Methods for reducing the oxidation level of cysteine residues in a secreted recombinantly-expressed protein during cell culture

Abstract

The present disclosure relates to methods for reducing the level of oxidation of cysteine residues in a recombinant polypeptide, such as an anti-IL-17 antibody, that has been recombinantly produced by mammalian cells during cell culture, e.g., production of secukinumab antibodies. Also provided are purified preparations of a recombinant polypeptide, such as an anti-IL-17 antibody, or antigen binding fragment thereof, produced by such methods, e.g., purified preparations of secukinumab. Also provided are purified preparations of a recombinant polypeptide produced by such methods, wherein the level of active recombinant polypeptide in the preparation is higher.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202610553586.7 (22) Application Date 2021.12.21 (30) Priority Data 63 / 129,091 2020.12.22 US (62) Divisional Application Data 202180087080.7 2021.12.21 (71) Applicant Novartis AG Address Switzerland (72) Inventors N. Buckkalinhaas H. Tsui D. Garcia M. Gobert J. Schulz (74) Patent Agency Beijing Zhongzi Law Firm 11247 Patent Attorney Gao Fei Huang Gesheng (51) Int.Cl. C07K 16 / 24 (2006.01) C12N 5 / 00 (2006.01) C12P 21 / 00 (2006.01) (54) Title of Invention: Method for Reducing Oxidation Levels of Cysteine ​​Residues in Secreted Recombinant Expression Proteins During Cell Culture (57) Abstract: This disclosure relates to a method for reducing the oxidation levels of cysteine ​​residues in recombinant polypeptides, such as anti-IL-17 antibodies, that have been recombined and produced from mammalian cells during cell culture (e.g., preparation of secukinumab antibodies). Purified formulations of recombinant polypeptides, such as anti-IL-17 antibodies or antigen-binding fragments thereof, produced by such methods are also provided, for example, purified formulations of secukinumab. Purified formulations of recombinant polypeptides produced by such methods are also provided, wherein the levels of active recombinant polypeptides in said formulations are high. Claims (2 pages), Description (27 pages), Sequence Listing (electronic publication), Drawings (4 pages) CN 122427280 A 2026.07.21 CN 1 22 42 72 80 A 1. A method for generating a recombinant polypeptide in fed-batch cell cultures, wherein the recombinant polypeptide is an antibody, namely secukinumab, wherein the method comprises the steps of: a. culturing mammalian cells in a cell culture medium comprising a basal medium and one or more feed media, wherein the basal medium contains a cys equivalent at a concentration of 0.3 g / L, and wherein the feed media contains a cys equivalent at a concentration of less than 0.8 g / L, and wherein the cumulative concentration of the cys equivalent in the cell culture medium is less than 0.4 g / L, wherein the mammalian cells are CHO cells; b. expressing the recombinant polypeptide; and c. recovering the recombinant polypeptide from the culture medium. 2. The method of claim 1, wherein the basal medium does not contain added cysteine, and the feed media contains cysteine ​​at a concentration of 0.66 g / L.3. The method of claim 1, wherein the basal culture medium does not contain added cysteine, and the feed culture medium contains cysteine ​​at a concentration of 0.33 g / L. 4. The method of claim 1, wherein the basal culture medium does not contain added cysteine, and the feed culture medium does not contain cysteine. 5. The method of any one of claims 1-4, the method comprising a downstream processing step of selective reduction, wherein the antibody is incubated with at least one reducing agent in a system to form a reducing mixture. 6. The method of any one of claims 1-5, the method further comprising the steps of: d. purifying the antibody; e. formulating the antibody for administration; f. packaging the antibody with a booklet. 7. The method according to any one of claims 1-6, wherein the recombinant polypeptide population recovered from the culture medium contains at least 10% higher levels of reduced free cysteine ​​compared to the recombinant polypeptide population recovered from the control culture medium, wherein the control culture medium comprises a control basal medium having a concentration greater than 0.4 g / L of cys equivalents and / or a control feed medium containing a concentration greater than 0.9 g / L of cys equivalents, and / or wherein the cumulative concentration of cys equivalents in the control cell culture is greater than 0.4 g / L. 8. The method according to any one of claims 1-7, wherein the method comprises producing a higher yield of recombinant polypeptides per mg active recombinant polypeptide / L culture medium compared to a control method, wherein the control method comprises culturing mammalian cells in a control cell culture medium comprising a control basal medium and one or more control feed media, wherein the control basal medium contains a concentration greater than 0.4 g / L of cys equivalents, and / or wherein the control feed medium contains a concentration greater than 0.9 g / L of cys equivalents, and / or wherein the cumulative concentration of cys equivalents in the control cell culture is greater than 0.4 g / L. 9. The method of claim 7 or 8, wherein the method comprises, as determined from used culture medium, producing a population of recombinant polypeptides having at least 61% reduced free cysteine.10. A method for producing a recombinant polypeptide from a mammalian cell culture, wherein the recombinant polypeptide is an antibody, namely secukinumab, and wherein the mammalian cell is a CHO cell, wherein the method comprises the steps of: a. culturing the mammalian cell in a culture containing a cell culture medium, wherein the cell culture medium contains a cys equivalent at a concentration of 0.3 g / L; b. exchanging a portion of the cell culture medium in the culture with fresh cell culture medium by perfusion, wherein the fresh cell culture medium contains a cys equivalent at a concentration of 0.3 g / L, and / or wherein the concentration of the accumulated cys equivalent added to the culture is less than 7 g / L / day, or less than 0.4 g / L / day; c. expressing the recombinant polypeptide; and d. recovering the recombinant polypeptide from the culture. 11. The method of claim 10, wherein the fresh culture medium does not contain added cysteine. 12. The method of claim 10, wherein the method comprises exchanging at least 50% of the cell culture medium daily by perfusion with fresh cell culture medium. 13. The method of any one of claims 10 to 12, wherein the recombinant polypeptide population recovered from the culture medium contains at least 10% higher levels of reduced free cysteine ​​compared to the recombinant polypeptide population recovered from the control culture medium, wherein the control culture medium contains a concentration greater than 0.4 g / L of cys equivalents, and / or wherein the cumulative concentration of cys equivalents added to the control cell culture is greater than 7 g / L / day, and / or greater than 0.4 g / L / day. 14. The method of any one of claims 10 to 13, wherein the method comprises producing a higher yield of recombinant polypeptides per mg active recombinant polypeptide / L culture medium compared to a control method, wherein the control method comprises culturing mammalian cells in a control cell culture medium containing a concentration greater than 0.4 g / L of cys equivalents, and / or wherein the cumulative concentration of cys equivalents in the control cell culture is greater than 7 g / L / day, and / or greater than 0.4 g / L / day. Claims 2 / 2 Page 3 CN 122427280 A Method for reducing the oxidation level of cysteine ​​residues in secreted recombinant expressed proteins during cell culture

[0001] This application is a divisional application of PCT application PCT / IB2021 / 062126, filed on December 21, 2021, entitled “Method for reducing the oxidation level of cysteine ​​residues in secreted recombinant expressed proteins during cell culture”, which entered the Chinese national phase on June 21, 2023, with application number 202180087080.7.Technical Field

[0002] This disclosure relates to a method for reducing the oxidation level of one or more cysteine ​​residues in a secreted recombinant expressed protein during cell culture, such as during the recombinant production of anti-IL-17 antibodies like secukinumab in mammalian cells. Background Art

[0003] Classical antibodies consist of two light chains (L) and two heavy chains (H), each with a molecular weight of about 25 kD and each heavy chain with a molecular weight of about 50 kD. The light and heavy chains are linked by disulfide bonds (LSSH), and the two LH units are further linked between the heavy chains by two disulfide bonds. The general formula for classical antibodies is L-SS-H(-SS-)2H-SS-L or simplified to H2L2 (HHLL). In addition to these conserved interchain disulfide bonds, there are also conserved intrachain disulfide bonds. Both types of disulfide bonds are important for the stability and behavior (e.g., affinity) of the antibody. Typically, disulfide bonds are generated by two cysteine ​​residues (Cys-SH) found at conserved positions in the antibody chain, which spontaneously form a disulfide bond (Cys-SS-Cys). The formation of disulfide bonds is determined by the redox potential of the environment and the presence of specialized enzymes in thiol-disulfide exchange. The internal disulfide bond (Cys-SS-Cys) stabilizes the three-dimensional structure of the antibody.

[0004] Antibodies containing one or more additional free cysteines (i.e., unpaired cysteines) exist. In some cases, one or more free cysteines are involved in antigen recognition and binding, for example, because of the presence of free cysteines in the complementarity-determining region of the antibody. For these antibodies, modification of free cysteines may have a negative impact on the activity and stability of the molecule and may lead to increased immunogenicity. Therefore, processing these antibodies can be difficult because the final product may contain a large amount of inactive, misfolded, and / or useless antibody material. Anti-IL-17 antibodies, such as secukinumab (i.e., AIN457), which are incorporated herein by reference in their entirety by US 20090280131, have a free cysteine ​​residue following cis-proline in the light chain complementarity-determining region (CDR) loop 3 (L-CDR3) (i.e., the eighth amino acid of L-CDR3 as shown in SEQ ID NO:6, which corresponds to amino acid 97 in the light chain variable region as shown in SEQ ID NO:10, hereinafter referred to as "CysL97"). To maintain full activity, the unpaired cysteine ​​residues of secukinumab cannot be masked by pairing with disulfides of other cysteine ​​residues or by oxidation with exogenous compounds (e.g., forming mixed disulfides with other proteins, derivatization with cellular metabolites (e.g., cysteine ​​or glutathione), and formation of sulfoxides by oxygen).Unfortunately, because secukinumab is manufactured using mammalian cells that secrete it into the cell culture medium, undesirable cell-based CysL97 modification does occur.

[0005] Similarly, engineering free cysteine ​​into antibody sequences can be used to facilitate site-directed conjugation of chemical linkers, drugs, labels, and / or other parts. For example, Junutula et al. (Nat. Biotechnol. [Nature Biotechnology], 2008, 26, 925–932) introduced engineered cysteine ​​into an anti-MUC16 antibody by mutating heavy chain alanine 114. The authors found that expression of the mutated antibody in Chinese hamster ovary (CHO) cells generated an antibody with an engineered cysteine ​​residue capped with either cysteine ​​or glutathione as a disulfide. Therefore, engineered cysteine ​​residues must be processed to remove undesirable cell-based modifications.

[0006] Methods for selectively reducing antibodies whose free cysteine ​​residues have been oxidized have been reported. For example, WO 2016 / 103146 ​​A1 discloses the reduction of oxidized CysL97 in a formulation of an IL-17 antibody that has been recombined and generated in mammalian cells. Specifically, downstream processing steps are applied, such as contacting the antibody-containing formulation with at least one reducing agent in a system to form a reducing mixture; and incubating the reducing mixture while maintaining an oxygen volumetric mass transfer coefficient (kLa) in the system < about 0.37 h⁻¹, where kLa is calculated by adapting a dissolved oxygen profile to a saturation profile. Similarly, Junutula et al. reported a procedure using a strong reducing agent (e.g., tris(2-carboxyethyl)phosphine [TCEP] or dithiothreitol [DTT]) to purify the reduced antibody and subsequently reoxidize the interchain disulfide bonds using Cu²⁺ or dehydroascorbic acid.

[0007] However, such downstream method steps may require expensive equipment, additional purification steps, and result in extended process lead time. Therefore, there is a need for improved methods that allow for faster and / or lower overall manufacturing. Consequently, methods in the upstream processing steps of recombinant antibody production can also be evaluated for optimization.

[0008] The culture of secretory mammalian cells for industrial applications, such as the expression of recombinant peptides, requires culture media that support growth and production. Such media must support high viable cell densities while also stimulating the synthesis and extracellular transport of bioproducts. Early efforts in culture media development have yielded basic formulations that maintain growth, viability, and cell function, but these basic formulations contain animal-derived components and complex components for batch culture modalities.Subsequent improvements included the development of serum-free and chemically defined (CD) media, the identification of key nutrients, growth factors, and potentially inhibitory or toxic cellular metabolites, and the optimization of nutrient delivery using fed-batch and perfusion culture techniques while minimizing the accumulation of unwanted waste products.

[0009] All cell culture media require similar basic nutrients to support cell growth. Amino acids are key components of cell culture media, and studies have confirmed that small changes in the amino acid composition of cell culture media can alter growth curves and titers. For example, Ghaffari et al. (Biotechnol Progress. 2020; 36:e2946) reported that maintaining the availability of the so-called non-essential amino acid cysteine ​​is a key process parameter for high-yield recombinant protein production in common CHO cell lines. However, cysteine ​​is readily oxidized under the pH and oxygen and metal enrichment conditions of typical cell culture media. However, cysteine ​​can also promote the undesirable oxidation of free cysteine ​​residues in recombinant proteins. Therefore, cysteine ​​feeding strategies often need to be highly optimized to obtain high yields of recombinant expressed proteins from CHO cells.

[0010] Many cell culture media and feeders are commercially available to provide key nutrients. However, to optimize the quality and yield of secretory recombinant peptides with reduced cysteine ​​residues in production, it remains necessary to tailor culture media and feeders specifically for recombinant peptides. Process optimization is complex due to the many parameters that can be varied in cell culture conditions, and even for commercially available recombinant peptides such as antibodies, optimized processes for industrial production are still needed. Summary of the Invention

[0011] Despite extensive research and optimization in the field of cell culture media, it has been surprisingly found that reducing the amount of cysteine ​​added to mammalian cell culture media to lower the concentration of cys equivalents in the medium can reduce undesirable modifications of free cysteine ​​to the expressed antibody. This reduction in the amount of undesirable free cysteine ​​modification can lead to increased antibody activity and / or avoid the need for subsequent antibody reduction and optional re-oxidation.

[0012] According to a first aspect, this disclosure provides a method for generating a recombinant polypeptide in a fed-batch cell culture, the method comprising the steps of: a. culturing mammalian cells in a cell culture medium comprising a basal medium and one or more feed media, wherein the basal medium contains a cys equivalent at a concentration of about 0.3 g / L, and wherein the feed media contains a cys equivalent at a concentration of less than about 0.8 g / L, and wherein the cumulative concentration of the cys equivalent in the cell culture medium is less than about 0.4 g / L; b. expressing the recombinant polypeptide; and c. recovering the polypeptide from the culture medium.

[0013] The accumulated cys equivalent in the cell culture medium is the total concentration of cysteine ​​and cystine in the cell culture medium derived from the basal medium and / or the feed medium. The accumulated cys equivalent concentration may be different at the start of the fed-batch method compared to the end of the method, for example, after several hours or days, and may vary during the method as feed medium is added to the cell culture medium. In one embodiment, the concentration of the cys equivalent in the cell culture medium at the start of the fed-batch method may be less than about 0.6 g / L. For example, less than about 0.5 g / L, less than about 0.4 g / L, and preferably less than about 0.3 g / L. During the fed-batch method, the concentration of the cys equivalent may vary due to the addition of 0.3 g / L to about 0.8 g / L of the cys equivalent to the cell culture medium (caused by the addition of cysteine ​​or cystine). To obtain this concentration of cys equivalent, the concentration of cysteine ​​in the cell culture medium added to the feed medium may be less than about 1 g / L. For example, the basal culture medium may not contain added cysteine, and the feed medium may contain cysteine ​​at a concentration of less than about 1.0 g / L, for example less than about 0.9 g / L, less than about 0.8 g / L, and preferably less than about 0.7 g / L. In one embodiment, the basal culture medium may not contain added cysteine, and the feed medium may contain cysteine ​​at a concentration of about 0.66 g / L. In another embodiment, the basal culture medium may not contain added cysteine, and the feed medium may contain cysteine ​​at a concentration of about 0.33 g / L. In yet another embodiment, the basal culture medium may not contain added cysteine, and the feed medium may also not contain cysteine.

[0014] At the end of the feed batch method as described herein, the concentration of accumulated CYS equivalents in the cell culture medium may be less than about 0.4 g / L. In standard fed-batch cell culture, where no alterations are made to reduce cys equivalents in the basal and / or fed culture media, the cumulative concentration of cys equivalents in the cell culture medium can be approximately 0.6 g / L.

[0015] In one embodiment, the recombinant polypeptide generated in the fed-batch cell culture is an antibody, preferably an anti-IL-17 antibody, i.e., secukinumab.

[0016] In one embodiment, the mammalian cells used in the fed-batch cell culture are selected from the group consisting of CHO cells, HEK cells, and SP2 / O cells. For example, the mammalian cells can be CHO cells selected from the group consisting of CHO-S, CHO K1, CHO pro3-, CHO DG44, CHO P12, or dhfr-CHO cell lines DUK-BII, DUXBI 1, or CHO-K1SV.

[0017] In a second aspect, the method described above for reducing the concentration of Cys equivalents in cell culture medium can be combined with a downstream processing step of selective reduction, wherein an antibody is incubated with at least one reducing agent in a system to form a reducing mixture, and the reducing mixture is incubated while maintaining an oxygen volumetric mass transfer coefficient (kLa) in the system < about 0.37 h⁻¹, wherein kLa is calculated by adapting a dissolved oxygen profile to a saturation profile. Preferably, the antibody is secukinumab. Such a downstream processing step for selectively reducing cysteine ​​residues at position CysL97 in an IL-17 antibody formulation is disclosed in WO 2016 / 103146 ​​A1.

[0018] The recombinant polypeptide produced according to the method disclosed herein can be prepared for administration to human patients by performing additional steps in preparing a pharmaceutical product. For example, in the case where the recombinant polypeptide is an antibody, the antibody must be purified and formulated with various excipients to provide a pharmaceutical composition suitable for administration to a patient. Additionally, the antibody can be packaged with a brochure containing instructions for administration to a patient. Such booklets can provide the dosage, route of administration, regimen, and total duration of treatment for the blocked antibody. Specification 3 / 27 pages 6 CN 122427280 A

[0019] In one aspect, the present invention provides a method for producing a recombinant polypeptide from a mammalian cell culture, the method comprising the steps of: a) culturing mammalian cells (e.g., selected from the group consisting of CHO cells, HEK cells, and SP2 / 0 cells) in a culture containing a cell culture medium, wherein the cell culture medium contains a cys equivalent at a reduced concentration compared to a control basal medium; b) replacing all or part of the cell culture medium in the culture with fresh cell culture medium by perfusion, wherein the fresh cell culture medium contains a cys equivalent at a reduced concentration compared to a control exchange medium; c) expressing the recombinant polypeptide; and d) recovering the polypeptide from the culture.

[0020] In some embodiments, the perfused cell culture medium comprises a cys equivalent at a concentration of about 0.1 g / L to less than about 0.6 g / L, about 0.2 g / L to less than about 0.5 g / L, about 0.25 g / L to less than about 0.4 g / L, or 0.3 g / L to about 0.4 g / L. In some embodiments, the fresh perfused cell culture medium comprises a cys equivalent at a concentration of about 0.1 g / L to less than about 1.1 g / L, about 0.2 g / L to less than about 0.9 g / L, about 0.25 g / L to less than about 0.6 g / L, or 0.3 g / L to about 0.4 g / L. In some embodiments, the culture medium comprises a cys equivalent at a concentration of about 0.3 g / L. In some embodiments, the fresh culture medium comprises a cys equivalent at a concentration of about 0.3 g / L.

[0021] In some embodiments, the cumulative cys equivalent added to the perfused culture is less than about 11 g / L, less than about 9 g / L, or less than about 7 g / L. In some embodiments, the cumulative cys equivalent added to the culture is about 3 g / L to less than about 11 g / L, about 4 g / L to less than about 11 g / L, about 5 g / L to less than about 11 g / L, about 3 g / L to less than about 9 g / L, about 4 g / L to less than about 9 g / L, about 5 g / L to less than about 9 g / L, or preferably about 5 g / L to less than about 7 g / L.

[0022] In some embodiments, the cumulative cys equivalent added to the perfused culture is less than about 1 g / L / day, less than 0.9 g / L / day, less than 0.7 g / L / day, less than 0.6 g / L / day, less than about 0.5 g / L / day, less than about 0.4 g / L / day, or less than about 0.3 g / L / day. In some embodiments, the cumulative cys equivalent added to the perfused culture is about 0.1 g / L / day to less than about 1 g / L / day, preferably about 0.2 g / L / day to less than about 0.6 g / L / day, more preferably about 0.2 g / L / day to less than 0.5 g / L / day, or about 0.3 g / L / day or 0.4 g / L / day.

[0023] The present invention also discloses the following specific technical solutions 1-24: 1. A method for generating recombinant polypeptides in fed-batch cell cultures, the method comprising the steps of: a. culturing mammalian cells in a cell culture medium comprising a basal culture medium and one or more feed cultures, wherein the basal culture medium contains a cys equivalent at a concentration of about 0.3 g / L, and wherein the feed culture medium contains a cys equivalent at a concentration of less than about 0.8 g / L, and wherein the concentration of the cumulative cys equivalent in the cell culture medium is less than about 0.4 g / L; b. expressing the recombinant polypeptide; and c. recovering the polypeptide from the culture medium.

[0024] 2. The method according to embodiment 1, wherein the basal culture medium does not contain added cysteine, and the feed culture medium contains cysteine ​​at a concentration of about 0.66 g / L.

[0025] 3. The method according to embodiment 1, wherein the basal culture medium does not contain added cysteine, and the feed culture medium contains cysteine ​​at a concentration of about 0.33 g / L.

[0026] 4. The method according to embodiment 1, wherein the basal culture medium does not contain added cysteine, and the feed culture medium does not contain cysteine.

[0027] 5. The method according to any one of the preceding embodiments, wherein the recombinant polypeptide is an antibody.

[0028] 6. The method according to embodiment 5, wherein the antibody is secukinumab.

[0029] 7. The method according to any one of the preceding embodiments, wherein the mammalian cells are selected from the group consisting of CHO cells, HEK cells and SP2 / O cells.

[0030] 8. The method according to any one of embodiments 5-7, wherein the method includes a downstream processing step of selective reduction, wherein the antibody is incubated with at least one reducing agent in a system to form a reducing mixture.

[0031] 9. The method according to any one of embodiments 5-8, further comprising the steps of: d. purifying the antibody; e. formulating the antibody for administration; f. packaging the antibody together with a booklet.

[0032] 10. The method according to any one of embodiments 1-4, wherein the recombinant polypeptide comprises at least one free cysteine.

[0033] 11. The method according to embodiment 10, wherein the recombinant polypeptide comprises at least one disulfide bond and at least one free cysteine.

[0034] 12. The method according to embodiment 11, wherein the recombinant polypeptide is an antibody, such as secukinumab.

[0035] 13. The method according to any one of the foregoing embodiments, wherein the recombinant polypeptide population recovered from the culture medium contains at least about 10% higher levels of reduced free cysteine ​​compared to the recombinant polypeptide population recovered from the control culture medium, wherein the control culture medium comprises a control basal culture medium having a concentration of more than about 0.4 g / L of cys equivalent and / or a control feed culture medium containing a concentration of more than about 0.9 g / L of cys equivalent, and / or wherein the cumulative concentration of cys equivalent in the control cell culture is more than about 0.4 g / L.

[0036] 14. The method according to any one of the preceding embodiments, wherein the method comprises producing a higher yield of recombinant polypeptides per mg of recombinant polypeptide / L of culture medium compared to a control method, wherein the control method comprises culturing mammalian cells in a control cell culture medium comprising a control basal culture medium and one or more control feed cultures, wherein the control basal culture medium comprises a concentration of cys equivalent greater than about 0.4 g / L, and / or wherein the control feed culture medium comprises a concentration of cys equivalent greater than about 0.9 g / L, and / or wherein the concentration of accumulated cys equivalent in the control cell culture is greater than about 0.4 g / L.

[0037] 15. The method according to embodiment 13 or 14, wherein the method comprises producing a population of recombinant polypeptides having at least 61% reduced free cysteine, as determined from used culture medium.

[0038] 16. A method for producing a recombinant polypeptide from a mammalian cell culture, the method comprising the steps of: a. culturing mammalian cells (e.g., selected from the group consisting of CHO cells, HEK cells, and SP2 / O cells) in a culture containing a cell culture medium, wherein the cell culture medium contains a cys equivalent at a concentration of about 0.3 g / L; b. exchanging a portion of the cell culture medium in the culture by perfusion with fresh cell culture medium, wherein the fresh cell culture medium contains a cys equivalent at a concentration of about 0.3 g / L, and / or wherein the cumulative concentration of cys equivalent added to the culture is less than about 7 g / L / day, or less than about 0.4 g / L / day; c. expressing the recombinant polypeptide; and d. recovering the polypeptide from the culture.

[0039] 17. The method of embodiment 16, wherein the fresh culture medium does not contain added cysteine.

[0040] 18. The method of embodiment 16, wherein the method comprises exchanging at least 50% of the cell culture medium daily by perfusion with fresh cell culture medium.

[0041] 19. The method according to embodiment 16, 17 or 18, wherein the recombinant polypeptide comprises at least one free cysteine. Specification 5 / 27 page 8 CN 122427280 A

[0042] 20. The method according to embodiment 19, wherein the recombinant polypeptide comprises at least one free cysteine ​​and at least one disulfide bond.

[0043] 21. The method according to any one of embodiments 16 to 20, wherein the recombinant polypeptide is an antibody, such as secukinumab.

[0044] 22. The method according to any one of embodiments 16 to 21, wherein the population of recombinant polypeptides recovered from the culture medium contains at least about 10% higher levels of reduced free cysteine ​​compared to the population of recombinant polypeptides recovered from a control culture medium containing a concentration greater than about 0.4 g / L of cys equivalent, and / or wherein the cumulative concentration of cys equivalent added to the control cell culture is greater than about 7 g / L / day, and / or greater than about 0.4 g / L / day.

[0045] 23. The method according to any one of embodiments 16 to 22, wherein the method comprises producing a higher yield of recombinant polypeptide based on mg recombinant polypeptide / L culture medium compared to a control method, wherein the control method comprises culturing mammalian cells in a control cell culture medium containing a concentration greater than about 0.4 g / L of cys equivalent, and / or wherein the cumulative concentration of cys equivalent in the control cell culture is greater than about 7 g / L / day, and / or greater than about 0.4 g / L / day.

[0046] 24. The method according to any one of embodiments 1 to 5, 7-22 or 23, wherein the method further comprises covalently modifying the reduced free cysteine ​​with a linker, label or drug. Brief Description of the Drawings

[0047] Figure 1 is a graph illustrating the activity of an antibody sample according to an embodiment.

[0048] Figure 2 shows the effect of reducing the cysteine / cystine concentration (cys equivalent) in the perfusion medium on antibody activity (%) over time (days) (x-axis). A greater reduction in the cysteine / cystine concentration compared to the baseline of the perfusion medium (y-axis) resulted in a greater retention of antibody activity.

[0049] Figure 3 shows that reducing the amount of cysteine ​​in the basal medium and / or the supplemental medium has almost no effect on the concentration (mg / ml; y-axis) of secukinumab over time (days; x-axis).

[0050] Figure 4 shows that the final culture concentration (mg / ml) of secukinumab on day 12 varied little between baseline (cell culture medium containing standard basal and supplemental media) and the basal and / or supplemental media of the three tested variants.

[0051] Figure 5 shows the activity (%) of antibody samples generated using the basal and / or supplemental media of the three tested variants compared to baseline (cell culture medium containing standard basal and supplemental media).

[0052] Figure 6 shows the activity (%) of antibody samples generated by perfusion culture using test perfusion medium without cysteine, thus reducing cys equivalents by 50% compared to control perfusion cell culture containing normal levels of cysteine ​​and cys equivalents. The cumulative cys equivalents added to the test perfused cell culture and the control perfused cell culture were approximately 5.875 g / L (approximately 0.31 g / L / day) and 11.642 g / L (0.61 g / L / day), respectively, with a complete culture medium exchange performed approximately once daily. Detailed Description

[0053] The purpose of this disclosure is to provide a method for reducing the oxidation level of cysteine ​​residues in recombinant peptides such as anti-IL-17 antibodies during cell culture, for example, during the recombinant production of secukinumab in mammalian cells.

[0054] The term “comprising” covers both “including” and “consisting of”, for example, a composition “comprising” X may consist of only X or may contain additional substances, such as X + Y. Specification 6 / 27 pages 9 CN 122427280 A

[0055] The term “about” in relation to the numerical value x means, for example, + / - 10%. When used before a range of numbers or a list of numbers, the term “about” applies to each number in the series. For example, the phrase “about 1-5” should be interpreted as “about 1 – about 5”, or the phrase “about 1, 2, 3, 4” should be interpreted as “about 1, about 2, about 3, about 4, etc.”

[0056] Based on the post-translational amino acid sequence, the relative molecular mass of secukinumab is 147,944 Daltons. This molecular weight (i.e., 147,944 Daltons) is used to calculate the secukinumab molar concentration values ​​and molar ratios throughout this disclosure. However, during production in CHO cells, C-terminal lysine residues are typically removed from each heavy chain. The relative molecular mass of secukinumab lacking C-terminal lysine residues in each heavy chain is 147,688 Daltons. Secukinumab formulations contain a mixture of molecules with and without C-terminal lysine residues on the heavy chain. The secukinumab molar concentration values ​​(and the ratios of these molar concentration values) used in this disclosure are therefore estimates, and the terms “about,” “approximately,” etc., regarding these values ​​at least cover this variation relative to molecular mass and the resulting calculations.

[0057] The word “substantially” does not exclude “completely,” for example, a composition “substantially free” of Y can be completely free of Y. Where necessary, the word “substantially” may be omitted in the definitions of this disclosure.

[0058] Large-scale cell culture can be used in various fermentation methods established, for example, in industrial biotechnology. Cell culture media according to the invention can be used, utilizing discontinuous and continuous cell culture methods, such as perfusion and chemostatic cultures. Discontinuous methods, including repeated fed-batch and repeated batch cultures, are a preferred embodiment. Generally, the methods and compositions of the invention relate to the production of secretory peptides through cell culture.

[0059] Batch cell culture includes fed-batch culture or simple batch culture. The term "fed-batch cell culture" refers to a cell culture in which cells and cell culture medium are initially supplied to a culture vessel, and additional culture nutrients are continuously or discretely fed to the culture during the culture process, with or without periodic cell and / or product harvesting before the culture is terminated. The term "simple batch culture" refers to a procedure in which all components (including cells and cell culture medium) for cell culture are supplied to a culture vessel at the beginning of the culture process. Preferably, the cells cultured in the cell culture medium according to the invention are CHO cells.

[0060] The term "cell culture medium" refers to an aqueous solution of nutrients that can be used to grow cells for an extended period of time. Typically, cell culture media contain the following components: an energy source, usually carbohydrates, preferably glucose; amino acids, preferably amino acids in the basic group, including all essential and non-essential amino acids; vitamins and / or other organic compounds required in low concentrations; free fatty acids; and inorganic compounds, including trace elements, inorganic salts, buffer compounds, and nucleosides and bases.

[0061] The term "growth medium" refers to the cell culture medium typically used during the expansion phase of the entire production process. The expansion phase is the first phase of the entire culture / production process, characterized primarily by high cell growth and less peptide production.The expansion phase is used to expand the cells, which means generating a sufficient number of cells in the exponential growth phase to inoculate the production bioreactor.

[0062] The term "production medium" refers to the cell culture medium typically used during the production phase of the entire production process. The production phase is the second phase of the entire culture / production process, which is used to generate a large quantity of product. During the production phase, the cells should remain viable and in production mode for as long as possible.

[0063] Due to safety and contamination concerns, the use of cell culture media in the pharmaceutical industry, for example for the production of recombinant polypeptides with therapeutic activity, generally does not allow the use of any animal-derived materials. Therefore, the cell culture medium according to the invention is preferably a serum- and / or protein-free medium. The term "serum- and / or protein-free medium" means a completely chemically defined medium that does not contain additives from animal sources such as tissue hydrolysates, fetal bovine serum, etc. Furthermore, it is preferable not to add proteins, especially growth factors such as insulin, transferrin, etc., to the cell cultures according to the invention. Preferably, the cell culture medium according to the invention is also not supplemented with hydrolyzed protein sources such as soybean, wheat, or rice peptone or yeast hydrolysates, etc., as per specification page 7 / 27, 10 CN 122427280 A.

[0064] The term “basal medium” is a culture medium used to culture cells directly and not as an additive to other culture media, but various components may be added to the basal medium. For example, if CHO cells are cultured in DMEM (a well-known, commercially available mammalian cell culture medium) and periodically fed with glucose or other nutrients, DMEM would be considered the basal medium. “Feed medium” is a culture medium used as a feed in cell culture, which may be a fed batch cell culture. Similar to the basal medium, the feed medium is designed based on the needs of the specific cells being cultured, and the feed medium may have a higher concentration of most, but not all, of the components of the basal medium. For example, some components, such as nutrients including amino acids or carbohydrates, may be about 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 400, 600, 800 times or even about 1000 times their normal concentration in the basal medium. Some components, such as salts, may be maintained at approximately the same concentration as the basal medium to keep the feed isotonic with the basal medium. Some components are added to maintain the physiological nature of the feed, and these components are added because they replenish nutrients to the culture.

[0065] The cell culture medium according to the invention can be used in various cell culture methods. Cell culture can be carried out in adherent culture, for example in monolayer culture or preferably in suspension culture.

[0066] There are no limitations on the polypeptides that can be generated from the cell culture and cell culture medium according to the invention. The polypeptides may be recombinant or non-recombinant.As used herein, the term "peptide" encompasses a molecule consisting of chains of more than two amino acids linked by peptide bonds; a molecule containing two or more such chains; or a molecule containing one or more such chains that are otherwise modified, for example, by glycosylation. The polypeptide may contain one or more natural disulfide bonds. The polypeptide may contain natural or engineered free cysteine ​​residues. The term polypeptide is intended to encompass proteins.

[0067] A preferred class of polypeptides produced according to the invention via cell cultures and cell culture media are recombinant antibodies.

[0068] As mentioned herein, the term "antibody" includes a complete antibody and any antigen-binding portion or single chain thereof. A naturally occurring "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The constant region of the light chain consists of a single domain CL. The VH and VL regions can be further subdivided into hypervariable regions, referred to as hypervariable regions or complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0069] As used herein, the term “antigen-binding fragment” of an antibody refers to a fragment of an antibody that retains the ability to specifically bind an antigen (e.g., IL-17). It has been shown that fragments of full-length antibodies can perform the antigen-binding function of an antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of antibody include Fab fragments, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of the antibody; dAb fragments consisting of VH domains (Ward et al., 1989, Nature [Nature] 341:544-546); and isolated CDRs. Exemplary antigen-binding sites include the CDRs of secukinumab listed in SEQ ID NOs: 1-6 and 11-13 (Table 1), preferably heavy chain CDR3.Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, these two domains can be linked using recombination methods via synthetic linkers that enable them to form a single protein chain, whereby the VL and VH regions pair to form a monovalent molecule (referred to as a single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242: 423-426; Huston et al., (1988) Proc. Natl. Acad . Sci. [Proceedings of the National Academy of Sciences] , 85: 5879-5883). Such single-chain antibodies are also intended to be covered within the scope of the term "antibody." The single-chain antibody and antigen-binding moiety are obtained using conventional techniques known to those skilled in the art.

[0070] As used herein, “isolated antibody” means an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to IL-17 is substantially free of antibodies that specifically bind to antigens other than IL-17). The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a formulation of an antibody molecule having a single molecular composition. As used herein, the term “human antibody” is intended to include antibodies having variable regions having both a framework region and a CDR region derived from human-derived sequences. “Human antibody” does not need to be produced by a human, human tissue, or human cell. Human antibodies disclosed herein may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro, N-nucleotide addition at the in vivo linker during antibody gene recombination, or somatic mutations in vivo). In some embodiments of the disclosed procedures and compositions, the IL-17 antibody is a human antibody, an isolated antibody, and / or a monoclonal antibody.

[0071] The term “IL-17” refers to IL-17A, formerly known as CTLA8, and includes wild-type IL-17A, polymorphic variants of IL-17A, and functional equivalents of IL-17A from different species (e.g., humans, mice, and monkeys). Functional equivalents of IL-17A according to this disclosure preferably have at least about 65%, 75%, 85%, 95%, 96%, 97%, 98%, or even 99% overall sequence identity with wild-type IL-17A (e.g., human IL-17A) and substantially retain the ability to induce IL-6 production in human dermal fibroblasts.

[0072] The term “KD” is intended to refer to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term “KD” is intended to refer to the dissociation constant, which is derived from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods well established in the art.Methods for determining the KD of an antibody include using surface plasmon resonance or using a biosensor system such as the Biacore® system. In some embodiments, the KD of an IL-17 antibody or antigen-binding fragment (e.g., secukinumab) for human IL-17 is about 100-250 pM.

[0073] The term “affinity” refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable region of the antibody “arm” interacts with the antigen at many sites via weak non-covalent forces; the more interactions, the stronger the affinity. Standard assays for assessing the binding affinity of antibodies for IL-17 of various species are known in the art, including, for example, ELISA, Western blotting, and RIA. The binding kinetics of antibodies (e.g., binding affinity) can also be assessed by standard assays known in the art, such as by Biacore analysis.

[0074] An antibody that “inhibits” one or more of these IL-17 functional properties (e.g., biochemical, immunochemical, cellular, physiological, or other biological activities) as known in the art and as defined by the methods described herein shall be understood to involve a statistically significant reduction in a specific activity relative to the specific activity observed in the absence of the antibody (or when a control antibody of unrelated specificity is present). An antibody that inhibits IL-17 activity results in a statistically significant reduction, for example, a reduction of at least about 10% of the measured parameter, a reduction of at least 50%, 80%, or 90%, and in some embodiments of the disclosed methods and compositions, the IL-17 antibody used can inhibit greater than 95%, 98%, or 99% of IL-17 functional activity.

[0075] Unless otherwise stated, according to this disclosure, the term “derivative” is used to define amino acid sequence variants and covalent modifications (e.g., PEGylation, deamidation, hydroxylation, phosphorylation, methylation, etc.) of an IL-17 antibody or its antigen-binding fragment (e.g., secukinumab) of a specific sequence (e.g., variable domain). "Functional derivatives" include molecules having the same qualitative biological activity as the disclosed IL-17 antibody. Functional derivatives include fragments and peptide analogs of the IL-17 antibody as disclosed herein. Fragments comprise regions within a polypeptide sequence according to this disclosure (e.g., specified sequences).The functional derivatives of the IL-17 antibody disclosed herein (e.g., functional derivatives of secukinumab) preferably contain VH and / or VL sequences (e.g., VH and / or VL sequences in Table 1) that have at least about 65%, 75%, 85%, 95%, 96%, 97%, 98%, or 99% overall sequence identity with the VH and / or VL sequences of the IL-17 antibody and its antigen-binding fragment disclosed herein (e.g., VH and / or VL sequences in Table 1), and substantially retain the ability to bind to human IL-17, or, for example, inhibit IL-6 production induced by IL-17 in human dermal fibroblasts.

[0076] The phrase “substantially identical” means that the relevant amino acid or nucleotide sequence (e.g., VH or VL domain) is identical or has non-substantially different (e.g., by conserved amino acid substitutions) compared to a specific reference sequence. Non-substantial differences include minor amino acid changes, such as substitutions of one or two amino acid sequences in a specific region (e.g., the VH or VL domain). In the case of antibodies, the second antibody has the same specificity and at least 50% of its affinity. Sequences substantially identical to those disclosed herein (e.g., having at least about 85% sequence identity) are also part of this application. In some embodiments, the sequence identity of a derivative IL-17 antibody (e.g., a derivative of secukinumab, such as a secukinumab biosimilar antibody) relative to the disclosed sequence can be about 90% or higher, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.

[0077] “Identity” of a natural polypeptide and its functional derivatives is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to residues of the corresponding natural polypeptide after sequence alignment and the introduction of vacancies (if desired) to achieve the maximum percentage of identity, and without regard to any conserved substitutions as part of sequence identity. N-terminal or C-terminal extensions and insertions should not be interpreted as a reduction in identity. The methods and computer programs used for alignment are well known. Percentage identity can be determined using standard alignment algorithms, such as the local alignment search tool (BLAST) described by Altshul et al. ((1990) J. Mol. Biol. [Journal of Molecular Biology], 215: 403 410); the algorithm by Needleman et al. ((1970) J. Mol. Biol. [Journal of Molecular Biology], 48: 444 453); or the algorithm by Meyers et al. ((1988) Comput. Appl. Biosci. [Computer Applications in Biological Sciences], 4: 11 17).A set of parameters can be a Blosum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5. Alternatively, the algorithm of E. Meyers and W. Miller ((1989) CABIOS [Computer Applications in the Biosciences], 4:11-17), which has been integrated into the ALIGN program (version 2.0), can be used to determine the percentage identity between two amino acid or nucleotide sequences using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4.

[0078] “One or more amino acids” means, for example, all naturally occurring L-α-amino acids and includes D-amino acids. The phrase “amino acid sequence variant” refers to a molecule whose amino acid sequence differs from that of the sequence according to this disclosure. Amino acid sequence variants of antibodies according to this disclosure, such as variants of a specific sequence, still possess the ability to bind to human IL-17 or, for example, the ability to inhibit IL-6 production induced by IL-17 in human dermal fibroblasts. Amino acid sequence variants include substitution variants (those variants that remove at least one amino acid residue and insert a different amino acid at the same position in the polypeptide according to the disclosure), insertion variants (those variants that insert one or more amino acids immediately adjacent to an amino acid at a specific position in the polypeptide according to the disclosure), and deletion variants (those variants that remove one or more amino acids in the polypeptide according to the disclosure).

[0079] The phrases “free cysteine,” “non-traditional cysteine,” and “unpaired cysteine” are interchangeable to refer to cysteine ​​that does not participate in the conserved antibody disulfide bond binding, or, in relation to a non-antibody polypeptide, to cysteine ​​that does not form a disulfide bond with another unpaired cysteine ​​in the polypeptide wild-type structure. Free cysteine ​​may be present in the antibody frame region or variable region (e.g., within the CDR). In secukinumab, the eighth amino acid of L-CDR3 as shown in SEQ ID NO:6 (which corresponds to amino acid 97 in the light chain variable region as shown in SEQ ID NO:10) (hereinafter referred to as CysL97) is free cysteine. Each secukinumab molecule contains two such free cysteine ​​residues – one in each VL domain.

[0080] As used herein, the term “selective reduction” refers to a method for selectively reducing CysL97 in a formulation of an IL-17 antibody that has been recombined and generated in mammalian cells, as disclosed in WO 2016 / 103146 ​​A1. Specifically, downstream processing steps are applied, such as contacting the antibody-containing formulation with at least one reducing agent in a system to form a reducing mixture; and incubating the reducing mixture while maintaining an oxygen volumetric mass transfer coefficient (kLa) in the system < about 0.37 h⁻¹, said kLa being calculated by adapting a dissolved oxygen profile to a saturation profile.

[0081] During recombinant peptide expression in a cell culture system, cysteine ​​may be included in the culture medium, or for example, in the basal medium during a fed-batch method and / or added to the culture vessel in the culture medium feed. Cysteine ​​refers to L-cysteine ​​rather than D-cysteine ​​and may be added in the form of a salt such as cysteine ​​monohydrate hydrochloride. Typically, monomeric cysteine ​​dimers immediately upon addition to the cell culture medium and thus exists only as a dimer of cystine. This redox reaction results in the formation of a disulfide bond between the two monomeric cysteine ​​molecules. The cysteine ​​concentration in the basal or fed culture medium of the present invention may be less than about 5.0, 4.0, 3.0, 2.5, 2.0, 1.5, 1.0, 0.90, 0.80, 0.70, 0.60, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, or 0.10 g / L. Alternatively, the basal culture medium and / or supplemental culture medium may be cysteine-free.

[0082] Cystine may also be present in the basal cell culture medium or added to the culture medium as a supplement, for example as part of a tyrosine-cysteine ​​stock solution. The cysteine ​​concentration in the basal or supplemental culture medium of the present invention may be less than about 5.0, 4.0, 3.0, 2.5, 2.0, 1.5, 1.0, 0.90, 0.80, 0.70, 0.60, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, or 0.10 g / L. Alternatively, the basal culture medium and / or supplemental culture medium may be cysteine-free.

[0083] Although cysteine ​​added to the culture medium is generally oxidized to cystine, some cysteine ​​added to the culture medium can be reduced to cysteine. Therefore, the figures given above for these concentrations refer to the actual concentration of cysteine ​​or cystine added to the culture medium, without needing to later determine how much of the substance may have been oxidized or reduced. Therefore, the term “cys equivalent” can be used in the context of the amount of cysteine ​​and / or cystine available to cells in the culture vessel. As used herein, this term refers to the total amount or concentration of cysteine ​​and cystine in the cell culture medium or feed medium. The cys equivalent will be the cysteine / cystine available to cells in the cell culture medium in the culture vessel, regardless of whether it originates from the basal medium and / or feed medium. Therefore, in culture vessels, such as cell culture media used in fed-batch methods, the concentration of total CYS equivalents can be less than about 5.0, 4.0, 3.0, 2.5, 2.0, 1.5, 1.0, 0.90, 0.80, 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, or 0.10 g / L.

[0084] Since, particularly in fed-batch methods, the cys equivalents in the cell culture medium can be derived from the basal medium and / or the feed medium, the term "cumulative cys equivalents" is used to refer to the total amount or total concentration of cys equivalents in the cell culture medium derived from the basal medium and / or the feed medium. The cumulative cys equivalents concentration can be different at the start of the fed-batch method compared to the end of the method, for example, after 5, 8, 10, 11, or 12 days, and can vary during the method as feed medium is added to the cell culture medium. In fed-batch cell culture methods under standard culture conditions, the concentration of cys equivalents in the cell culture medium can range from about 0.5 g / L to about 0.6 g / L or from about 4 mM to about 5 mM. In the method according to this disclosure, the concentration of cys equivalents in the cell culture medium at the start of the fed-batch method can be less than about 0.6 g / L or less than about 4.5 mM. For example, less than about 0.5 g / L, less than about 0.4 g / L and preferably less than about 0.3 g / L, or less than about 3.5 mM, less than about 3.0 mM and preferably less than about 2.5 mM. During the fed-batch method, the concentration of the cys equivalent can vary due to the addition of 0.3 g / L to about 0.8 g / L of the cys equivalent to the cell culture medium (caused by the addition of cysteine ​​or cystine or both). To achieve this concentration of the cys equivalent, the concentration of cysteine ​​added to the cell culture medium can be less than about 1 g / L. For example, the basal medium may not contain added cysteine, and the fed-batch medium may contain cysteine ​​at a concentration of less than about 1.0 g / L, for example less than about 0.9 g / L, less than about 0.8 g / L, and preferably less than about 0.7 g / L. In one embodiment, the basal medium does not contain added cysteine, and the feed medium contains cysteine ​​at a concentration of about 0.66 g / L. In another embodiment, the basal medium does not contain added cysteine, and the feed medium contains cysteine ​​at a concentration of about 0.33 g / L. In yet another embodiment, the basal medium does not contain added cysteine, and the feed medium does not contain cysteine.

[0085] The feed medium can be added to the fed-batch cell culture at different points in the culture process. For example, the feed medium can be added to the cell culture medium daily during the culture period, or the feed medium can be added after an initial period of two or three days, and then added daily thereafter. During the fed-batch cell culture method, the feed medium can be added once, twice, three times, four times, five times, six times, seven times, etc.

[0086] At the end of the fed-batch cell culture method as described herein, the concentration of accumulated cys equivalents in the cell culture medium may be less than about 0.4 g / L or less than about 3 mM. In standard fed-batch cell culture, where no changes are made to reduce cys equivalents in the basal medium and / or feed medium, the concentration of accumulated cys equivalents in the cell culture medium may be about 0.6 g / L or about 5 mM. The concentration of accumulated cys equivalents in standard (e.g., fed-batch) cell culture medium may also be referred to as the baseline.

[0087] In perfusion culture, fresh cell culture medium can be added to the cell culture via perfusion-mediated medium exchange. The exchange can be continuous or discontinuous (e.g., performed at different points in the culture period). For example, fresh medium can be continuously exchanged with cell culture medium in the culture during the culture period, or perfusion exchange can begin after an initial period of two or three days and then be continuously exchanged. In some embodiments, perfusion is performed under conditions sufficient to replace at least 50%, preferably 75%, more preferably 99%, or about 100% of the cell culture medium per day of culture.

[0088] Therefore, the total volume of culture medium consumed during perfusion batch culture can be much higher than that of fed batch culture. Consequently, the cumulative cys equivalent added to the perfused culture can be correspondingly higher, while still achieving the lower oxidation levels of free cysteine ​​provided by the methods and compositions described herein. For example, 1000 L of perfused batch culture with approximately 100% culture medium exchange per day can be cultured for 19 days, consuming a total volume of culture medium of approximately 19,000 L. However, the total culture volume can be maintained at approximately 1000 L. In such embodiments, the cumulative cys equivalent added to the control perfused culture can be greater than about 7 g / L culture volume, greater than about 8 g / L, greater than about 10 g / L, or greater than about 11 g / L, or about 11 g / L. In some embodiments, the cumulative cys equivalent added to the control perfusion culture may be 11 g / L or about 11.5 g / L. Similarly, the cumulative cys equivalent added to 1000 L of perfusion culture for 19 days (with approximately 100% of the medium replaced daily and cultured under reduced cysteine ​​and / or reduced cys equivalent conditions) may be less than about 7 g / L culture volume, 6.5 g / L, 6 g / L, or about 5.8 g / L of cumulative cys equivalent.

[0089] Therefore, with respect to perfusion batch culture, reduced cys equivalent conditions can be characterized by reduced cumulative cys equivalent conditions normalized to the number of culture days.Therefore, for example, under control conditions with approximately 0.6 g / L cys equivalent in the starting and exchange media, and with complete media exchange approximately once daily for 19 days of perfused culture, the normalized cumulative cys equivalent could be approximately 0.6 g / L / day. Similarly, under experimental conditions with approximately 0.3 g / L cys equivalent (or less) in the starting and exchange media, and with complete media exchange approximately once daily for 19 days of perfused culture, the normalized cumulative cys equivalent could be less than 0.6 g / L / day, such as approximately 0.3 g / L / day.

[0090] In some embodiments, secretory recombinant proteins with free cysteine ​​are produced by perfusion batching by culturing mammalian cells in a basal perfusion medium containing less than 0.62 g / L cys equivalent and by continuously or discontinuously exchanging (e.g., by perfusion) all or part of the cell culture medium with perfusion exchange medium, adding less than 0.62 g / L cys equivalent to the culture daily. In some cases, the method includes adding approximately 0.2 g / L cys equivalents to less than 1 g / L cys equivalents to the culture daily (e.g., via perfusion exchange). In some cases, the method includes adding approximately 0.2 g / L cys equivalents to less than 0.9 g / L cys equivalents to the culture daily (e.g., via perfusion exchange). In some cases, the method includes adding approximately 0.2 g / L cys equivalents to less than 0.6 g / L cys equivalents to the culture daily (e.g., via perfusion exchange). In some cases, the method includes adding approximately 0.2 g / L cys equivalents to less than 0.5 g / L cys equivalents to the culture daily (e.g., via perfusion exchange). In some cases, the method includes adding approximately 0.2 g / L of cys equivalent to less than 0.4 g / L of cys equivalent to the culture daily (e.g., via perfusion exchange).

[0091] In some embodiments, secretory recombinant proteins with free cysteine ​​are produced by perfusion batching by culturing mammalian cells in a basal perfusion medium containing less than 0.62 g / L of cys equivalent and continuously or discontinuously exchanging (e.g., via perfusion) all or part of the cell culture medium with an exchange medium containing less than 1.1 g / L of cys equivalent. In some cases, the perfusion exchange medium contains less than 1 g / L, less than 0.9 g / L, less than 0.6 g / L, less than 0.5 g / L, less than about 0.4 g / L, or about 0.3 g / L of cys equivalent. In some embodiments, the basal perfusion medium contains less than 0.6 g / L or about 0.3 g / L of cys equivalent.

[0092] In some embodiments, the basal perfusion medium contains about 0.2 g / L of cysteine ​​to less than about 0.6 g / L of cys equivalent. In some embodiments, the basal perfusion medium contains about 0.25 g / L of cysteine ​​to less than about 0.5 g / L of cys equivalent. In some embodiments, the basal perfusion medium contains about 0.3 g / L of cysteine ​​to less than about 0.4 g / L of cys equivalent. In some embodiments, the perfusion exchange medium contains about 0.2 g / L of cysteine ​​to less than about 1.1 g / L of cys equivalent. In some embodiments, the perfusion exchange medium contains about 0.25 g / L of cysteine ​​to less than about 0.9 g / L of cys equivalent. In some embodiments, the perfusion exchange medium contains about 0.3 g / L of cysteine ​​to less than about 0.6 g / L of cys equivalent. In some embodiments, the perfusion basal medium contains no or substantially no cysteine. In some embodiments, the perfusion exchange medium contains no or substantially no cysteine. In some embodiments, the perfusion basal medium and / or perfusion exchange medium are free of or substantially free of cysteine. In some embodiments, the perfusion basal medium and perfusion exchange medium are the same or substantially the same. A substantially cysteine-free production (e.g., perfusion basal or exchange production or fed-batch basal or fed-batch) medium comprises a medium in which residual amounts of cysteine ​​are present due to the presence of residual amplification medium, cysteine ​​production by host cells, and / or cysteine ​​reduction during production culture. A substantially cysteine-free basal medium, exchange medium, or fed-batch medium contains less than 0.1 g / L of cysteine, preferably less than 0.05 g / L, more preferably less than 0.01 g / L.

[0093] In some embodiments, activity is measured by a cystamine-CEX (cation exchange chromatography) method. The cystamine-CEX method involves derivatizing an antibody with cystamine (2,2'-dithiobis(ethylamine)) followed by analytical separation using cation exchange chromatography (CEX). Because the activity of the antibodies disclosed herein (e.g., secukinumab) is reduced if CysL97 is in its oxidized form, cystamine-derived CysL97 is used as a representative of antibody activity. Cystamine derivatization results in the addition of a positive charge to each free Cys97 residue. The resulting secukinumab derivatized form (e.g., +2, +1 charge) can then be separated from the underrivatized form and quantified via CEX. Theoretically, a cystamine-derived secukinumab molecule with two cystamines bound to unpaired Cys97 residues on both light chains can be considered to have 100% biological activity. A cystamine-derived secukinumab molecule with one cystamine added to an unpaired Cys97 residue on one of the light chains can be considered to have 50% biological activity.Cystamine-derived secukinumab molecules without any cystamine binding to the molecule can be considered biologically inactive. The level of cystamine derivatization in the antibody formulation (e.g., a secukinumab antibody formulation), compared to the theoretical maximum level of cystamine derivatization in that formulation (e.g., expressed as a percentage of the theoretical maximum), can then be used as a measure of formulation activity.

[0094] Briefly, cystamine-CEX can be performed as follows. First, the antibody sample (50 μg) is treated with carboxypeptidase B (1:40, w:w) to remove C-terminal lysine from the heavy chain, and then derivatized with 4 mM cystamine in 5 mM sodium acetate and 0.5 mM EDTA (pH 4.7) at room temperature for 2 hours. Derivatization is terminated by adding 2 μL of 1 M phosphate. The cystamine-derived antibody sample is then subjected to CEX using a ProPac™ WCX-10 analytical column (4 mm × 250 mm, Dionex). Separation was performed using a gradient of 12.5 mM to 92.5 mM sodium chloride in 25 mM sodium phosphate (pH 6.0) at a flow rate of 1.0 ml / min. Absorption at 220 nm was recorded using a UV detector (Agilent HPLC 1200).

[0095] IL-17 antibody and its antigen-binding fragment

[0096] In one embodiment, the IL-17 antibody or its antigen-binding fragment comprises at least one immunoglobulin heavy chain variable domain (VH) containing hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO:1, CDR2 has the amino acid sequence SEQ ID NO:2, and CDR3 has the amino acid sequence SEQ ID NO:3. In one embodiment, the IL-17 antibody or its antigen-binding fragment comprises at least one immunoglobulin light chain variable domain (VL') containing hypervariable regions CDR1', CDR2', and CDR3', wherein CDR1' has the amino acid sequence SEQ ID NO: 4, CDR2' has the amino acid sequence SEQ ID NO: 5, and CDR3' has the amino acid sequence SEQ ID NO: 6. In one embodiment, the IL-17 antibody or its antigen-binding fragment comprises at least one immunoglobulin heavy chain variable domain (VH) containing hypervariable regions CDR1-x, CDR2-x, and CDR3-x, wherein CDR1-x has the amino acid sequence SEQ ID NO: 11, CDR2-x has the amino acid sequence SEQ ID NO: 12, and CDR3-x has the amino acid sequence SEQ ID NO: 13.

[0097] In one embodiment, the IL-17 antibody or its antigen-binding fragment comprises at least one immunoglobulin VH domain and at least one immunoglobulin VL domain, wherein: a) the VH domain comprises (e.g., sequentially): i) hypervariable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence SEQ ID NO: 1, CDR2 has the amino acid sequence SEQ ID NO: 2, and CDR3 has the amino acid sequence SEQ ID NO: 3; or ii) hypervariable regions CDR1-x, CDR2-x, and CDR3-x, wherein CDR1-x has the amino acid sequence SEQ ID NO: 11, CDR2-x has the amino acid sequence SEQ ID NO: 12, and CDR3-x has the amino acid sequence SEQ ID NO: 13; and b) the VL domain comprises (e.g., sequentially) hypervariable regions CDR1', CDR2', and CDR3', wherein CDR1' has the amino acid sequence SEQ ID NO: 4, CDR2' has the amino acid sequence SEQ ID NO: 13, and CDR3' has the amino acid sequence SEQ ID NO: 14. 5, and the CDR3' has the amino acid sequence SEQ ID NO: 6.

[0098] In one embodiment, the IL-17 antibody or its antigen-binding fragment comprises: a) an immunoglobulin heavy chain variable domain (VH) comprising the amino acid sequence listed in SEQ ID NO: 8; b) an immunoglobulin light chain variable domain (VL) comprising the amino acid sequence listed in SEQ ID NO: 10; c) an immunoglobulin VH domain comprising the amino acid sequence listed in SEQ ID NO: 8 and an immunoglobulin VL domain comprising the amino acid sequence listed in SEQ ID NO: 10; d) an immunoglobulin VH domain comprising the hypervariable regions listed in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; e) an immunoglobulin VL domain comprising the hypervariable regions listed in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; f) an immunoglobulin VH domain comprising the hypervariable regions listed in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; g) comprising SEQ ID NO: 1. The immunoglobulin VH domain of the hypervariable region listed in SEQ ID NO: 2 and SEQ ID NO: 3 and the immunoglobulin VL domain containing the hypervariable region listed in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; or h) the immunoglobulin VH domain containing the hypervariable region listed in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 and the immunoglobulin VL domain containing the hypervariable region listed in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0099] For ease of reference, the amino acid sequence of the hypervariable region of the secukinumab monoclonal antibody is provided in Table 1 below, based on the Kabat definition and as determined by X-ray analysis using the method of Chothia and colleagues.

[0100] Table 1. Amino acid sequence of the hypervariable region of secukinumab antibody, specification 14 / 27 pages, 17 CN 122427280 A

[0101] In a preferred embodiment, the constant region domain preferably further comprises a suitable human constant region domain, for example, as described in “Sequences of Proteins of Immunological Interest” (Kabat EA et al., US Department of Health and Human Services, Public Health Service, National Institutes of Health). The DNA encoding the VL of secukinumab is listed in SEQ ID NO: 9. The DNA encoding the VH of secukinumab is listed in SEQ ID NO: 7.

[0102] In some embodiments, the IL-17 antibody or its antigen-binding fragment (e.g., secukinumab) comprises the three CDRs of SEQ ID NO: 10. In other embodiments, the IL-17 antibody or its antigen-binding fragment comprises the three CDRs of SEQ ID NO: 8. In other embodiments, the IL-17 antibody or its antigen-binding fragment comprises three CDRs of SEQ ID NO: 10 and three CDRs of SEQ ID NO: 8. The CDRs of SEQ ID NO: 8 and SEQ ID NO: 10 can be found in Table 1. Free cysteine ​​in the light chain (CysL97) can be seen in SEQ ID NO: 6.

[0103] In some embodiments, the IL-17 antibody or its antigen-binding fragment comprises the light chain of SEQ ID NO: 14. In other embodiments, the IL-17 antibody or its antigen-binding fragment comprises the heavy chain of SEQ ID NO: 15 (with or without a C-terminal lysine). In other embodiments, the IL-17 antibody or its antigen-binding fragment comprises the light chain of SEQ ID NO: 14 and the heavy chain of SEQ ID NO: 15 (with or without a C-terminal lysine). In some embodiments, the IL-17 antibody or its antigen-binding fragment comprises three CDRs of SEQ ID NO: 14. In other embodiments, the IL-17 antibody or its antigen-binding fragment comprises the three CDRs of SEQ ID NO: 15 (page 15 / 27, CN 122427280 A NO: 15). In other embodiments, the IL-17 antibody or its antigen-binding fragment comprises the three CDRs of SEQ ID NO: 14 and the three CDRs of SEQ ID NO: 15.The CDRs of SEQ ID NO: 14 and SEQ ID NO: 15 can be found in Table 1. A complete set of sequences is listed in the table.

[0104] The highly variable region can be linked to any type of frame region, but is preferably human. Suitable frame regions are described in Kabat EA et al. (ibid.). The preferred heavy chain frame is the human heavy chain frame, such as the frame of the secukinumab antibody. This frame is composed, for example, of FR1 (amino acids 1 to 30 of SEQ ID NO: 8), FR2 (amino acids 36 to 49 of SEQ ID NO: 8), FR3 (amino acids 67 to 98 of SEQ ID NO: 8), and FR4 (amino acids 117 to 127 of SEQ ID NO: 8). Considering the hypervariable region of secukinumab identified by X-ray analysis, another preferred heavy chain framework is composed of the FR1-x (amino acids 1 to 25 of SEQ ID NO: 8), FR2-x (amino acids 36 to 49 of SEQ ID NO: 8), FR3-x (amino acids 61 to 95 of SEQ ID NO: 8), and FR4 (amino acids 119 to 127 of SEQ ID NO: 8) regions sequentially. Similarly, the light chain framework is composed of the FR1' (amino acids 1 to 23 of SEQ ID NO: 10), FR2' (amino acids 36 to 50 of SEQ ID NO: 10), FR3' (amino acids 58 to 89 of SEQ ID NO: 10), and FR4' (amino acids 99 to 109 of SEQ ID NO: 10) regions sequentially.

[0105] In one embodiment, the IL-17 antibody or its antigen-binding fragment (e.g., secukinumab) is selected from human IL-17 antibodies, which at least comprise: a) an immunoglobulin heavy chain or a fragment thereof, the immunoglobulin heavy chain or the fragment thereof comprising a variable domain and a constant portion of a human heavy chain or a fragment thereof, the variable domain comprising, in sequence, hypervariable regions CDR1, CDR2, and CDR3; wherein CDR1 has the amino acid sequence SEQ ID NO: 1, CDR2 has the amino acid sequence SEQ ID NO: 2, and CDR3 has the amino acid sequence SEQ ID NO: 3; and b) an immunoglobulin light chain or a fragment thereof, the immunoglobulin light chain or the fragment thereof comprising a variable domain and a constant portion of a human light chain or a fragment thereof, the variable domain comprising, in sequence, hypervariable regions CDR1', CDR2', and CDR3', wherein CDR1' has the amino acid sequence SEQ ID NO: 4, CDR2' has the amino acid sequence SEQ ID NO: 5, and CDR3' has the amino acid sequence SEQ ID NO: 6.

[0106] In one embodiment, the IL-17 antibody or its antigen-binding fragment is selected from a single-chain antibody or its antigen-binding fragment containing an antigen-binding site, the antigen-binding site comprising: a) a first domain comprising hypervariable regions CDR1, CDR2 and CDR3 in sequence, wherein CDR1 has an amino acid sequence SEQ ID NO: 1, CDR2 has an amino acid sequence SEQ ID NO: 2 and CDR3 has an amino acid sequence SEQ ID NO: 3; and b) a second domain comprising hypervariable regions CDR1', CDR2' and CDR3' in sequence, wherein CDR1' has an amino acid sequence SEQ ID NO: 4, CDR2' has an amino acid sequence SEQ ID NO: 5 and CDR3' has an amino acid sequence SEQ ID NO: 6; and c) a peptide linker that binds to the N-terminus of the first domain and the C-terminus of the second domain or binds to the C-terminus of the first domain and the N-terminus of the second domain.

[0107] Alternatively, the IL-17 antibody or its antigen-binding fragment used in the disclosed methods may comprise a derivative of an IL-17 antibody listed herein by sequence (e.g., a PEGylated variant of secukinumab). Alternatively, the VH or VL domain of the IL-17 antibody or its antigen-binding fragment used in the disclosed methods may have a VH or VL domain substantially identical to the VH or VL domains listed herein (e.g., those listed in SEQ ID NO: 8 and 10). The human IL-17 antibody disclosed herein may comprise a heavy chain substantially identical to the heavy chain listed in SEQ ID NO: 15 (with or without a C-terminal lysine) and / or a light chain substantially identical to the light chain listed in SEQ ID NO: 14. The human IL-17 antibody disclosed herein may comprise: a heavy chain containing the heavy chain of SEQ ID NO: 15 (with or without a C-terminal lysine) and a light chain containing the light chain of SEQ ID NO: 14. The human IL-17 antibody disclosed herein may comprise: a) a heavy chain comprising a variable domain having an amino acid sequence substantially identical to that shown in SEQ ID NO: 8 and a constant portion of a human heavy chain; and b) a light chain comprising a variable domain having an amino acid sequence substantially identical to that shown in SEQ ID NO: 10 and a constant portion of a human light chain. Specification 16 / 27 pages 19 CN 122427280 A

[0108] Alternatively, the IL-17 antibody or its antigen-binding fragment used in the disclosed method may be an amino acid sequence variant of the reference IL-17 antibody listed herein (provided it contains CysL97).This disclosure also includes IL-17 antibodies or antigen-binding fragments thereof (e.g., secukinumab), wherein only a few (e.g., 1-10) amino acid residues in one or more of the VH or VL domains of secukinumab (but not CysL97) are typically altered; for example, by mutation, such as site-directed mutagenesis of the corresponding DNA sequence. In the case of all such derivatives and variants, IL-17 antibodies or antigen-binding fragments thereof are capable of inhibiting the activity of about 1 nM (= 30 ng / ml) of human IL-17 by 50% at concentrations of about 50 nM or less, about 20 nM or less, about 10 nM or less, about 5 nM or less, about 2 nM or less, or more preferably about 1 nM or less of the molecule, as measured against hu-IL-17-induced IL-6 production in human dermal fibroblasts, as described in Example 1 of WO 2006 / 013107.

[0109] In some embodiments, an IL-17 antibody or its antigen-binding fragment (e.g., secukinumab) binds to epitopes of mature human IL-17, which include Leu74, Tyr85, His86, Met87, Asn88, Val124, Thr125, Pro126, Ile127, Val128, and His129. In some embodiments, an IL-17 antibody (e.g., secukinumab) binds to epitopes of mature human IL-17, which include Tyr43, Tyr44, Arg46, Ala79, and Asp80. In some embodiments, an IL-17 antibody (e.g., secukinumab) binds to epitopes of an IL-17 homodimer having two mature human IL-17 chains, the epitopes being Leu74, Tyr85, His86, Met87, Asn88, Val124, Thr125, Pro126, Ile127, Val128, His129 on one chain and Tyr43, Tyr44, Arg46, Ala79, Asp80 on the other chain. The residue numbering scheme used to define these epitopes is based on the residues that are the first amino acid of the mature protein (i.e., IL-17A, lacking the 23-amino acid N-terminal signal peptide and starting with glycine). The sequence of the immature IL-17A is listed in Swiss-Prot entry Q16552. In some embodiments, the IL-17 antibody has a KD of approximately 100-200 pM. In some embodiments, the IL-17 antibody has an IC50 of about 0.4 nM for in vitro neutralization of the biological activity of about 0.67 nM human IL-17A. In some embodiments, the absolute bioavailability of the IL-17 antibody administered subcutaneously (sc) ranges from about 60% to about 80%, for example about 76%.In some embodiments, the IL-17 antibody (such as secukinumab) has an elimination half-life of about 4 weeks (e.g., about 23 to about 35 days, about 23 to about 30 days, for example, about 30 days). In some embodiments, the IL-17 antibody (such as secukinumab) has a Tmax of about 7 to 8 days.

[0110] Particularly preferred IL-17 antibodies or antigen-binding fragments thereof used in the disclosed methods are human antibodies, particularly secukinumab as described in Examples 1 and 2 of WO 2006 / 013107. Secukinumab is a recombinant high-affinity, fully human monoclonal anti-human interleukin-17A (IL-17A, IL-17) antibody of the IgG1 / κ isotype, which is currently in clinical trials for the treatment of immune-mediated inflammatory conditions. Secukinumab (see, for example, WO 2006 / 013107 and WO 2007 / 117749) has a very high affinity for IL-17, i.e., a KD of about 100-200 pM, and an IC50 of about 0.4 nM for in vitro neutralization of the biological activity of about 0.67 nM human IL-17A. Therefore, secukinumab inhibits the antigen at a molar ratio of about 1:1. This high binding affinity makes secukinumab antibodies particularly suitable for therapeutic applications. In addition, secukinumab has been determined to have a very long half-life of about 4 weeks, which allows for extended dosing intervals, a special property when treating chronic lifelong conditions such as rheumatoid arthritis.

[0111] This document discloses methods for preparing the above-described IL-17 antibodies and their antigen-binding fragments (e.g., secukinumab). The disclosed methods facilitate formulation of antibodies (e.g., IL-17 antibodies, such as secukinumab) to reduce costs. An antibody “formulation” refers to a composition (e.g., a solution) having multiple antibody molecules. “Formulation” includes any liquid composition containing an IL-17 antibody or an antigen-binding fragment thereof. Thus, a formulation may contain an IL-17 antibody or an antigen-binding fragment thereof (e.g., secukinumab) in, for example, in water or a buffer, in a column elution buffer, in a dialysis buffer, etc. In some embodiments, the initial formulation of the antibody is contained in a library of IL-17 antibodies or an antigen-binding fragment thereof (e.g., secukinumab) in a buffer (e.g., Tris, such as 1 mM–1 M Tris, pH 6.0–8.0) or WFI.Specification 17 / 27 pages 20 CN 122427280 A

[0112] In some embodiments of the above method, the IL-17 antibody or its antigen-binding fragment comprises: i) an immunoglobulin heavy chain variable domain (VH) comprising the amino acid sequence listed in SEQ ID NO: 8; ii) an immunoglobulin light chain variable domain (VL) comprising the amino acid sequence listed in SEQ ID NO: 10; iii) an immunoglobulin VH domain comprising the amino acid sequence listed in SEQ ID NO: 8 and an immunoglobulin VL domain comprising the amino acid sequence listed in SEQ ID NO: 10; iv) an immunoglobulin VH domain comprising the hypervariable regions listed in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in sequence; v) an immunoglobulin VL domain comprising the hypervariable regions listed in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in sequence; vi) an immunoglobulin VL domain comprising SEQ ID NO: 11, SEQ ID NO: 122427280 A in sequence; The method comprises: 1) the immunoglobulin VH domain of the hypervariable region listed in SEQ ID NO: 12 and SEQ ID NO: 13; vii) the immunoglobulin VH domain of the hypervariable region listed in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the immunoglobulin VL domain of the hypervariable region listed in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, in sequence; and viii) the immunoglobulin VH domain of the hypervariable region listed in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, and the immunoglobulin VL domain of the hypervariable region listed in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, in sequence. In the disclosed method, the IL-17 antibody or its antigen-binding fragment is a human antibody of the IgG1 isotype. In some embodiments of the disclosed method, the antibody is secukinumab.

[0113] Recombinant antibody production

[0114] The production of recombinant peptides is traditionally divided into two main steps: upstream (cell culture and target peptide synthesis) and downstream (peptide purification and formulation into active pharmaceutical ingredient or drug product).

[0115] More specifically, preparations of monoclonal antibodies or their antigen-binding fragments can be recombinantly generated from any mammalian cell line using any mammalian cell line, such as Chinese hamster ovary (CHO) cells, mouse myeloma NSO cells, young hamster kidney (BHK) cells, human embryonic kidney cell line HEK-293, human retinal cell line Per.C6 (Crucell, NL, Netherlands), and HKB11 cell clone (derived from a hybrid cell fusion of HEK 293S and Burkitt lymphoma line 2B8). “Recombinantly generated from mammalian cells” means that antibody production has been achieved in mammalian cells using recombinant DNA technology.

[0116] CHO cells are currently the most widely used mammalian host in biological and medical research, particularly for expressing human therapeutic proteins, and it has been reported that approximately 70% of recombinant therapeutic proteins are generated in CHO cell systems. Although they require expensive culture media and grow relatively slowly compared to E. coli and yeast expression systems, CHO cells can achieve more precise protein glycosylation, assembly, and folding, much like human cells. Therefore, CHO cells are a preferred production host for some proteins whose activity is closely related to post-translational modifications. CHO cells also efficiently synthesize large molecules that cannot be actively expressed in prokaryotic hosts. Suitable CHO cell lines include, for example, CHO-S (Invitrogen, Carlsbad, CA, USA), CHO Kl (ATCC CCL-61), CHO pro3-, CHO DG44, CHO P12 or dhfr-CHO cell lines DUK-BII (Urlaub G and Chasin LA (1980) PNAS 77(7): 4216-4220), DUXBI 1 (Simonsen CC and Levinson AD (1983) PNAS 80(9): 2495-2499) or CHO-K1SV (Lonza, Basel, Switzerland). Many CHO cell-derived products have received regulatory approval, such as erythropoietin (Epogen; Amgen), TNFα receptor fusion (Enbrel; Amgen), anti-HER2 antibody (Herceptin; Genentech), anti-TNFα antibody (Humira; AbbVie), and anti-VEGF antibody (Avastin; Genentech).

[0117] For the industrial production of recombinant proteins, the most common culture modes used in biomanufacturing are fed-batch and perfusion.The use of one technology or another depends on different factors related to the protein or the host (Kadouri and Spier, (1997) Cytotechnology [Cell Technology] 24: 89-98), in which cells are attached to a carrier for culture or suspension culture. One of the most common methods is batch bioreactors, in which cells grow and produce after inoculation until they reach limitations due to culture medium consumption and cell density begins to decrease. A second very common method is fed-batch, in which nutrient limitation is prevented by adding highly concentrated feed at different time points during culture. Thus, the culture duration is longer than batch mode, and the final productivity is higher. For continuous methods of continuous feeding of culture medium and continuous removal of harvest, one of the simplest methods is the chemostat method, in which culture medium is added at a constant flow rate and the bioreactor contents are removed at the same flow rate, with no cells retained (Henry O et al., (2008) Biotechnol. Prog. [Advances in Biotechnology], 921-931). An alternative continuous method is perfusion, in which there is a constant inflow and outflow, but the cells are now retained within the bioreactor. The current industry standard for producing stable proteins such as monoclonal antibodies is the fed-batch method in stirred-tank bioreactors up to 20 kL. These culture vessels can provide very high mixing and mass transfer rates and also offer high flexibility in working volume and can be used for different cell types and operating modes (Rodrigues ME et al., (2010) Biorechnol. Prog. [Advances in Biotechnology] 26: 332-51).

[0118] Since the beginning of biomanufacturing, culture medium development has been the most important aspect of cell culture development and optimization, primarily for process performance, but secondly, and more importantly, for safety reasons. First cell culture media were prepared using products of animal origin (Yao and Asayama, (2017) Reprod. Med. Biol. [Reproductive Medicine and Biology], 16: 99-117). Patient outcomes involve exposure to numerous risk factors such as viruses and prions, and the risk of infection is significant, especially for chronic diseases, due to ongoing drug exposure (Grillberger L et al., (2009) Biotechnol. J. [Journal of Biotechnology], 4: 186-201). Process inconsistencies due to batch heterogeneity are also a driving force behind the reduction of animal or even plant-derived culture medium components, and even today, significant efforts are being made to optimize chemically defined media that can enhance cell growth.

[0119] Chemically defined culture media are now commercially available, and most large biomanufacturing companies have developed their own formulations. For example, highly concentrated feeds can be challenging because the physical properties of some compounds can limit solubility or stability. Further optimization of CHO cell culture media and process parameters, particularly for the commercial manufacture of monoclonal antibodies and other recombinant peptides, has led to significant increases in cell density and protein expression, in some cases titers exceeding 10 g / L (Li F et al., (2010) MAbs [Monoclonal Antibodies], 2(5): 466–479; Lu F et al., (2013) Biotechnol Bioeng. [Biotechnology and Bioengineering], 110(1): 191–205; Xing Z et al., (2011) Process Biochem. [Process Biochemistry], 46(7): 1432-9). Several companies specializing in cell culture media have developed and optimized base and feed media combinations specifically for recombinant CHO manufacturing processes (Thermo Fisher Scientific, Waltham, MA, USA; GE Healthcare, Waukesha, WI, USA; MilliporeSigma, St. Louis, MO, USA; Lonza, Basel, Switzerland; Irvine Scientific, Santa Ana, CA, USA).

[0120] The formulations of these commercially available media are often proprietary; however, all cell culture media require similar essential nutrients that are necessary to support life and cell growth. Water, along with carbon, nitrogen, and phosphate sources, certain amino acids, fatty acids, vitamins, trace elements, and salts, is supplied at concentrations based on the cell's chemical composition, the computational amount required to achieve the desired cell density, and an understanding of the rate of nutrient consumption. This allows for the replenishment of key components to maintain and prolong cell viability.Specifically, amino acids are key components in CHO cell culture media, especially chemically defined media, and studies have shown that small changes in the amino acid composition of cell culture media can alter growth curves and titers, and can also significantly affect the glycosylation pattern of products (Fan Y et al., (2015) Biotechnol. Bioeng. [Biotechnology Specification 19 / 27 pages 22 CN 122427280 A Biotechnology and Bioengineering], 112(3): 521-35).

[0121] Generally speaking, amino acids can be classified into non-essential amino acids and essential amino acids. Non-essential amino acids can be synthesized by mammalian cells, while essential amino acids cannot be synthesized by cells and therefore must be supplied as components of cell culture media. Both non-essential and essential amino acids can have a significant impact on the growth of CHO cells, and it has been shown that optimizing the relative concentrations of non-essential and essential amino acids in the culture medium formulation can improve the production rate of recombinant monoclonal antibodies (Parampalli A et al., (2007) Cytotechnology [Cell Technology], 54(1): 57-68). Essential amino acids include histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine, and in most cases, all essential amino acids are required in CHO cell culture media.

[0122] Non-essential amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine. Although non-essential amino acids can be synthesized by mammalian cells in the culture, most cell culture media still contain most or all of these amino acids to support cell growth and peptide production. Most non-essential amino acids can have a significant impact on the cell culture process.

[0123] Specifically, cysteine ​​(an amino acid containing only thiol groups) is a specific non-essential amino acid in monoclonal antibody production. The formation of disulfide bridges between thiol groups on cysteine ​​residues supports the folding of tertiary and quaternary structures of CHO cell structural proteins and recombinant antibody products. Cysteine ​​restriction can be lethal and irreversible for CHO cell growth and may lead to decreased cell viability. In a study by Ghaffari et al. (2020), the effects of glutamine, asparagine, and cysteine ​​restriction on cell growth, metabolism, antibody production, and product glycosylation were investigated in three Chinese hamster ovary (CHO) cell lines (CHO-DXB11, CHO-K1SV, and CHO-S). Cysteine ​​restriction was detrimental to cell proliferation and productivity in all three CHO cell lines. Of the three amino acid restrictions studied, cysteine ​​restriction had the most detrimental effects on culture growth and productivity, as well as mAb glycosylation.Cysteine ​​has low solubility and can be a limiting factor in discontinuous feeding protocols commonly used at industrial scales, particularly for high cell concentrations. Ghaffari et al. investigated the duration of cysteine ​​restriction tolerance in CHO-DXB11 cells initially grown in BIOGRO medium without cysteine. Cysteine ​​concentrations were then restored to 0.4 mM by adding a concentrated cysteine ​​solution on day 1 or 2 of culture. If cysteine ​​levels were restored on day 1, cells were maintained for an additional day during lag phase and then resumed growth on day 2; by day 5, these cultures reached concentrations similar to the control. Restoring cysteine ​​levels after 2 days of cysteine ​​restriction proved ineffective, and cells did not grow. (Ghaffari N et al., (2020) Biotech. Prog. [Advances in Biotechnology], 36: e2946). Conversely, cysteine ​​concentrations >1 mM can be toxic to mammalian cells, likely due to lipid peroxidation and the formation of hydroxyl radicals, which can be further accelerated in the presence of copper (Ritacco FV et al. (2018) Biotechnol. Prog. [Advances in Biotechnology], 34(6): 1407-26). In mammals, the cysteine ​​pool is regulated by the liver, but this regulatory mechanism is absent in CHO cells, and the cysteine ​​concentration in the culture medium needs to be carefully designed and controlled for use in cell culture methods (Stipanuk MH et al., (2006) J. Nutr. [Journal of Nutrition], 136(6): 1652S-59S).

[0124] Recombinant peptide formulations (e.g., formulations of IL-17 antibodies or their antigen-binding fragments) used in the methods described herein can be recombinantly generated from any mammalian cell line using any mammalian cell line. Preferably, the mammalian cell line is a CHO cell. Recombinant peptides, such as anti-IL-17 antibodies or their antigen-binding fragments, can be produced in continuous manufacturing systems or by adding feed to the culture medium using fed-batch systems. As described above, the anti-IL-17 antibody secukinumab contains a free, unpaired cysteine ​​residue involved in antigen recognition and binding. This free cysteine ​​residue is found after cis-proline in the 3 ring of the light chain complementarity-determining region (CDR), specifically the eighth amino acid of L-CDR3 as shown in SEQ ID NO:6, which corresponds to amino acid 97 in the light chain variable region as shown in SEQ ID NO:10, and is referred to as "CysL97". To maintain full activity, this free cysteine ​​residue cannot be masked by pairing with disulfide oxide bonds of other cysteine ​​residues or by oxidation with exogenous compounds.Furthermore, this modification of free cysteine ​​can negatively impact the activity and stability of the antibody and can lead to increased immunogenicity. Therefore, processing of secukinumab can be challenging because the final product may contain significant amounts of inactive antibody material. However, because secukinumab is manufactured using mammalian cells, particularly CHO cells, cell-based modifications of CysL97 do occur, which can affect yield and antibody activity.

[0125] As discussed above, the term “cys equivalent” refers to cysteine ​​and cystine available to cells in the culture medium in the culture vessel, regardless of their origin from the basal medium and / or feed medium. Surprisingly, it has been found that reducing the amount of cys equivalent in the secukinumab cell culture growth medium leads to a reduction in the modification of free cysteine ​​CysL97. This, in turn, results in an increase in the amount of active antibody produced during the production process, i.e., an increase in product quality. Contrary to established studies (e.g., Ghaffari et al., ibid.), a reduction in cysteine ​​in the production medium and feed medium has no negative impact on the yield of secukinumab.

[0126] Purification of Recombinant Peptides

[0127] A purification step is necessary to obtain a substantially homogeneous formulation of the recombinant peptide produced according to the cell culture methods described herein. As a first step, the culture medium or lysis buffer is typically centrifuged to remove granular cell debris. The resulting peptide can be conveniently purified by hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Other techniques for protein purification can also be used, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin agarose, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation.

[0128] Pharmaceutical Compositions, Administration, and Kits

[0129] Pharmaceutical compositions are provided herein that comprise a combination of the recombinant peptide as described herein with one or more pharmaceutically acceptable excipients, diluents, or carriers. To prepare a pharmaceutical or sterile composition comprising the molecules of this disclosure, the molecules are mixed with a pharmaceutically acceptable carrier or excipient. The phrase "pharmaceutically acceptable" means approved by a regulatory agency of the U.S. federal or state government or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals and, more particularly, in humans. The term "pharmaceutical composition" means a mixture of at least one active ingredient (e.g., an antibody or fragment disclosed herein) and at least one pharmaceutically acceptable excipient, diluent, or carrier. "Drug" means a substance intended for medical treatment.

[0130] Pharmaceutical compositions of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, for example, Hardman et al., (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis et al. (eds) (1993) Pharmaceutical Dosage Forms: General Medications, Marcel Dekker, New York; Lieberman et al. (eds) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, New York; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, New York; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY. Specification 21 / 27 pages 24 CN 122427280 A

[0131] The selection of an administration regimen for a therapeutic agent depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of target cells in the biological matrix. In some embodiments, the administration regimen maximizes the amount of therapeutic agent delivered to the patient, consistent with an acceptable level of side effects. Therefore, the quantity of biological products delivered depends in part on the specific entity and the severity of the condition being treated.Guidelines for selecting appropriate doses of antibodies, cytokines, and small molecules are available (see, for example, Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.), (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.), (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al., (2003) New England Journal of Medicine 348:601-608; Milgrom et al., (1999) New England Journal of Medicine. [New England Journal of Medicine] 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. [New England Journal of Medicine] 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. [New England Journal of Medicine] 342:613-619; Ghosh et al. (2003) New Engl. J. Med. [New England Journal of Medicine] 348:24-32; Lipsky et al. (2000) New Engl. J. Med. [New England Journal of Medicine] 343:1594-1602).

[0132] This disclosure also covers kits for treating patients with pathological disorders mediated by IL-17, such as autoimmune diseases or inflammatory disorders or conditions. Such kits contain a therapeutically effective amount of antibody produced according to the methods described herein and a packaging booklet indicating the recommended dosing regimen for the anti-IL-17 antibody against the patient. Preferably, the antibody is an anti-IL-17 antibody, such as secukinumab. Additionally, such kits may include tools for administering the antibody (e.g., autoinjector, syringe and vial, pre-filled syringe, pre-filled pen) and instructions for use. The kit may also include instructions for administering the anti-IL-17 antibody to treat the patient.Such instructions can provide the dosage, route of administration, regimen, and total duration of treatment for the blocked antibody. The phrase “tool for administration” is used to refer to any available tool for systemic administration of the drug to a patient, including but not limited to pre-filled syringes, vials and syringes, injection pens, autoinjectors, IV drips and bags, infusion pumps, patches, infusion bags, and needles. Using such items, a patient can self-administer the drug (i.e., administer the drug without the assistance of a physician) or a physician can administer the drug.

[0133] Details of one or more embodiments of this disclosure are set forth in the appended specification. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, preferred methods and materials are now described. Other features, objectives, and advantages of this disclosure will be apparent from the specification and the claims. In the specification and the appended claims, the singular form includes the plural referent unless the context clearly indicates otherwise. Unless otherwise defined, 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 pertains. All patents and publications cited in this specification are incorporated herein by reference. The following examples are presented to more fully illustrate preferred embodiments of the disclosure. These examples should in no way be construed as limiting the scope of the disclosed patient problems as defined by the appended claims.

[0134] Examples

[0135] The following experiments are intended to further illustrate the invention as defined in this application.

[0136] Example 1

[0137] A set of experiments was designed to determine whether the oxidation of cys97 in secukinumab occurs extracellularly.

[0138] The purified secukinumab active pharmaceutical ingredient (API) was percolated with cell culture medium containing a standard amount of cysteine / cystine (Amicon Ultracel tubes, page 22 / 27 of the specification). The solution was then diluted in the culture medium to a concentration of 1.5 g / L to maintain consistency with the bioreactor titer. The solution was then incubated at 37°C. Finally, samples were taken at 24 and 48 hours for cystamine-CEX to determine the percentage of free cys97.

[0139] Table 2 shows that the percentage of free cys97 decreased significantly after incubation in the culture medium, indicating that cys97 can be oxidized in an extracellular environment containing cysteine / cystine after secukinumab secretion. This understanding leads to the hypothesis that cys97 oxidation may occur due to the presence of cysteine / cystine in the cell culture medium.

[0140] Table 2. Incubation of secukinumab active pharmaceutical ingredient with cell culture medium

[0141] The results confirmed that cysteine / cystine in the cell culture medium oxidized cys97.

[0142] The secukinumab active pharmaceutical ingredient was incubated together with culture media containing different amounts of cysteine / cystine.Figure 1 shows that a lower cysteine / cystine level in the culture medium reduces the oxidation of cys97. This culture medium incubation study indicates that cysteine / cystine does indeed oxidize cys97 on secukinumab.

[0143] Example 2

[0144] Previous observations have shown that incubation of secukinumab in a culture medium such as perfusion medium at 37°C results in a decrease in antibody activity over time. To determine whether the level of cysteine / cystine (i.e., cys equivalent) in the culture medium could mitigate the decrease in activity, secukinumab eluent was incubated in different culture medium variants based on standard perfusion medium to study the effect of culture medium components.

[0145] To minimize the dilution effect when adding the sample to the culture medium, the starting solution was percolated in perfusion medium. The resulting solution was added to perfusion medium to achieve a final volume of 50 ml and a final protein concentration of approximately 1.5 g / L to simulate a typical antibody bioreactor titer. The solution was incubated at 37°C (standard bioreactor temperature) for two days. After 24 and 48 hours, 25 ml samples were taken and antibodies were captured. The activity of all samples was analyzed by cystamine-CEX. In standard perfusion medium, cysteine ​​was supplied in the form of cysteine ​​monohydrate hydrochloride, and cystine was supplied by a stock solution containing tyrosine and cystine.

[0146] Secukinumab was incubated in three medium variants to study the effect of medium composition: Table 3. Specification 23 / 27 pages 26 CN 122427280 A

[0147] As shown in Figure 2, when incubated in baseline medium, the activity of secukinumab decreased from about 98% to 25% within 2 days. By reducing the amount of cys equivalent by 75%, the activity decrease was reduced to 60%. In the absence of any cys equivalent, the activity decrease was further reduced to only 83%. In addition, the removal of trace elements did not show a significant improvement, as the activity decreased from 96% to 84%.

[0148] Example 3

[0149] To determine the effect of cysteine / cystine (i.e., cys equivalent) variations in a fed-batch reactor method, an experiment was designed based on the standard principle of CHO cell expression of secukinumab. The cysteine ​​concentrations in both the basal and fed media were different. The concentration of cystine added from the tyrosine / cystine stock solution did not change. Table 4 below shows the tested media variants for which fed-batch cell culture was run for 10 days. Even with reductions or removal of cysteine ​​from either the basal or fed media, cystine from the tyrosine / cystine stock solution remained in the medium. Therefore, the total cys equivalents for the baseline and medium variants are also shown in Table 4.

[0150] Table 4. Specification 24 / 27 pages 27 CN 122427280 A

[0151] As shown in Figure 3, changing the content of cys equivalent in the basal medium and the culture medium feed produced similar cell growth and secukinumab expression titers (mg / ml) for all variants (including baseline). Only minor changes in the final antibody titer (mg / ml) were detected with the culture medium variants (from approximately 2.4 mg / ml for variant 1 to approximately 2.6 mg / ml for variant 3), as shown in Figure 4.

[0152] Figure 5 is a graph showing the activity (%) of secukinumab expressed using different culture medium variants. The activity of secukinumab expressed in cell culture medium with reduced cys equivalent was higher than 80%. In contrast, the activity of secukinumab expressed in baseline cell culture medium was approximately 60%.

[0153] As demonstrated in this experiment, reducing and / or removing the cys equivalent from the basal and / or fed culture media had no effect on the yield of secukinumab in the fed batch process, and instead produced an antibody product with higher activity. Therefore, these results indicate that secukinumab expressed in cell culture media at reduced concentrations of the cys equivalent mitigates undesirable cell-based modifications of CysL97, thereby improving product quality.

[0154] Example 4

[0155] An integrated API manufacturing method using 1000 L-scale high cell density perfusion batch (HDPB) culture with approximately one reactor volume of perfusion medium per day was suitable for producing secukinumab from CHO cells. The peak viable cell density (VCD) of the HDPB production process was close to 16 million cells / mL and the process lasted approximately 19 days. Compared to fed-batch production media, the HDPB production media (including the concentrations of components such as manganese, Prönkel F68, glucose, glutamine, cysteine, and NaCl) were minimally adjusted to ensure growth robustness and desired product quality. No new components were introduced. The HDPB bioreactor volumetric productivity was approximately 1.2 g / L / day (or 22.8 g / L accumulation titer).

[0156] During the evaluation of candidate media formulations, as measured by cystamine CEX, it was determined that reducing the cysteine ​​concentration increased the bioactivity of secukinumab. As shown in Tables 5 and 6 and Figure 6, in laboratory-scale experimental harvest libraries, by reducing the cysteine ​​concentration in the perfusion medium by 50%, the bioactivity increased by approximately 6%–8%. In addition to the effects determined in the upstream process, the proposed cysteine ​​effect was also confirmed from the downstream perspective due to its direct impact on the reduction step.

[0157] Table 5. Perfusion medium

[0158] Table 6. Perfusion results

[0159] Compared with other conditions, the lower the cysteine ​​concentration, the lower the acidity value, which is also considered beneficial.

[0160] Table 7. Sequence Listing Specification 26 / 27 Page 29 CN 122427280 A Specification 27 / 27 Page 30 CN 122427280 A Figure 1 Specification Figure 1 / 4 Page 31 CN 122427280 A Figure 2 Specification Figure 2 / 4 Page 32 CN 122427280 A Figure 3 Figure 4 Specification Figure 3 / 4 Page 33 CN 122427280 A Figure 5 Figure 6 Specification Figure 4 / 4 Page 34 CN 122427280 A Abstract The present disclosure relates to methods for reducing the oxidation level of cysteine ​​residues in recombinant polypeptides such as anti-IL-17 antibodies during cell culture (e.g., a preparation of secukinumab antibodies) that have been recombinantly produced by mammalian cells. Also provided are purified preparations of recombinant polypeptides such as anti-IL-17 antibodies or antigen-binding fragments thereof produced by Such methods, eg, purified preparations of secukinumab. Also provided are purified preparations of recombinant polypeptides produced by such methods wherein the level of active recombinant polypeptide in the preparation is high.

Claims

1. A method for generating a recombinant polypeptide in fed-batch cell cultures, wherein the recombinant polypeptide is an antibody, namely secukinumab, wherein the method comprises the following steps: a. Culturing mammalian cells in a cell culture medium comprising a basal medium and one or more feed media, wherein the basal medium contains a cys equivalent at a concentration of 0.3 g / L, and wherein the feed media contains a cys equivalent at a concentration of less than 0.8 g / L, and wherein the cumulative concentration of the cys equivalent in the cell culture medium is less than 0.4 g / L, wherein the mammalian cells are CHO cells. b. Expression of the recombinant polypeptide and c. Recover the recombinant polypeptide from the culture medium.

2. The method according to claim 1, wherein the basal culture medium does not contain added cysteine, and the supplemental culture medium contains cysteine ​​at a concentration of 0.66 g / L.

3. The method according to claim 1, wherein the basal culture medium does not contain added cysteine, and the feed culture medium contains cysteine ​​at a concentration of 0.33 g / L.

4. The method of claim 1, wherein the basal culture medium does not contain added cysteine, and the supplemental culture medium does not contain cysteine.

5. The method according to any one of claims 1-4, the method comprising a downstream processing step of selective reduction, wherein the antibody is incubated with at least one reducing agent in the system to form a reducing mixture.

6. The method according to any one of claims 1-5, further comprising the following steps: d. Purify the antibody; e. Prepare the antibody for administration. f. Package the antibody together with the booklet.

7. The method according to any one of claims 1-6, wherein the recombinant polypeptide population recovered from the culture medium contains at least 10% higher levels of reduced free cysteine ​​compared to the recombinant polypeptide population recovered from the control culture medium, wherein the control culture medium comprises a control basal culture medium having a concentration of greater than 0.4 g / L of cys equivalents and / or a control feed culture medium containing a concentration of greater than 0.9 g / L of cys equivalents, and / or wherein the cumulative concentration of cys equivalents in the control cell culture is greater than 0.4 g / L.

8. The method according to any one of claims 1-7, wherein the method comprises producing a higher yield of recombinant polypeptide based on mg active recombinant polypeptide / L culture medium compared to a control method, wherein the control method comprises culturing mammalian cells in a control cell culture medium comprising a control basal culture medium and one or more control feed cultures, wherein the control basal culture medium comprises a cys equivalent at a concentration greater than 0.4 g / L, and / or wherein the control feed culture medium comprises a cys equivalent at a concentration greater than 0.9 g / L, and / or wherein the cumulative concentration of the cys equivalent in the control cell culture is greater than 0.4 g / L.

9. The method of claim 7 or 8, wherein the method comprises, as determined from used culture medium, producing a population of recombinant polypeptides having at least 61% reduced free cysteine.

10. A method for producing a recombinant polypeptide from a mammalian cell culture, wherein the recombinant polypeptide is an antibody, namely secukinumab, and wherein the mammalian cell is a CHO cell, wherein the method comprises the following steps: a. The mammalian cells are cultured in a culture containing a cell culture medium, wherein the cell culture medium contains a cys equivalent at a concentration of 0.3 g / L; b. A portion of the cell culture medium in the culture is replaced by perfusion with fresh cell culture medium containing a concentration of 0.3 g / L of cys equivalent, and / or wherein the cumulative concentration of cys equivalent added to the culture is less than 7 g / L / day or less than 0.4 g / L / day; c. Expression of the recombinant polypeptide and d. Recover the recombinant polypeptide from the culture.

11. The method of claim 10, wherein the fresh culture medium does not contain added cysteine.

12. The method of claim 10, wherein the method comprises exchanging at least 50% of the cell culture medium daily by perfusion with fresh cell culture medium.

13. The method according to any one of claims 10 to 12, wherein the recombinant polypeptide population recovered from the culture medium contains at least 10% higher levels of reduced free cysteine ​​compared to the recombinant polypeptide population recovered from the control culture medium, the control culture medium containing a concentration of cys equivalent greater than 0.4 g / L, and / or wherein the cumulative concentration of cys equivalent added to the control cell culture is greater than 7 g / L / day, and / or greater than 0.4 g / L / day.

14. The method according to any one of claims 10 to 13, wherein the method comprises producing a higher yield of recombinant polypeptide based on mg active recombinant polypeptide / L culture medium compared to a control method, wherein the control method comprises culturing mammalian cells in a control cell culture medium containing a concentration greater than 0.4 g / L of cys equivalent, and / or wherein the cumulative concentration of cys equivalent in the control cell culture is greater than 7 g / L / day, and / or greater than 0.4 g / L / day.

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