JP-7856652-B2 - Method for reducing the oxidation level of cysteine residues in secreted recombinant expression proteins during cell culture.

Inventors

• ブソ カリンハス，ヌノ
• キュイ，フアンチュン
• ガルシア，デイビッド
• ゴエベル，マティアス
• シュルツ，ジョセフ

Assignees

• ノバルティス アーゲー

Dates

Publication Date

20260511

Application Date

20211221

Priority Date

20201222

Claims (14)

1. A method for producing recombinant polypeptides under fed-batch cell culture, wherein the recombinant polypeptide is an antibody, the antibody is secukinumab, and the method is a. A step of culturing mammalian cells in a cell medium comprising a basic medium and one or more feed media, wherein the basic medium contains a cys equivalent at a concentration of 0.3 g/L, the feed media contains a cys equivalent at a concentration of less than 0.8 g/L, the cumulative concentration of cys equivalent in the cell medium is less than 0.4 g/L, and the mammalian cells are CHO cells ; b. The step of expressing the recombinant polypeptide, c. A step of recovering the recombinant polypeptide from the culture medium, Methods that include...
2. The method according to claim 1, wherein the basic medium does not contain the added cysteine, and the feed medium contains cysteine at a concentration of 0.66 g/L.
3. The method according to claim 1, wherein the basic medium does not contain the added cysteine, and the feed medium contains cysteine at a concentration of 0.33 g/L.
4. The method according to claim 1, wherein the basic culture medium does not contain added cysteine, and the feed medium does not contain cysteine.
5. The method according to any one of claims 1 to 4, wherein the method comprises a downstream processing step of selective reduction, wherein the antibody is incubated in system with at least one reducing agent to form a reduction mixture.
6. d. The step of purifying the antibody; e. The step of formulating the antibody for administration, f. The step of packaging the antibody together with a leaflet, The method according to any one of claims 1 to 5 , further comprising:
7. The method according to any one of claims 1 to 6, wherein the population of recombinant polypeptides recovered from the culture medium contains at least 10 % higher levels of reduced free cysteine compared to the population of recombinant polypeptides recovered from the control medium, and the control medium comprises a control basic medium having a cys equivalent at a concentration greater than 0.4 g/L and/or a control feed medium having a cys equivalent at a concentration greater than 0.9 g /L, and/or the cumulative concentration of cys equivalent in the control cell culture is greater than 0.4 g/L.
8. The method comprises producing a higher yield of recombinant polypeptides in mg units of active recombinant polypeptides per liter of medium compared to a control method comprising culturing mammalian cells in a control cell medium containing a control basic medium and one or more control feed media. The method according to any one of claims 1 to 7, wherein the control basic medium contains a cys equivalent at a concentration greater than 0.4 g/L, and/or the control feed medium contains a cys equivalent at a concentration of 0.9 g /L, and/or the cumulative concentration of cys equivalent in the control cell culture is greater than 0.4 g/L.
9. The method according to claim 7 or 8 , wherein the method, when assayed from the consumed culture medium, produces a population of recombinant polypeptides having at least 61% reduced free cysteine.
10. A method for producing recombinant polypeptides by mammalian cell culture, wherein the recombinant polypeptide is an antibody, the antibody is secukinumab, the mammalian cells are CHO cells, and the method is a. A step of culturing mammalian cells in a culture medium comprising a cell medium, wherein the cell medium comprises a cys equivalent at a concentration of 0.3 g/L; b. A step of replacing a portion of the cell medium in the culture with fresh cell medium by perfusion, wherein the fresh cell medium contains a cys equivalent at a concentration of 0.3 g/L and/or the cumulative concentration of cys equivalent added to the culture is less than 7 g/L or less than 0.4 g/L/day; c. The step of expressing the recombinant polypeptide, d. A step of recovering the recombinant polypeptide from the culture, Methods that include...
11. The method according to claim 10 , wherein the new culture medium does not contain added cysteine.
12. The method according to claim 10 , wherein the method comprises replacing at least 50% of the cell culture medium with fresh cell culture medium by daily perfusion culture.
13. The method according to any one of claims 10 to 12, wherein the population of recombinant polypeptides recovered from the culture medium contains at least 10 % higher levels of reduced free cysteine compared to the population of recombinant polypeptides recovered from the control medium, the control medium contains a cys equivalent at a concentration greater than 0.4 g/L, and/or the cumulative concentration of cys equivalent added to the control cell culture is greater than 7 g /L and/or greater than 0.4 g /L/day.
14. The method comprises producing a higher yield of recombinant polypeptides in mg units of active recombinant polypeptides per liter of medium compared to a control method which involves culturing mammalian cells in a control cell medium. The method according to any one of claims 10 to 13, wherein the control cell medium contains a cys equivalent at a concentration greater than 0.4 g /L, and/or the cumulative concentration of the cys equivalent in the control cell medium is greater than 7 g/L and/or greater than 0.4 g /L/day.

Description

This disclosure relates to a method for reducing the oxidation level of one or more cysteine residues in secreted recombinant expression proteins during recombinant production of anti-IL-17 antibodies, such as secukinumab, by mammalian cells in cell culture. Background of Disclosure Classical antibodies consist of two light chains (L) each with a molecular weight of approximately 25 kD and two heavy chains (H) each with a molecular weight of approximately 50 kD. The light and heavy chains are linked by a disulfide bond (L-S-S-H), and the two LH units are further linked by two disulfide bonds between the heavy chains. The general formula for a classical antibody is L-SS-H(-SS-) 2H -SS-L or simply H2L2 ( HHLL ). In addition to these conserved interchain disulfide bonds, conserved intrachain disulfide bonds also exist. Both types of disulfide bonds are important for the stability and behavior (e.g., affinity) of the antibody. Generally, the disulfide bond is brought about by two cysteine residues (Cys-SH) found at conserved positions in the antibody chain, thereby spontaneously forming a disulfide bond (Cys-S-S-Cys). The formation of disulfide bonds is determined by the redox potential of the environment and the presence of enzymes specialized in thiol-disulfide exchange. Internal disulfide bonds (Cys-S-S-Cys) stabilize the three-dimensional structure of antibodies. Antibodies containing additional free cysteine (i.e., unpaired cysteine) exist. In some cases, one or more free cysteine are involved in antigen recognition and binding, for example, because they are located within the complementarity-determining region of the antibody. In these antibodies, modification with free cysteine can have a negative effect on the molecular activity and stability and may lead to increased immunogenicity. As a result, processing these antibodies can be difficult because the final product may contain inactive, misfolded, and/or useless antibody material in real mass. U.S. Patent Application Publication No. 20090280131 (which is incorporated herein by reference in its entirety) provides an anti-IL-17 antibody, for example, secukinumab (i.e., AIN457), which has a free cysteine residue after cis-proline in the light chain complementarity-determining region (CDR) 3 loop (L-CDR3) (i.e., amino acid 8 of L-CDR3 as described in SEQ ID NO: 6, corresponding to amino acid 97 of the light chain variable region described in SEQ ID NO: 10, hereafter referred to as "CysL97"). To maintain full activity, the unpaired cysteine residue of secukinumab cannot be shielded by oxidative disulfide pairing with other cysteine residues or by oxidation by exogenous compounds (e.g., formation of mixed disulfides with other proteins, derivatization by cellular metabolites (e.g., cysteine or glutathione), and formation of sulfoxides by oxygen). Unfortunately, since secukinumab is produced using mammalian cells that secrete secukinumab into the cell culture medium, unwanted cell-based modifications of CysL97 occur. Similarly, designing free cysteine into antibody sequences may be useful for facilitating site-specific conjugation of chemical linkers, drugs, labels, and/or other parts. For example, Junutula et al. (Nat. Biotechnol., 2008, 26, 925-932) introduced designed cysteine into an anti-MUC16 antibody by mutation at heavy-chain alanine 114. The authors found that expression of the mutant antibody in Chinese hamster ovary (CHO) cells produced antibodies containing the designed cysteine residue capped as a disulfide with cysteine or glutathione. Therefore, processing of the designed cysteine residue must remove unwanted cell-based modifications. Methods have been reported for selectively reducing antibodies that have oxidation of free cysteine residues. For example, the reduction of oxidized CysL97 in the preparation of recombinantly produced IL-17 antibodies by mammalian cells is disclosed in International Publication No. 2016/103146A1. Specifically, downstream processing steps are applied, such as contacting the antibody-containing formulation with at least one reducing agent in system to form a reduction mixture; incubating the reduction mixture while maintaining an in-system volume oxygen mass transfer coefficient (kLa * ) < approximately 0.37 h⁻¹ (wherein kLa * is calculated by fitting the dissolved oxygen curve to a saturation curve). Similarly, Junutula et al. have reported a procedure utilizing a potent reducing agent (e.g., tris(2-carboxyethyl)phosphine [TCEP] or dithiothreitol [DTT]), purification of the reductive antibody, and subsequent reoxidation of interchain disulfide bonds using Cu²⁺ or dehydroascorbic acid. However, such downstream process steps can require expensive equipment, further purification steps, and result in extended process lead times. Therefore, there is a need for improved processes that enable a faster and/or more cost-effective overall manufacturing process. For this reason, methods in the upstream processing steps of rec