CN116583536A - Method for producing protein - Google Patents

Abstract

The present disclosure provides a novel method of controlling the glycosylation profile of a protein during production. The present disclosure also provides a novel method for increasing protein production while controlling the glycosylation profile of the protein.

Description

Method for producing protein

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application No. 63/066,127, filed 8/14/2020, and U.S. provisional application No. 63/199,547, filed 1/2021, each of which is incorporated herein by reference in its entirety.

Reference to sequence Listing submitted electronically via EFS-WEB

The contents of the electronically submitted sequence listing of ASCII text files submitted with the present application (name: 3338_205PC02_seqlising_ST25. Txt; size: 25,078 bytes; and date of creation: 2021, 8, 10) are incorporated herein by reference in their entirety.

Background

The market for protein therapeutics has grown significantly and the pace of development has increased. However, maintaining desirable quality attributes (e.g., glycosylation) while maintaining quantity, reducing production costs, and providing production flexibility is a challenge to the industry. In order to continue to meet market demand, efficient production-scale production of protein therapeutics is required.

Glycosylation is one of the most abundant of all protein post-translational modifications (PTMs). It results from the addition of sugar residues to protein side chains to form glycoproteins. Mammalian glycoprotein oligosaccharides are usually composed of a limited number of monosaccharides, but their structure is very diverse, mainly because they often form complex branching patterns.

Glycosylation plays an important role in many specific biological functions including immune defense, fertilization, viral replication, parasitic infection, cell growth, inflammation and cell-cell adhesion. For pharmaceutical glycoproteins, glycosylation affects protein conformational stability, clearance, protection from proteolysis, and improves protein solubility. Since different glycoforms may have different biological properties, the ability to monitor and control glycosylation during production is critical to the quality of the biopharmaceutical molecule.

However, glycosylation naturally occurs as a degree of heterogeneity and can be affected by many different factors (e.g., expression system, process conditions, medium composition, feeding scheme, purification process, or any combination thereof). Thus, there is a need to improve the protein manufacturing process, including culturing and/or purification, while maintaining consistent glycosylation patterns of the protein.

Disclosure of Invention

The present disclosure relates to a method of increasing the yield of a protein and/or controlling the glycosylation of a protein during a protein production phase, the method comprising culturing cells capable of expressing the protein in a bioreactor under suitable conditions during a protein induction phase, wherein the suitable conditions comprise a pH set point between about 7.1 and about 7.2. In some aspects, the pH set point is about 7.15. In some aspects, the suitable conditions further include an initial temperature set point between about 35 ℃ and about 37 ℃, a second temperature set point between about 32 ℃ and about 34 ℃, and a third temperature set point between about 30 ℃ and about 32 ℃. In some aspects, the suitable conditions further comprise a temperature of at least about 0.5X10 ⁶ Individual cells/mL and about 1X10 ⁶ An initial Viable Cell Density (VCD) set point between individual cells/mL. In some aspects, the suitable conditions include: (a) An initial temperature set point of about 36 ℃, a second temperature set point of about 33 ℃, and a third temperature set point of about 31 ℃; (b) a pH setpoint of about 7.15; and (c) about 0.70X10 ⁶ Initial Viable Cell Density (VCD) set point of individual cells/mL.

The present disclosure relates to a method of controlling cell growth rate, cell viability, viable cell density, and/or cell titer to produce a protein, the method comprising culturing the cells in a bioreactor at a pH set point of about 7.15 during a protein induction phase. In some aspects, the suitable conditions further include: (i) An initial temperature set point of about 36.0 ℃ and a second temperature set point of less than about 36 ℃; (ii) An initial temperature set point of less than about 36.5 ℃ and a final temperature set point of about 31 ℃; or (iii) an initial temperature set point of less than about 36.5 ℃, a second temperature set point of about 33 ℃, and a final temperature set point of less than about 33 °c A temperature set point. In some aspects, the suitable conditions further comprise culturing the cells at an initial temperature set point of about 36 ℃, a second temperature set point of about 33 ℃, and a final temperature set point of about 31 ℃. In some aspects, the suitable conditions further comprise about 0.70X10 ⁶ Initial Viable Cell Density (VCD) set point of individual cells/mL.

The present disclosure also relates to methods of increasing yield of and/or controlling glycosylation of betaxolol during a protein production phase, comprising culturing cells capable of expressing betaxolol in a bioreactor under suitable conditions, wherein the suitable conditions comprise: (a) An initial temperature set point of about 36 ℃, a second temperature set point of about 33 ℃, and a third temperature set point of about 31 ℃; (b) a pH setpoint of about 7.15; and (c) about 0.70X10 ⁶ Initial Viable Cell Density (VCD) set point of individual cells/mL.

In some aspects, the suitable conditions further comprise a first feed time of about 80 hours. In some aspects, the third or final temperature set point occurs between about 204 hours and about 276 hours. In some aspects, the third or final temperature set point occurs about 204 hours, about 216 hours, about 228 hours, about 240 hours, about 252 hours, about 264 hours, or about 276 hours after the initial temperature set point. In some aspects, the third final temperature set point is about 31 ℃ and occurs after about 240 hours. In some aspects, the second temperature set point occurs between about 72 hours and about 168 hours. In some aspects, the second temperature set point occurs at about 72 hours, about 78 hours, about 84 hours, about 90 hours, about 96 hours, about 102 hours, about 108 hours, about 114 hours, about 120 hours, about 126 hours, about 132 hours, about 138 hours, about 144 hours, about 150 hours, about 156 hours, about 162 hours, or about 168 hours. In some aspects, the second temperature set point is about 33 ℃ after about 140 hours.

In some aspects, the conditions increase the protein yield by at least 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, or at least about 400% as compared to a reference method without the suitable conditions.

In some aspects, the suitable conditions further comprise manganese in the bioreactor. In some aspects, the manganese concentration is from about 1.6 parts per billion (ppb) to about 15ppb. In some aspects, the manganese concentration is from about 3ppb to about 6ppb.

In some aspects, the methods reduce the rate of cell growth. In some aspects, the methods control cell viability. In some aspects, the cell viability is displayed at about 10.0x10 ⁶ Individual cells/mL and about 15.0x10 ⁶ Average peak Viable Cell Density (VCD) between individual cells/mL. In some aspects, the method controls titer. In some aspects, the titer exhibits a final titer between about 1.50g/L and about 3.5 g/L. In some aspects, the titer exhibits a final titer of greater than about 2.00 g/L.

In some aspects, the methods control the glycosylation profile of the protein. In some aspects, the glycosylation profile includes one or more N-linked glycans. In some aspects, the glycosylation profile is measured during the protein production phase. In some aspects, the glycosylation profile is measured about every 1 day. In some aspects, the glycosylation profile is measured when the cell culture is harvested. In some aspects, the N-linked glycans comprise: G0F, G1F, G2F, S1G1F, S1G2F, S G2F or any combination thereof. In some aspects, the protein comprises a CTLA4 domain. In some aspects, the protein is a fusion protein. In some aspects, the fusion protein comprises an Fc portion. In some aspects, the protein is betanaproxen. In some aspects, the protein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID No. 5. In some aspects, the one or more N-linked glycans are located at one or more residues selected from Asn76, asn108, and/or Asn207 of the betanaproxen. In some aspects, the one or more N-linked glycans comprise sialic acid and have a molar ratio of NANA of from about 4 to about 10. In some aspects, the one or more N-linked glycans comprise sialic acid and have a molar ratio of NANA of from about 5 to about 9, from about 5.5 to about 8.5, from about 5.8 to about 6.7, from about 5.2 to about 7.5, from about 6 to about 8, from about 6.2 to about 7.4, or from about 5 to about 6. In some aspects, the NANA molar ratio is about 6.8. In some aspects, the glycosylation profile is analyzed via an N-linked carbohydrate profiling method. In some aspects, the glycosylation profile includes one or more O-linked glycans. In some aspects, the O-linked glycans are located at residues Ser129, ser130, ser136 and/or Ser 139.

In some aspects, the method is performed as a fed-batch culture process. In some aspects, glucose and/or galactose is supplemented into the feed medium in the bioreactor. In some aspects, the feed medium is added to the bioreactor periodically. In some aspects, the feed medium is added to the bioreactor about every 24 hours.

In some aspects, the method is performed as a perfusion process. In some aspects, the cell is a mammalian cell. In some aspects, the mammalian cell is a Chinese Hamster Ovary (CHO) cell. In some aspects, the mammalian cell is a CHO-K1 cell, a CHO-DXB11 cell, or a CHO-DG44 cell.

The disclosure also relates to a method of analyzing glycans of CTLA4-Fc fusion proteins, the method comprising measuring one or more N-linked glycans attached to one or more asparagine residues in CTLA4 proteins, wherein one of the glycans comprises G0F, G1F, G2F, S1G1F, S1G2F and/or S2G2F. In some aspects, the glycans are measured via ultra-high performance liquid chromatography-fluorescence detection (UPLC-FLR). In some aspects, the Fc domain of the CTLA4-Fc fusion protein is cleaved prior to the measurement. In some aspects, the Fc domain of the CTLA4-Fc fusion protein is not cleaved prior to the measurement.

In some aspects, the protein is produced by the methods described herein. In some aspects, the protein comprises a CTLA4-Fc fusion protein. In some aspects, the protein is betanaproxen. In some aspects, the cells are produced by the methods described herein. In some aspects, the cell is a mammalian cell. In some aspects, the cell is a Chinese Hamster Ovary (CHO) cell. In some aspects, the cell is a CHO-K1 cell, a CHO-DXB11 cell or a CHO-DG44 cell.

The present disclosure also relates to a bioreactor for producing a protein produced by the methods described herein. In some aspects, the bioreactor comprises the cells and cell culture media described herein, wherein the bioreactor is maintained at a pH of about 7.15. In some aspects, the bioreactor is maintained at: (a) An initial temperature set point of about 36 ℃, a second temperature set point of about 33 ℃, and a third temperature set point of about 31 ℃; (b) a pH setpoint of about 7.15; and (c) about 0.70X10 ⁶ Initial Viable Cell Density (VCD) set point of individual cells/mL. In some aspects, the cell culture medium further comprises manganese.

Drawings

Fig. 1A shows the operating parameters of three processes (process a, process B, and process X). Fig. 1B and 1C show further process parameters of process a, process B, process X.

FIG. 2 shows sialic acid (NANA) molar ratios of cell cultures measured during the 16 day upstream production period of culture.

Fig. 3A and 3B show full-scale and enlarged representative chromatograms of the N-glycan spectral analysis of the betanaproxen Reference Standard (RS) elution, with specific glycans (G0F, G1F, G2F, S1G1F and S2G 2F) labeled.

Fig. 4A and 4B show full-scale and enlarged representative chromatograms of the N-glycan spectral analysis of the betanaproxen RS elution, with the glycan S1G1F ("s1g1f_2") eluting into two peaks.

Fig. 5 shows an overview of the upstream production process of betaxolol.

Fig. 6A and 6B show exemplary structures of G0F (mannose-3-N-acetylglucosamine-4-fucose), G1F (mannose-3-N-acetylglucosamine-4-galactose-1-fucose), G2F (mannose-3-N-acetylglucosamine-4-galactose-2-fucose), S1G1F (monosialylated mannose-3-N-acetylglucosamine-4-galactose-1-fucose), S1G2F (monosialylated mannose-3-N-acetylglucosamine-4-galactose-2-fucose), S1G3F (monosialylated mannose-3-N-acetylglucosamine-4-galactose-3-fucose), and S2G2F (disialylated mannose-3-N-acetylglucosamine-4-galactose-2-fucose).

Detailed Description

The present disclosure relates to methods of increasing the yield of a protein in a cell while maintaining a desired property (e.g., glycosylation pattern) of the protein. In particular, the present disclosure shows the unexpected effect of pH set points during protein production in a bioreactor, especially in the protein induction phase. In some aspects, the method of increasing yield comprises culturing cells in a bioreactor during a protein induction phase under suitable conditions, including, but not limited to, adjusting temperature, setting pH, using a specific living cell density, or any combination thereof.

I.Definition of the definition

The term "and/or" as used herein is considered a specific disclosure of each of two specified features or components with or without the other. Thus, the terms "and/or" as used herein in terms such as "a and/or B" are intended to include "a and B", "a or B", "a" (alone), and "B" (alone). Also, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following aspects: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

It should be appreciated that any aspect described herein, whether described in the language "comprising," is also provided with other similar aspects described as "consisting of … …" and/or "consisting essentially of … ….

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For example, concise Dictionary of Biomedicine and Molecular Biology, juo, pei-Show, 2 nd edition, 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 3 rd edition, 1999, academic press; and Oxford Dictionary of Biochemistry And Molecular Biology, revisions, 2000, oxford university press, provide those skilled in the art with a general explanation of many of the terms used in this disclosure.

Units, prefixes, and symbols are all expressed in terms of their international units System (SI) acceptability. Numerical ranges include numbers defining the ranges. The headings provided herein are not limitations of the various aspects of the disclosure which can be had by reference to the specification as a whole. Accordingly, by referring to the specification in general, the terms defined immediately below are more fully defined.

The use of alternatives (e.g., "or") should be understood to mean either, both, or any combination thereof. As used herein, the indefinite article "a" or "an" is to be understood to mean "one or more" of any recited or enumerated component.

The term "about" or "consisting essentially of … …" refers to a value or composition that is within acceptable error limits for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" or "consisting essentially of … …" may mean within 1 or more than 1 standard deviation. Alternatively, "about" or "consisting essentially of … …" may mean a range of up to 20%. Furthermore, in particular with respect to biological systems or processes, the term may mean up to one order of magnitude of value or up to 5 times the value. When a particular value or composition is provided in the application and claims, unless otherwise indicated, it should be assumed that the meaning of "about" or "consisting essentially of … …" is within an acceptable error of that particular value or composition.

As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer and (where appropriate) fractional (e.g., one tenth and one hundredth) values of any integer within the recited range.

As used herein, the term "CTLA4 extracellular domain" refers to a protein domain comprising all or part of the amino acid sequence shown in SEQ ID No. 1 that binds to B7-1 (CD 80) and/or B7-2 (CD 86). In some aspects, the CTLA4 extracellular domain can comprise a polypeptide having an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 1. The CTLA4 extracellular domain is represented by the following sequence:

SEQ ID NO. 1[ CTLA4 extracellular domain ] MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD

As used herein, the terms "CTLA4-Ig" or "CTLA4-Ig molecule" or "CTLA4-Ig protein" or "CTLA4-Fc" are used interchangeably and refer to a protein molecule comprising at least a CTLA4-Ig polypeptide having a CTLA4 extracellular domain and an immunoglobulin constant region or portion thereof. In some aspects, for example, the CTLA4-Ig polypeptide comprises at least the amino acid sequence of SEQ ID NO. 2. In certain aspects, the CTLA4 extracellular domain and immunoglobulin constant region or portion thereof can be wild-type or mutated or modified. The mutated CTLA4-Ig polypeptide is a CTLA4-Ig polypeptide comprising a mutated CTLA4 extracellular domain. The mutated CTLA4Ig molecule comprises at least a mutated CTLA4-Ig polypeptide. In some aspects, the CTLA4 extracellular domain and immunoglobulin constant region or portion thereof can be mammalian (including human or mouse). In some aspects, the mutated CTLA4 extracellular domain can have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the CTLA4 extracellular domain set forth in any one or more of SEQ ID NOs 2, 3, 4, 5, 6, 7 or 8. The polypeptide may further comprise an additional protein domain. CTLA4-Ig molecules may refer to monomers of CTLA4-Ig polypeptides, and may refer to multimeric forms of the polypeptides, such as dimers, tetramers, hexamers, and the like (or other high molecular weight materials). CTLA4-Ig molecules are also able to bind CD80 and/or CD 86. Examples of CTLA4-Ig and fragments (e.g., betazepine) are shown in SEQ ID NOs 2, 3, 4, 5, 6, 7 and 8. In some aspects, the betanaproxen is a combination of SEQ ID NOs 2, 3, 4, 5, 6, 7 and 8.

SEQ ID NO:2[CTLA4 ^A29YL104E Ig amino acid sequence]

MGVLLTQRTLLSLVLALLFPSMASMAMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO. 3[ amino acids 25-383 of SEQ ID NO: 2]

MAMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO. 4[ amino acids 26-383 of SEQ ID NO: 2]

AMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO 5[ amino acids 27-383 of SEQ ID NO 2]

MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO. 6[ amino acids 25-382 of SEQ ID NO: 2]

MAMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO. 7[ amino acids 26-382 of SEQ ID NO: 2]

AMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO. 8[ amino acids 27-382 of SEQ ID NO: 2]

MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO 9[ CTLA4 extracellular domain with mutations A29Y and L104E ]

MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD

As used herein, the term "soluble CTLA4" means a molecule that can circulate in vivo or CTLA4 that does not bind to a cell membrane. For example, soluble CTLA4 can include CTLA4-Ig comprising CTLA4 extracellular region linked to Ig.

As used herein, the term "dimer" refers to a CTLA4-Ig protein or CTLA4-Ig molecule composed of two CTLA4-Ig polypeptides or monomers linked or joined together. The linkage between monomers or dimers may be non-covalent, or both (e.g., one or more disulfide bonds). The CTLA4-Ig protein or CTLA4-Ig molecule, which is composed of two identical monomers, is a homodimer. CTLA4-Ig homodimers also encompass molecules comprising two monomers that may differ slightly in sequence. Homodimers encompass dimers in which monomers linked together have substantially the same sequence. Monomers contained in homodimers have comparable structural homology. For example, the sequence differences may be due to N-terminal processing modifications of the monomers.

As used herein, the term "glutamate" is used interchangeably with "glutamic acid".

As used herein, asn76, asn108 (CTLA 4 region) and Asn207 (Fc region) refer to specific glycosylation sites present on a betanaproxen molecule. These markers correspond to asparagine 76, asparagine 108 and asparagine, respectively, and correspond to the residues in SEQ ID NO. 5. Typically, five-character codes are used to define carbohydrate structure categories, e.g., P2100, P2120, P2121, P3131, and P4142. With respect to this labeling scheme, the first character (P) in the code defines the released carbohydrate as an N-linked structure containing a trimannosyl core structure. The second character indicates the number of N-acetylglucosamine (GlcNAc) units attached to the core. The third character (0 or 1) indicates whether there is fucose (Fuc) attached to the first GlcNAc of the core. The fourth character indicates the amount of galactose (Gal) attached to the core. The fifth character indicates the number of SA (N-acetylneuraminic acid or N-glycolylneuraminic acid) attached to the carbohydrate. P2100 may also be denoted G0F. P2110 may also be denoted as G1F. P2120 may also be denoted G2F. P2121 may also be denoted S1G2F. P2122 may also be denoted S2G2F. P3131 may also be denoted S1G3F. P4142 may also be denoted S2G4F. Representative graphs of these glycans can be seen in fig. 6A and 6B.

As used herein, the term "purified" refers to a composition comprising a protein (e.g., CTLA4-Ig molecule) or a selected protein population (e.g., CTLA4-Ig molecule) that is removed from its natural environment (e.g., isolated) and is at least 90% free, 91% free, 92% free, 93% free, 94% free, 95% free, 96% free, 97% free, 98% free, 99% free, 99.5% free, or 99.9% free of other components of natural association, such as cellular material or culture medium. "purified" may also refer to a composition comprising a protein (e.g., CTLA4-Ig molecule) or a selected population of proteins (e.g., CTLA4-Ig molecule) that is removed from its natural environment and is at least 60%, 65%, 70%, 75%, 80% or 85% free of other components of natural association, such as cellular material or culture medium. For example, with respect to recombinantly produced proteins, such as CTLA4-Ig protein molecules, the term "purified" may also refer to compositions comprising proteins (e.g., CTLA4-Ig protein molecules) that are removed from their production environment such that the protein molecule is at least 90% free, 91% free, 92% free, 93% free, 94% free, 95% free, 96% free, 97% free, 98% free, 99% free, 99.5% free, or 99.9% free of protein molecules other than the polypeptide of SEQ ID NO:2 or mutant polypeptide of SEQ ID NO: 2. "purified" does not exclude mixtures of proteins (e.g., CTLA4-Ig molecules (e.g., dimers)) with other variant proteins (e.g., CTLA4-Ig molecules (e.g., tetramers)). "purified" does not exclude pharmaceutically acceptable excipients or carriers in combination with a protein (e.g., a CTLA4-Ig molecule) that has been removed from its natural environment.

As used herein, the term "large scale process" is used interchangeably with the term "industrial scale process". The term "culture vessel" is used interchangeably with "bioreactor", "reactor" and "tank". The bioreactor used on an industrial scale may be at least 2,000l, at least 5,000l, at least 10,000l, at least 15,000l, at least 20,000l, at least 25,000L or any size suitable for the large production scale required for the production of industrial supplies.

"liquid culture" refers to cells (e.g., bacterial, plant, insect, yeast, or animal cells) grown on a support or grown in suspension in a liquid nutrient medium.

"seed culture" refers to a cell culture grown for use in inoculating a larger volume of medium. Seed cultures may be used to inoculate a larger volume of medium in order to expand the number of cells grown in the culture (e.g., cells grown in suspension).

As used herein, the terms "medium" and "cell culture medium" and "feed medium" and "fermentation medium" refer to a nutrient solution used to grow and or maintain cells, particularly mammalian cells. Without limitation, these solutions typically provide at least one component from one or more of the following classes: (1) Energy sources, typically in the form of carbohydrates such as glucose; (2) All essential amino acids, and typically a basic set of twenty amino acids plus cysteine; (3) Vitamins and/or other organic compounds required at low concentrations; (4) free fatty acids or lipids, such as linoleic acid: and (5) trace elements, wherein trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations (typically in the micromolar range). The nutrient solution may be optionally supplemented with one or more components from any of the following classes: (1) Hormones and other growth factors such as serum, insulin, transferrin and epidermal growth factor; (2) salts such as magnesium, calcium and phosphate; (3) buffers such as HEPES; (4) Nucleosides and bases such as adenosine, thymidine and hypoxanthine; (5) Proteins and tissue hydrolysates, such as peptones or peptone mixtures obtainable from purified gelatin, plant material or animal by-products; (6) antibiotics such as gentamicin; (7) Cytoprotective agents such as pluronic polyols (pluronic polyols); and (8) galactose. Commercially available media such as Ham's F (Sigma), minimal essential media ((MEM), (Sigma)), RPMI-1640 (Sigma) and Du's modified eagle Medium ((DMEM), (Sigma)) are suitable for culturing host cells. In addition, any of the media described in the following documents may be used as the medium for the host cells: ham et al, meth.Enz.58:44 (1979), barnes et al, anal.biochem.102:255 (1980). Any other necessary supplements may also be included in the appropriate concentrations.

As used herein, "culturing" refers to growing one or more cells in vitro under defined or controlled conditions. Examples of culture conditions that may be defined include temperature, gas mixture, time and medium formulation.

As used herein, "expansion" refers to the purpose of culturing one or more cells in vitro for the purpose of obtaining a greater number of cells in culture.

As used herein, "temperature set point" refers to a temperature setting of a bioreactor or other upstream processing vessel for growing cells and/or producing protein products. The temperature set point may be established at the beginning of the cell culture, in which case it may also be referred to as the "initial temperature set point". Sequence numbers (i.e., second temperature set point or subsequent third temperature set point) may be used to refer to subsequent temperature changes after the initial temperature set point during cell culture. The final temperature set point prior to downstream processing may also be referred to as the "final temperature set point". In some cases, the process may include an initial temperature set point, a second temperature set point, and a third (and final) temperature set point.

As used herein, unless otherwise indicated, "set point" refers to an initial condition setting of a bioreactor or other upstream processing vessel for growing cells and/or producing protein products. The set point is established at the beginning of cell culture. Subsequent changes in conditions after the set point during cell culture may occur due to changes in cell culture medium conditions during growth. For example, the set point may be a pH set point. In some aspects, the set point is a temperature set point. In some aspects, the set point may be maintained throughout the cell culture process. In other aspects, the set point may be maintained until a different set point is set. In other aspects, the set point may be changed to another set point.

As used herein, "population" refers to a group of two or more molecules ("population of molecules") or cells ("population of cells") characterized by the presence or absence of one or more measurable or detectable characteristics. In a homogeneous population, the molecules or cells in the population are characterized by the same or substantially the same characteristics (e.g., cells of a clonal cell line). In a heterogeneous population, a molecule or cell in the population is characterized by at least one identical or substantially identical property, wherein the cell or molecule can also exhibit a non-identical property (e.g., a protein population CTLA4-Ig molecule having a substantially similar average sialic acid content but having a dissimilar mannose content).

As used herein, "high molecular weight aggregates" are used interchangeably with "high molecular weight species" or "HMW" and refer to CTLA4-Ig molecules comprising at least three CTLA4-Ig monomers. For example, the high molecular weight aggregates can be tetramers, pentamers, or hexamers.

As used herein, "protein a" refers to a protein of about 42kDa that binds very strongly to the Fc portion of an immunoglobulin, and its use in antibody purification is well known in the art. Protein a has been widely used in the art for purification. (Boyle et al, 1993; hou et al, 1991). When applied to protein a, the term "residual" or "rPA" refers to any remaining protein a present in the mixture due to its use in further upstream purification of the protein or antibody of interest in the manufacturing process.

As used herein, "protein yield" or "yield" refers to the total amount of a protein of interest harvested from a culture process, i.e., a bioreactor cell culture process. "protein yield" or "yield" may also refer to the total amount of protein of interest recovered after the methods disclosed herein, and may be measured by mass (i.e., grams).

As used herein, "titer" or "protein titer" refers to the concentration of a protein of interest in a solution. For example, a measurement of the concentration of the protein of interest in an upstream culture process (i.e., bioreactor) is indicative of the protein titer at that time. Titer can be measured in a fixed volume in a concentration form (e.g., mg/ml).

As used herein, "glycosylation content" refers to the amount of N-linked or O-linked sugar residues that are covalently attached to a protein molecule (e.g., glycoprotein, like CTLA4-Ig molecule).

As used herein, "glycosylation" refers to the addition of complex oligosaccharide structures to a protein at specific sites within a polypeptide chain. Glycosylation of proteins and subsequent processing of added carbohydrates can affect the folding and structure of the protein, the stability of the protein (including the half-life of the protein), and the functional properties of the protein. Protein glycosylation can be divided into two classes, depending on the sequence context in which the modification occurs: o-linked glycosylation and N-linked glycosylation. The O-linked polysaccharide is linked to hydroxyl groups, typically to the hydroxyl groups of serine or threonine residues. O-glycans were not added to each serine and threonine residue. The O-linked oligosaccharides are typically mono-or bi-antennary, i.e. they comprise one or at most two branches (antennary) and comprise one to four different kinds of sugar residues, which are added one by one. The N-linked polysaccharide is attached to the asparagine at the amide nitrogen. Only asparagine, which is part of one of two tripeptide sequences (asparagine-X-serine or asparagine-X-threonine (where X is any amino acid other than proline)), is the target of glycosylation. The N-linked oligosaccharides may have one to four branches, known as mono-antennary, di-antennary, tri-antennary, tetra-antennary. The structure of the sugar residues found in the N-linked oligosaccharides and the O-linked oligosaccharides are different. Despite this difference, the terminal residues on each branch of the N-linked polysaccharide and the O-linked polysaccharide can be modified by sialic acid molecules, a modification known as sialic acid termination. Sialic acid is a common name for a unique family of nine-carbon monosaccharides, which can be linked to other oligosaccharides. Two family members are N-acetylneuraminic acid (abbreviated as Neu5Ac, neuAc or NANA) and N-glycolylneuraminic acid (abbreviated as Neu5Gc or NGNA). The most common form of sialic acid in humans is NANA. N-acetylneuraminic acid (NANA) is the predominant sialic acid species present in CTLA4-Ig molecules. However, it should be noted that small but detectable levels of N-glycolylneuraminic acid (NGNA) are also present in CTLA4-Ig molecules. Furthermore, the methods described herein can be used to determine the moles of sialic acid for both NANA and NGNA, thus determining and reporting the levels of both NANA and NGNA for CTLA4-Ig molecules. N-linked oligosaccharides and O-linked oligosaccharides have different numbers of branches that provide different numbers of positions to which sialic acid molecules can be attached. The N-linked oligosaccharides may provide up to four sialic acid attachment sites, while the O-linked oligosaccharides may provide two sialic acid attachment sites.

As used herein, the term "molar ratio of sialic acid to protein" or "MR" is calculated and given in moles of sialic acid molecule per mole of protein (CTLA 4-Ig molecule) or dimer.

As used herein, the term "glycoprotein" refers to a protein that is modified by the addition of one or more carbohydrates, including the addition of one or more sugar residues.

As used herein, the term "sialylation" refers to the addition of sialic acid residues to proteins (including glycoproteins).

As used herein, the term "glycoprotein subtype" refers to a molecule characterized by its carbohydrate and sialic acid content, as determined by isoelectric focusing (IEF) gel electrophoresis or other suitable method, to distinguish between different proteins in a mixture by their molecular weight, charge, and/or other characteristics. For example, each distinct band observed on an IEF gel represents a molecule having a specific isoelectric point (pI) and therefore the same net total charge. Glycoprotein subtypes can be distinct bands observed on IEF gels, where each band can be a population of molecules with a specific pI.

The term "β polypeptide" as used herein refers to a mutant CTLA4-Ig polypeptide that (1) comprises the amino acid sequence of SEQ ID No. 1 (wherein the amino acid at position 29 is mutated to tyrosine and position 10 4 to glutamic acid, optionally with various additional mutations) and an immunoglobulin constant region or a portion thereof: and (2) is capable of binding to CD80 and/or CD 86. In some aspects, for example, the β polypeptide comprises at least CTLA4 ^A29YL104E The amino acid sequence of the extracellular domain of Ig (as shown in SEQ ID NO: 9). Non-limiting examples of beta polypeptides include betanaproxen and SEQ ID NO. 2-8. In certain aspects, the immunoglobulin constant region or portion thereof may be wild-type or mutated or modified. In certain aspects, the immunoglobulin constant region or portion thereof may be mammalian (including human or mouse). Further non-limiting examples of beta polypeptides include beta 0 polypeptides comprising one or more amino acid mutations in an immunoglobulin constant region or a portion thereof (e.g., substitution of cysteine 120 of SEQ ID NO: 2), and further mutated beta polypeptides comprising one or more of amino acid positions 25, 30, 93, 96, 103, or 105 of SEQ ID NO: 1. The beta polypeptide molecule comprises a beta polypeptide. Beta polypeptide molecules may refer to monomeric and multimeric forms of beta polypeptides, such as dimers, tetramers, hexamers, and the like. For example, betanaproxen comprises a beta polypeptide molecule. Beta polypeptide molecules are further described in U.S. patent No. 10,508,144 issued at 12/17 2019, which is hereby incorporated by reference in its entirety.

"efficacy" refers to a measure of response as a function of ligand concentration. For example, agonist potency is quantified as the concentration of ligand that produces half the maximum effect (EC ₅₀ ). Non-limiting pharmacological definition of potency includes affinity and components of potency, where potency is the ability of a drug to elicit a response once bound. Efficacy is related to affinity, but efficacy and affinity are different measures of drug action.

As used herein, "pharmaceutically acceptable carrier" refers to a vehicle for a pharmacologically active agent. The carrier aids in delivering the active agent to the target site without terminating the function of the agent. Non-limiting examples of suitable forms of carrier include solutions, creams, gels, gel emulsions, jellies (jellies), pastes, lotions, ointments, sprays, ointments, powders, solid mixtures, aerosols, emulsions (e.g., water-in-oil or oil-in-water), aqueous solutions of gels, aqueous solutions, suspensions, liniments, tinctures, and patches suitable for topical application.

As used herein, the phrase "pharmaceutically acceptable composition" (or "pharmaceutical composition") refers to a composition that is acceptable for pharmaceutical administration (e.g., administration to a human). Such compositions may include, in addition to any one or more active agents, substances that are impurities at levels not exceeding those acceptable for pharmaceutical administration (such levels including the absence of such impurities), and may include pharmaceutically acceptable excipients, vehicles, carriers, and other inactive ingredients, e.g., to formulate such compositions for administration. For example, a pharmaceutically acceptable CTLA4-Ig composition can include MCP-1 or DNA, so long as the agents are at a level acceptable for administration to a human.

"drug substance" is an active pharmaceutical ingredient contained in a pharmaceutical composition. The term "drug substance" includes active pharmaceutical ingredients in solution and/or buffered form. A "pharmaceutical product" is a pharmaceutical composition containing a drug substance formulated for pharmaceutical administration. For purposes of assays contained in examples and elsewhere herein where drug substances and/or drug products may be mentioned, exemplary drug substances and drug products that may be assayed are as follows.

Exemplary pharmaceutical products of CTLA4Ig molecules include:

composition of lyophilized CTLA4-Ig protein (250 mg/vial) drug product

Component (A)	Quantity (mg/vial)
		CTLA4-Ig proteins	262.5
Maltose monohydrateArticle (B)	525
		Sodium dihydrogen phosphate monohydrate	18.1
Sodium chloride	15.3
		Hydrochloric acid	Adjust to pH 7.5
Sodium hydroxide	Adjust to pH 7.5

As used herein, the term "seeding" refers to the addition of cells to a culture medium to initiate culture.

As used herein, the term "induction" or "induction phase" or "growth phase" of a cell culture refers to the initial inoculation of a bioreactor at the beginning of an upstream cell culture and includes an exponential cell growth phase (e.g., log phase) in which cells divide primarily rapidly. During this phase, the rate of increase of viable cell density was higher than at any other time point.

As used herein, the term "production phase" of a cell culture refers to the period of time during which cell growth is quiescent or maintained at a near constant level. The density of living cells remains approximately constant over a given period of time. Log cell growth has ceased and protein production is the primary activity during the production phase. The medium at this point is typically supplemented to support continued protein production and to obtain the desired glycoprotein product.

As used herein, the term "expression" or "expression" is used to refer to transcription and translation occurring within a cell. The expression level of the product gene in the host cell may be determined based on the amount of the corresponding mRNA present in the cell or the amount of the protein encoded by the product gene produced by the cell or both.

As used herein, "N-linked glycans" refer to protein modifications in which glycans are linked to glycoconjugates via nitrogen linkages. The receptor for glycans is a selected asparagine residue of the polypeptide chain that enters the periplasm or ER lumen, respectively. Oligosaccharide transferases (central enzymes of the N-glycosylation pathway) catalyze the formation of N-glycosidic linkages of oligosaccharides with the side-chain amides of asparagine residues designated by the consensus sequence N-X-S/T. All eukaryotic N-glycans share a common core sequence Manα1-3 (Manα1-6) Manβ1-4GlcNAcβ1-Asn-X-Ser/Thr and are classified into three types: (1) oligomannose type, wherein only Man residues extend the core; (2) A complex wherein GlcNAc initiates an "antennary" extended core; and (3) heterozygous, wherein Man extends the Manα1-6 arm of the core and one or both GlcNAc extends the Manα1-3 arm.

As used herein, "N-linked glycosylation" refers to the attachment of an oligosaccharide to a nitrogen atom (typically N4 of an asparagine residue). N-glycosylation can occur on secreted or membrane-bound proteins, mainly in eukaryotes and archaebacteria. A detailed review of biosynthetic pathways and enzymes for the production of N-linked Glycans (e.g., high mannose oligosaccharides) is described in Stanley et al, "N-glycanes" in Essentials of Glycobiology, editions Varki, cummings, and Eskho, cold Spring Harbor Press,2009.

As used herein, the terms "fed-batch", "fed-batch culture" or "fed-batch culture process" refer to a method of culturing cells in which additional components are provided to the culture at some time after the start of the culture process. The fed-batch culture may be started using a basal medium. The medium that provides the culture with additional components at some time after the start of the culture process is a feed medium. The fed-batch culture is usually stopped at some point and the cells and/or components in the medium are harvested and optionally purified.

As used herein, "perfusion" or "perfusion culture process" refers to a continuous flow of physiological nutrient solution through or past a population of cells at a steady rate. Since perfusion systems generally involve retention of cells within the culture unit, perfusion culture typically has a relatively high cell density, but culture conditions are difficult to maintain and control. In addition, since cells are grown to a high density and remain in the culture unit at the high density, the growth rate generally decreases continuously over time, resulting in a late exponential phase or even a resting phase of cell growth. Such continuous culture strategies typically involve culturing mammalian cells (e.g., anchorage-independent cells) during the production phase in a continuous cell culture system, expressing the polypeptide and/or virus of interest.

As used herein, the term "mass spectrometry" refers to a sensitive technique for detecting, identifying, and quantifying molecules based on their mass-to-charge ratio (m/z). The electric field is used to separate ions according to their mass-to-charge ratio (m/z), integer ratio of mass to charge (z), as they pass along the central axes of parallel and equidistant poles or rods, such as quadrupoles, which comprise four poles or rods. Each rod is applied with two voltages, one of which is a fixed direct current and the second of which is an alternating current circulating at superimposed radio frequencies. The magnitude of the applied electric field may be ordered such that only ions having a particular m/z ratio can pass through the quadrupole and then be detected. Ions with all other m/z values are deflected onto the trajectories, which will cause them to collide with quadrupole rods and discharge, or ejected from the mass analyzer field and removed via vacuum. Quadrupoles are commonly referred to as dedicated detectors because only ions with a particular m/z are stable in the quadrupole at any time. Those ions having a stable trajectory are generally referred to as having a non-collisional, resonant or stable trajectory.

Typically, for experiments on triple quadrupole mass spectrometers, the first quadrupole (Q1) is set to pass only ions of a given m/z (precursor ions) of the desired chemical species in the sample. The second quadrupole (i.e., Q2 or collision cell) is used to fragment ions passing through Q1. The third quadrupole (Q3) is set to pass only ions (fragment ions) having a specified m/z corresponding to the expected fragmentation products of the expected chemical species to the detector. In some aspects, the sample is ionized in a mass spectrometer to generate one or more protonated or deprotonated molecular ions. In some aspects, one or more of the protonated or deprotonated molecules are singly charged, doubly charged, tri-charged, or higher. In some aspects, the mass spectrometer is a triple quadrupole mass spectrometer. In some aspects, the resolution for Q1 and Q3 is a unit resolution. In other aspects, the resolution for Q1 and Q3 is different. In other aspects, the resolution for Q1 is higher than the unit resolution of Q3.

As used herein, the term "fluorophore" refers to a fluorescent compound that can re-emit light upon excitation by light. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several pi bonds. Two commonly used fluorophores are 2-AB (2-aminobenzamide) and 2-AA (anthranilic acid or 2-aminobenzoic acid). Other fluorophores include PA (2-aminopyridine), AMAC (2-aminoacridine ketone), ANDS (7-amino-1, 3-naphthalene disulfonic acid), ANTS (8-amino naphthalene-1, 3, 6-trisulfonic acid), APTS (9-amino pyrene-1, 4, 6-trisulfonic acid), and 3- (acetamido) -6-aminoacridine.

As used in this disclosure, "glycan profile" is understood to be any defined set of values that can be used for quantitative results of glycans compared to a reference value or profile derived from another sample or group of samples. For example, the glycan profile of a sample from a protein sample may be significantly different from the glycan profile of a sample from an alternative source. Glycan profile can help predict or expect a Pharmacodynamic (PD) or Pharmacokinetic (PK) therapeutic effect of a protein by comparing the profile to a reference or standard profile. The reference and sample glycan spectra may be generated by any analytical instrument capable of detecting glycans, such as mass spectrometry. The one or more N-glycans may be galactose (Gal), N-acetylgalactosamine (GalNAc), galactosamine (GalN), glucose (Glc), N-acetylglucosamine (GlcNAc), glucosamine (GlcN), mannose (Man), N-acetylmannosamine (ManNAc), mannosamine (ManN), xylose (Xyl), N-acetylneuraminic acid (Neu 5 Ac), N-glycolylneuraminic acid (Neu 5 Gc), 2-keto-3-deoxypelargonic acid (Kdn), fucose (Fuc), glucuronic acid (GlcA), iduronic acid (IdoA), galacturonic acid (GalA), mannuronic acid (ManA), or any combination thereof.

As used herein, the phrase "one or more working solutions" refers to solutions used in the process. Non-limiting examples of working solutions include buffers.

As used herein, "reference material" refers to a material that is used as a standard in a method. For example, a reference material may be used as a standard against which an experimental sample is compared.

As used herein, "reference method" or "reference process" refers to a process or method that uses the same method (except for the conditions used for process X) to produce the same protein. For example, the reference process or method may be the process described in example 1 (process a), example 1B (process B) and/or example 1C (process C). As used herein, a reference method may be used as a baseline against which the methods of the present disclosure are compared.

The absence of such a substance is considered without providing a lower limit to the range of amounts of the substance.

As used herein, the temperatures recited with respect to cell culture refer to temperature settings on an instrument that regulates bioreactor temperature. Of course, the temperature of the liquid culture itself will be the temperature set on the instrument regulating the temperature of the bioreactor. Where temperature refers to a cell culture maintained on a shelf in an incubator, then temperature refers to the shelf temperature of the incubator.

II method for improving protein production

The present disclosure provides methods of improving the protein production phase in a cell culture system to increase the yield of protein. Thus, the methods disclosed herein are capable of improving cell culture performance, i.e., any desired improvement in cell culture performance as a result of the methods of the invention. For example, enhanced cell culture performance includes, but is not limited to, any one or more of the following: increased protein production; protein titer increases; increased cell specific productivity; an increase in maximum cell density; the high molecular weight material is reduced; the monomer material increases; cell viability is enhanced; precipitation in the medium and/or CDFM was reduced: overall product quality enhancement as determined by, for example, glycosylation profile and size exclusion chromatography: and overall batch-to-batch consistency enhancement. In some aspects, the methods comprise culturing cells capable of expressing the protein in a bioreactor under suitable conditions in a protein induction phase and/or a protein production phase, wherein the suitable conditions include, but are not limited to, adjustment of an initial temperature set point, adjustment of a second temperature set point, adjustment of a final temperature set point, adjustment of feed time, adjustment of a pH set point, adjustment of initial cell density, or any combination thereof. In some aspects, the disclosure provides methods of increasing protein production in a cell comprising culturing the cell in a bioreactor under suitable conditions during a protein induction phase, wherein the suitable conditions comprise a pH set point of about 7.15.

The methods of the present disclosure can be used to create reactor conditions that increase protein production. In some aspects, the conditions increase the protein yield by at least 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, or at least about 400% as compared to a reference method that does not employ the suitable conditions, wherein the suitable conditions comprise adjusting the pH set point to, for example, about 7.15. In some aspects, the suitable conditions further include modulation of initial viable cell density (e.g., 0.70X10 ⁶ Individual cells/mL), and/or adjustments of the initial, second, and final temperature set points (e.g., temperature set points of 36 ℃, 33 ℃, and 31 ℃). In some aspects, the conditions increase protein production by at least 150%. In some aspects, the conditions increase protein production by at least 160%. In some aspects, the conditions increase protein production by at least 170%. In some aspects, the conditions increase protein production by at least 180%. In some aspects, the conditions provide for protein production At least 190% higher. In some aspects, the conditions increase protein production by at least 200%. In some aspects, the conditions increase protein production by at least 210%. In some aspects, the conditions increase protein production by at least 220%. In some aspects, the conditions increase protein production by at least 230%. In some aspects, the conditions increase protein production by at least 240%. In some aspects, the conditions increase protein production by at least 250%. In some aspects, the conditions increase protein production by at least 260%. In some aspects, the conditions increase protein production by at least 270%. In some aspects, the conditions increase protein production by at least 280%. In some aspects, the conditions increase protein production by at least 290%. In some aspects, the conditions increase protein production by at least 300%. In some aspects, the conditions increase protein production by at least 310%. In some aspects, the conditions increase protein production by at least 320%. In some aspects, the conditions increase protein production by at least 330%. In some aspects, the conditions increase protein production by at least 340%. In some aspects, the conditions increase protein production by at least 350%. In some aspects, the conditions increase protein production by at least 360%. In some aspects, the conditions increase protein production by at least 370%. In some aspects, the conditions increase protein production by at least 380%. In some aspects, the conditions increase protein production by at least 390%. In some aspects, the conditions increase protein production by at least 400%.

In some aspects, the methods increase protein production by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold as compared to a reference method (e.g., a pH set point of about 7.15) that does not employ suitable conditions.

In some aspects, suitable conditions include adjustment of the pH set point (to, for example, about 7.15), adjustment of the initial viable cell density (for example 0.70X10) ⁶ Individual cells/mL) and/or adjustment of initial, second, and final temperature set points (e.g., 36 ℃, 33 ℃ c)And a temperature set point of 31 ℃), wherein the method increases protein production by about 2-fold to about 10-fold, for example about 2-fold to about 5-fold, about 3-fold to about 5-fold, or about 2-fold to about 4-fold. In some aspects, the methods of the invention increase protein production by about 2-fold to about 3-fold. In some aspects, the methods of the invention increase protein production by about 3-fold to about 4-fold. In some aspects, the methods of the invention increase protein production by about 4-fold to about 5-fold. In some aspects, the methods of the invention increase protein production by about 5-fold to about 6-fold. In some aspects, the methods of the invention increase protein production by about 6-fold to about 7-fold. In some aspects, the methods of the invention increase protein production by about 7-fold to about 8-fold. In some aspects, the methods of the invention increase protein production by about 8-fold to about 9-fold.

The methods of the present disclosure may also be used to increase the total protein output as part of the total host cell protein, thereby achieving greater protein production per batch. In some aspects, the methods of the present disclosure can also be used to increase the overall protein output with a desired glycosylation pattern based on changes in the residence time of the protein in the golgi apparatus and exposure to the glycosylase. In some aspects, the conditions increase protein production of a protein having a desired glycosylation profile by at least 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, or at least about 400% as compared to a reference method that does not employ the suitable conditions; such as adjustment of the pH set point (e.g., pH of about 7.15), adjustment of the initial viable cell density (e.g., 0.70X10) ⁶ Individual cells/mL) and/or adjustments of the initial, second, and final temperature set points (e.g., temperature set points of 36 ℃, 33 ℃, and 31 ℃).

The methods of the present disclosure can also be used to control viable cell density or peak viability during protein productionCell density. In some aspects, "controlling the viable cell density or peak viable cell density" means maintaining the viable cell density within certain ranges in cells/mL. In some aspects, cell viability is displayed at about 5x10 ⁶ Individual cells/mL to about 21x10 ⁶ Average peak Viable Cell Density (VCD) between individual cells/mL. In some aspects, cell viability is displayed at about 6x10 ⁶ Individual cells/mL to about 20x10 ⁶ Average peak Viable Cell Density (VCD) between individual cells/mL. In some aspects, cell viability is displayed at about 7x10 ⁶ Individual cells/mL to about 19x10 ⁶ Average peak Viable Cell Density (VCD) between individual cells/mL. In some aspects, cell viability is displayed at about 8x10 ⁶ Individual cells/mL to about 18x10 ⁶ Average peak Viable Cell Density (VCD) between individual cells/mL. In some aspects, cell viability is displayed at about 9x10 ⁶ Individual cells/mL to about 17x10 ⁶ Average peak Viable Cell Density (VCD) between individual cells/mL. In some aspects, cell viability is displayed at about 10x10 ⁶ Individual cells/mL to about 16x10 ⁶ Average peak Viable Cell Density (VCD) between individual cells/mL. In some aspects, cell viability is displayed at about 10x10 ⁶ Individual cells/mL to about 15x10 ⁶ Average peak Viable Cell Density (VCD) between individual cells/mL. In some aspects, cell viability is displayed at about 11x10 ⁶ Individual cells/mL to about 15x10 ⁶ Average peak Viable Cell Density (VCD) between individual cells/mL. In some aspects, cell viability is displayed at about 12x10 ⁶ Individual cells/mL to about 14x10 ⁶ Average peak Viable Cell Density (VCD) between individual cells/mL.

In some aspects, the cell viability exhibits about 6x10 ⁶ Average peak Viable Cell Density (VCD) of individual cells/mL. In some aspects, the cell viability exhibits about 8x10 ⁶ Average peak Viable Cell Density (VCD) of individual cells/mL. In some aspects, the cell viability exhibits about 10x10 ⁶ Average peak Viable Cell Density (VCD) of individual cells/mL. In some aspects, the cell viability exhibits about 10.5x10 ⁶ Average peak Viable Cell Density (VCD) of individual cells/mL. In some aspects, the cell viability exhibits about 11x10 ⁶ Average peak Viable Cell Density (VCD) of individual cells/mL. In some aspects, the cell viability exhibits about 11.5x10 ⁶ Average peak Viable Cell Density (VCD) of individual cells/mL. In some aspects, the cell viability exhibits about 12x10 ⁶ Average peak Viable Cell Density (VCD) of individual cells/mL. In some aspects, the cell viability exhibits about 12.5x10 ⁶ Average peak Viable Cell Density (VCD) of individual cells/mL. In some aspects, the cell viability exhibits about 13x10 ⁶ Average peak Viable Cell Density (VCD) of individual cells/mL. In some aspects, the cell viability exhibits about 13.5x10 ⁶ Average peak Viable Cell Density (VCD) of individual cells/mL. In some aspects, the cell viability exhibits about 14x10 ⁶ Average peak Viable Cell Density (VCD) of individual cells/mL. In some aspects, the cell viability exhibits about 14.5x10 ⁶ Average peak Viable Cell Density (VCD) of individual cells/mL. In some aspects, the cell viability exhibits about 15x10 ⁶ Average peak Viable Cell Density (VCD) of individual cells/mL.

The methods of the present disclosure may also be used to control and/or monitor protein titers. The titer can be measured at one or more time points (i.e., during the protein production phase or the protein induction phase) after culturing the cells in the bioreactor for a period of time (to induce protein production in the bioreactor). Titer can also be measured at the time of harvest of the culture prior to downstream processing. In some aspects, the titer is measured during the protein induction phase at least about one week after the start of the protein induction phase. In some aspects, during the protein induction phase, the titer is measured at least about 10 days, at least about two weeks, or at least about three weeks after starting the protein induction phase. In some aspects, the titer is measured after a period of about 14 days from the start of the protein induction phase. In some aspects, the titer exhibits an average day 14 titer between about 1.5g/L and about 3.5 g/L. In some aspects, the titer exhibits an average day 14 titer between about 1.5g/L and about 3 g/L. In some aspects, the titer exhibits an average day 14 titer between about 2g/L and about 3 g/L. In some aspects, the titer exhibits an average day 14 titer between about 2g/L and about 2.5 g/L. In some aspects, the titer exhibits an average day 14 titer between about 2.5g/L and about 3 g/L. In some aspects, the titer exhibits an average day 14 titer of about 2 g/L. In some aspects, the titer exhibits an average day 14 titer of about 2.5 g/L. In some aspects, the titer exhibits an average day 14 titer of about 3 g/L. In some aspects, the titer exhibits an average day 14 titer of about 3.5 g/L.

In some aspects, the cell capable of expressing a protein (e.g., a recombinant protein) is a mammalian cell. In some aspects, the cell is a eukaryotic cell. In some aspects, the cell is a mammalian cell. In some aspects, the cells are selected from the group consisting of Chinese Hamster Ovary (CHO) cells, HEK293 cells, mouse myeloma (NS 0), baby hamster kidney cells (BHK), monkey kidney fibroblasts (COS-7), motor-bison bovine kidney cells (Madin-Darby bovine kidney cell, MDBK), and any combination thereof. In one aspect, the cell is a chinese hamster ovary cell. In some aspects, the cell is an insect cell, such as a spodoptera frugiperda (Spodoptera frugiperda) cell. In other aspects, the cell is a mammalian cell. Such mammalian cells include, but are not limited to CHO, VERO, BHK, hela, MDCK, HEK T3, W138, BT483, hs578T, HTB2, BT2O and T47D, NS0, CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, hss78Bst, HEK-293T, hepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, BMT10 and HsS Bst cells. In some aspects, the mammalian cell is a CHO cell. In some aspects, the CHO cell is CHO-DG44, CHOZN, CHO/dhfr-, CHOK1SV GS-KO or CHO-S. In some aspects, the CHO cell is CHO-DG4. In some aspects, the CHO cell is CHOZN. Other suitable CHO cell lines disclosed herein include CHO-K (e.g., CHO K1), CHO pro3-, CHO P12, CHO-K1/SF, DUXB11, CHO DUKX, PA-DUKX, CHO pro5, DUK-BII or derivatives thereof.

IIA.pH

In some aspects, the methods of the present disclosure relate to improving or controlling protein production by changing the pH of the process. In some aspects, the methods involve changing the pH set point of the protein production phase during cell culture. The regulation of intracellular pH is a fundamental physiological process that is important for cell growth and metabolism. Since intracellular pH has a broad influence on the transport of nutrients and hormones and enzymatic reactions in cells, cells use a large amount of energy for cytoplasmic pH regulation. In addition, pH plays a role in the rate and spectrum of glycosylation of proteins produced by cells. The present disclosure provides unexpected effects on protein yield and/or protein glycosylation by controlling pH set points in the protein production stage.

In some aspects, the pH set point useful in the present disclosure is between about 7.1 and about 7.2. In some aspects, the pH set point is about 7.15. In some aspects, the pH set point is 7.25. In some aspects, the pH set point is 7.15. In some aspects, the pH set point is 7.05. In some aspects, the pH set point is 7.0. In some aspects, the pH set point is 6. In some aspects, the pH set point is 6.1. In some aspects, the pH set point is 6.2. In some aspects, the pH set point is 6.3. In some aspects, the pH set point is 6.4. In some aspects, the pH set point is 6.5. In some aspects, the pH set point is 6.6. In some aspects, the pH set point is 6.7. In some aspects, the pH set point is 6.8. In some aspects, the pH set point is 6.9.

IIB temperature

The temperature of the production vessel (e.g., bioreactor) may be another important aspect of biological production, as the temperature of the bioreactor plays a role in cell growth, viable cell density, cell life, and/or glycosylation activity of the glycosylase within the cell. Temperature changes can significantly affect the rate of intracellular enzymatic reactions, denature proteins, and/or have other effects on cell culture. For example, cells can be cultured at an initial temperature set point (e.g., 37 ℃) to promote maximum viable cell density, and then the temperature can be modified to another temperature set point (i.e., a second temperature set point or a final temperature set point) to extend cell life or enhance desired glycosylation activity within the cell. One or more temperature set points may be used during various stages of the upstream production process to improve the overall cell density, protein yield, or protein glycosylation profile of the protein of interest. In some aspects, the methods involve one or more temperature adjustments during protein production. The temperature adjustment may be a decrease in operating temperature during the manufacturing process. The temperature adjustment may also be an increase in operating temperature during the manufacturing process. In some aspects, the methods of the present disclosure use at least one, at least two, at least three, or at least four temperature adjustments during the manufacturing process.

The methods of the present disclosure also relate to controlling cell growth rate, cell viability, viable cell density, and/or cell titer to produce a protein. The initial temperature set point is important to create reactor conditions that favor cell expansion and cell growth during the logarithmic growth phase. After the initial log phase, the cell expansion conditions are reduced using a second temperature set point that is lower than the initial set point to prevent overgrowth of the cell culture that would result in undesirable cell density and subsequent loss of total cell viability. In some aspects, the methods of the present disclosure involve culturing cells in a bioreactor at an initial temperature set point of about 36 ℃ during the induction phase, then culturing cells at a second temperature set point of 33 ℃, and finally culturing cells at a final temperature set point of 31 ℃. In some aspects, the methods of the present disclosure use at least two, at least three, at least four, or at least five temperature setpoints, e.g., an initial temperature setpoint, a final setpoint, or more setpoints after the initial temperature setpoint but before the final setpoint. In some aspects, the methods of the present disclosure use at least three temperature setpoints, namely, an initial temperature setpoint, a second temperature setpoint, and a final temperature setpoint.

In some aspects, the initial temperature set point of the inventive process is about 37 ℃ and the second temperature set point is less than about 36 ℃. In some aspects, the initial temperature set point is about 36 ℃ and the second temperature set point is lower than the first temperature set point, e.g., about 35 ℃, about 34 ℃, about 33 ℃, about 32 ℃, or about 31 ℃. In some aspects, the initial temperature set point is about 37 ℃ and the second temperature set point is less than about 34 ℃. In some aspects, the initial temperature set point is about 36 ℃ and the second temperature set point is less than about 35 ℃, about 34 ℃, or about 33 ℃. In some aspects, the initial temperature set point is less than about 36.5 ℃ and the final temperature set point is about 31 ℃. In some aspects, the initial temperature set point is about 36.0 ℃ and the final temperature set point is about 31 ℃. In some aspects, the initial temperature set point is less than about 35.5 ℃ and the final temperature set point is about 31 ℃. In some aspects, the initial temperature set point is less than about 35.0 ℃ and the final temperature set point is about 31 ℃. In some aspects, the initial temperature set point is less than about 36.5 ℃, the second temperature set point is about 33 ℃, and the final temperature set point is less than about 33 ℃ or about 32 ℃. In some aspects, the initial temperature set point is about 36.0 ℃, the second temperature set point is about 33 ℃, and the final temperature set point is less than about 33 ℃ or about 32 ℃. In some aspects, the initial temperature set point is about 36.0 ℃, the second temperature set point is about 33 ℃, and the final temperature set point is less than about 32 ℃. In some aspects, the initial temperature set point is about 36.0 ℃, the second temperature set point is about 33 ℃, and the final temperature set point is about 31 ℃.

The method of the present disclosure may include an initial temperature set point, a second temperature set point, and a third and/or final set point. The initial, second, and third and/or final temperature set points are used to further control steady state cell density, control the percentage of living cells, manage cell cycle division of the culture, and/or alter the glycosylation rate and glycosylation profile of the produced protein during the manufacturing process. In some aspects, the third temperature set point is lower than the second temperature set point. In some aspects, the initial temperature set point is a temperature between 34 ℃ and 37 ℃, e.g., 34 ℃, 35 ℃, 36 ℃, or 37 ℃: the second temperature set point is a temperature between 32 ℃ and 34 ℃, e.g., 32 ℃, 33 ℃, or 34 ℃: and a final temperature set point is a temperature between 30 ℃ and 32 ℃, e.g., 30 ℃, 31 ℃, or 32 ℃, wherein the second temperature set point is lower than the first temperature set point and the final temperature set point is lower than the second temperature set point. In some aspects, the initial temperature set point is a temperature between 34 ℃ and 37 ℃, e.g., 34 ℃, 35 ℃, 36 ℃, or 37 ℃: the second temperature set point is a temperature between 32 ℃ and 34 ℃, e.g., 32 ℃, 33 ℃, or 34 ℃: and the final temperature set point is a temperature between 30 ℃ and 32 ℃, e.g., 30 ℃, 31 ℃, or 32 ℃, wherein the first temperature set point is not 37 ℃, the second temperature set point is not 34 ℃, and/or the final temperature set point is not 32 ℃.

The method of the present disclosure may include an initial temperature set point, a second temperature set point, a third temperature set point, optionally a fourth temperature set point, optionally a fifth temperature set point, and optionally a sixth temperature set point. These fourth, fifth and sixth temperature setpoints are used to further control steady state cell density, control the percentage of living cells, manage cell cycle division of the culture and/or alter the glycosylation rate and glycosylation profile of the produced protein during the manufacturing process. In some aspects, the method further comprises setting an optional fourth temperature set point, an optional fifth temperature set point, an optional sixth temperature set point, wherein the optional fourth temperature set point, the fifth temperature set point, and/or the sixth temperature set point is lower than the third temperature set point. In some aspects, the method further comprises setting an optional fourth temperature set point, an optional fifth temperature set point, and an optional sixth temperature set point, wherein the optional fourth temperature set point, the fifth temperature set point, and/or the sixth temperature set point is higher than the third temperature set point. In some aspects, the fourth, fifth, or sixth temperature set point, the fifth temperature set point, and/or the sixth temperature set point is about 30 ℃, about 31 ℃, about 32 ℃, about 33 ℃, about 34 ℃, about 35 ℃, about 36 ℃, about 37 ℃, about 38 ℃, about 39 ℃, or about 40 ℃. In some aspects, the fourth, fifth, or sixth temperature set point, the fifth temperature set point, and/or the sixth temperature set point is about 30 ℃. In some aspects, the fourth, fifth, or sixth temperature set point is about 31 ℃. In some aspects, the fourth, fifth, or sixth temperature set point is about 32 ℃. In some aspects, the fourth, fifth, or sixth temperature set point is about 33 ℃. In some aspects, the fourth, fifth, or sixth temperature set point is about 34 ℃. In some aspects, the fourth, fifth, or sixth temperature set point is about 35 ℃. In some aspects, the fourth, fifth, or sixth temperature set point is about 36 ℃. In some aspects, the fourth, fifth, or sixth temperature set point is about 37 ℃. In some aspects, the fourth, fifth, or sixth temperature set point is about 38 ℃. In some aspects, the fourth, fifth, or sixth temperature set point is about 39 ℃. In some aspects, the fourth, fifth, or sixth temperature set point is about 40 ℃.

In addition to the temperature set point, the methods of the present disclosure also involve modifying the time setting of the temperature set point to make a temperature transition within a particular time window. The timing of the temperature transition is important to control the cell density, cell growth and protein production characteristics of the cell culture during the upstream process. In some aspects, the methods of the present disclosure relate to a method of increasing the production of a protein in a cell, the method comprising culturing the cell in a bioreactor under suitable conditions, wherein the suitable conditions comprise (i) an initial temperature set point of 36.0 ℃ and a second temperature set point of less than about 36 ℃; (ii) An initial temperature set point of less than about 36.5 ℃ and a final temperature set point of about 31 ℃; or (iii) an initial temperature set point of less than about 36.5 ℃, a second temperature set point of about 33 ℃, and a final temperature set point of less than about 33 ℃. In some aspects, the methods of the present disclosure relate to a method of increasing the production of a protein in a cell, the method comprising culturing the cell in a bioreactor under suitable conditions, wherein the suitable conditions comprise (i) an initial temperature set point of 36.0 ℃ and a second temperature set point of less than 34 ℃; (ii) An initial temperature set point below 36.5 ℃ and a final temperature set point of 31 ℃; or (iii) an initial temperature set point below 36.5 ℃, a second temperature set point at 32 ℃, and a final temperature set point below 32 ℃.

In some aspects, the cells are cultured in a bioreactor under suitable conditions including (i) an initial temperature set point above about 35 ℃ but below about 37 ℃ and a second temperature set point above about 32 ℃ but below about 34 ℃, (ii) an initial temperature set point above about 35.5 ℃ but below about 37.5 ℃ and a second temperature set point above about 32 ℃ but below about 34 ℃, and (iii) an initial temperature set point above about 35 ℃ but below about 37 ℃, a second temperature set point above about 32 ℃ but below about 34 ℃, and a final temperature set point above about 30 ℃ but below about 32 ℃.

The methods of the present disclosure can also be used to increase the production of protein in a cell by adjusting the temperature set point in the bioreactor after the initial temperature set point. In some aspects, the initial temperature set point is above about 35 ℃ and below 37 ℃, such as about 36 ℃; the second temperature set point is above about 32 ℃ and below about 34 ℃, about 33 ℃; and the third temperature set point is above about 30 ℃ and below about 32 ℃, such as about 31 ℃, wherein the second temperature set point occurs between about 120 hours and about 168 hours, such as about 5 days, about 6 days, or about 7 days, after the initial temperature set point. In some aspects, the initial temperature set point is above about 35 ℃ and below 37 ℃, such as about 36 ℃; the second temperature set point is above about 32 ℃ and below about 34 ℃, about 33 ℃; and the third temperature set point is greater than about 30 ℃ and less than about 32 ℃, such as about 31 ℃, wherein the second temperature set point occurs between about 84 hours, about 90 hours, about 96 hours, about 102 hours, about 108 hours, about 114 hours, about 120 hours, about 126 hours, about 132 hours, about 138 hours, about 144 hours, about 150 hours, about 156 hours, about 162 hours, or about 168 hours after the initial temperature set point. In some aspects, the second temperature set point occurs from about 72 hours to about 168 hours. In some aspects, the second temperature set point occurs from about 96 hours to about 168 hours. In some aspects, the second temperature set point occurs from about 96 hours to about 144 hours.

In some aspects, the initial temperature set point is above about 35 ℃ and below 37 ℃, such as about 36 ℃; the second temperature set point is above about 32 ℃ and below about 34 ℃, about 33 ℃; and the third temperature set point is greater than about 30 ℃ and less than about 32 ℃, such as about 31 ℃, wherein the second temperature set point occurs about 120 hours, 5 days after the initial temperature set point. In some aspects, the initial temperature set point is above about 35 ℃ and below 37 ℃, such as about 36 ℃; the second temperature set point is above about 32 ℃ and below about 34 ℃, about 33 ℃; and the third temperature set point is greater than about 30 ℃ and less than about 32 ℃, such as about 31 ℃, wherein the second temperature set point occurs about 144 hours, 6 days after the initial temperature set point. In some aspects, the initial temperature set point is above about 35 ℃ and below 37 ℃, such as about 36 ℃; the second temperature set point is above about 32 ℃ and below about 34 ℃, about 33 ℃; and the third temperature set point is greater than about 30 ℃ and less than about 32 ℃, such as about 31 ℃, wherein the second temperature set point occurs about 168 hours, 7 days after the initial temperature set point. In some aspects, the second temperature set point occurs about 192 hours, e.g., 8 days, after the initial temperature set point.

The method of the present disclosure is also used to control the transition time setting to the final temperature set point. In some aspects, the initial temperature set point is above about 35 ℃ and below 37 ℃, such as about 36 ℃; the second temperature set point is above about 32 ℃ and below about 34 ℃, about 33 ℃; and the final temperature set point is greater than about 30 ℃ and less than about 32 ℃, such as about 31 ℃, wherein the final temperature set point occurs from about 168 hours to about 312 hours, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days or about 13 days, from 7 days to 12 days, from 8 days to 13 days, from 9 days to 13 days, from 8 days to 12 days, from 8 days to 11 days after the initial temperature set point. In some aspects, the initial temperature set point is above about 35 ℃ and below 37 ℃, such as about 36 ℃; the second temperature set point is above about 32 ℃ and below about 34 ℃, about 33 ℃; and the final temperature set point is above about 30 ℃ and below about 32 ℃, such as about 31 ℃, wherein the final temperature set point occurs from about 192 hours to about 300 hours. In some aspects, the final temperature set point occurs from about 216 hours to about 288 hours. In some aspects, the final temperature set point occurs from about 216 hours to about 264 hours.

IIC viable cell Density

The initial Viable Cell Density (VCD) or seed density of the cells at the beginning of the bioreactor process can be an important aspect of the upstream growth process, as the initial Viable Cell Density (VCD) affects the steady state and/or maximum viable cell density of the cell culture during production. A higher initial cell density can result in a much higher number of cells generated during the initial growth phase of the upstream process and ultimately can result in faster cell death and shorter bioreactor run times. Bioreactor production processes can be shortened, but in some cases post-translational modifications to proteins produced by the process can be affected, as shorter bioreactor run times can result in changes in residence time of the protein in the golgi apparatus, thus altering post-translational modifications, such as changes in glycosylation patterns of the protein. Since initial cell density may have a broad impact on overall yield and mass output of protein, initial viable cell density is an important parameter for upstream protein production.

In some aspects, the methods of the present disclosure involve seeding a bioreactor with an initial Viable Cell Density (VCD). In some aspects, the initial Viable Cell Density (VCD) set point is about 0.45X10 ⁶ Individual cells/mL and about 1.35X10 ⁶ Between individual cells/mL. In some aspects, the initial Viable Cell Density (VCD) set point is about 0.45X10 ⁶ Individual cells/mL and about 1.3X10 ⁶ Between individual cells/mL. In some aspects, the initial Viable Cell Density (VCD) set point is about 0.45X10 ⁶ Individual cells/mL and about 1.25X10 ⁶ Between individual cells/mL. In some aspects, the initial Viable Cell Density (VCD) set point is about 0.45X10 ⁶ Individual cells/mL and about 1.2x10 ⁶ Between individual cells/mL. In some aspects, the initial Viable Cell Density (VCD) set point is about 0.45X10 ⁶ Individual cells/mL and about 1.15X10 ⁶ Between individual cells/mL. In some aspects, the initial Viable Cell Density (VCD) set point is about 0.45X10 ⁶ Individual cells/mL and about 1.1X10 ⁶ Between individual cells/mL. In some aspects, the initial Viable Cell Density (VCD) set point is about 0.45X10 ⁶ Individual cells/mL and about 1.05X10 ⁶ Between individual cells/mL. In some aspects, the initial Viable Cell Density (VCD) set point is about 0.5X10 ⁶ Individual cells/mL and about 1.0X10 ⁶ Between individual cells/mL. In some aspects, the initial Viable Cell Density (VCD) set point is about 0.55X10 ⁶ Individual cells/mL and about 1.0X10 ⁶ Between individual cells/mL. In some aspects, the initial Viable Cell Density (VCD) set point is about 0.50X10 ⁶ Individual cells/mL and about 0.90X10 ⁶ Between individual cells/mL. In some aspects, the initial Viable Cell Density (VCD) set point is about 0.55X10 ⁶ Individual cells/mL and about 0.90X10 ⁶ Between individual cells/mL. In some aspects, the initial Viable Cell Density (VCD) set point is about 0.55X10 ⁶ Individual cells/mL and about 0.85X10 ⁶ Between individual cells/mL. In some aspects, the initial Viable Cell Density (VCD) set point is about 0.55X10 ⁶ Individual cells/mL and about 0.8X10 ⁶ Between individual cells/mL. In some aspects, the initial Viable Cell Density (VCD) set point is about 0.6X10 ⁶ Individual cells/mL and about 1.0X10 ⁶ Between individual cells/mL. In some aspects, the initial Viable Cell Density (VCD) set point is about 0.6X10 ⁶ Individual cells/mL and about 0.75X10 ⁶ Between individual cells/mL. In some aspects, the initial Viable Cell Density (VCD) set point is about 0.65X10 ⁶ Individual cells/mL and about 0.75X10 ⁶ Between individual cells/mL.

IID time for cell feed supplement

The methods of the present disclosure can also be accomplished by varying the cell feed time to affect or control the growth conditions of the cells. The timing of the feed process is important to produce the desired growth characteristics (e.g., cell density) of the cell culture production process. The optimal feeding strategy in a bioreactor depends on the structure of the reaction kinetics and the interactions between different reactions, such as protein synthesis and post-translational modification (i.e. glycosylation) of these proteins. Overfeeding and under-feeding the cell population are both detrimental to cell growth and product formation, and thus the timing of the feeding can be important to ensure maximum product yield. Insufficient feeding of the culture can lead to nutrient depletion and cell death, while excessive feeding can lead to nutrient excess, increased osmolality and cellular stress in dense cellular environments, and undesirable post-translational modification of the protein of interest.

In some aspects, the cell feed time is from about 24 hours to about 100 hours after induction. In some aspects, the cell feed time is from about 48 hours to about 100 hours after induction. In some aspects, the cell feed time is from about 48 hours to about 72 hours after induction. In some aspects, the cell feed time is from about 48 hours to about 96 hours after induction. In some aspects, the cell feed time is from about 72 hours to about 96 hours after induction. In some aspects, the cell feed time is from about 66 hours to about 84 hours after induction. In some aspects, the cell feed time is about 24 hours after induction. In some aspects, the cell feed time is about 48 hours after induction. In some aspects, the cell feed time is about 66 hours after induction. In some aspects, the cell feed time is about 72 hours after induction. In some aspects, the cell feed time is about 84 hours after induction. In some aspects, the cell feed time is about 96 hours after induction.

In some aspects, the first cell feed time is about 80 hours after induction, and one or more subsequent feed times occur about 24 hours thereafter. In some aspects, the subsequent feeding time is 104 hours post-induction, 128 hours post-induction, 152 hours post-induction, 176 hours post-induction, 200 hours post-induction, 224 hours post-induction, 248 hours post-induction, 272 hours post-induction, 296 hours post-induction, 320 hours post-induction, 344 hours post-induction, 368 hours post-induction, 392 hours post-induction, and/or 416 hours post-induction. In some aspects, the first cell feed time is about 74 hours to about 86 hours after induction, and one or more subsequent feed times occur about 24 hours thereafter. In some aspects, the subsequent feeding time is from about 98 hours to about 110 hours after induction. In some aspects, the subsequent feeding time is from about 122 hours to about 134 hours after induction. In some aspects, the subsequent feeding time is from about 146 hours to about 158 hours after induction. In some aspects, the subsequent feeding time is from about 170 hours to about 182 hours after induction. In some aspects, the subsequent feeding time is from about 194 hours to about 206 hours after induction. In some aspects, the subsequent feeding time is from about 218 hours to about 230 hours after induction. In some aspects, the subsequent feeding time is from about 242 hours to about 254 hours after induction. In some aspects, the subsequent feeding time is from about 266 hours to about 278 hours after induction. In some aspects, the subsequent feeding time is from about 290 hours to about 302 hours after induction. In some aspects, the subsequent feeding time is from about 314 hours to about 326 hours after induction. In some aspects, the subsequent feeding time is from about 338 hours to about 350 hours after induction. In some aspects, the subsequent feeding time is from about 362 hours to about 374 hours after induction. In some aspects, the subsequent feeding time is from about 386 hours to about 396 hours after induction. In some aspects, the subsequent feeding time is from about 410 hours to about 422 hours after induction. In some aspects, the subsequent feed time is according to fig. 1B.

IIE combinations of conditions

In some aspects, the methods of the present disclosure include any combination of the conditions listed above. In some aspects, the method comprises two or more conditions selected from: (i) An initial temperature set point between about 35 ℃ and about 37 ℃, such as about 36 ℃, a second temperature set point between 32 ℃ and about 34 ℃, such as about 33 ℃, and a third temperature set point between about 30 ℃ and about 32 ℃, such as about 31 ℃; (ii) a pH setpoint of 7.15; and (iii) at about 0.65X10 ⁶ Individual cells/mL and about 0.75X10 ⁶ An initial Viable Cell Density (VCD) set point between individual cells/mL.

In some aspects, the method includes (i) an initial temperature set point between about 35 ℃ and about 37 ℃, e.g., about 36 ℃, a second temperature set point between 32 ℃ and about 34 ℃, e.g., about 33 ℃, and a third temperature set point between about 30 ℃ and about 32 ℃, e.g., about 31 ℃, and (ii) a pH set point of 7.15.

In some aspects, the method includes (i) an initial temperature set point of about 36 ℃, a second temperature set point of about 33 ℃ and a third temperature set point of about 31 ℃, and (ii) at about 0.65X10 ⁶ Individual cells to about 0.75X10 ⁶ Between individual cells (e.g. 0.70X10 ⁶ Individual cells) initial Viable Cell Density (VCD) set point.

In some aspects, the method includes (i) an initial temperature set point between about 35 ℃ and about 37 ℃, e.g., about 36 ℃, a second temperature set point between 32 ℃ and about 34 ℃, e.g., about 33 ℃, and a third temperature set point between about 30 ℃ and about 32 ℃, e.g., about 31 ℃; (ii) a pH setpoint of 7.15; and (iii) at about 0.65X10 ⁶ Individual cells to about 0.75X10 ⁶ Between individual cells (e.g. 0.70X10 ⁶ Individual cells) initial Viable Cell Density (VCD) set point.

In some aspects, the method includes (i) an initial temperature set point between about 35 ℃ and about 37 ℃, e.g., about 36 ℃, a second temperature set point between 32 ℃ and about 34 ℃, e.g., about 33 ℃, and a third temperature set point between about 30 ℃ and about 32 ℃, e.g., about 31 ℃; (ii) a pH setpoint of 7.15; (iii) At about 0.65X10 ⁶ Individual cells to about 0.75X10 ⁶ Between individual cells (e.g. 0.70X10 ⁶ Individual cells) initial Viable Cell Density (VCD) set point: and (iv) a first feed time of between about 66 to about 84 hours.

In some aspects, the method includes (i) an initial temperature set point between about 35 ℃ and about 37 ℃, e.g., about 36 ℃, a second temperature set point between 32 ℃ and about 34 ℃, e.g., about 33 ℃, and a third temperature set point between about 30 ℃ and about 32 ℃, e.g., about 31 ℃; (ii) a pH setpoint of 7.15; (iii) At about 0.65X10 ⁶ Individual cells to about 0.75X10 ⁶ Between individual cells (e.g. 0.70X10 ⁶ Individual cells) initial Viable Cell Density (VCD) set point: and (iv) a first feed time of about 80 hours.

In some aspects, the method comprises (i) an initial temperature set point between about 35 ℃ and about 37 ℃, such as about 36 ℃, at 32 ℃ and about 34 DEG CA second temperature set point therebetween, e.g., about 33 ℃, and a third temperature set point between about 30 ℃ and about 32 ℃, e.g., about 31 ℃; (ii) a pH setpoint of 7.15; (iii) At about 0.65X10 ⁶ Individual cells to about 0.75X10 ⁶ Between individual cells (e.g. 0.70X10 ⁶ Individual cells) initial Viable Cell Density (VCD) set point: and (iv) a first feed time of between about 66 to about 84 hours.

In some aspects, the method includes (i) an initial temperature set point between about 35 ℃ and about 37 ℃, e.g., about 36 ℃, a second temperature set point between 32 ℃ and about 34 ℃, e.g., about 33 ℃, and a third temperature set point between about 30 ℃ and about 32 ℃, e.g., about 31 ℃; (ii) a pH setpoint of 7.15; (iii) At about 0.65X10 ⁶ Individual cells to about 0.75X10 ⁶ Between individual cells (e.g. 0.70X10 ⁶ Individual cells) initial Viable Cell Density (VCD) set point: and (iv) a first feed time of about 80 hours.

IIF culture medium

Cells producing the protein of interest may be grown in a cell culture medium. As used herein, the term "culture medium" (used interchangeably with "culture medium") refers to a nutritional composition that helps to maintain, propagate, and/or differentiate cells. The term "culture medium" refers to any medium capable of supporting the growth, maintenance, propagation or expansion of cells in an artificial in vitro environment outside of a multicellular organism or tissue. Cell culture media can be optimized for a particular cell culture use, including, for example, cell culture growth media formulated to promote cell growth, or cell culture production media formulated to promote recombinant protein production. The medium provides standard inorganic salts (such as zinc, iron, magnesium, calcium and potassium), trace elements, vitamins, energy sources, buffer systems and essential amino acids. Exemplary media include, but are not limited to, du's modified Du's medium, RPMI 1640, minimal essential medium-alpha (MEM-alpha), du's modified Ig's medium (DMEM), DME/F12, alpha MEM, igE's basal medium with Irvine BSS, high glucose DMEM with L-glutamine, high glucose DMEM without L-glutamine, low glucose DMEM without L-glutamine, DMEM with L-glutamine; F12:1, GMEM (Grassgo MEM), L-glutamine-containing GMEM, grasss complete insect medium, FBS-free Grasss insect medium, F-10, F-12, L-glutamine-containing Ham's F-10, L-glutamine-containing Ham's F-12, HEPES-and L-glutamine-containing IMDM, HEPES-containing IMDM without L-glutamine, IPL-41 insect medium, L-15 (Leibovitz) without L-glutamine or phenol red (2X), L-15 (Leibovitz) free of L-glutamine, mcCoy's 5A modified medium, medium 199, MEM eagle free of L-glutamine or phenol red (2X), MEM eagle-Ireger BSS containing L-glutamine, MEM eagle-Er BSS containing no L-glutamine, MEM eagle-Hanks BSS containing no L-glutamine, NCTC-109 containing L-glutamine, richter's CM medium containing L-glutamine, HEPES, RPMI 1640 with L-glutamine and/or penicillin-streptomycin, RPMI 1640 with L-glutamine, RPMI 1640 without L-glutamine, schneider's insect medium or any other medium suitable for use in the methods of the invention. In addition, media as described herein include, but are not limited to, chemically defined media, media containing aqueous products, and simple media.

The methods of the present disclosure may be performed in a variety of vessel types to accommodate a variety of protein production strategies. The methods of the present disclosure may involve fed-batch culture. Fed-batch culture is a method of culturing cells in which additional components are provided to the culture at some time after the start of the culture process. The fed-batch culture may be started using a basal medium. The medium that provides the culture with additional components at some time after the start of the culture process is a feed medium. The fed-batch culture is usually stopped at some point and the cells and/or components in the medium are harvested and optionally purified. The methods of the present disclosure may involve perfusion culture. Perfusion culture involves a continuous flow of physiological nutrient solution at a steady rate through or past a population of cells. Since perfusion systems generally involve retention of cells within the culture unit, perfusion culture typically has a relatively high cell density, but culture conditions are difficult to maintain and control. In addition, since cells are grown to a high density and remain in the culture unit at the high density, the growth rate generally decreases continuously over time, resulting in a late exponential phase or even a resting phase of cell growth. In some aspects, the methods of the present disclosure relate to batch culture processes. In some aspects, the methods of the present disclosure relate to fed-batch culture processes. In some aspects, the methods of the present disclosure relate to perfusion culture processes.

In some aspects, a medium suitable for use in the methods of the invention may be supplemented with a feed medium. In some aspects, the feed medium is a chemically-defined feed medium. In some aspects, a chemically-defined feed medium (or CDFM) refers to a medium containing one or more nutrients, the chemical composition and relative concentration of which are known and which begin to be added to the medium at some time after inoculation. CDFM is sometimes used interchangeably with "concentrated feed medium", "enriched medium", "highly concentrated feed medium" or "super concentrated feed medium". CDFM is supplied continuously or in discrete increments to the culture vessel during the culture, to the culture medium, with or without periodic cell and/or product harvest prior to termination of the culture. CDFM can be formulated alone to contain a unique blend of enriched amounts of amino acids, vitamins, trace minerals, and organic compounds for use as a feed medium for cell culture media. Alternatively, commercially available CDFM may be used. Some examples of commercially available CDFM include, but are not limited to IS CHO feed-CD (Irvine Scientific), BALANCD ^TM CHO feed medium (1-3) (Irvine Scientific), IS-CHO-V ^TM (Irvine Scientific) IS-CHO-CD XP with hydrolyzed blend ^TM (Irvine Scientific), CHO feed bioreactor supplement (Sigma-Aldrich), CHO CD EFFICIENT FEED ^TM B nutritional supplements (Life Technologies).

In some aspects, the cells used in the present invention are prokaryotic, yeast, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative organisms or gram-positive organisms, e.g. Enterobacteriaceae (Enterobacteriaceae) such as Escherichia (e.g. E.coli), enterobacter (Enterobacter), erwinia (Erwinia), klebsiella (Klebsiella), proteus (Proteus), salmonella (Salmonella) such as Salmonella typhimurium (Salmonella typhimurium), serratia (Serratia) such as Serratia marcescens (Serratia marcescans) and Shigella, and Bacillus (Bacillus) such as Bacillus subtilis (B.sub.and Bacillus licheniformis (B.licheniformis) such as Bacillus licheniformis 41P disclosed in DD 266,710 published 4 month 12 in 1989, pseudomonas such as Streptomyces and Streptomyces sp. A suitable E.coli cloning host is E.coli 294 (ATCC 31,446), but other strains such as E.coli B, E.coli X1776 (ATCC 31,537) and E.coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative only and not limiting.

In certain embodiments, the cell is a eukaryotic microorganism, such as a filamentous fungus or yeast. Saccharomyces cerevisiae (Saccharomyces cerevisiae) or Saccharomyces cerevisiae are the most commonly used among lower eukaryotic host microorganisms. However, many other genera, species and strains are generally available and useful herein, such as schizosaccharomyces pombe (Schizosaccharomyces pombe); kluyveromyces hosts such as Kluyveromyces lactis (K.lactis), kluyveromyces fragilis (K.fragilis) (ATCC 12,424), kluyveromyces bulgaricus (K.bulgaricus) (ATCC 16,045), kluyveromyces weissei (K.winkeramii) (ATCC 24,178), kluyveromyces walteri (K.wati) (ATCC 56,500), kluyveromyces drosophila (K.drosophila) (ATCC 36,906), kluyveromyces thermotolerans (K.thermals) and Kluyveromyces marxianus (K.marxianus); yarrowia (EP 402,226); pichia pastoris (EP 183,070); candida (Candida); trichoderma reesei (Trichoderma reesia) (EP 244,234); neurospora crassa (Neurospora crassa); schwanniomyces (Schwanniomyces), such as Schwanniomyces western (Schwanniomyces occidentalis); and filamentous fungi, such as, for example, neurospora (Neurospora), penicillium (Penicillium), curvularia (Tolypocladium) and Aspergillus (Aspergillus) hosts, such as Aspergillus nidulans (A. Nidulans) and Aspergillus niger (A. Niger).

In some aspects, the cells are derived from multicellular organisms. In particular embodiments, the cells are invertebrate cells from plant and insect cells. Non-limiting examples include (but may also utilize) cells derived from: spodoptera frugiperda (caterpillar)), aedes aegypti (mosquito), aedes albopictus (mosquito), drosophila melanogaster (Drosophila melanogaster) (drosophila melanogaster), silkworm (Bombyx mori), cotton, corn, potato, soybean, petunia (petunia), tomato, and tobacco.

In some aspects, the cell is a mammalian cell. For example, the cells are chinese hamster ovary cells (CHO cells) (including DHFR-CHO cells described in Urlaub and Chasin, (1980) PNAS USA 77:4216-4220), used with DHFR selectable markers, 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. Other non-limiting examples of mammalian cell lines are the SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney lines (293 cells or 293 cells subcloned for suspension culture growth, 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.Natl. Acad. Sci.usa 77:4216 (1980)); mouse Sertoli cells (TM 4, mather, biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV 1, ATCC CCL 70); african green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); rat hepatocytes (BRL 3a, atcc crl 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (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 the human hepatoma line (Hep G2), the entire teachings of which are incorporated herein by reference.

In some aspects, the cells are transformed with an expression vector or cloning vector to produce a product or portion thereof, and cultured as appropriate to induce a promoter, select transformants, or amplify a gene encoding the desired sequence. In some aspects, standard molecular biology techniques are used to prepare recombinant expression vectors, transfect cells, select transformants, culture cells, and recover the product from the culture medium. In some aspects, the cell culture media described herein can be used as media for hybridoma cells, monoclonal antibody-producing cells, virus-producing cells, transfected cells, cancer cells, and/or recombinant peptide-producing cells.

The cells of the present disclosure may be cultured under suitable conditions for a suitable period of time, the conditions depending on the type or types of cells being cultured and the products produced. In some aspects, the cells are cultured for about two days to about fourteen days. In some aspects, the cells are cultured for about four days to about ten days.

In some aspects, the cell culture is a suspension culture or an adherent culture. The term "suspension culture" refers to cells in a culture in which most or all of the cells in the culture are present in suspension and few or no cells in the culture vessel attach to the surface of the vessel or another surface within the vessel (adherent cells). A "suspension culture" may have greater than about 50%, 60%, 65%, 75%, 85%, or 95% of the cells suspended without adhering to surfaces on or in the culture vessel. The term "adherent culture" refers to cells in a culture in which most or all of the cells in the culture are attached to the surface of a vessel or are present at another surface within the vessel, while few or no cells in the culture vessel are in suspension. An "adherent culture" may have greater than 50%, 60%, 65%, 75%, 85%, or 95% cell adherence.

The medium used to produce the recombinant protein may include one or more metal ions suitable for use in cell culture media. In some aspects, the metal ion that may be included in the cell culture is, but is not limited to, ag ⁺ 、Au ³⁺ 、Cd ²⁺ 、Cu ²⁺ 、Ga ³⁺ 、In ^{3 +} 、Ni ²⁺ 、Pd ²⁺ 、Zn ²⁺ And Mn of ²⁺ . In some aspects, the concentration of certain metal ions (e.g., manganese) at the beginning of a bioreactor process can affect growth and/or steady state cell culture kinetics during production. In addition, metal ions (e.g., manganese) can affect the flux of various enzymes involved in the glycosylation of the produced protein in the bioreactor. In particular, manganese is a known cofactor for several glycosyltransferases in mammalian cells, including N-acetyl-glucosaminyl-transferase and beta-1, 4-galactosyl-transferase. Thus, without being bound by any theory, the modulation of manganese concentration during the bioreactor process may affect the glycosylation profile and glycosylation profile of the protein. In some aspects, manganese is fed into a bioreactor, i.e., a fed-batch bioreactor. In some aspects, manganese is replenished into the bioreactor at the beginning of or during the protein induction phase.

In some aspects, the manganese concentration of the methods of the invention is at least about 30mg/mL, at least about 50mg/mL, at least about 80mg/mL, at least about 100mg/mL, at least about 110mg/mL, at least about 120mg/mL, at least about 130mg/mL, at least about 140mg/mL, at least about 150mg/mL, at least about 160mg/mL, at least about 170mg/mL, at least about 180mg/mL, at least about 190mg/mL, at least about 200mg/mL, at least about 220mg/mL, at least about 240mg/mL, at least about 260mg/mL, at least about 280mg/mL, or at least about 300mg/mL. In some aspects, the methods of the present disclosure involve adjusting the manganese concentration to about 30-300mg/mL. In some aspects, the manganese concentration is from about 30mg/mL to about 280mg/mL. In some aspects, the manganese concentration is from about 50mg/mL to about 280mg/mL. In some aspects, the manganese concentration is from about 50mg/mL to about 260mg/mL. In some aspects, the manganese concentration is from about 70mg/mL to about 260mg/mL. In some aspects, the manganese concentration is from about 90mg/mL to about 240mg/mL. In some aspects, the manganese concentration is from about 120mg/mL to about 240mg/mL. In some aspects, the manganese concentration is from about 120mg/mL to about 220mg/mL. In some aspects, the manganese concentration is from about 140mg/mL to about 220mg/mL. In some aspects, the manganese concentration is from about 140mg/mL to about 200mg/mL. In some aspects, the manganese concentration is from about 160mg/mL to about 200mg/mL. In some aspects, the manganese concentration is from about 160mg/mL to about 180mg/mL. In some aspects, the manganese concentration is about 160mg/mL, or about 180mg/mL.

Manganese concentration can also be measured during the upstream cell culture process. Manganese can also be measured during the protein production phase or the protein induction phase, i.e. directly in the bioreactor. In some aspects, manganese is present at a concentration of about 1-20 parts per billion (ppb). In some aspects, manganese is present at a concentration of about 1.6ppb to about 15 ppb. In some aspects, manganese is present at a concentration of about 2ppb to about 10 ppb. In some aspects, manganese is present at a concentration of about 2ppb to about 6 ppb. In some aspects, manganese is present at a concentration of about 2ppb to about 4 ppb. In some aspects, manganese is present at a concentration of about 1ppb to about 3 ppb. In some aspects, manganese is present at a concentration of about 1ppb to about 2 ppb. In some aspects, manganese is present at a concentration of about 3ppb to about 10 ppb. In some aspects, manganese is present at a concentration of about 3ppb to about 6 ppb. In some aspects, the manganese is present at a concentration of about 1.3 ppb. In some aspects, the manganese is present at a concentration of about 1.6 ppb. In some aspects, the manganese is present at a concentration of about 2 ppb. In some aspects, manganese is present at a concentration of about 3 ppb. In some aspects, manganese is present at a concentration of about 4 ppb. In some aspects, manganese is present at a concentration of about 5 ppb. In some aspects, manganese is present at a concentration of about 6 ppb.

The present disclosure also includes proteins produced by the methods of the present invention. In some aspects, the protein produced by the methods of the invention is a CTLA4-Fc fusion protein. In some aspects, the protein comprises betanaproxen. In some aspects, the proteins produced by the methods of the invention are glycosylated. In some aspects, the glycosylated proteins are shown elsewhere herein.

In some aspects, the disclosure includes cells cultured by the methods of the invention. A bioreactor useful in the methods of the invention is also provided. In some aspects, the bioreactor is maintained at: (a) An initial temperature set point of about 36 ℃, a second temperature set point of about 33 ℃, and a third temperature set point of about 31 ℃; (b) a pH setpoint of about 7.15; and (c) about 0.70X10 ⁶ Initial Viable Cell Density (VCD) set point of individual cells/mL.

Glycosylation profile

The methods and conditions of the present disclosure can be used to influence, maintain, control and/or modify the glycosylation profile of proteins produced by the methods of the present invention. In some aspects, the methods control the glycosylation profile of the protein. In some aspects, the glycosylation profile of the protein comprises one or more N-linked glycans.

The methods of the present disclosure may be implemented or validated via glycan analysis using various methods, including glycan release assays. The first step in glycan analysis of glycoconjugates, such as glycoproteins, is to release the saccharide from the molecule to which they are attached. The N-linked glycans on glycoproteins can be released by amidases such as peptide-N-glycosidase F (PNGase F). Most methods for analyzing oligosaccharides from biological sources require a glycan derivatization step: the glycans can be derivatized to introduce chromophores or fluorophores to facilitate detection after chromatographic or electrophoretic separation. Derivatization may also be used to attach charged or hydrophobic groups at the reducing end to enhance glycan separation and mass spectrometry detection. In addition, derivatization steps (e.g., hypermethylation) aim to stabilize sialic acid residues, improve mass spectrometry sensitivity, and support detailed structural characterization by (tandem) mass spectrometry.

In the specific case of betanaproxen, after derivatization of the glycans, immunoglobulin degrading enzymes such as those from streptococcus pyogenes (Streptococcus pyogenes) (IdeS) can be used to digest the hinge region of IgG at a specific site directly below the hinge region, thereby producing a CTLA4 fragment and two Fc fragments. The isolated fragments can then be subjected to glycan release, derivatization and glycan analysis, wherein the Asn76 and Asn108 sites (CTLA 4 region) can be analyzed together, and the Asn207 (Fc region) site can be analyzed separately.

Mass spectrometry (mass spectrometry) ("MS" or "mass-spec") is an analytical technique for measuring mass-to-charge ratio ions. This can be achieved by: the sample was ionized and particles of different masses were separated and their relative abundance was recorded by measuring the intensity of the ion flow. A typical mass spectrometer comprises three parts: an ion source, a mass analyzer, and a detector system. The ion source is the part of the mass spectrometer that ionizes the analyte substance (analyte). The ions are then transported by a magnetic or electric field to a mass analyzer, which separates the ions according to their mass-to-charge ratio (m/z). Many mass spectrometers use two or more mass analyzers for tandem mass spectrometry (MS/MS). The detector records the charge induced or current generated as the ions pass or hit the surface. The mass spectrum is the result of measuring the signal generated in the detector while scanning the m/z ions with a mass analyzer.

Various N-linked glycans can be present in the glycosylation profile of the protein. In some aspects, the N-linked glycans comprise G0F, G1F, G2F, S1G1F, S1G2F, S G2F, S1G3F and/or S2G4F. Representative diagrams of G0F, G1F, G2F, S1G1F, S G2F, S1G3F and S2G4F can be seen in FIGS. 6A-6B. In some aspects, the methods of the disclosure relate to measuring glycosylation patterns during the protein induction phase after day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 9, day 20, or day 21 beginning at the protein induction phase. In some aspects, the methods of the disclosure relate to measuring glycosylation profile after day 7 from the beginning of the protein induction phase. In some aspects, the methods of the disclosure relate to measuring glycosylation profile after day 14, beginning at the protein induction phase. In some aspects, the methods of the disclosure relate to measuring glycosylation profile after day 21, beginning at the protein induction stage. In some aspects, the methods of the present disclosure relate to measuring glycosylation profile at the time of harvesting a cell culture.

In some aspects, the glycosylated protein produced by the methods of the invention is a CTLA4 protein. CTLA4 molecules or CTLA4 extracellular domains can be fused to Fc, wherein the molecules are referred to as CTLA4-Fc or CTLA4-Ig. "Fc region" (fragment crystallizable region), "Fc domain" or "Fc" refers to the C-terminal region of the heavy chain of an antibody that mediates binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or binding to the first component (C1 q) of the classical complement system. Thus, the Fc region comprises the constant region of the antibody in addition to the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, igA and IgD antibody isotypes, the Fc region comprises two identical protein fragments derived from the second (CH 2) and third (CH 3) constant domains of the two heavy chains of the antibody; igM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. IgG isotypes fall into the following subclasses in certain species: igG1, igG2, igG3 and IgG4 in humans, and IgG1, igG2a, igG2b and IgG3 in mice. For IgG, the Fc region comprises the immunoglobulin domains CH2 and CH3 and the hinge between the CH1 and CH2 domains. As defined herein, although the definition of the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of a human IgG heavy chain is defined as extending from amino acid residue D221 of IgG1, amino acid residue V222 of IgG2, amino acid residue L221 of IgG3, and amino acid residue P224 of IgG4 to the carboxy terminus of the heavy chain, wherein numbering is according to the Kabat numbering scheme. The CH2 domain of the human IgG Fc region extends from amino acid 237 to amino acid 340, and the CH3 domain is located on the C-terminal side of the CH2 domain in the Fc region, i.e., it extends from amino acid 341 to amino acid 447 or 446 (if no C-terminal lysine residues are present) or 445 (if no C-terminal glycine and lysine residues are present) of the IgG. As used herein, an Fc region may be a native sequence Fc, including any allotypic variant or variant Fc (e.g., a non-naturally occurring Fc). The methods of the present disclosure can also be used to produce proteins comprising CTLA4 domains. The methods of the present disclosure can also be used to generate CTLA4 domains fused to an Fc portion. In some aspects, the protein is a fusion protein. In some aspects, the fusion protein comprises an Fc portion. In some aspects, the protein is betanaproxen. In some aspects, the protein comprises a sequence selected from SEQ ID Nos 2-9. In some aspects, the protein comprises SEQ ID NO. 2. In some aspects, the protein comprises SEQ ID NO. 3. In some aspects, the protein comprises SEQ ID NO. 4. In some aspects, the protein comprises SEQ ID NO. 5. In some aspects, the protein comprises SEQ ID NO. 6. In some aspects, the protein comprises SEQ ID NO. 7. In some aspects, the protein comprises SEQ ID NO. 8. In some aspects, the protein comprises SEQ ID NO 9.

The CTLA4-Ig fusion proteins produced by the methods of the invention can comprise one or more mutations. In some aspects, the CTLA4-Ig fusion protein is (a) a CTLA4-Ig fusion protein having the amino acid sequence of SEQ ID NO 8 (methionine at amino acid position 27 and glycine at amino acid position 382); (b) A CTLA4-Ig fusion protein having the amino acid sequence of SEQ ID No. 5 (methionine at amino acid position 27 and lysine at amino acid position 383); (c) A CTLA4-Ig fusion protein having the amino acid sequence of SEQ ID No. 7 (alanine at amino acid position 26 and glycine at amino acid position 382); (d) A CTLA4-Ig fusion protein having the amino acid sequence of SEQ ID No. 4 (alanine at amino acid position 26 and lysine at amino acid position 383); (e) A CTLA4-Ig fusion protein having the amino acid sequence of SEQ ID No. 6 (methionine at amino acid position 25 and glycine at amino acid position 382): or (f) a CTLA4-Ig fusion protein having the amino acid sequence of SEQ ID NO. 3 (methionine at amino acid position 25 and lysine at amino acid position 383). In some aspects, the CTLA4-Ig fusion protein is (a) about 90% of CTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID No. 2 beginning with methionine at residue 27; (b) About 10% of the CTLA4-Ig polypeptide comprises the amino acid sequence of SEQ ID NO. 2 starting with alanine at residue number 26; (c) About 4% of the CTLA4-Ig polypeptide comprises the amino acid sequence of SEQ ID NO. 2 ending with a lysine at residue number 383; (d) About 96% of the CTLA4-Ig polypeptide comprises the amino acid sequence of SEQ ID NO. 2 ending with glycine at residue number 382; and optionally (e) less than about 1% of the CTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO. 2 beginning with methionine at residue number 25.

The proteins of the present disclosure have glycosylation sites. Glycosylation is a process that involves the addition of complex oligosaccharide structures to proteins at specific sites within the polypeptide chain. Glycosylation of proteins and subsequent processing of added carbohydrates can affect the folding and structure of the protein, the stability of the protein (including the half-life of the protein), and the functional properties of the protein. Protein glycosylation can be divided into two classes, depending on the sequence context in which the modification occurs: o-linked glycosylation and N-linked glycosylation. The O-linked polysaccharide is linked to hydroxyl groups, typically to the hydroxyl groups of serine or threonine residues. O-glycans were not added to each serine and threonine residue. The O-linked oligosaccharides are typically mono-or bi-antennary, i.e. they contain one or at most two branches (antennary) and one to four different kinds of sugar residues, which are added one by one. The N-linked polysaccharide is attached to the asparagine at the amide nitrogen. Only asparagine, which is part of one of two tripeptide sequences (asparagine-X-serine or asparagine-X-threonine (where X is any amino acid other than proline)), is the target of glycosylation. The N-linked oligosaccharides may have one to four branches, known as mono-antennary, di-antennary, tri-antennary, tetra-antennary. In some aspects, the one or more N-linked glycans are located at one or more asparagine residues selected from Asn76, asn108, and/or Asn207 of betanaproxen.

In some aspects, the methods comprise culturing the protein under any of the conditions disclosed herein, e.g., conditions such that the initial temperature set point is between about 35 ℃ and about 37 ℃, e.g., about 36 ℃, the second temperature set point is between 32 ℃ and about 34 ℃, e.g., about 33 ℃, and the third temperature set point is between about 30 ℃ and about 32 ℃, e.g., about 31 ℃, wherein the protein has an N-linked glycan, e.g., G2F, at residue Asn 108.

The methods of the present disclosure may also be used to characterize, analyze, or control the sialic acid content of a protein. In some aspects, the one or more N-linked glycans are sialic acids and have a molar ratio of between about 5 to about 9, from about 5.5 to about 8.5, from about 5.8 to about 6.7, from about 5.2 to about 7.5, from about 6 to about 8, from about 6.2 to about 7.4, or from about 5 to about 6. In some aspects, the one or more N-linked glycans are sialic acids and have a molar ratio of between about 3 to about 9, from about 3.5 to about 8.5, from about 4.5 to about 7.5, from about 5.5 to about 7.4, from about 6.0 to about 7.4, or from about 6.2 to about 7.4. In some aspects, the one or more N-linked glycans are sialic acid and have a molar ratio of NANA of between about 4 to about 7. In some aspects, the one or more N-linked glycans are sialic acid and have a molar ratio of NANA of between about 5 to about 8. In some aspects, the one or more N-linked glycans are sialic acid and have a molar ratio of NANA of between about 6.2 to about 7.4.

The methods of the present disclosure may also be used to analyze O-linked glycans. In some aspects, the glycosylation profile includes one or more O-linked glycans. In some aspects, the O-linked glycans are located at residues Ser129, ser130, ser136 and/or Ser 139.

The methods of the present disclosure can also be used to analyze the biantennary glycans of CTLA4-Fc fusion proteins, comprising measuring one or more N-linked glycans attached to one or more asparagine residues in the CTLA4 protein, wherein one of the biantennary glycans is G2F. In some aspects, one or more N-linked glycans attached to one or more asparagine residues in CTLA4 protein are measured, wherein one of the biantennary glycans is G0F. In some aspects, the biantennary glycans are selected from G0F, G1F, G2F, S1G1F, S1G2F and/or S2G2F. Liquid chromatography can be used to analyze glycans of the present disclosure. Specific glycoproteins may exhibit carbohydrate heterogeneity. Several levels of heterogeneity can be observed: the glycosylation sites can vary from fully occupied to unoccupied, and any particular site can be filled with many different oligosaccharide structures, each of which can be modified by a sialic acid molecule (e.g., NANA or NGNA).

The carbohydrate content of the proteins of the present disclosure can be analyzed by methods known in the art, including those described in the examples herein. Several methods for glycosylation analysis are known in the art and can be used in the context of the present disclosure. These methods provide information about the identity and composition of the oligosaccharides attached to the resulting peptides. Methods of carbohydrate analysis useful in connection with the present disclosure include, but are not limited to, lectin chromatography; high performance anion exchange chromatography combined with pulsed amperometric detection (HPAEC-PAD) using high pH anion exchange chromatography to separate oligosaccharides based on charge; NMR; mass spectrometry; HPLC; porous graphitized carbon (GPC) chromatography.

The method for releasing oligosaccharides comprises the following steps: 1) Enzymatic processes, which are generally carried out using peptide-N-glycosidase F/endo- α -galactosidase: 2) Beta-elimination methods, which use harsh alkaline environments to release structures that are predominantly O-linked; and 3) chemical methods using anhydrous hydrazine to release both N-linked oligosaccharides and O-linked oligosaccharides. The analysis method may comprise one or more of the following steps: 1. the samples were dialyzed against deionized water to remove all buffer salts, then cold dried: 2. releasing the intact oligosaccharide chains with anhydrous hydrazine; 3. treating the intact oligosaccharide chains with anhydrous methanolic HCl to release the individual monosaccharides as O-methyl derivatives; 4. n-acetylating any primary amino groups; 5. derivatization to produce the per-O-trimethylsilylmethyl glycoside: 6. separating the derivative by capillary gas-liquid chromatography (GLC) on a CP-SIL8 column; 7. identifying individual glycoside derivatives by GLC and retention time of mass spectra compared to known standards; the individual derivatives were quantified by FID with an internal standard (13-O-methyl-D-glucose). In some aspects, the biantennary glycans are measured via ultra-high performance liquid chromatography and fluorescence detection (UPLC-FLR). In some aspects, the Fc domain of the CTLA4-Fc fusion protein is cleaved prior to the measurement. In some aspects, the Fc domain of the CTLA4-Fc fusion protein is not cleaved prior to the measurement. In some aspects, the protein is run through a viral inactivation process. In some aspects, the viral inactivation process is run with 0.5% Triton X-100.

IV pharmaceutical composition

The proteins produced by the methods of the present disclosure may be further formulated to be suitable for human administration, e.g., pharmaceutical compositions. Compositions acceptable for pharmaceutical administration may include, in addition to any one or more active agents, substances that are impurities at levels not exceeding acceptable levels for pharmaceutical administration (such levels including the absence of such impurities), and may include pharmaceutically acceptable excipients, vehicles, carriers, and other inactive ingredients, e.g., to formulate such compositions for administration. For example, a pharmaceutically acceptable CTLA4-Ig composition can include MCP-1 or DNA, so long as the agents are at a level acceptable for administration to a human.

The disclosure also provides any of the CTLA4-Ig molecules described as a lyophilized mixture. The formulation comprising CTLA4-Ig to be lyophilized may further comprise three basic components: (1) one or more additional active ingredients (e.g., immunosuppressants) comprising other proteins or small molecules, (2) one or more excipients, and (3) one or more solvents. Excipients include pharmaceutically acceptable agents to provide good lyophile characteristics (bulking agents) and to provide lyoprotection and/or cryoprotection of the protein ("stabilizers"), to maintain pH (buffers), and proper conformation of the protein during storage so as to maintain substantial retention of biological activity (including stability of the active ingredient, such as stability of the protein). With respect to excipients, examples of formulations may include one or more of the following: one or more buffers, one or more fillers, one or more protein stabilizers, and one or more antimicrobial agents. Sugar or polyols can be used as non-specific protein stabilizers in solution as well as during freeze-thawing and freeze-drying. The polymers can be used to stabilize proteins in solution as well as during freeze thawing and freeze drying. One popular polymer is serum albumin, which has been used as both a cryoprotectant and a lyoprotectant. In one aspect, the present disclosure provides an albumin-free formulation. Various salts may be used as fillers. Illustrative salt fillers include, for example, naCl, mgCl2, and CaCl2.

Certain amino acids may be used as cryoprotectants and/or lyoprotectants and/or bulking agents. Amino acids that may be used include, but are not limited to, glycine, proline, 4-hydroxyproline, L-serine, sodium glutamate, alanine, arginine, and lysine hydrochloride. Many buffers can be selected in the formulation that cover a wide pH range. Buffers include, for example, acetate, citrate, glycine, histidine, phosphate (sodium or potassium), diethanolamine and Tris. Buffers encompass those reagents that maintain the pH of the solution within an acceptable range prior to lyophilization. In one aspect, the disclosure provides lyophilized CTLA4-Ig mixtures comprising at least 90%, 95%, 99% or 99.5% CTLA4-Ig dimers comprising any of the sequences according to any of SEQ ID No. 1-9. In one aspect, the disclosure provides lyophilized CTLA4-Ig mixtures comprising at least 90%, 95%, 99% or 99.5% CTLA4-Ig dimer and no more than 5%, 4%, 3%, 2% or 1% CTLA4-Ig tetramer. In another aspect, the disclosure provides lyophilized CTLA4-Ig mixtures comprising at least 90%, 95%, 99%, or 99.5% CTLA4-Ig dimer, and no more than 5%, 4%, 3%, 2%, or 1% CTLA4-Ig tetramer, and no more than 2%, 1.5%, 1.0%, 0.8%, 0.5%, or 0.3% CTLA4-Ig monomer. In a further aspect, the disclosure provides lyophilized CTLA4-Ig mixtures comprising at least 8.0 moles of sialic acid per mole of CTLA4-Ig dimer or CTLA4-Ig molecule. In another aspect, the disclosure provides lyophilized CTLA4-Ig mixture comprising: from about 15 to about 35 moles GlcNac per mole CTLAIg molecule or dimer; from about 1 to about 5 moles GalNac per mole CTLA4-Ig dimer or CTLA4-Ig molecule; from about 5 moles to about 20 moles galactose per mole of CTLA4-Ig dimer or CTLA4-Ig molecule; from about 2 to about 10 moles of fucose per mole of CTLA4-Ig dimer or CTLA4-Ig molecule; and/or from about 5 to 15 moles mannose per mole of CTLA4-Ig dimer or CTLA4-Ig molecule.

In some aspects, pharmaceutical compositions comprising CTLA4-Ig molecules (e.g., betanaproxen) can be provided as sterile white or off-white lyophilized powders for intravenous administration. The lyophilisate (lyophile) can be reconstituted with a suitable fluid to obtain a clear to slightly opalescent colorless to pale yellow solution having a pH ranging from 7.2 to 7.8. Suitable fluids for reconstitution of the lyophilisate include SWFI, 0.9% NS or D5W. Each single-use vial of CTLA4-Ig molecule (betanaproxen) may also contain: sodium dihydrogen phosphate (34.5 mg), sodium chloride (5.8 mg), and sucrose (500 mg).

V. therapeutic methods

Compositions prepared by the methods of the present disclosure are useful for treating a variety of diseases. The present disclosure provides a method for inhibiting T cell proliferation (or activation) comprising contacting a T cell with an effective amount of a CTLA4-Ig composition of the disclosure. The present disclosure provides a method for inhibiting an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the disclosure. The present disclosure provides a method for inducing immune tolerance to an antigen in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the disclosure. The present disclosure provides a method for treating inflammation in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the disclosure. The present disclosure provides a method for treating rheumatoid arthritis comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the disclosure.

The present disclosure provides a method for treating psoriasis in a subject, comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the disclosure. The present disclosure provides a method for treating or preventing allergy in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the disclosure. The present disclosure provides a method for treating or preventing graft versus host disease in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the disclosure. The present disclosure provides a method for treating or preventing transplant organ rejection in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the disclosure.

The present disclosure provides a method for treating crohn's disease in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the disclosure. The present disclosure provides a method for treating type I diabetes in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the disclosure.

The present disclosure provides a method for treating oophoritis in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the present disclosure. The present disclosure provides a method for treating glomerulonephritis in a subject comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the disclosure. The present disclosure provides a method for treating allergic encephalomyelitis in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the present disclosure.

The present disclosure provides a method for treating myasthenia gravis in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the present disclosure. Thus, in certain aspects of the disclosure, the disclosure provides CTLA4-Ig molecules produced by cell lines in the production methods described herein, for treating T cell-related diseases or disorders, including, but not limited to, generally any T cell-dependent lymphoproliferative disease or disorder and any T cell-dependent autoimmune disease or disorder, and more particularly: t cell lymphoma, T cell acute lymphoblastic leukemia, testicular central T cell lymphoma, benign lymphocytic vasculitis, graft Versus Host Disease (GVHD), immune disorders associated with graft rejection, psoriasis, inflammation, allergies, oophoritis, glomerulonephritis, encephalomyelitis, hashimoto thyroiditis, graves ' disease, addison's disease, primary myxoedema, pernicious anemia, autoimmune atrophic gastritis, rheumatoid arthritis, insulin dependent diabetes mellitus, goodpasture's syndrome, myasthenia gravis, pemphigus, sympathogenic ophthalmitis, autoimmune uveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic active hepatitis, scleroderma, polymyositis, and mixed connective tissue disease.

The present disclosure provides a method for inhibiting T cell proliferation (or activation) comprising contacting a T cell with an effective amount of a CTLA4-Ig composition of the disclosure, with or without another agent (such as methotrexate). The present disclosure provides a method for inhibiting an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the disclosure alone or in combination with methotrexate. The present disclosure provides a method for inducing immune tolerance to an antigen in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the disclosure in combination with methotrexate.

In some aspects, the CTLA4-Ig molecule (e.g., betanaproxen) is indicated for use in preventing organ rejection in adult patients receiving kidney transplantation. In some aspects, the CTLA4-Ig molecule (e.g., betanaproxen) will be used in combination with a bastard-induced, mycophenolate mofetil and a corticosteroid. In some aspects, the CTLA4-Ig molecule (e.g., betanaproxen) is used only in EBV seropositive patients.

Various aspects of the disclosure are described in more detail in the following subsections. The present disclosure is further illustrated by the following examples, which should not be construed as further limiting.

Examples

Example 1

Process A and process B referencing method

Betazepine is a genetically engineered fusion protein consisting of the functional binding domain of human cytotoxic T lymphocyte antigen-4 (CTLA-4) and the Fc domain of human monoclonal immunoglobulins of the IgG1 class. Two amino acid modifications were made in the B7 binding region of CTLA-4 domain, namely leucine to glutamic acid at position 104 and alanine to tyrosine at position 29, to produce betahistine. Betazepine consists of 2 homologous glycosylated polypeptide chains of approximately 46kDa each, which are covalently linked by a single interchain disulfide bond. Betazepine is produced in large scale cell culture using a Chinese Hamster Ovary (CHO) cell line and is initiated by thawing frozen vials from MWCB. The cultures were propagated in a series of shake flask cultures. These cultures were then transferred to a cell bag bioreactor to generate sufficient cell numbers to seed a series of seed bioreactors, followed by a production bioreactor. The production bioreactor is harvested based primarily on the target Sialic Acid (SA) to beta-seprin molar ratio. Finally, the cell culture harvest is clarified in preparation for downstream processing. Representative growth conditions and processes can be seen in fig. 1A, 1B, and 1C. Fig. 1A shows the reactor conditions for the methods of process a and process B under the heading "process a" and "process B". Fig. 1B shows the feed time settings for the growth of the upstream bioreactor of process a and process B, and fig. 1C shows the feed strategy parameters for process a and process B. The results of the sialic acid molar ratio analysis of process a and process B can be seen in figure 2. Process a and process B resulted in higher protein titers but during production resulted in significantly lower sialic acid (NANA) molar ratios, as shown in fig. 2. Since sialic acid molar ratio is a key quality attribute for the production of betanaproxen, process X was subsequently developed.

Example 2

Process C reference method

After seed culture expansion, 0.8x10 was used in 5000L production bioreactor in Process C ⁶ The initial cell density of individual cells/mL, the temperature setting at 37 ℃ and the pH set point of about 7.05 produced betanaproxen. After the culture reached a incubation time of 144 hours, the bioreactor temperature set point was reduced from 37 ℃ to 34 ℃. The acceptable range of sialic acid to betahistine molar ratio at harvest is from about 5.8 to about 6.7 and betahistine is produced at titers of from about 0.34g/L to about 0.82 g/L.

Example 3

CTLA4-Ig 5,000L bioreactor Process-Process X

To maintain the glycosylation profile shown in process C, but also to increase protein yield and/or titre, we produced process X as shown herein. Tables 1 and 2 below summarize the upstream processes and in-process controls (IPCs) defined for the steps of the production bioreactor of betanaproxen. By utilizing a strategy of operating ranges in a set of layered processes with acceptable ranges, action limits, or alarm ranges, the criticality is classified by Critical Process Parameters (CPP), process Parameters (PP), critical Process Attributes (CPA), and Performance Attributes (PA) to ensure consistent monitoring and control of the drug substance manufacturing process.

The seed bioreactor step of betanaproxen manufacture described in table 1 provides sufficient cell culture biomass to seed the production bioreactor. Samples were taken daily from the seed bioreactor to monitor cell growth, percent cell viability, and metabolite and nutrient concentrations. Robust growth and viability of the cell culture used to inoculate the production bioreactor is important to ensure consistent production of the betanapin fusion protein. The general process flow for the seed bioreactor step and the production bioreactor step of the betanaproxen manufacture is shown in fig. 5.

TABLE 1 seed reactor

The reactor conditions used during the production of the production bioreactor stage are described in table 2 below and fig. 1A and 1B. Specifically, the initial temperature was set to 36 ℃ in order to maintain high cell viability and sialic acid (NANA) content. The production bioreactor was adjusted to a pH set point of 7.15±0.1 in order to increase growth and sialic acid (NANA) content. During production, the temperature was adjusted to about 33 ℃ about 240 hours after induction of the production bioreactor, as detailed in fig. 1A and table 2. This temperature adjustment is performed in order to maintain the titer throughout the production run in the bioreactor. Furthermore, as described in fig. 1A and table 2, the temperature was further reduced to 31 ℃ about 240 hours after induction, in order to further maintain the titer during the production run. Cultures were harvested at approximately 240-420 hours to ensure that the desired sialic acid (NANA) molar ratio range of 6.2 to 7.4 was achieved, as depicted in fig. 2. Further reaction parameters are detailed in Table 2. Process X shows a production titer of greater than 2.0g/L, at least 2 times the titer of process C (see example 2), and the protein produced by process X has a much higher sialic acid molar ratio (NANA) compared to process a and process B, as shown in fig. 2.

TABLE 2 production bioreactor conditions.

* The pressure can exceed 12psig during and 15 minutes after inoculation.

* Additional 4.5ppm additions may be added as needed to reduce foaming of the cell culture.

Example 4

Glycosylation analysis of Betacalcp-Process X

Immunoglobulin degrading enzyme (IdeS) from streptococcus pyogenes is a unique enzyme that digests the hinge region of IgG at a specific site just below the hinge region, thereby producing CTLA4 fragment and two Fc fragments of betanaproxen. Complete protein digestion of the betahistine Drug Substance (DS) occurs within 1 hour at 37 ℃. The digested sample was then purified by elution using a protein a spin column. The resin binds the Fc fragment while allowing CTLA4 fragment to flow through. Using AgilentThe CTLA4 fragment was prepared by an InstantPC N-glycan release and fluorescent labelling kit and analyzed on an ultra-high performance liquid chromatography system-fluorescence detection (UPLC-FLR).

UPLC is a chromatographic technique that operates at higher pressures than High Performance Liquid Chromatography (HPLC) and separates due to the compatibility of the compounds and the characteristics of the column used. In this method, the purified N-glycans of the CTLA4 region of Betazeep DS are fluorescently labeled and analyzed by UPLC. The glycans were then separated using a gradient method and an amide column using the difference in analyte polarity. The amount of fluorescently labeled N-glycans in the purified CTLA4 fragment is determined by area%. This method is suitable for determining the amount of fluorescently labeled N-glycans present in the CTLA4 region of a betaxopril DS sample. Preparation of beta in 25mM sodium phosphate, 10mM sodium chloride (pH 7.5) Sipran DS. The method was carried out on a Waters Acquity UPLC system configured with Waters Acquity UPLC glycans BEH amide @1.7 μm,2.1mm x 150 mm) column and Waters Acquity FLR detector. Chromatographic separation parameters for analysis are shown in table 3:

TABLE 3 Table 3

The full-scale and enlarged representative chromatograms of the N-glycan profile eluted by the betahistine Reference Standard (RS), in which specific glycans (G0F, G1F, G2F, S G1F and S2G 2F) are labeled, can be seen in fig. 3A and 3B, while the full-scale and enlarged representative chromatograms of the N-glycan profile eluted by the betahistine RS, in which glycans S1G1F elute as two peaks, can be seen in fig. 4A and 4B. Various glycans are shown in fig. 6A and 6B.

***

Throughout the present application, various publications are referenced in parentheses, either by author name and date, or by patent number or patent publication number. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the date of the present disclosure as of the state of the art as known to the skilled artisan as of course described and claimed herein. However, citation of a reference herein shall not be construed as an admission that such reference is prior art to the present disclosure.

SEQUENCE LISTING

<110> Bai Shi Guibao Co

<120> method for producing protein

<130> 3338.205PC02

<150> US 63/066,127

<151> 2020-08-14

<150> US 63/199,547

<151> 2021-01-07

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> CTLA4 Extracellular Domain

<400> 1

Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile

1 5 10 15

Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val

20 25 30

Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys

35 40 45

Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser

50 55 60

Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln

65 70 75 80

Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu

85 90 95

Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile

100 105 110

Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp

115 120

<210> 2

<211> 383

<212> PRT

<213> Artificial Sequence

<220>

<223> CTLA4A29YL104E-Ig amino acid sequence

<400> 2

Met Gly Val Leu Leu Thr Gln Arg Thr Leu Leu Ser Leu Val Leu Ala

1 5 10 15

Leu Leu Phe Pro Ser Met Ala Ser Met Ala Met His Val Ala Gln Pro

20 25 30

Ala Val Val Leu Ala Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu

35 40 45

Tyr Ala Ser Pro Gly Lys Tyr Thr Glu Val Arg Val Thr Val Leu Arg

50 55 60

Gln Ala Asp Ser Gln Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met

65 70 75 80

Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser

85 90 95

Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp

100 105 110

Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr

115 120 125

Tyr Glu Gly Ile Gly Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu

130 135 140

Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys Ser Ser Asp Lys Thr His

145 150 155 160

Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Ser Ser Val

165 170 175

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

180 185 190

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

195 200 205

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

210 215 220

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

225 230 235 240

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

245 250 255

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

260 265 270

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

275 280 285

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

290 295 300

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

305 310 315 320

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

325 330 335

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

340 345 350

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

355 360 365

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

370 375 380

<210> 3

<211> 359

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acids 25-383 of SEQ ID NO: 2

<400> 3

Met Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg

1 5 10 15

Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Tyr Thr

20 25 30

Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu

35 40 45

Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp

50 55 60

Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr

65 70 75 80

Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val

85 90 95

Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr

100 105 110

Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu

115 120 125

Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro

130 135 140

Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

145 150 155 160

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

165 170 175

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

180 185 190

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

195 200 205

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

210 215 220

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

225 230 235 240

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

245 250 255

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

260 265 270

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

275 280 285

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

290 295 300

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

305 310 315 320

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

325 330 335

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

340 345 350

Leu Ser Leu Ser Pro Gly Lys

355

<210> 4

<211> 358

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acids 26-383 of SEQ ID NO: 2

<400> 4

Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly

1 5 10 15

Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Tyr Thr Glu

20 25 30

Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val

35 40 45

Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp

50 55 60

Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile

65 70 75 80

Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu

85 90 95

Leu Met Tyr Pro Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr Gln

100 105 110

Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro

115 120 125

Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu

130 135 140

Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

145 150 155 160

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

165 170 175

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

180 185 190

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

195 200 205

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

210 215 220

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

225 230 235 240

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

245 250 255

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

260 265 270

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

275 280 285

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

290 295 300

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

305 310 315 320

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

325 330 335

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

340 345 350

Ser Leu Ser Pro Gly Lys

355

<210> 5

<211> 357

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acids 27-383 of SEQ ID NO: 2

<400> 5

Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile

1 5 10 15

Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Tyr Thr Glu Val

20 25 30

Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys

35 40 45

Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser

50 55 60

Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln

65 70 75 80

Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu

85 90 95

Met Tyr Pro Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr Gln Ile

100 105 110

Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys

115 120 125

Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu

130 135 140

Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

145 150 155 160

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

165 170 175

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

180 185 190

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

195 200 205

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

210 215 220

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

225 230 235 240

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

245 250 255

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

260 265 270

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

275 280 285

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

290 295 300

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

305 310 315 320

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

325 330 335

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

340 345 350

Leu Ser Pro Gly Lys

355

<210> 6

<211> 358

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acids 25-382 of SEQ ID NO: 2

<400> 6

Met Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg

1 5 10 15

Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Tyr Thr

20 25 30

Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu

35 40 45

Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp

50 55 60

Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr

65 70 75 80

Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val

85 90 95

Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr

100 105 110

Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu

115 120 125

Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro

130 135 140

Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

145 150 155 160

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

165 170 175

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

180 185 190

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

195 200 205

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

210 215 220

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

225 230 235 240

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

245 250 255

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

260 265 270

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

275 280 285

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

290 295 300

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

305 310 315 320

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

325 330 335

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

340 345 350

Leu Ser Leu Ser Pro Gly

355

<210> 7

<211> 357

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acids 26-382 of SEQ ID NO: 2

<400> 7

Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly

1 5 10 15

Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Tyr Thr Glu

20 25 30

Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val

35 40 45

Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp

50 55 60

Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile

65 70 75 80

Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu

85 90 95

Leu Met Tyr Pro Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr Gln

100 105 110

Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro

115 120 125

Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu

130 135 140

Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

145 150 155 160

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

165 170 175

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

180 185 190

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

195 200 205

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

210 215 220

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

225 230 235 240

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

245 250 255

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

260 265 270

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

275 280 285

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

290 295 300

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

305 310 315 320

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

325 330 335

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

340 345 350

Ser Leu Ser Pro Gly

355

<210> 8

<211> 356

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acids 27-382 of SEQ ID NO: 2

<400> 8

Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile

1 5 10 15

Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Tyr Thr Glu Val

20 25 30

Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys

35 40 45

Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser

50 55 60

Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln

65 70 75 80

Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu

85 90 95

Met Tyr Pro Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr Gln Ile

100 105 110

Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys

115 120 125

Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu

130 135 140

Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

145 150 155 160

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

165 170 175

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

180 185 190

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

195 200 205

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

210 215 220

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

225 230 235 240

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

245 250 255

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

260 265 270

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

275 280 285

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

290 295 300

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

305 310 315 320

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

325 330 335

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

340 345 350

Leu Ser Pro Gly

355

<210> 9

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> CTLA4 Extracellular Domain with A29Y and L104E Mutations

<400> 9

Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile

1 5 10 15

Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Tyr Thr Glu Val

20 25 30

Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys

35 40 45

Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser

50 55 60

Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln

65 70 75 80

Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu

85 90 95

Met Tyr Pro Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr Gln Ile

100 105 110

Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp

115 120

Claims (68)

1. A method of increasing the yield of a protein and/or controlling glycosylation of the protein during a protein production phase, the method comprising culturing cells capable of expressing the protein in a bioreactor under suitable conditions during a protein induction phase, wherein the suitable conditions comprise a pH set point between about 7.1 and about 7.2.

2. The method of claim 1, wherein the pH set point is about 7.15.

3. The method of claim 1 or 2, wherein the suitable conditions further comprise an initial temperature set point between about 35 ℃ and about 37 ℃, a second temperature set point between about 32 ℃ and about 34 ℃, and a third temperature set point between about 30 ℃ and about 32 ℃.

4. The method of any one of claims 1 to 3, wherein the suitable conditions further comprise a temperature of about 0.5X10 ⁶ Individual cells/mL and about 1X 10 ⁶ An initial Viable Cell Density (VCD) set point between individual cells/mL.

5. The method of any one of claims 1 to 4, wherein the suitable conditions comprise:

a. an initial temperature set point of about 36 ℃, a second temperature set point of about 33 ℃, and a third temperature set point of about 31 ℃;

b. A pH set point of about 7.15; and

c. about 0.70X10 ⁶ Initial viable cell density of individual cells/mL(VCD) setpoint.

6. A method of controlling cell growth rate, cell viability, viable cell density, and/or cell titer to produce a protein, the method comprising culturing the cells in a bioreactor at a pH set point of about 7.15 during a protein induction phase.

7. The method of claim 6, wherein the suitable conditions further comprise (i) an initial temperature set point of about 36.0 ℃ and a second temperature set point of less than about 36 ℃; (ii) An initial temperature set point of less than about 36.5 ℃ and a final temperature set point of about 31 ℃; or (iii) an initial temperature set point of less than about 36.5 ℃, a second temperature set point of about 33 ℃, and a final temperature set point of less than about 33 ℃.

8. The method of claim 6, wherein the suitable conditions further comprise incubating the cells at an initial temperature set point of about 36 ℃, a second temperature set point of about 33 ℃, and a final temperature set point of about 31 ℃.

9. The method of any one of claims 6 to 8, wherein the suitable conditions further comprise about 0.70X10 ⁶ Initial Viable Cell Density (VCD) set point of individual cells/mL.

10. A method of increasing yield of and/or controlling glycosylation of betaxolol during a protein production phase, the method comprising culturing cells capable of expressing betaxolol in a bioreactor under suitable conditions, wherein the suitable conditions comprise:

a. an initial temperature set point of about 36 ℃, a second temperature set point of about 33 ℃, and a third temperature set point of about 31 ℃;

b. a pH set point of about 7.15; and

c. about 0.70X 10 ⁶ Initial Viable Cell Density (VCD) set point of individual cells/mL.

11. The method of any one of claims 1 to 10, wherein the suitable conditions further comprise a first feed time of about 80 hours.

12. The method of any one of claims 1 to 11, wherein the third or final temperature set point occurs between about 204 hours and about 276 hours.

13. The method of claim 12, wherein the third or final temperature set point occurs about 204 hours, about 216 hours, about 228 hours, about 240 hours, about 252 hours, about 264 hours, or about 276 hours after the initial temperature set point.

14. The method of any one of claims 1 to 13, wherein the third final temperature set point is about 31 ℃ and occurs after about 240 hours.

15. The method of any one of claims 1 to 14, wherein the second temperature set point occurs between about 72 hours and about 168 hours.

16. The method of claim 15, wherein the second temperature set point occurs at about 72 hours, about 78 hours, about 84 hours, about 90 hours, about 96 hours, about 102 hours, about 108 hours, about 114 hours, about 120 hours, about 126 hours, about 132 hours, about 138 hours, about 144 hours, about 150 hours, about 156 hours, about 162 hours, or about 168 hours.

17. The method of any one of claims 1 to 16, wherein the second temperature set point is about 33 ℃ after about 140 hours.

18. The method of any one of claims 1 to 17, wherein the conditions increase the protein yield by at least 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, or at least about 400% as compared to a reference method without the suitable conditions.

19. The method of any one of claims 1 to 18, wherein the suitable conditions further comprise manganese in the bioreactor.

20. The method of claim 19, wherein the manganese is present at a concentration of from about 1.6 parts per billion (ppb) to about 15 ppb.

21. The method of claim 19, wherein the manganese is present at a concentration of from about 3ppb to about 6 ppb.

22. The method of any one of claims 1 to 21, wherein the method reduces the rate of cell growth.

23. The method of any one of claims 1 to 22, wherein the method controls cell viability.

24. The method of claim 23, wherein the cell viability is displayed at about 10.0x 10 ⁶ Individual cells/mL and about 15.0x10 ⁶ Average peak Viable Cell Density (VCD) between individual cells/mL.

25. The method of any one of claims 1 to 24, wherein the method controls titre.

26. The method of claim 25, wherein the titer exhibits a final titer between about 1.50g/L and about 3.5 g/L.

27. The method of claim 25, wherein the titer exhibits a final titer of greater than about 2.00 g/L.

28. The method of any one of claims 1 to 27, wherein the method controls the glycosylation profile of the protein.

29. The method of claim 28, wherein the glycosylation profile comprises one or more N-linked glycans.

30. The method of claim 28 or 29, wherein the glycosylation profile is measured during the protein production phase.

31. The method of claim 30, wherein the glycosylation profile is measured about every 1 day.

32. The method of any one of claims 29 to 31, wherein the glycosylation profile is measured when the cell culture is harvested.

33. The method of any one of claims 29 to 32, wherein the N-linked glycans comprise: G0F, G1F, G2F, S1G1F, S1G2F, S G2F or any combination thereof.

34. The method of any one of claims 1 to 9 or 11 to 33, wherein the protein comprises a CTLA4 domain.

35. The method of any one of claims 1 to 9 or 11 to 34, wherein the protein is a fusion protein.

36. The method of claim 35, wherein the fusion protein comprises an Fc portion.

37. The method of any one of claims 1 to 9 or 11 to 36, wherein the protein is betanaproxen.

38. The method of any one of claims 1 to 37, wherein the protein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID No. 5.

39. The method of any one of claims 29 to 38, wherein the one or more N-linked glycans are located at one or more residues selected from Asn76, asn108 and/or Asn207 of betanaproxen.

40. The method of any one of claims 29 to 39, wherein the one or more N-linked glycans comprise sialic acid and have a molar ratio of NANA of from about 4 to about 10.

41. The method of any one of claims 29 to 40, wherein the one or more N-linked glycans comprise sialic acid and have a molar ratio of NANA of from about 5 to about 9, from about 5.5 to about 8.5, from about 5.8 to about 6.7, from about 5.2 to about 7.5, from about 6 to about 8, from about 6.2 to about 7.4, or from about 5 to about 6.

42. The method of claim 41, wherein the NANA molar ratio is about 6.8.

43. The method of any one of claims 28 to 42, wherein the glycosylation profile is analyzed via an N-linked carbohydrate profiling method.

44. The method of any one of claims 28 to 43, wherein the glycosylation profile comprises one or more O-linked glycans.

45. The method of claim 44, wherein the O-linked glycans are located at residues Ser129, ser130, ser136 and/or Ser 139.

46. The method of any one of claims 1 to 45, which is performed as a fed-batch culture process.

47. The method of any one of claims 1 to 46, wherein glucose and/or galactose is supplemented into a feed medium in the bioreactor.

48. The method of claim 47, wherein the feed medium is added to the bioreactor periodically.

49. The method of claim 48, wherein the feed medium is added to the bioreactor about every 24 hours.

50. The method of any one of claims 1 to 45, which is performed as a perfusion process.

51. The method of any one of claims 1 to 50, wherein the cell is a mammalian cell.

52. The method of claim 51, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

53. The method of claim 52, wherein said mammalian cell is a CHO-K1 cell, a CHO-DXB11 cell or a CHO-DG44 cell.

54. A method of assaying glycans of CTLA4-Fc fusion proteins, the method comprising measuring one or more N-linked glycans attached to one or more asparagine residues in CTLA4 proteins, wherein one of the glycans comprises G0F, G1F, G2F, S1G1F, S1G2F and/or S2G2F.

55. The method of claim 54, wherein the glycans are measured via ultra-high performance liquid chromatography-fluorescence detection (UPLC-FLR).

56. The method of claim 54 or 55, wherein the Fc domain of the CTLA4-Fc fusion protein is cleaved prior to the measuring.

57. The method of claim 54, wherein the Fc domain of the CTLA4-Fc fusion protein is not cleaved prior to the measuring.

58. A protein produced by the method of any one of claims 1 to 57.

59. A protein produced by the method of any one of claims 1 to 9 and 11 to 57, wherein the protein comprises a CTLA4-Fc fusion protein.

60. The protein of claim 59, wherein the protein is betanaproxen.

61. A cell produced by the method of any one of claims 1 to 57.

62. A cell produced by the method of any one of claims 1 to 50 and 54 to 57, wherein the cell is a mammalian cell.

63. The cell of claim 62, wherein the cell is a Chinese Hamster Ovary (CHO) cell.

64. The cell of claim 63, wherein the cell is a CHO-K1 cell, a CHO-DXB11 cell or a CHO-DG44 cell.

65. A bioreactor for producing a protein produced according to the method of any one of claims 1 to 57.

66. A bioreactor comprising the cells and cell culture medium of any one of claims 61 to 64, wherein the bioreactor is maintained at a pH of about 7.15.

67. The bioreactor of claim 66, wherein said bioreactor is maintained at:

a. an initial temperature set point of about 36 ℃, a second temperature set point of about 33 ℃, and a third temperature set point of about 31 ℃;

b. a pH set point of about 7.15; and

c. about 0.70X 10 ⁶ Initial Viable Cell Density (VCD) set point of individual cells/mL.

68. The bioreactor according to claim 66 or 67, wherein said cell culture medium further comprises manganese.