etc.
  • the support is prepared from a native polymer, such as cross-linked carbohydrate material, such as agarose, agar, cellulose, dext an, chitosan, konjac, carrageenan, gellan, alginate etc.
  • the support can be porous, and ligands are then coupled to the external surfaces as well as to the pore surfaces.
  • Such native polymer supports can be prepared according to standard methods, such as inverse suspension gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964).
  • the support can be prepared from a synthetic polymer, such as cross-linked synthetic polymers, e.g. styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters, vinyl amides etc.
  • Such synthetic polymers can be produced according to standard methods, see e.g. "Styrene based polymer supports developed by suspension polymerization" (R Arshady: Chimica e L'Industria 70(9), 70-75 (1988)).
  • Porous native or synthetic polymer supports are also available from commercial sources, such as Amersham Biosciences, Uppsala, Sweden.
  • compositions suitable for administration to a subject can be incorporated into pharmaceutical compositions suitable for administration to a subject.
  • the pharmaceutical composition comprises a protein of the invention and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • isotonic agents e.g., sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion.
  • the protein compositions of the invention can be incorporated into a pharmaceutical composition suitable for parenteral admimstration.
  • the protein can be prepared as an injectable solution containing, e.g., 0.1-250 mg/mL antibody.
  • the injectable solution can be composed of either a liquid or lyophilized dosage form in a flint or amber vial, ampule or pre-filled syringe.
  • the buffer can be L-histidine approximately 1-50 mM, (optimally 5-10 mM), at pH 5.0 to 7.0 (optimally pH 6.0).
  • Other suitable buffers include but are not limited to sodium succinate, sodium citrate, sodium phosphate or potassium phosphate.
  • Sodium chloride can be used to modify the toxicity of the solution at a concentration of 0-300 mM (optimally 150 mM for a liquid dosage form).
  • Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%).
  • Other suitable cryoprotectants include trehalose and lactose.
  • Bulking agents can be included for a lyophilized dosage form, principally 1-10% mannitol (optimally 24%).
  • Stabilizers can be used in both liquid and lyophilized dosage forms, principally 1-50 mM L-methionine (optimally 5- 10 mM).
  • the pharmaceutical composition includes the protein at a dosage of about 0.01 mg kg-10 mg/kg.
  • the dosages of the protein include approximately 1 rag/kg administered every other week, or approximately 0.3 mg/kg administered weekly. A skilled practitioner can ascertain the proper dosage and regime for administering to a subject.
  • the compositions of this invention may be in a variety of forms.
  • liquid, semi-solid and solid dosage forms such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • liquid solutions e.g., injectable and infusible solutions
  • dispersions or suspensions tablets, pills, powders, liposomes and suppositories.
  • Typical compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies.
  • One mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • the protein is administered by intravenous infusion or injection, in another aspect, the protein is administered by intramuscular or subcutaneous injection.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration.
  • Sterile injectable solutions can be prepared by incorporating the active compound (i.e., protein, antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the methods of preparation are vacuum drying and spray-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, e.g., monostearate salts and gelatin.
  • the protein of the present invention can be administered by a variety of methods known in the art, one route/mode of administration is subcutaneous injection, intravenous injection or infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
  • the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed. ; Marcel Dekker, Inc., New York, 1978, the entire teaching of which
  • a protein of the invention may be orally administered, e.g., with an inert diluent or an assimilable edible carrier.
  • the compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet.
  • the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Supplementary active compounds can also be incorporated into the compositions.
  • a protein of the invention is co-formulated with and/or co-administered with one or more additional therapeutic agents that are useful for treating disorders.
  • an antibody or antibody portion of the invention may be co-formulated and/or co-administered with one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules).
  • one or more antibodies of the invention may be used in combination with two or more of the foregoing therapeutic agents.
  • Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.
  • the protein of the invention when used as part of a combination therapy, a lower dosage of protein may be desirable than when the protein alone is administered to a subject (e.g., a synergistic therapeutic effect may be achieved through the use of combination therapy which, in turn, permits use of a lower dose of the protein to achieve the desired therapeutic effect).
  • the protein of the invention can be used alone or in combination with an additional agent, e.g., a therapeutic agent, said additional agent being selected by the skilled artisan for its intended purpose.
  • the additional agent can be a therapeutic agent art-recognized as being useful to treat the disease or condition being treated by the protein of the present invention.
  • the additional agent also can be an agent which imparts a beneficial attribute to the therapeutic composition, e.g., an agent which effects the viscosity of the composition.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In certain embodiments it is especially 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 suited as unitary dosages for the mammalian subjects to be treated; each unit comprising a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of a protein of the invention is 0.01-20 mg/kg, or 1- 10 mg/kg, or 0.3-1 mg kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. 6. EXAMPLES
  • Production of recombinant proteins by host cells can result in product- related charge heterogeneities present in the population of proteins produced by the cells.
  • the presence of acidic species in the population of proteins is an example of a product-related charge heterogeneity.
  • Control of the amount of acidic species present in the population of proteins produced by the host cells can be accomplished by modifying the culture conditions of the host cells.
  • adalimumab producing cell lines Three adalimumab producing cell lines, one mAbl producing cell line and one mAb2 producing were employed in the studies covered here.
  • adalimumab producing cell lines cells were cultured in their respective growth media (chemically defined media (media 1) or a hydrolysate based media (media 2 or media 3)) in a combination of vented non-baffled shake flasks (Corning) on a shaker platform at 110 RPM (cell line 1),180 RPM (cell line 2), 140 RPM (cell line 3) and 10L or 20L wave bags (GE).
  • RPM cell line 1
  • GE 10L or 20L wave bags
  • mAbl producing cell line cells were cultured in chemically defined growth media (media 1) in a combination of vented non-baffled shake flasks (Corning) on a shaker platform at 130 RPM and 20L wave bags (GE). Cultures were propagated in a 36°C, 5% C0 2 incubator to obtain the required number of cells to be able to initiate production stage cultures.
  • media 1 chemically defined growth media
  • GE vented non-baffled shake flasks
  • Growth and production media were prepared from either a chemically defined media formulation (media 1) or hydrolysate-based medium formulations (media 2 and media 3).
  • media 1 the media (IVGN GIA-1, a proprietary basal media formulation from Invitrogen) was supplemented with L-glutamine, sodium bicarbonate, sodium chloride, and methotrexate solution.
  • Production media consisted of all the components in the growth medium, excluding methotrexate.
  • both growth and production medium were also supplemented with insulin.
  • the growth medium were also supplemented with insulin.
  • the growth media was composed of PFCHO (proprietary chemically defined formulation from SAFC), Dextrose, L-Glutamine, L-Asparagine, HEPES, Poloxamer 188, Ferric Citrate, Recombinant Human insulin, Yeastolate (BD), Phytone Peptone (BD), Mono- and Di-basic Sodium Phosphate, Sodium Bicarbonate, Sodium Chloride and methotrexate.
  • Production media consisted of all the components listed in the growth medium, excluding methotrexate.
  • the growth media was composed of OptiCHO (Invitrogen), L-Glutamine, Yeastolate (BD), Phytone Peptone (BD) and methotrexate. Production media consisted of all the components listed in the growth medium, excluding methotrexate.
  • Amino acids used for the experiments were reconstituted in Milli-Q water to make a lOOg/L stock solution, which was subsequently supplemented to both growth and production basal media. After addition of amino acids, media was brought to a pH similar to unsupplemented (control) media using 5N hydrochloric acid/5N NaOH, and it was brought to an osmolality similar to unsupplemented (control) media by adjusting the concentration of sodium chloride.
  • Calcium Chloride Dihydrate (Sigma or Fluka) used for the experiments were reconstituted in Milli-Q water to make a stock solution, which was subsequently supplemented to the production basal media. After addition of calcium chloride, media was brought to a pH similar to non-supplemented (control) media using 6N hydrochloric acid/5N NaOH, and it was brought to an osmolality similar to non- supplemented (control) media by adjusting the concentration of sodium chloride.
  • Niacinamide (Sigma or Calbiochem) used for the experiments were reconstituted in Milli-Q water to make a stock solution, which was subsequently supplemented to the production basal media. After addition of niacinamide, media was brought to a pH similar to non-supplemented (control) media using 6N hydrochloric acid/5N NaOH, and it was brought to an osmolality similar to non- supplemented (control) media by adjusting the concentration of sodium chloride. All media was filtered through Corning 1L filter systems (0.22 ⁇
  • Cultures were run in either batch or fed-batch mode. In the batch mode, cells were cultured in the respective production medium. 1.25% (v/v) of 40% glucose stock solution was fed when the media glucose concentration reduced to less than 3 g/L. In the fed-batch mode, cultures were run with either the IVGN feed (proprietary chemically defined feed formulation from Invitrogen) as per the following feed schedule - (4% (v/v) - day 6, day 7, and day 8, respectively) along with 10X Ex-Cell PFCHO feed (proprietary chemically defined formulation) - 3% (v/v) on day 3. The cultures were also fed with 1.25% (v/v) of 40% glucose stock solution when the glucose concentration was below 3.0 g/L
  • the mobile phases used were lOmM Sodium Phosphate dibasic pH 7.5 (Mobile phase A) and lOmM Sodium Phosphate dibasic, 500 mM Sodium Chloride pH 5.5 (Mobile phase B).
  • a binary gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100%B: 22-28 min; 94% A, 6% B: 28-34 min) was used with detection at 280 nm.
  • the mobile phases used were 20 mM (4- Acid Monohydrate (MES) pH 6.5 (Mobile phase A) and 20 mM MES, 500 mM Sodium Chloride pH 6.5 (Mobile phase B).
  • Quantitation is based on the relative area percent of detected peaks.
  • the peaks that elute at relative residence time earlier than the main peak corresponding to the drug product are together represented as the acidic peaks ( Figure 1). Lysine-C peptide mapping for MGO quantification
  • Typical trypsin digestion employed almost universally for peptide mapping cuts a denatured, reduced and alkylated protein at the carboxyl side of the two basic amino acids, lysine and arginine.
  • Methylglyoxal is a small molecule metabolite derived as a glycolysis byproduct which can modify arginine residues.
  • a modification of an arginine prevents trypsin from cutting this site and results in a mis- cleavage.
  • the challenge of qiiantifying the amount of MGO modified peptide is that it is not compared to an equivalent non-modified peptide but rather two parental cleaved peptides which will likely have different ionization potential than the modified peptide.
  • arginine was tested in several experimental systems covering multiple cell lines, media and monoclonal antibodies. Following is a detailed description of two representative experiments where two different adalimumab producing cell lines were cultured in a chemically defined media (media 1). Cell line 2 was cultured in media 1 with different total amounts of arginine (1 (control), 1.25, 1.5, 2, 3, 5, 9 g/1). The cultures were performed in shake flasks in batch format with only glucose feed as described in the materials and methods. The cells grew to maximum viable cell densities (VCD) in the range of 18- 22 x 10 6 cells/ml for the different conditions tested.
  • VCD maximum viable cell densities
  • Cell line 3 was cultured in media 1 with different total amounts of arginine (1 (control), 3, 5, 7, 9 g/1). The cultures were performed in shake flasks in batch format with only glucose feed as described in the materials and methods. The cells grew to maximum viable ceil densities (VCD) in the range of 7-10 x 10 6 cells/ml for the different conditions tested. The growth and viability profiles were comparable between the different test conditions, although a slight decrease in viable cell density and viability profiles was observed in samples with the 9 g/1 arginine condition ( Figure 6, 7). The product titer was also comparable between all conditions ( Figure 8).
  • lysine was tested in several experimental systems covering multiple cell lines, media and monoclonal antibodies. Following is a detailed description of two representative experiments where two different cell lines were cultured in a chemically defined media (media 1) for the production of adalimumab.
  • Cell line 2 was cultured in media 1 with different total concentrations of lysine (1 (control), 5, 7, 9, 11 g/1). The cultures were performed in shake flasks in batch format with only glucose feed as described in the materials and methods. The cells grew to maximum viable cell densities (VCD) in the range of 17-23 x 10 6 cells/ml for the different conditions tested. A slight dose dependent decrease in viable cell density profile was observed in all samples with respect to the control sample ( Figure 17). The viability profiles were comparable between the conditions ( Figure 1
  • the cultures were performed in shake flasks in batch format with only glucose feed as described in the materials and methods.
  • the cells grew to maximum viable cell densities (VCD) in the range of 9.5-1 1.5 x 10 6 cells/ml for the different conditions tested.
  • VCD viable cell densities
  • the growth and viability profiles were comparable between the different test conditions, although a slight decrease in viable cell density and viability profiles was observed in samples with higher lysine concentrations than that in the control sample ( Figure 21, 22).
  • On Days 10, 1 1 and 12 of culture samples were collected for titer analysis (Figure 23). The titers for all conditions were comparable.
  • protein A eluate samples from a representative set of lysine supplementation experiments were pre- treated with the enzyme carboxypeptidase before WCX-10.
  • One set of samples from adalimumab experiment and another set of samples from a mAB2 experiment were used for this analysis.
  • the carboxypeptidase treatment of the samples resulted in the cleavage of the C-terminal lysine residues as demonstrated by the conversion of Lysl/Lys2 to Lys 0 in each of these samples (data not shown here).
  • the acidic species quantified in these samples corresponded to an aggregate sum of acidic species that would be expected to also include those species that may have previously shifted corresponding to the lysine variant shift and perhaps gone unaccounted for in the samples that were not treated with carboxypeptidase prior to WCX-10.
  • a dose dependent reduction in acidic species was observed in the carboxypeptidase treated samples with increasing concentration of lysine for the adalimumab samples from 26.8% in the non-supplemented sample to 21.1% in the 10 g/1 Lysine supplemented sample, a reduction of 5.7% in total acidic species (Figure 30). Similar results were also observed for the mA2 samples ( Figure 31). This suggests that the acidic species reduction described here is not completely attributed to a probable shift of the acidic species corresponding to the lysine redistribution.
  • histidine was tested in several experimental systems covering multiple cell lines, media and monoclonal antibodies. Following is a detailed description of two representative experiments where two different cell lines were cultured in a chemically defined media (media 1) for the production of adalimumab.
  • Cell line 2 was cultured in media 1 with different total concentrations of histidine (0 (control), 4, 6, 8, 10 g/1). The cultures were performed in shake flasks in batch format with only glucose feed as described in the materials and methods. The cells grew to maximum viable cell densities (VCD) in the range of 12-22 x 10 6 cells/ml for the different conditions tested, A dose dependent decrease in viable cell density profile was observed with the lOg 1 histidine condition having significant reduction in growth ( Figure 32). A corresponding effect on viability was also observed ( Figure 33). On Days 10, 1 1 and 12 of culture samples were collected for titer analysis and reported for the harvest day for each sample ( Figure 34). There was a small dose dependent decrease in titers for conditions with histidine supplementation.
  • Cell line 3 was cultured in media 1 with different total concentrations of histidine (0 (control), 2, 4, 6, 8 g/1). The cultures were performed in shake flasks in batch format with only glucose feed as described in the materials and methods. The cells grew to maximum viable cell densities (VCD) in the range of 6-10 x 10 6 cells/ml for the different conditions tested. A dose dependent decrease in viable cell density profile was observed in all conditions with histidine concentrations higher than that in the control ( Figure 36). The viability profiles were more comparable between conditions with this cell line ( Figure 37). On Day 12 of culture, samples were collected for titer analysis ( Figure 38). The titers for all conditions were comparable.
  • VCD viable cell densities
  • ornithine was tested in several experimental systems covering multiple cell lines, media and monoclonal antibodies. Following is a detailed description of two representative experiments where two different cell lines were employed in a chemically defined media (media 1) for the production of adalimumab.
  • Cell line 2 was cultured in media 1 with different total concentrations of ornithine (0 (control), 4, 6, 8, 10 g/1). The cultures were performed in shake flasks in batch format with only glucose feed as described in the materials and methods. The cells grew to maximum viable cell densities (VCD) in the range of 15-22 x 10 6 cells/ml for the different conditions tested. A slight decrease in viable cell density with ornithine supplementation was observed ( Figure 47). Corresponding differences in the viability profiles were also observed ( Figure 48). On Day 11 of culture, samples were collected for titer analysis (Figure 49). The titers for all conditions were comparable.
  • VCD maximum viable cell densities
  • Cell line 3 was cultured in media 1 supplemented with different total concentrations of ornithine (0 (control), 2, 4, 6, 8 g 1). The cultures were performed in shake flasks in batch format with only glucose feed as described in the materials and methods. The cells grew to maximum viable cell densities (VCD) in the range of 9.5- 1 1.5 x 10 6 cells/ml for the different conditions tested. The viable cell density and viability profiles were comparable ( Figure 51, 52). On Day 12 of culture, samples were collected for titer analysis ( Figure 53).
  • the increase of the amino acid (arginine, lysine) concentration in basal media may also be combined with choice of when to harvest a culture to achieve optimal reduction in total acidic species.
  • a study was earned out in 3L bioreactors with cell line 1 (producing adalimumab) in media 1. Two sets of conditions were tested: Control condition (Arginine l /1, Lysine 1 g/1); Test condition 1 (Arginine 3 g 1, Lysine 5g/l). Cell growth, viability and titer profiles were comparable between the conditions ( Figure 63, 64, 65). A small amount of cell culture harvests were collected every day from day 4 to day 10 from each of the reactors and submitted for protein A purification and WCX-10 analysis.
  • the percentage of acidic species in the control condition increased from 12.1% (on day 4) to 24.6% (on daylO) ( Figure 66).
  • the percentage of acidic species in the test condition 1 was lower than that observed in the control condition at each corresponding culture day.
  • the percentage of acidic species in the test condition also increased from 8.7% (day 4) to 18.8% (day 10).
  • the rate of increase in acidic species with culture duration also correlated with the drop in viability for both conditions, with a sharp increase on day 8.
  • choice of harvest day/harvest viability can be used in combination to achieve a desired acidic species reduction.
  • the increase of the amino acid (arginine, lysine) concentration in basal media may be combined with process pH modulation to achieve further reduction in total acidic species.
  • a study was carried out in 3L bioreactors with cell line 1 (producing adalimumab) in media 1.
  • Three sets of conditions were tested in duplicates: Control condition (Arginine (lg 1), Lysine (lg 1), pH 7.1->6.9 in 3 days, pH 6.9 thereafter); Test condition 1 (Arginine (3g/l), Lysine (3g/l), pH 7.1->6.9 in 3 days, pH 6.9 thereafter); Test condition 2 (Arginine (3g/3), Lysine (3g/l), pH 7.1->6.8 in 3 days, pH 6.8 thereafter).
  • Cell line 3 was cultured in media 1 with different total concentrations of calcium (0.14, 0.49, 0.84, 1.19, 1.54, 1.89 g/1). The cultures were performed in shake flasks in batch format with only glucose feed as described in the materials and methods. The cells grew to maximum viable cell densities (VCD) in the range of 9.5- 10.5 x 10 6 cells/ml for the different conditions tested. The viable cell density and viability profiles for all test conditions were comparable ( Figure 75, 76). On Day 1 1 of culture, samples were collected for titer analysis. The harvest titers for all conditions were comparable ( Figure 77).
  • VCD viable cell densities
  • niacinamide addition may also be used independent of the other supplements as demonstrated in the experiments below for two mAbs: adalimumab and mAbl .
  • adalimumab Cell line 1 was cultured in media 1 supplemented with different amounts of niacinamide (0, 0.2, 0.4, 0.8 and 1.6 mM). The cultures were performed in shake flasks in hatch format with only glucose feed as described in the materials and methods. The cells grew to maximum viable cell densities (VCD) in the range of 8.5-11 x 10 6 cells/ml for the different conditions tested.
  • VCD viable cell densities
  • a mAB2 producing cell line was cultured in media 1 supplemented with different amounts of niacinamide (0, 0.1, 0.5, 1.0, 3.0 and 6.0 mM).
  • the cultures were performed in shake flasks in batch format with only glucose feed as described in the materials and methods.
  • the cells grew to maximum viable cell densities (VCD) in the range of 14-21.5 x 10 6 cells/ml for the different conditions tested.
  • VCD maximum viable cell densities
  • a slight decrease in the viable cell density profile was observed for the conditions with 3.0 mM and 6.0 mM niacinamide concentrations (Figure 90).
  • the viability profiles for all test conditions were comparable ( Figure 91). On Day 12 of culture samples were collected for titer analysis ( Figure 92).
  • Types of acidic species variants reduced by supplementation of culture medium with additives may be used to specifically reduce particular acidic variants within the larger fraction of total acidic species.
  • Table 4 a summary of the extent of some of the sub-species of the acidic species fraction have been presented for a representative set of experiments for adalimumab.
  • the methods presented in this section may also be used for reduction of sub-species that include, but not limited to, AR1, AR2 and MGO (methylglyoxal) modified product variants.
  • Table 4 Summary of types of acidic species variants reduced in cultures supplemented with medium additives
  • the different experiments above demonstrate that supplementation of cell culture medium with supplemental amounts of amino acids, calcium chloride and niacinamide enhances product quality by decreasing the amount of acidic species in the culture harvest.
  • the amino acids included in the study were arginine, lysine, ornithine and histidine and belong to group of amino acids that are basic,
  • the study covered examples from multiple cell lines/molecules, in shake flasks and bioreactors and in batch and fed-batch culture formats. A dose dependent effect in the extent of reduction of acidic species with increasing concentrations of the supplements was observed.
  • the possibility to supplement these medium additives individually or in suitable combinations for acidic species reduction was also demonstrated.
  • adalimumab producing CHO cell lines and a mAB2 producing cell line were employed in the studies covered here.
  • adalimumab producing cell line 3 was cultured in chemically defined growth media (media 1) in a combination of vented shake flasks on a shaker platform @140 rpm and 20L wave bags. Cultures were propagated in a 36°C, 5% C0 2 incubator to obtain the required number of cells to be able to initiate production stage cultures.
  • adalimumab producing cell line 1 was cultured in a hydrolysate based growth media (media 2) in a combination of vented shake flasks on a shaker platform @ 110 rpm and 20L wavebags in a 35°C, 5% C0 2 incubator.
  • the culture might be transferred into a seed reactor with pH 7.1, 35°C and 30% DO.
  • the culture would be adapted to either media lor media 2 by propagated in a lOL or 20L wavebag for 7 - 13 days with one or two passages before initiating production stage cultures.
  • mAb2 producing cells were cultured in media 1 in a combination of vented non-baffled shake flasks (Corning) on a shaker platform at 140 RPM and 20L wave bags (GE). Cultures were propagated in a 35°C, 5% C0 2 incubator to obtain the required number of cells to be able to initiate production stage cultures.
  • Cell culture media
  • Media 1 the chemical defined growth or production media, was prepared from basal IVGN CD media (proprietary formulation).
  • the proprietary media was supplemented with L- glutamine, sodium bicarbonate, sodium chloride, and methotrexate solution.
  • Production media consisted of all the components in the growth medium, excluding methotrexate.
  • the medium was also supplemented with insulin.
  • lOmM or 5mM of Galactose (Sigma, G5388) and 0.2 ⁇ or ⁇ of Manganese (Sigma, Ml 787) were supplemented into production medium for cell line 3 or 1, respectively. Osmolality was adjusted by the concentration of sodium chloride. All media was filtered through filter systems (0.22 ⁇ PES) and stored at 4°C until usage.
  • Media 2 is the hydrolysate based media, which contains basal proprietary media, Bacto TC Yeastolate and Phytone Peptone.
  • Production cultures were initiated in 3L Bioreactors (Applikon). The bioreactors (1.5-2.0L working volume) were run at the following conditions (except for the different experimental conditions): 35°C, 30% DO (dissolved oxygen), 200 rpm, pH profile from 7.1 to 6.9 in three days and pH 6.9 thereafter. In all experiments, the cells were transferred from the wavebag to the production stage at a split ratio of 1 :5.6 (except raAb2 with a ratio of 1 :5). When the media glucose concentration reduced to less than 3 g/L, approximately 1.25% (v/v) of 40% glucose stock solution was fed
  • the harvest procedure of reactors involved centrifugation of the culture sample at 3,000 RPM for 30 min and storage of supernatant in PETG bottles at -80°C before submission for protein A purification and WCX-10 analysis.
  • the WCX-10 method used for mAb B used different buffers.
  • the mobile phases used were 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).
  • Quantitation is based on the relative area percent of detected peaks.
  • the peaks that elute at relative residence time earlier than the main peak corresponding to the drag product are together represented as the acidic peaks.
  • the present study describes a process for reducing and controlling levels of acidic species in antibody preparations.
  • the invention provides a method for reducing the acidic variant content in clarified harvest, as well as a method for reducing the formation rate of acidic species in clarified harvest.
  • the method involves adding additives like various amino acids to clarified harvest or adjusting the pH of the clarified harvest using acidic substances.
  • Clarified harvest is liquid material containing a monoclonal antibody of interest that has been extracted from a fermentation bioreactor after undergoing centrifugation to remove large solid particles and subsequent filtration to remove finer solid particles and impurities from the material. Clarified harvest was used for low pH treatment studies described herein. Clarified harvest was also used for the experiments to study the effect of amino acid concentration on the presence of acidic species in clarified harvest., and for acid type- pH treatment studies described herein. Different batches of mAB-B and mAb-C clarified harvest material were employed for experiments to study the effect of amino acid and low pH treatment studies on the presence of acidic species described herein.
  • the clarified harvest material was first adjusted to pH 4 using 3M citric acid.
  • the material at pH 4 was then agitated for 60 minutes before adjusting the pH to a target pH of 5, 6 or 7 with 3M sodium hydroxide.
  • the material was then agitated for a further 60 minutes.
  • the samples were then subjected to centrifugation at 7300 x g for 15 minutes in a Sorvall Evolution RC with an SLA-3000 centrifuge bowl.
  • the supematants obtained from the centrifuged material were then depth filtered using B1HC depth filters (Millipore) followed by 0.22 ⁇ sterile filters.
  • the filtrates of different pH were then subjected to holding for different period of time for evaluating the formation rate of acidic variants.
  • the material to study the effect of arginine on acidic species was prepared in two ways. For lower target arginine concentrations of 5mM, lOmM, 30mM and lOOmM, they were made by adding the appropriate amount of 0.5M arginine stock buffer at pH 7 (pH adjusted with acetic acid) to attain the target arginine concentrations needed.
  • arginine concentrations For higher target arginine concentrations of 50mM, 100mM, 300mM, 500mM, 760mM, 1M and 2M, they were made by adding the appropriate amount of arginine (solid) to the samples to attain the target arginine concentrations, with subsequent titration to a final pH of 7 using glacial acetic acid. Arginine was adjusted to a final concentration of lOOmM using the two methods to determine if the method of preparation would result in different effects. For all the experiments, following the arginine addition, treated clarified harvests were held at room temperature for the indicated duration followed by purification with Protein A column and analysis of charge variants.
  • the material to study the effect of histidine was prepared with target concentrations of 5mM, lOmM, 30mM 50mM, l OOmM, 200mM and 250mM.
  • the samples were prepared by adding the appropriate amount of histidine (solid) to the samples to attain the target histidine concentrations, with subsequent titration to a final pH of 7 using glacial acetic acid.
  • the sample preparation scheme is shown in Figure 108.
  • the material to study the effect of Lysine was prepared with target concentrations of 5mM, lOmM, 30mM 50mM, lOOmM, 200mM, 300mM, 500mM and 1 OOOmM.
  • the samples were prepared by adding the appropriate amount of lysine hydrochloride (solid) to the samples to attain the target Lysine concentrations, with subsequent titration to a final pH of 7 using hydrochloric acid.
  • the sample preparation scheme is shown below in Figure 109.
  • the material to study the effect of methionine was prepared with target concentrations of 5mM, lOmM, 30mM 50mM, lOOmM, 200m and 300mM.
  • the samples were prepared by adding the appropriate amount of methionine (solid) to the samples to attain the target methionine concentrations, with subsequent titration to a final pH of 7 using glacial acetic acid.
  • the sample preparation scheme is shown in Figure 11.0.
  • the samples were prepared by adding the appropriate amount of amino acid (solid) to the samples to attain the target amino acid concentrations as shown in Table 6, with subsequent titration to a final pH of 7 using glacial acetic acid.
  • the sample preparation scheme is shown below in Figure 1 1 1.
  • the samples were prepared by adding the appropriate amount of additive to the samples to attain the target amino acid concentrations as show in Table 2, with subsequent titration to a final pH of 7 using glacial acetic acid.
  • the mAb B hydrolysate clarified harvest was used to study the effect of the aforementioned methods.
  • the mAb C hydrolysate clarified harvest was used to study the effect of the aforementioned methods.
  • the effects of acid type, clarified harvest pH and arginine content on acidic variant reduction were evaluated in this study.
  • the samples were prepared in triplicates on 3 consecutive days to target arginine concentrations of either OmM (no arginine added) or 500mM, then titrated with either glacial acetic acid, phosphoric acid, 3M citric acid or 6M hydrochloric acid to target pH values of either 5, 6 or 7.
  • One other sample was prepared by adding a 2M arginine acetate pH 7 stock buffer to clarified harvest to attain a target arginine concentration of 500mM,
  • the sample preparation scheme is shown in Figure 113.
  • Protein A purification of the samples was performed using a 5mL rProtein A FF Hitrap column (GE Healthcare) at lOg D2E7/L resin loading and a operating flow rate of 3.4mL/min. 5 column volumes (CVs) of equilibration (IX PBS pH 7.4) is followed by loading of the sample, then washing of the column with equilibration buffer to remove non-specifically bound impurities, followed by elution of the protein with 0.1M Acetic acid, 0.15M sodium chloride.
  • the eluate samples were collected and neutralized to pH 6.9-7.2 with 1M Tris pH 9.5 at 45-75 minutes after collection. The samples were then frozen at - 80°C for at least one day before thawing and subjecting to WCX-10 analysis.
  • Quantitation is based on the relative area percent of detected peaks.
  • the peaks that elute at relative residence time less than that of the dominant Lysine 0 peak are together represented as the acidic variant peaks (A ). 6.3.2 Results
  • Figure 115 shows that the low pH treatment with subsequent neutralization to pH 5 or 6 reduces the rate of acidic variant formation over time. However, there is no significant reduction in initial acidic variant content as shown in Figure 114.
  • Figure 1 16 and Figure 117 show the results of the arginine treatment.
  • Figure 116, 117 shows that the sample preparation method resulted in different levels of acidic species in clarified harvest. Adding a 0.5M Arginine pH 7 stock buffer tends to increase acidic species, while adding pure arginine with subsequent acetic acid titration to pH 7 reduced acidic variants at arginine concentrations of greater than lOOmM. Moreover, the effect due to treatment method is demonstrated when comparing the two lOOmM arginine samples, which show an absolute difference of 1% in acidic variants between the two methods.
  • Figure 1 18 shows that the rate of acidic variant formation decreases with increasing arginine concentration in clarified harvest, plateauing at around concentrations of 500mM arginine and higher.
  • the two methods of sample preparation does not result in significantly different formation rate of acidic variants.
  • Figure 120 Similar to arginine treatment effect, as shown in Figure 128, when histidine was added to clarified harvest with subsequent pH neutralization with acetic acid, acidic variants were reduced at histidine concentrations higher than 50mM. Figure 120 shows that the rate of acidic variants formation decreases with increasing Histidine concentration in clarified harvest, plateauing at around concentrations of 200mM Histidine and higher.
  • Figure 122 Similar to arginine treatment effect, as shown in Figure 128, when lysine was added to clarified harvest with subsequent pH neutralization with acetic acid, acidic variants were significantly reduced by ⁇ 1 % or more.
  • Figure 132 shows that the rate of acidic variants formation decreases with increasing lysine concentration in clarified harvest.
  • Figures 125 and 146 The results of the treatments with the various amino acids are summarized below in Figures 125 and 146. As shown in Figure 125, the addition of 14 amino acids including arginine, histidine, lysine and methionine resulted in lower amounts of acidic variant content in clarified harvest. The addition of sodium acetate or the use of acetic acid also caused a reduction in acidic variant content as well.
  • Figure 126 shows that the rate of acidic variants formation is reduced by several amino acids including arginine, histidine, lysine, aspartic acid, glutamic acid, and leucine.
  • Figures 136, 137, 138, and 139 The results obtained from the study are summarized below in Figures 136, 137, 138, and 139.
  • Figures 136, 137 indicate that when the acid used is of higher concentration, there is an decrease in acidic variant content in hydrolysate clarified harvest as compared to a lower concentration acid being used.
  • Figures 138, 139 show that when the clarified harvest is subjected to base neutralization to pH 7 after being treated with low pH, there is an increase in acidic variant content.
  • the figures also show that the Fractogel resin is better able to clear acidic variants than Mabselect Sure.
  • Antibody acidic species in clarified harvest can be reduced by adding additives such as arginine or histidine to clarified harvest at concentrations of more than lOOmM and 50mM, respectively. It can also be achieved by pH adjustment of the clarified harvest to pH 6 or pH 5. In addition, the rate of acidic variant formation can be reduced through the use of arginine or histidine in a concentration dependent manner, or by low pH treatment of the clarified harvest.
  • control or reduction in the amount of acidic species present in the population of proteins obtained at end of cell culture can be accomplished by modifying the exchange rate of fresh medium into the bioreactor (or removal of spent medium with product antibody out of the bioreactor).
  • adalimumab producing CHO cell line was employed in the study covered here.
  • the vial was cultured in a chemically defined growth media (media 1) in a series of vented shake flasks on a shaker platform at 110 rpm in a 35°C, 5% C0 2 incubator. Cultures were propagated to obtain a sufficient number of cells for inoculation of the perfusion cultibag.
  • the perfusion culture was carried out with the Sartorius BIOSTAT M 20 optical perfusion system (SN# 00582
  • the perfusion bag was run with a working culture volume of 1.5L and operation conditions of; pH: 7.00, dissolved oxygen 30%, 25 rpm, 35°C, an air overlay of 0.3 slpm and a C0 2 overlay of 15sccm. pH control was initiated on day three of the culture. pH was controlled with 0.5M sodium hydroxide and C0 2 additions.
  • Perfusion was carried out by 'harvesting' spent culture through an integrated 1.2 ⁇ filter integrated into the perfusion cultibag. Fresh media was added to the culture through a feed line at the same rate as the harvest. Perfusion began on day four of the process at a rate of 1.0 exchanges per day (ex/day). The perfusion rate was adjusted throughout the run to accommodate glucose needs, lactate accumulation and sampling plans. Perfusion cell-free harvest samples were collected at perfusion rates of 1.5, 3.0 and 6.0 exchange volumes/day on day 5-6 of perfusion. A fresh harvest bag was used for each harvest sample. The samples were then purified using protein A and analyzed using WCX- 10 assay.
  • the perfusi on culture was ended on day 8 of the process.
  • the mobile phases used were lOmM Sodium Phosphate dibasic pH 7.5 (Mobile phase A) and lOmM Sodium Phosphate dibasic, 500 mM Sodium Chloride pH 5.5 (Mobile phase B).
  • a binary gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100%B: 22-28 min; 94% A, 6% B: 28-34 min) was used with detection at 280 nm.
  • the WCX-10 method used for mAb2 samples used different buffers.
  • the mobile phases used were 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).
  • Adalimumab producing cell line 1 was cultured in media 1 and the cultures were carried out as described in the materials and methods. As described in table 9, the exchange rates were modified over a period of 24 hrs between day 5 and day 6 to explore the influence of medium exchange rates on the extent of acidic species. At a continuous medium exchange rate of 1.5 volumes/day, the product antibody in spent medium was collected in a harvest bag over a period of 17 hrs. The harvest bag was then exchanged with a new bag and the old bag was transferred to 4C. Subsequently and in succession, the medium exchange rates were increased to 3 and 6 volumes/day and the product harvest was collected over a time period of 5 and 2 hrs, respectively.
  • the current invention provides a method for reducing acidic species for a given protein of interest.
  • adalimumab was prepared using a combination of supplementation of arginine and lysine to cell culture as shown in this invention along with AEX and CEX purification technologies (described in the U.S. patent application having attorney reference no. 082254.0236) to produce a Low-AR and High-AR sample with a final AR of 2.5% and 6.9%, respectively. Both samples were incubated in a controlled environment at 25°C and 65% relative humidity for 10 weeks, and the AR measured every two weeks.
  • Figure 142 shows the growth of AR for each sample over the 10 week incubation.
  • Upstream and Downstream process technologies e.g., cell culture and chromatographic separations, of the inventions disclosed in the following applications can be combined together or combined with methods in the art to provide a final target AR value or achieve a %AR reduction, as well as to, in certain embodiments, reduce product related substances and/or process related impurities.
  • Upstream methods for AR reduction include, but are not limited to those described in the instant application.
  • Downstream methods for AR reduction include, but are not limited to, those described in the U.S. patent application having attorney reference no. 082254.0236.
  • Exemplary technologies disclosed in the referenced applications include, but are not limited to: cell culture additives & conditions; clarified harvest additives and pH/salt conditions; mixed mode media separations; anion exchange media separations; and cation Exchange media separations.
  • the instant example demonstrates the combined effect of one or more of these technologies in achieving a target AR value or AR reduction, thereby facilitating the preparation of an antibody material having a specific charge heterogeneity. Additional examples of combinations of downstream technologies and upstream technologies are provided in the referenced applications.
  • the combination of upstream and downstream methods involves the reduction of acidic species in 3L bioreactor cell cultures supplemented with arginine (2 g/1) and lysine (4g/l) as has been previously demonstrated in the instant application.
  • the results of that strategy are summarized in Table 10.
  • the total acidic species was reduced from 20.5% in the control sample to 10.2% in sample from cultures that were supplemented with the additives.
  • Adalimumab producing cell line 1 was cultured in media 1 (chemically defined media) supplemented with amino acid arginine (2g/l) and lysine (4 g/1) in a 300L bioreactor.
  • Adalimumab was purified by a Protein A chromatography step 0 followed with a low pH viral inactivation step.
  • the filtered viral inactivated material was buffer exchanged and loaded onto a Capto Adhere column.
  • the flowthrough of Capto Adhere material was then purified with a HIC column with bind/elute mode as well as Flow Through mode.
  • AR reduction was achieved primarily with MM step, with some contribution from other steps.
  • the table also 5 shows that additional product related substances such as aggregates and process related impurities such as HCP can be effectively reduced employing these combined technologies.
  • the MM method further reduced the AR levels, by 2.26%. Therefore upstream technologies for reduction can be combined with downstream technologies to achieve AR levels/ AR reduction.
  • patent applications designated by the following attorney docket numbers are incorporated herein by reference in their entireties for all purposes: 082254.0104; 082254.0236; 082254.0238; 082254.0242; and 082254.0243.

Abstract

La présente invention concerne le domaine de la production et de la purification des protéines et, en particulier, des compositions et des procédés qui permettent de réguler la quantité d'espèces acides exprimées par des cellules hôtes, ainsi que des compositions et des procédés qui permettent de réguler la quantité d'espèces acides présentes dans des préparations purifiées.
PCT/US2013/031485 2012-04-20 2013-03-14 Procédés de culture cellulaire pour réduire des espèces acides WO2013158275A1 (fr)

Priority Applications (10)

Application Number Priority Date Filing Date Title
BR112015017307A BR112015017307A2 (pt) 2013-03-14 2013-10-18 composições de espécies com baixa acidez e métodos para produção e uso das mesmas
CA2926384A CA2926384A1 (fr) 2013-03-14 2013-10-18 Compositions d'especes faiblement acides et leurs procedes de production et d'utilisation
SG11201504260UA SG11201504260UA (en) 2013-03-14 2013-10-18 Low acidic species compositions and methods for producing and using the same
KR1020157029562A KR20150129033A (ko) 2013-03-14 2013-10-18 저 산성 종 조성물 및 이의 제조 및 사용 방법
CA2899308A CA2899308C (fr) 2013-03-14 2013-10-18 Compositions d'especes d'adalimumab a faible teneur en acide et utilisations associees
EP13786072.2A EP2836515A1 (fr) 2013-03-14 2013-10-18 Compositions d'espèces faiblement acides et leurs procédés de production et d'utilisation
CA2926301A CA2926301A1 (fr) 2013-03-14 2013-10-18 Compositions d'especes faiblement acides et leurs procedes de production et d'utilisation
PCT/US2013/065749 WO2014158231A1 (fr) 2013-03-14 2013-10-18 Compositions d'espèces faiblement acides et leurs procédés de production et d'utilisation
AU2013384204A AU2013384204B2 (en) 2013-03-14 2013-10-18 Low acidic species compositions and methods for producing and using the same
HK15107319.2A HK1206755A1 (en) 2013-03-14 2015-07-30 Low acidic species compositions and methods for producing and using the same

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201261636511P 2012-04-20 2012-04-20
US61/636,511 2012-04-20

Publications (2)

Publication Number Publication Date
WO2013158275A1 WO2013158275A1 (fr) 2013-10-24
WO2013158275A9 true WO2013158275A9 (fr) 2013-12-19

Family

ID=48045064

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2013/031485 WO2013158275A1 (fr) 2012-04-20 2013-03-14 Procédés de culture cellulaire pour réduire des espèces acides

Country Status (1)

Country Link
WO (1) WO2013158275A1 (fr)

Families Citing this family (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2702077A2 (fr) 2011-04-27 2014-03-05 AbbVie Inc. Procédé de contrôle du profil de galactosylation de protéines exprimées de manière recombinante
WO2013158279A1 (fr) 2012-04-20 2013-10-24 Abbvie Inc. Procédés de purification de protéines pour réduire des espèces acides
US9067990B2 (en) 2013-03-14 2015-06-30 Abbvie, Inc. Protein purification using displacement chromatography
US9181572B2 (en) 2012-04-20 2015-11-10 Abbvie, Inc. Methods to modulate lysine variant distribution
US9249182B2 (en) 2012-05-24 2016-02-02 Abbvie, Inc. Purification of antibodies using hydrophobic interaction chromatography
US9512214B2 (en) 2012-09-02 2016-12-06 Abbvie, Inc. Methods to control protein heterogeneity
AU2013309506A1 (en) 2012-09-02 2015-03-12 Abbvie Inc. Methods to control protein heterogeneity
WO2014143205A1 (fr) 2013-03-12 2014-09-18 Abbvie Inc. Anticorps humains qui se lient au tnf-alpha et leurs procédés de préparation
US9017687B1 (en) 2013-10-18 2015-04-28 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same using displacement chromatography
US9499614B2 (en) 2013-03-14 2016-11-22 Abbvie Inc. Methods for modulating protein glycosylation profiles of recombinant protein therapeutics using monosaccharides and oligosaccharides
WO2014159579A1 (fr) 2013-03-14 2014-10-02 Abbvie Inc. Anticorps anti-tnfα ayant mutés et leurs procédés d'utilisation
WO2015051293A2 (fr) 2013-10-04 2015-04-09 Abbvie, Inc. Utilisation d'ions métalliques pour moduler les profils de glycosylation des protéines dans le cas de protéines recombinées
US8946395B1 (en) 2013-10-18 2015-02-03 Abbvie Inc. Purification of proteins using hydrophobic interaction chromatography
US9181337B2 (en) 2013-10-18 2015-11-10 Abbvie, Inc. Modulated lysine variant species compositions and methods for producing and using the same
US9085618B2 (en) 2013-10-18 2015-07-21 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
WO2015073884A2 (fr) 2013-11-15 2015-05-21 Abbvie, Inc. Compositions de protéines de liaison génétiquement glycomodifiées
EP3347049A4 (fr) * 2015-09-08 2019-01-30 Waters Technologies Corporation Méthode de chromatographie multidimensionnelle pour l'analyse de conjugués anticorps-médicament (cam)
WO2017122157A1 (fr) * 2016-01-14 2017-07-20 Dr. Reddy’S Laboratories Limited Procédé de culture cellulaire
GB201708655D0 (en) * 2017-05-31 2017-07-12 Ucb Biopharma Sprl Cell culture methods
US20210171901A1 (en) * 2017-11-16 2021-06-10 Life Technologies Corporation Streamlined methods for making liquid media
EP3492582A1 (fr) 2017-12-01 2019-06-05 UCB Biopharma SPRL Procédés de culture cellulaire
GB202012991D0 (en) 2020-08-20 2020-10-07 Ucb Biopharma Sprl Cell culture processes
EP4263597A2 (fr) * 2020-12-18 2023-10-25 Kiniksa Pharmaceuticals, Ltd. Compositions de protéines et leurs procédés de production et d'utilisation
GB202105424D0 (en) 2021-04-16 2021-06-02 UCB Biopharma SRL Cell culture processes
CN115521919B (zh) * 2022-11-04 2023-04-11 广州誉衡生物科技有限公司 一种调节pd-1抗体和lag-3抗体酸性电荷异构体的cho细胞培养方法

Family Cites Families (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USRE30985E (en) 1978-01-01 1982-06-29 Serum-free cell culture media
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4634665A (en) 1980-02-25 1987-01-06 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US5179017A (en) 1980-02-25 1993-01-12 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4510245A (en) 1982-11-18 1985-04-09 Chiron Corporation Adenovirus promoter system
US4560655A (en) 1982-12-16 1985-12-24 Immunex Corporation Serum-free cell culture medium and process for making same
US4657866A (en) 1982-12-21 1987-04-14 Sudhir Kumar Serum-free, synthetic, completely chemically defined tissue culture media
GB8308235D0 (en) 1983-03-25 1983-05-05 Celltech Ltd Polypeptides
US4534972A (en) 1983-03-29 1985-08-13 Miles Laboratories, Inc. Protein compositions substantially free from infectious agents
DD266710A3 (de) 1983-06-06 1989-04-12 Ve Forschungszentrum Biotechnologie Verfahren zur biotechnischen Herstellung van alkalischer Phosphatase
US4767704A (en) 1983-10-07 1988-08-30 Columbia University In The City Of New York Protein-free culture medium
US4879231A (en) 1984-10-30 1989-11-07 Phillips Petroleum Company Transformation of yeasts of the genus pichia
US5168062A (en) 1985-01-30 1992-12-01 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter-regulatory DNA sequence
GB8516415D0 (en) 1985-06-28 1985-07-31 Celltech Ltd Culture of animal cells
US4968615A (en) 1985-12-18 1990-11-06 Ciba-Geigy Corporation Deoxyribonucleic acid segment from a virus
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US4927762A (en) 1986-04-01 1990-05-22 Cell Enterprises, Inc. Cell culture medium with antioxidant
GB8610600D0 (en) 1986-04-30 1986-06-04 Novo Industri As Transformation of trichoderma
US5476996A (en) 1988-06-14 1995-12-19 Lidak Pharmaceuticals Human immune system in non-human animal
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
AU632065B2 (en) 1988-09-23 1992-12-17 Novartis Vaccines And Diagnostics, Inc. Cell culture medium for enhanced cell growth, culture longevity and product expression
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
EP0402226A1 (fr) 1989-06-06 1990-12-12 Institut National De La Recherche Agronomique Vecteurs de transformation de la levure yarrowia
US5427908A (en) 1990-05-01 1995-06-27 Affymax Technologies N.V. Recombinant library screening methods
GB9015198D0 (en) 1990-07-10 1990-08-29 Brien Caroline J O Binding substance
ATE185601T1 (de) 1990-07-10 1999-10-15 Cambridge Antibody Tech Verfahren zur herstellung von spezifischen bindungspaargliedern
US5122469A (en) 1990-10-03 1992-06-16 Genentech, Inc. Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins
ATE164395T1 (de) 1990-12-03 1998-04-15 Genentech Inc Verfahren zur anreicherung von proteinvarianten mit geänderten bindungseigenschaften
EP0575485A1 (fr) 1991-03-01 1993-12-29 Dyax Corp. Procede de developpement de mini-proteines de liaison
IE921169A1 (en) 1991-04-10 1992-10-21 Scripps Research Inst Heterodimeric receptor libraries using phagemids
DE4122599C2 (de) 1991-07-08 1993-11-11 Deutsches Krebsforsch Phagemid zum Screenen von Antikörpern
US6914128B1 (en) 1999-03-25 2005-07-05 Abbott Gmbh & Co. Kg Human antibodies that bind human IL-12 and methods for producing
AU2002309063B8 (en) 2001-05-11 2008-04-24 Kyowa Kirin Co., Ltd. Artificial human chromosome containing human antibody lambda light chain gene
GB201112429D0 (en) * 2011-07-19 2011-08-31 Glaxo Group Ltd Antigen-binding proteins with increased FcRn binding

Also Published As

Publication number Publication date
WO2013158275A1 (fr) 2013-10-24

Similar Documents

Publication Publication Date Title
US9683033B2 (en) Cell culture methods to reduce acidic species
US9708400B2 (en) Methods to modulate lysine variant distribution
WO2013158275A9 (fr) Procédés de culture cellulaire pour réduire des espèces acides
US9708399B2 (en) Protein purification using displacement chromatography
US9249182B2 (en) Purification of antibodies using hydrophobic interaction chromatography
US20130336957A1 (en) Novel purification of human, humanized, or chimeric antibodies using protein a affinity chromatography
US20120238730A1 (en) Integrated approach to the isolation and purification of antibodies
CA2932207A1 (fr) Isolation et purification d'anticorps au moyen de chromatographie d'affinite de proteine a
WO2014142882A1 (fr) Purification de protéine utilisant une chromatographie de déplacement
WO2022133191A2 (fr) Compositions de protéines et leurs procédés de production et d'utilisation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13713638

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13713638

Country of ref document: EP

Kind code of ref document: A1