Method for reducing the oxidation level of cysteine ​​residues in secreted recombinant expression proteins during cell culture.

Abstract

The present disclosure relates to methods for reducing the level of oxidation of cysteine ​​residues in recombinant polypeptides, such as anti-IL-17 antibodies (e.g., formulations of secukinumab antibodies), that have been recombinantly produced by mammalian cells in cell culture. Also provided are purified preparations of recombinant polypeptides, such as anti-IL-17 antibodies or antigen-binding fragments thereof, e.g., purified preparations of secukinumab, produced by such methods. Also provided are purified preparations of recombinant polypeptides produced by such methods, wherein the preparations have elevated levels of active recombinant polypeptide.

Description

Technical Field

[0001] The present disclosure relates to a method for reducing the oxidation level of one or more cysteine residues in a secreted recombinant expression protein during cell culture, for example, during the recombinant production of an anti-IL-17 antibody such as secukinumab by mammalian cells.

Background Art

[0002] Background of Disclosure Classical antibodies consist of two light chains (L) each having a molecular weight of about 25 kD and two heavy chains (H) each having a molecular weight of about 50 kD. The light and heavy chains are linked by disulfide bonds (L-S-S-H), and the two LH units are further linked by two disulfide bonds between the heavy chains. The general formula of a classical antibody is L-SS-H(-SS-)2H-SS-L or simply H2L2 (HHLL). In addition to these conserved inter-chain disulfide bonds, conserved intra-chain disulfide bonds also exist. Both types of disulfide bonds are important for the stability and behavior (e.g., affinity) of the antibody. Generally, disulfide bonds are brought about by two cysteine residues (Cys-SH) found at conserved positions within the antibody chains, whereby a disulfide bond (Cys-S-S-Cys) is spontaneously formed. The formation of disulfide bonds is determined by the redox potential of the environment and by the presence of enzymes specialized in thiol-disulfide exchange. The internal disulfide bond (Cys-S-S-Cys) stabilizes the three-dimensional structure of the antibody.

[0003] 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 the free cysteine ​​is located within the complementarity-determining region of the antibody. In the case of these antibodies, modification with free cysteine ​​may have a negative effect on the activity and stability of the molecule 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), having 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 described as SEQ ID NO: 6, corresponding to amino acid 97 of the light chain variable region described as SEQ ID NO: 10, hereafter referred to as "CysL97"). To maintain full activity, the unpaired cysteine ​​residues 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.

[0004] 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 with 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.

[0005] 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 IL-17 antibodies recombinantly produced by mammalian cells is disclosed in International Publication No. 2016 / 103146A1. Specifically, the antibody-containing preparation is brought into contact with at least one reducing agent in system to form a reduction mixture; the reduction mixture is incubated while the volume oxygen mass transfer coefficient (kLa) in system is <approximately 0.37 h⁻¹. * )(the kLa * Downstream processing steps such as maintaining the dissolved oxygen curve (which is calculated by fitting the dissolved oxygen curve to the saturation curve) are applied. Similarly, Junutula et al. have described a procedure utilizing a strong reducing agent (e.g., tris(2-carboxyethyl)phosphine [TCEP] or dithiothreitol [DTT]), purification of the reducing antibody, and subsequent Cu 2+ Alternatively, we have reported on the reoxidation of interchain disulfide bonds using dehydroascorbic acid.

[0006] However, such downstream process steps may 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 recombinant antibody production may be further evaluated for optimization.

[0007] The culture of secreted mammalian cells for industrial applications such as recombinant polypeptide expression 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 biological products. Early media development efforts have yielded basic formulations for sustained growth, viability, and cellular function, although these may include animal-derived components and complex components used in batch culture modes. Subsequent improvements have included the development of serum-free and synthetic (CD) media, the identification of critical nutrients, growth factors, and potentially inhibitory or toxic cellular metabolites, and the use of fed-batch and perfusion culture techniques, all aimed at optimizing nutrient delivery while minimizing the accumulation of unwanted waste.

[0008] All cell media require similar basic nutrients to support cell proliferation. Amino acids are major components in cell media, and studies have shown that small changes in the amino acid composition of cell media can alter growth characteristics and titers. For example, Ghaffari et al. (Biotechnol Progress. 2020;36:e2946) reported that maintaining the efficacy of the so-called non-essential amino acid cysteine ​​is a critical process parameter for high-yield recombinant protein production in common CHO cell lines. However, cysteine ​​is readily oxidized under typical cell medium pH and oxygen and metal-rich conditions. Moreover, cysteine ​​can promote the unwanted oxidation of free cysteine ​​residues in recombinant proteins. Therefore, cysteine ​​supply methods typically require a high degree of optimization to achieve high-yield recombinant expression of proteins from CHO cells.

[0009] Many cell culture media and culture feeds are commercially available to supply essential nutrients. However, the need to adjust the media and feeds for recombinant polypeptides remains to optimize the quality and yield of secreted recombinant polypeptides containing reduced cysteine ​​residues during production. Optimizing the process is complex, even for commercially available recombinant polypeptides such as antibodies, using numerous parameters that can be modified under cell culture conditions, and the need for processes optimized for industrial production still exists. [Overview of the Initiative]

[0010] Despite extensive research and optimization in the field of cell media, it has been surprisingly found that unwanted modifications of free cysteine ​​in expressed antibodies can be reduced by decreasing the amount of cysteine ​​added to mammalian cell media and lowering the concentration of cys equivalents in the medium. Such a reduction in the amount of unwanted modifications of free cysteine ​​can lead to improved antibody activity and / or avoid the need for subsequent antibody reduction and / or optional reoxidation.

[0011] According to a first aspect, the present disclosure relates to a method for producing recombinant polypeptides in fed-batch cell culture, 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 concentration of cys equivalent of approximately 0.3 g / L, the feed media contains a concentration of cys equivalent of less than approximately 0.8 g / L, and the cumulative concentration of cys equivalent in the cell medium is less than approximately 0.4 g / L; b. A step of expressing a recombinant polypeptide, c. A step of recovering polypeptides from the culture medium, This provides a method that includes [something].

[0012] The cumulative cys equivalent in the cell medium is the total concentration of cysteine ​​and cystine in the cell medium derived from the base medium and / or feed medium. The cumulative cys equivalent may be at a different concentration at the start of the fed-batch process compared to, for example, at the end of the process after a period of several hours or several days, and may change during the process if the feed medium is added to the cell medium. In one embodiment, the concentration of cys equivalent in the cell medium at the start of the fed-batch process 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, preferably less than about 0.3 g / L. During the fed-batch process, the concentration of cys equivalent may change from about 0.3 g / L to about 0.8 g / L due to the addition of cys equivalent to the cell medium (due to the addition of either cysteine ​​or cystine). To obtain this concentration of cys equivalent, the concentration of cysteine ​​added to the cell medium in the feed medium may be less than about 1 g / L. For example, the basic medium may not contain added cysteine, and the feed medium may contain a cysteine ​​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, preferably less than about 0.7 g / L. In one embodiment, the basic medium may not contain added cysteine, and the feed medium may contain a cysteine ​​concentration of about 0.66 g / L. In another embodiment, the basic medium may not contain added cysteine, and the feed medium may contain a cysteine ​​concentration of about 0.33 g / L. In a further embodiment, the basic medium may not contain added cysteine, and the feed medium may also not contain cysteine.

[0013] At the end of the fed-batch process as described herein, the cumulative concentration of CYS equivalents in the cell medium may be less than approximately 0.4 g / L. In a standard fed-batch cell culture where no modifications have been made to reduce CYS equivalents in the basic medium and / or feed medium, the cumulative concentration of CYS equivalents in the cell medium may be approximately 0.6 g / L.

[0014] In one embodiment, the recombinant polypeptide produced under fed-batch cell culture is an antibody, preferably the anti-IL-17 antibody secukinumab.

[0015] In one embodiment, the mammalian cells used under red batch cell culture are selected from the group consisting of CHO cells, HEK cells, and SP2 / 0 cells. For example, the mammalian cells may be CHO cells selected from the group consisting of CHO-S, CHOKl, CHO pro3-, CHO DG44, CHO P12, or dhfr-CHO cell lines DUK-BII, DUXBI1, or CHO-K1SV.

[0016] In a second embodiment, the above method for reducing the concentration of cys equivalent in cell culture medium can be combined with a downstream processing step of selective reduction, wherein the antibody is incubated with at least one reducing agent in the system to form a reduction mixture, and the reduction mixture is incubated while the volume oxygen mass transfer coefficient (kLa) in the system is < approximately 0.37 h⁻¹ * ) maintain the kLa * This is calculated by fitting the dissolved oxygen curve to the saturation curve. Preferably, the antibody is secukinumab. Such downstream processing steps for selectively reducing the cysteine ​​residue at the CysL97 position in the preparation of IL-17 antibody are disclosed in International Publication No. 2016 / 103146A1.

[0017] Recombinant polypeptides produced according to the methods of this disclosure can be prepared for administration to human patients by carrying out further steps for preparing pharmaceuticals. For example, if the recombinant polypeptide is an antibody, it is necessary to purify the antibody and formulate it with various excipients to provide a pharmaceutical composition suitable for administration to a patient. Furthermore, the antibody can be packaged with a leaflet containing instructions for use for administration to a patient. Such a leaflet can provide dosage, route of administration, regimen, and total duration of treatment for the use of the inclusion antibody.

[0018] In one embodiment, the present invention provides a method for producing recombinant polypeptides by mammalian cell culture, comprising the steps of: a) culturing mammalian cells (selected from the group consisting of, for example, CHO cells, HEK cells, and SP2 / 0 cells) in a culture medium comprising a cell medium containing a reduced concentration of cys equivalent compared to a control basic medium; b) replacing all or part of the cell medium under culture with fresh cell medium by perfusion, wherein the fresh cell medium contains a reduced concentration of cys equivalent compared to a control exchange medium; c) expressing recombinant polypeptides; and d) recovering polypeptides from the culture.

[0019] In some embodiments, the perfused cell medium contains a concentration of cys equivalent of approximately 0.1 g / L to less than approximately 0.6 g / L, approximately 0.2 g / L to less than approximately 0.5 g / L, approximately 0.25 g / L to less than approximately 0.4 g / L, or 0.3 g / L to approximately 0.4 g / L. In some embodiments, the fresh perfused cell medium contains a concentration of cys equivalent of approximately 0.1 g / L to less than approximately 1.1 g / L, approximately 0.2 g / L to less than approximately 0.9 g / L, approximately 0.25 g / L to less than approximately 0.6 g / L, or 0.3 g / L to approximately 0.4 g / L. In some embodiments, the medium contains a concentration of cys equivalent of approximately 0.3 g / L. In some embodiments, the fresh medium contains a concentration of cys equivalent of approximately 0.3 g / L.

[0020] In some embodiments, the cumulative cys equivalent added to the perfusion 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 11 g / L, about 4 g / L to less than 11 g / L, about 5 g / L to less than 11 g / L, about 3 g / L to less than 9 g / L, about 4 g / L to less than 9 g / L, about 5 g / L to less than 9 g / L, or preferably about 5 g / L to less than 7 g / L.

[0021] In some embodiments, the cumulative cys equivalent added to the perfusion 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 perfusion 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 or 0.4 g / L / day. [Brief explanation of the drawing]

[0022] [Figure 1] This is a graph showing the activity of antibody samples according to the embodiment. [Figure 2] This graph shows the effect on antibody activity (%) by reducing cysteine / cystine concentration (cys equivalent) under several days of variation in perfusion medium (x axis). A greater reduction in cysteine / cystine concentration results in better maintenance of antibody activity compared to baseline in the perfusion medium (y axis). [Figure 3] This study demonstrates that reducing the amount of cysteine ​​in the basic medium and / or feed medium has little effect on the concentration of secukinumab produced over time (days; x-axis) (mg / ml; y-axis). [Figure 4] This shows that the final culture concentration (mg / ml) of secukinumab on day 12 shows little change between baseline (cell medium including standard basic and feed media) and the three variants of the basic and / or feed media used in the study. [Figure 5] This shows the activity (%) of antibody samples produced using three variants of the basic medium and / or feed medium under test, compared to baseline (cell medium including standard basic medium and feed medium). [Figure 6]The activity (%) of antibody samples produced by perfusion culture using a test perfusion medium that does not contain cysteine ​​and therefore has a 50% reduction in cys equivalent compared to a control perfusion cell medium containing normal levels of cysteine ​​and cys equivalent. The cumulative cys equivalent added to the test and control perfusion cell cultures, with approximately one complete medium change per day, was approximately 5.875 g / L (approximately 0.31 g / L / day) and 11.642 g / L (0.61 g / L / day), respectively. [Modes for carrying out the invention]

[0023] The object of this disclosure is to provide a method for reducing the oxidation level of cysteine ​​residues in recombinant polypeptides such as anti-IL-17 antibodies during cell culture, for example, during the recombinant production of secukinumab by mammalian cells.

[0024] The term "contains" encompasses both "contains" and "consists of," for example, a composition "contains" X may consist solely of X, or it may contain something additional (e.g., X + Y).

[0025] The term "approximately" in relation to a number x means, for example, + / - 10%. When used before a range of numbers or a list of numbers, the term "approximately" applies to each number in the series; for example, the expression "approximately 1 to 5" should be interpreted as "approximately 1 to approximately 5," or for example, the expression "approximately 1, 2, 3, 4" should be interpreted as "approximately 1, approximately 2, approximately 3, approximately 4, etc."

[0026] The relative molecular weight of secukinumab, based on its post-translational amino acid sequence, is 147,944 daltons. This molecular weight (i.e., 147,944 daltons) is used throughout this disclosure in the calculation of molar concentrations and molar ratios of secukinumab. However, during production in CHO cells, the C-terminal lysine is generally removed from each heavy chain. The relative molecular weight of secukinumab lacking C-terminal lysine from each heavy chain is 147,688 daltons. Secukinumab formulations contain mixtures of molecules in the presence and absence of C-terminal lysine residues on the heavy chains. Therefore, the molar concentrations of secukinumab used in this disclosure (and the ratios using these molar concentrations) are estimates, and terms such as “approximate” and “approximate” relating to these values ​​encompass at least this difference in the relative molecular weight and the resulting calculations.

[0027] The word “substantially” does not exclude “completely.” For example, a composition that “substantially does not contain” Y may not contain Y at all. Where necessary, the word “substantially” may be omitted from the definitions in this disclosure.

[0028] Large-scale cell culture can be used, for example, by various fermentation processes established in industrial biotechnology. Discontinuous and continuous cell culture processes, such as perfusion and chemostats, can be utilized with cell culture media according to the present invention. Discontinuous processes, including repeated fed-add and repeated batch processes, are a preferred embodiment. In general, the methods and compositions of the present invention are aimed at the production of polypeptides secreted by cell culture.

[0029] Batch cell culture includes fed-batch culture and simple batch culture. The term “fed-batch cell culture” refers to a cell culture in which cells and cell medium are initially supplied to a culture vessel, and additional culture nutrients are continuously or incrementally supplied to the culture during the culture process, in or out of the presence of periodic cells, and / or the product is collected before termination of the culture. The term “simple batch culture” refers to a procedure in which all components of the cell culture, including cells and cell medium, are supplied to the culture vessel at the start of the culture process. Preferably, the cells cultured in cell medium according to the present invention are CHO cells.

[0030] The term "cell medium" refers to an aqueous solution of nutrients that can be used to grow cells over a long period of time. Typically, cell mediums contain the following components: usually carbohydrate compounds, preferably glucose; amino acids, preferably a set of basic amino acids, e.g., all essential and non-essential amino acids; vitamins and / or other organic compounds (as needed in low concentrations); free fatty acids; and inorganic compounds, e.g., trace elements, inorganic salts, buffer compounds and nucleosides, and energy sources that act as bases.

[0031] The term "growth medium" typically refers to the cell culture medium used during the expansion phase of the entire production process. The expansion phase is the initial period of the entire culture / production process, characterized primarily by high cell growth and lower polypeptide production. The expansion phase means generating a sufficient number of cells in the logarithmic growth phase to contribute to the goal of expanding the cells and inoculating them into the production bioreactor.

[0032] The term "production medium" typically refers to the cell culture medium used during the production phase of the entire production process. The production phase is the second phase of the entire culture / production process, contributing to the goal of producing a large quantity of product. Throughout the production phase, cells need to be maintained in a viable and proliferative mode as much as possible.

[0033] For example, in the pharmaceutical industry's use of cell media for producing recombinant polypeptides with therapeutic activity, the use of any animal-derived material is generally not permitted due to safety and contamination concerns. Therefore, the cell media according to the present invention are preferably serum and / or protein-free media. The term "serum and / or protein-free media" refers to a completely synthetic medium that does not contain additives from animal sources such as tissue hydrolysates or fetal bovine serum. Furthermore, proteins, especially growth factors such as insulin, and transferrin are also preferably not added to the cell cultures according to the present invention. Preferably, the cell media according to the present invention are also free from hydrolyzable protein sources such as soy, wheat, or rice peptone or yeast hydrolysates.

[0034] The term "basic medium" refers to a medium used to culture cells that is used alone and directly, and not used as an additive to other media, although various components may be added to the basic medium. For example, if CHO cells are cultured in DMEM, which is a well-known, commercially available medium for mammalian cells and is periodically supplied with glucose or other nutrients, then DMEM would be considered a basic medium. "Feed medium" refers to a medium used as a feed under cell culture, which may be a fed-batch cell culture. Feed mediums, like basic mediums, are designed based on the need to culture specific cells. Feed mediums may have much, but not all, of the components of the basic medium at higher concentrations. For example, some components, such as nutrients including amino acids or carbohydrates, may be present at concentrations approximately 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 400, 600, 800, or even 1000 times higher than their normal concentrations in the basic medium. For example, some components, such as salts, may be maintained at approximately the same concentration as the basic medium, and the isotonicity of the feed may be maintained by the basic medium. Some components may be added to maintain the physiology of the feed, and some may be added to supplement nutrients in the culture.

[0035] The cell culture medium according to the present invention can be used in various cell culture processes. The culturing of cells can be carried out in adherent culture, for example, monolayer culture or preferably suspension culture.

[0036] The polypeptides that can be produced from cell cultures and cell culture media according to the present invention are not limited. The polypeptides can be recombinant or non-recombinant. As used herein, the term "polypeptide" refers to a molecule consisting of a chain of three or more amino acids linked by peptide bonds; a molecule containing two or more such chains; for example, a molecule containing one or more such chains further modified, such as by glycosylation. A polypeptide can contain one or more native disulfide bonds. A polypeptide can contain native or modified free cysteines. The term polypeptide is intended to encompass proteins.

[0037] A preferred class of polypeptides produced by cell cultures and cell culture media according to the present invention are recombinant antibodies.

[0038] As used herein, the term "antibody" includes a complete antibody and any antigen-binding portion or single chain thereof. A "antibody" of natural origin 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 V H 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 V L and a light chain constant region. The light chain constant region consists of one domain, CL. V H and V L regions can be further subdivided into regions of higher variability called hypervariable regions or complementarity-determining regions (CDRs) that interrupt more conserved regions called framework regions (FRs). Each V H and V LIt consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: 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 the antibody can mediate the binding of the immunoglobulin 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.

[0039] The antibody term "antigen-binding fragment," as used herein, refers to a fragment of an antibody that retains the ability to specifically bind to an antigen (e.g., IL-17). It has been shown that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody. An example of a binding fragment encompassed by the term "antigen-binding portion" of an antibody is the Fab fragment, i.e., V L , V H , a monovalent fragment consisting of CL and CH1 domains; a divalent fragment containing the F(ab)2 fragment, i.e., two Fab fragments linked by disulfide bridges in the hinge region; V H and Fd fragment consisting of the CH1 domain; V of a single arm of the antibody L and V H Fv fragment consisting of domains; V H Examples include dAb fragments consisting of domains (Ward et al., 1989 Nature 341:544-546) and isolated CDRs. Exemplary antigen-binding moieties include secukinumab CDRs, preferably heavy chain CDR3, as described in SEQ ID NOs: 1-6 and 11-13 (Table 1). Furthermore, the two domains of the Fv fragment, V L and V H These are encoded by other genes, but these are a single protein chain (where V L Region and V HThese can be linked using recombinant methods by synthetic linkers that allow the regions to pair up and behave as a monovalent molecule (known as single-chain Fv (scFv); see, for example, Bird et al., (1988) Science, 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci., 85:5879-5883). Such single-chain antibodies are also intended to be included in the term “antibody.” Single-chain antibodies and antigen-binding moieties can be obtained using conventional techniques known to those skilled in the art.

[0040] As used herein, “isolated antibody” refers to an antibody that substantially does not contain other antibodies having different antigen specificities (for example, an isolated antibody that specifically binds to IL-17 substantially does not contain antibodies that specifically bind to antigens other than IL-17). As used herein, the terms “monoclonal antibody” or “monoclonal antibody composition” refer to the preparation of an antibody molecule of single molecular composition. As used herein, the term “human antibody” is intended to include antibodies whose framework and CDR region have a variable region derived from a human sequence. A “human antibody” does not need to be produced by a human, human tissue, or human cell. Human antibodies in this disclosure may include amino acid residues not encoded by a human sequence (for example, mutations introduced by random or site-directed mutagenesis in vitro, by N-nucleotide addition at a junction in vivo during recombination of the antibody gene, or by somatic mutation in vivo). In some embodiments of the disclosed processes and compositions, the IL-17 antibody is a human antibody, an isolated antibody, and / or a monoclonal antibody.

[0041] The term "IL-17" refers to IL-17A, previously known as CTLA8, and includes wild-type IL-17A, polymorphic variants of IL-17A, and functional equivalents of IL-17A from various 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%, and 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 by human dermal fibroblasts.

[0042] The term “K D The term "K" is intended to refer to the dissociation rate of a specific antibody-antigen interaction. D When used herein, " is K d K against a The ratio (i.e., K d / K a It is intended to refer to the dissociation constant obtained from ) and expressed as molar concentration (M). K for antibodies D The value can be determined using methods established in the art. K for antibodies D The value can be determined using methods well established in the art. Antibody K D Methods for determining the K2 are by using surface plasmon resonance or by using a biosensor system such as the Biacore® system. In some embodiments, an IL-17 antibody or antigen-binding fragment, such as secukinumab, is used to determine the K2 D It has.

[0043] 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 numerous sites through weak non-covalent forces; the more interactions there are, the stronger the affinity. Standard assays for evaluating the binding affinity of antibodies to various species of IL-17 are known in the art, including, for example, ELISA, Western blotting, and RIA. Antibody binding kinetics (e.g., binding affinity) can also be evaluated by standard assays known in the art, such as Biacore analysis.

[0044] It will be understood that antibodies that "inhibit" one or more of these IL-17 functional properties (e.g., biochemical, immunochemical, cellular, physiological, or other biological activity) determined according to methodologies known in the art and described herein are associated with a statistically significant reduction in that particular activity compared to what is observed in the absence of the antibody (or in the presence of a control antibody of irrelevant specificity). Antibodies that inhibit IL-17 activity result in a statistically significant reduction of at least about 10%, at least 50%, 80%, or 90% of the parameter being measured, and in certain embodiments of the disclosed methods and compositions, the IL-17 antibodies used can inhibit IL-17 functional activity by more than 95%, 98%, or 99%.

[0045] The term “derivative” is used, unless otherwise indicated, to define, in this disclosure, for example, specific sequences (e.g., variable domains), amino acid sequence variants and covalent modifications (e.g., pegylation, deamidation, hydroxylation, phosphorylation, methylation, etc.) of the IL-17 antibody or its antigen-binding fragment, for example, secukinumab. “Functional derivatives” include molecules having qualitative biological activity common to the disclosed IL-17 antibody. Functional derivatives include fragments and peptide analogs of the IL-17 antibody disclosed herein. Fragments include, for example, regions within the sequence of a polypeptide of a specific sequence, as disclosed herein. Functional derivatives of the IL-17 antibody disclosed herein (e.g., functional derivatives of secukinumab) are VH and / or V L Array (for example, V in Table 1) H and / or V L V (sequence) having at least approximately 65%, 75%, 85%, 95%, 96%, 97%, 98%, or 99% overall sequence identity H and / or V L It is preferable that the substance contains a domain that substantially retains the ability to bind to human IL-17, or inhibits, for example, IL-17-induced IL-6 production in human dermal fibroblasts.

[0046] The expression "substantially identical" refers to the related amino acid or nucleotide sequence (e.g., V H or V L This means that a domain is either identical to a specific reference sequence or has substantial differences (e.g., due to conserved amino acid substitutions). Substantial differences include specific regions (e.g., V H or V L This includes minor amino acid changes, such as one or two substitutions in the 5-amino acid sequence of the domain. In the case of an antibody, the second antibody has the same specificity and at least 50% affinity. Sequences substantially identical to the sequences disclosed herein (e.g., at least about 85% sequence identity) are also part of this application. In some embodiments, the sequence identity of derivative IL-17 antibodies (e.g., derivatives of secukinumab, e.g., secukinumab biosimilar antibodies) may be about 90% or more, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, relative to the disclosed sequences.

[0047] "Identity" with respect to natural polypeptides and their functional derivatives is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to the residues of the corresponding natural polypeptide after the sequence has been aligned as necessary and gaps introduced to obtain the greatest possible percentage identity, and no conservative substitutions are considered part of sequence identity. Neither N- or C-terminal extensions nor insertions are interpreted as reducing identity. Methods and computer programs for alignment are well known. Percent identity can be determined by standard alignment algorithms, e.g., the Basic Local Alignment Search Tool (BLAST) described by Altshul et al. ((1990) J.Mol.Biol., 215:403 410); the algorithm of Needleman et al. ((1970) J.Mol.Biol., 48:444 453); or the algorithm of Meyers et al. ((1988) Comput.Appl.Biosci., 4:11 17). The set of parameters can be a Blosum 62 score matrix with 12 gap penalties, 4 gap length penalties, and 5 frameshift gap penalties. Percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17), which is incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, 12 gap length penalties, and 4 gap penalties.

[0048] "Amino acids" refers to all naturally occurring L-α-amino acids, including, for example, D-amino acids. The expression "amino acid sequence variant" refers to a molecule whose amino acid sequence differs somewhat from that of the sequence in this disclosure. For example, an amino acid sequence variant of the antibody in this disclosure of a particular sequence may still retain the ability to bind to human IL-17 or inhibit, for example, IL-17-induced IL-6 production in human dermal fibroblasts. Amino acid sequence variants include substitution variants (in which at least one amino acid residue in the polypeptide in this disclosure is removed and another amino acid is inserted in the same position), insertion variants (in which one or more amino acids are inserted directly next to an amino acid at a particular position in the polypeptide in this disclosure), and deletion variants (in which one or more amino acids are removed from the polypeptide in this disclosure).

[0049] The terms “free cysteine,” “non-traditional cysteine,” and “unpaired cysteine” are interchangeable and refer to cysteine ​​that does not participate in the preservation of antibody disulfide bonds, or, in relation to non-antibody polypeptides, cysteine ​​that does not form a disulfide bond with another unpaired cysteine ​​in the wild-type structure of the polypeptide. Free cysteine ​​can be present in the antibody framework region or variable region (e.g., within the CDR). In secukinumab, amino acid 8 of L-CDR3, described as SEQ ID NO: 6, is free cysteine, corresponding to amino acid 97 of the light chain variable region described as SEQ ID NO: 10 (hereinafter referred to as CysL97). Each molecule of secukinumab contains two such free cysteine ​​residues (V L (One per domain)

[0050] The term “selective reduction,” as used herein, refers to a method for selectively reducing CysL97 in the preparation of IL-17 antibodies recombinantly produced by mammalian cells, as disclosed in International Publication No. 2016 / 103146A1. Specifically, for example, a formulation containing the antibody is brought into contact with at least one reducing agent in a system to form a reduction mixture; the reduction mixture is incubated while the volume oxygen mass transfer coefficient (kLa) in the system is <approximately 0.37 h⁻¹. *)(the kLa * Downstream processing steps are applied, such as maintaining the dissolved oxygen curve (which is calculated by fitting the dissolved oxygen curve to the saturation curve).

[0051] Cysteine ​​can be added to the culture medium during recombinant polypeptide expression in a cell culture system, or, for example, during a fed-batch process, or / or added to the culture vessel's medium feed. Cysteine ​​refers to L-cysteine ​​rather than D-cysteine ​​and can be added in the form of a salt, such as cysteine ​​hydrochloride monohydrate. Typically, monomeric cysteine ​​exists exclusively in the dimer form of cystine, as it immediately forms dimers upon addition to the cell medium. This redox reaction leads to the formation of a disulfide bond between the two monomeric cysteine ​​molecules. The concentration of cysteine ​​in the basic or feed medium of the present invention may be less than about 5.0, about 4.0, about 3.0, about 2.5, about 2.0, about 1.5, about 1.0, about 0.90, about 0.80, about 0.70, about 0.60, about 0.50, about 0.45, about 0.40, about 0.35, about 0.30, about 0.25, about 0.20, about 0.15, or about 0.10 g / L. Alternatively, the basic medium and / or feed medium may not contain cysteine.

[0052] Furthermore, cystine may be present in the basic cell medium, or it may be added to the medium as a medium feed, for example, as part of a tyrosine-cystine stock solution. The concentration of cysteine ​​in the basic or feed medium of the present invention may be less than about 5.0, about 4.0, about 3.0, about 2.5, about 2.0, about 1.5, about 1.0, about 0.90, about 0.80, about 0.70, about 0.60, about 0.50, about 0.45, about 0.40, about 0.35, about 0.30, about 0.25, about 0.20, about 0.15, or about 0.10 g / L. Alternatively, the basic medium and / or feed medium may not contain cysteine.

[0053] Cysteine ​​added to culture media is usually oxidized to form cystine, but some of the cystine added to culture media may be reduced to form cysteine; therefore, the numbers given above for these concentrations refer to the concentration of cysteine ​​or cystine without any subsequent determination of the proportion that may actually be added to the culture media and oxidized or reduced. Thus, the term "cys equivalent" can be used in relation to the amount of cysteine ​​and / or cystine available to cells in a culture vessel. When used herein, this term refers to the total amount or concentration of cysteine ​​and cystine in the cell medium or medium feed. The cys equivalent is the cysteine / cystine available to cells in the cell medium within the culture vessel, regardless of whether it originates from the base medium and / or feed medium. Therefore, in culture vessels, for example in cell media in a fed-batch process, the concentration of total cys equivalent may be less than approximately 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.

[0054] Since cys equivalents in cell culture media, particularly during the fed-batch process, may originate from the basal medium and / or feed medium, the term "cumulative cys equivalents" is used to refer to the total amount or concentration of cys equivalents in cell culture media derived from the basal medium and / or feed medium. The cumulative cys equivalents may have different concentrations compared to the start of the fed-batch process and the end of the process, for example, after 5, 8, 10, 11, or 12 days, and may change during the process as feed medium is added to the cell culture media. In a fed-batch cell culture process under standard culture conditions, the concentration of cys equivalents in cell culture media may be in the range of about 0.5 g / L to about 0.6 g / L or about 4 mM to about 5 mM. In a process according to this disclosure, the concentration of cys equivalents in cell culture media at the start of the fed-batch process may be less than about 0.6 g / L or less than about 4.5 mM. For example, the concentration is 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 process, the concentration of the cys equivalent may change from about 0.3 g / L to about 0.8 g / L due to the addition of the cys equivalent to the cell medium (due to the addition of cysteine ​​or cystine, or both). To obtain this concentration of the cys equivalent, the concentration of cysteine ​​added to the cell medium in the feed medium may be less than about 1 g / L. For example, the basic medium may not contain the added cysteine, and the feed medium may contain a cysteine ​​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 basic medium does not contain the added cysteine, and the feed medium contains a cysteine ​​concentration of about 0.66 g / L. In another embodiment, the basic medium does not contain added cysteine, and the feed medium contains a cysteine ​​concentration of approximately 0.33 g / L. In yet another embodiment, the basic medium does not contain added cysteine, and the feed medium does not contain cysteine.

[0055] Feed medium can be added to fed-batch cell cultures at various points throughout the culture period. For example, feed medium can be added to the cell culture on a daily basis during the culture period, or it can be added after an initial period of 2 or 3 days, and then on a daily basis thereafter. Feed medium can be added once, twice, three times, four times, five times, six times, seven times, etc., during the fed-batch cell culture process.

[0056] At the end of the fed-batch cell culture process as described herein, the cumulative concentration of CYS equivalent in the cell medium may be less than approximately 0.4 g / L or less than approximately 3 mM. In a standard fed-batch cell culture where no modifications are made to reduce the CYS equivalent in the basic medium and / or feed medium, the cumulative concentration of CYS equivalent in the cell medium may be approximately 0.6 g / L or approximately 5 mM. This concentration of cumulative CYS equivalent in a standard, e.g., fed-batch, cell medium can also be referred to as the baseline.

[0057] In perfusion culture, new cell medium can be added to the cell culture by perfusion-mediated medium exchange. The exchange can be continuous or discontinuous (e.g., performed at various points throughout the culture period). For example, new medium can be exchanged with the cell medium under continuous culture conditions throughout the culture period, or perfusion exchange can be initiated after an initial period of two or three days and then continued. 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 medium per day of culture.

[0058] Therefore, the total volume of culture medium consumed during perfusion batch culture can be much higher than that of fed-batch culture. Thus, the cumulative cys equivalent added to the perfusion culture can be similarly higher, while the oxidation of free cysteine ​​provided by the methods and compositions described herein can be achieved at a lower level. For example, a 1000L perfusion batch culture with about 100% medium change per day can be cultured for 19 days if the total volume of culture medium consumed is about 19,000L. However, the total culture volume can be maintained at about 1000L. In such embodiments, the cumulative cys equivalent added to the control perfusion culture may be greater than about 7 g / L, 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 or 11.5 g / L, or about 11 or 11.5 g / L. Similarly, a cumulative cys equivalent added to a 1000L perfusion culture for 19 days, with approximately 100% of the medium replaced per day, and cultured under conditions of reduced cysteine ​​and / or reduced cys equivalent, may be a cumulative cys equivalent with a culture volume of less than approximately 7 g / L, 6.5 g / L, 6 g / L, or approximately 5.8 g / L.

[0059] Therefore, in relation to perfusion batch culture, the conditions for reduced cys equivalent may be characterized by conditions for reduced cumulative cys equivalent normalized by the number of culture days. For example, if a 19-day perfusion culture is cultured under control conditions of approximately 0.6 g / L of cys equivalent in the initial and replacement media, with approximately one complete change of medium volume per day, the normalized cumulative cys equivalent may be approximately 0.6 g / L / day. Similarly, if a 19-day perfusion culture is cultured under test conditions of approximately 0.3 g / L (or less) of cys equivalent in the initial and replacement media, with approximately one complete change of medium volume per day, the normalized cumulative cys equivalent may be less than 0.6 g / L / day, for example, approximately 0.3 g / L / day.

[0060] In some embodiments, secreted recombinant proteins having free cysteine ​​are produced in a perfusion batch process by culturing mammalian cells in a basic perfusion medium containing less than 0.62 g / L of cys equivalent, adding less than 0.62 g / L of cys equivalent to the culture daily, and continuously or discontinuously (e.g., by perfusion) exchanging all or part of the cell medium with perfusion exchange medium. In some cases, the process involves adding approximately 0.2 g / L of cys equivalent to less than 1 g / L of cys equivalent to the culture daily (e.g., by perfusion exchange). In some cases, the process involves adding approximately 0.2 g / L of cys equivalent to less than 0.9 g / L of cys equivalent to the culture daily (e.g., by perfusion exchange). In some cases, the process involves adding approximately 0.2 g / L of cys equivalent to less than 0.6 g / L of cys equivalent to the culture daily (e.g., by perfusion exchange). In some cases, the process involves adding a cys equivalent of approximately 0.2 g / L to less than 0.5 g / L to the culture daily (e.g., by perfusion exchange). In other cases, the process involves adding a cys equivalent of approximately 0.2 g / L to less than 0.4 g / L to the culture daily (e.g., by perfusion exchange).

[0061] In some embodiments, secreted recombinant proteins containing free cysteine ​​are produced in a perfusion batch process by culturing mammalian cells in a basic perfusion medium containing less than 0.62 g / L of cys equivalent and continuously or discontinuously (e.g., by perfusion) replacing all or part of the cell 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 basic perfusion medium contains less than 0.6 g / L or about 0.3 g / L of cys equivalent.

[0062] In some embodiments, the basic 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 basic 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 basic 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 basic medium does not contain cysteine ​​or is substantially cysteine-free. In some embodiments, the perfusion-exchange medium is cysteine-free or substantially cysteine-free. In some embodiments, the perfusion-basic medium and / or perfusion-exchange medium are cysteine-free or substantially cysteine-free. In some embodiments, the perfusion-basic medium and the perfusion-exchange medium are identical or substantially identical. A substantially cysteine-free production medium (e.g., perfusion-basic or exchange-productive or fed-basic or feed) includes a medium in which residual cysteine ​​is present due to the presence of residual expansion medium, cysteine ​​production by host cells, and / or reduction of cystine during production culture. A substantially cysteine-free basic, exchange, or feed medium contains less than 0.1 g / L of cysteine, preferably less than 0.05 g / L, and more preferably less than 0.01 g / L.

[0063] In some embodiments, activity is measured by the cystamine-CEX (cation exchange chromatography) method. The cystamine-CEX method involves derivatization of the antibody with cystamine (2,2'-dithiobis(ethylamine)) followed by analytical separation using cation exchange chromatography (CEX). Since the activity of the antibodies disclosed herein (e.g., secukinumab) decreases when CysL97 is in the oxidized form, derivatization of CysL97 with cystamine serves as a surrogate for measuring antibody activity. Derivatization with cystamine results in the addition of one positive charge per free Cys97 residue. The resulting derivatized form of secukinumab (e.g., +2, +1 charge) can then be separated from the underivative form and quantified by CEX. A cystamine-derivativeized secukinumab molecule, with two cystamines bound to unpaired Cys97 on both light chains, may theoretically be considered to have 100% biological activity. A secukinumab molecule derivatized with cystamine, with one cystamine bonded to an unpaired Cys97 on one side of the light chain, may be considered to have 50% biological activity. A secukinumab molecule derivatized with cystamine, without any cystamine bonded to it, may be considered biologically inactive. The level of cystamine derivatization in an antibody formulation (e.g., a secukinumab antibody formulation) may then be used as a measure of the formulation's activity by comparing it to the theoretical maximum level of cystamine derivatization in that formulation (e.g., expressed as the theoretical maximum percentage).

[0064] In short, cystamine-CEX may be performed as follows: The antibody sample (50 μg) is first treated with carboxypeptidase B (1:40, w:w) to remove C-terminal lysine from the heavy chain, and then derivatized for 2 hours with 4 mM cystamine in 5 mM sodium acetate and 0.5 mM EDTA at pH 4.7 and room temperature. Derivatization is stopped by the addition of 2 μL of 1 M phosphate. CEX is performed on the cystamine-derivativeized antibody sample using a ProPac® WCX-10 analytical column (4 mm × 250 mm, Dionex). A gradient of 25 mM sodium phosphate and 12.5 mM to 92.5 mM sodium chloride at pH 6.0 is used for separation at a flow rate of 1.0 ml / min. Absorption at 220 nm is recorded by a UV detector (Agilent HPLC 1200).

[0065] IL-17 antibody and its antigen-binding fragment In one embodiment, the IL-17 antibody or its antigen-binding fragment comprises at least one immunoglobulin heavy chain variable domain (V) including a hypervariable region CDR1 having amino acid sequence number 1, CDR2 having amino acid sequence number 2, and CDR3 having amino acid sequence number 3. H ) comprises. In one embodiment, the IL-17 antibody or its antigen-binding fragment comprises at least one immunoglobulin light chain variable domain (V) including high-frequency variable regions CDR1', CDR2', and CDR3'. L’ ) comprises, wherein CDR1' has amino acid sequence number 4, CDR2' has amino acid sequence number 5, and CDR3' has amino acid sequence number 6. In one embodiment, the IL-17 antibody or its antigen-binding fragment comprises at least one immunoglobulin heavy chain variable domain (V) including high-frequency variable regions CDR1-x, CDR2-x, and CDR3-x. H ), wherein CDR1-x has amino acid sequence number 11, CDR2-x has amino acid sequence number 12, and CDR3-x has amino acid sequence number 13.

[0066] In one embodiment, the IL-17 antibody or its antigen-binding fragment is at least one immunoglobulin VH Domain and at least one immunoglobulin V L The domain includes, where a) V H The domain includes (for example, in order) i) high-frequency variable regions CDR1, CDR2 and CDR3 (wherein CDR1 has amino acid sequence number 1, CDR2 has amino acid sequence number 2, and CDR3 has amino acid sequence number 3), or ii) high-frequency variable regions CDR1-x, CDR2-x and CDR3-x (wherein CDR1-x has amino acid sequence number 11, CDR2-x has amino acid sequence number 12, and CDR3-x has amino acid sequence number 13), and b) V L The domain includes (for example, in order) high-frequency variable regions CDR1', CDR2', and CDR3' (wherein CDR1' has amino acid sequence number 4, CDR2' has amino acid sequence number 5, and CDR3' has amino acid sequence number 6).

[0067] In one embodiment, the IL-17 antibody or its antigen-binding fragment is a) an immunoglobulin heavy chain variable domain (V) containing the amino acid sequence shown as SEQ ID NO: 8. H ), b) an immunoglobulin light chain variable domain (V) containing the amino acid sequence shown as Sequence ID No. 10 L ), c) Immunoglobulin V containing the amino acid sequence shown as SEQ ID NO: 8 H Immunoglobulin V containing the domain and the amino acid sequence shown as SEQ ID NO: 10 L Domain, d) Immunoglobulin V containing the high-frequency variable region indicated as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 H Domain, e) Immunoglobulin V containing the highly variable region shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 L Domain, f) Immunoglobulin V containing the high-frequency variable region indicated as SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 H Domain, g) Immunoglobulin V containing the high-frequency variable region indicated as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 HImmunoglobulin V containing the domain and the high-frequency variable region indicated as SEQ ID NOs: 4, 5, and 6. L Immunoglobulin V containing a domain, or a high-frequency variable region indicated as sequence numbers 11, 12, and 13. H Immunoglobulin V containing the domain and the high-frequency variable region indicated as SEQ ID NOs: 4, 5, and 6. L Includes the domain.

[0068] For ease of reference, Table 1 below provides the amino acid sequences of the high-frequency variable regions of secukinumab monoclonal antibodies, based on the Kabat definition, determined by X-ray analysis, and using the methods of Chothia and collaborators.

[0069] [Table 1]

[0070] In a preferred embodiment, the constant region domain preferably also includes a suitable human constant region domain, as described, for example, “Sequences of Proteins of Immunological Interest”, Kabat EA et al, US Department of Health and Human Services, Public Health Service, National Institute of Health. The DNA encoding the VL of secukinumab is described in Sequence ID No. 9. H The DNA encoding this is described in sequence number 7.

[0071] In some embodiments, the IL-17 antibody or its antigen-binding fragment (e.g., secukinumab) contains three CDRs of SEQ ID NO: 10. In other embodiments, the IL-17 antibody or its antigen-binding fragment contains three CDRs of SEQ ID NO: 8. In other embodiments, the IL-17 antibody or its antigen-binding fragment contains 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 ​​(CysL97) in the light chain can be found in SEQ ID NO: 6.

[0072] In some embodiments, the IL-17 antibody or its antigen-binding fragment includes the light chain of SEQ ID NO: 14. In other embodiments, the IL-17 antibody or its antigen-binding fragment includes the heavy chain of SEQ ID NO: 15 (in the presence or absence of C-terminal lysine). In other embodiments, the IL-17 antibody or its antigen-binding fragment includes the light chain of SEQ ID NO: 14 and the heavy chain of SEQ ID NO: 15 (in the presence or absence of C-terminal lysine). In some embodiments, the IL-17 antibody or its antigen-binding fragment includes the three CDRs of SEQ ID NO: 14. In other embodiments, the IL-17 antibody or its antigen-binding fragment includes the three CDRs of SEQ ID NO: 15. In other embodiments, the IL-17 antibody or its antigen-binding fragment includes 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. The complete sequence sets can be found in Table 7.

[0073] High-frequency variable regions can be associated with any type of framework region, but those of human origin are preferred. Preferred framework regions are described by Kabat EA et al, ibid. A preferred heavy chain framework is the human heavy chain framework, e.g., the heavy chain framework of the secukinumab antibody. This consists, in order, for example, the FR1 (amino acids 1-30 of SEQ ID NO: 8), FR2 (amino acids 36-49 of SEQ ID NO: 8), FR3 (amino acids 67-98 of SEQ ID NO: 8), and FR4 (amino acids 117-127 of SEQ ID NO: 8). Considering the high-frequency variable regions of secukinumab determined by X-ray analysis, another preferred heavy chain framework consists, in order, the FR1-x (amino acids 1-25 of SEQ ID NO: 8), FR2-x (amino acids 36-49 of SEQ ID NO: 8), FR3-x (amino acids 61-95 of SEQ ID NO: 8), and FR4 (amino acids 119-127 of SEQ ID NO: 8). Similarly, the light chain framework consists of the FR1' (amino acids 1-23 of SEQ ID NO: 10), FR2' (amino acids 36-50 of SEQ ID NO: 10), FR3' (amino acids 58-89 of SEQ ID NO: 10), and FR4' (amino acids 99-109 of SEQ ID NO: 10), respectively.

[0074] In one embodiment, the IL-17 antibody or its antigen-binding fragment (e.g., secukinumab) is selected from a human IL-17 antibody comprising, in order a) a variable domain including high-frequency variable regions CDR1, CDR2, and CDR3 and the constant region of a human heavy chain or a fragment thereof (the CDR1 has amino acid sequence number 1, the CDR2 has amino acid sequence number 2, and the CDR3 has amino acid sequence number 3), and, in order b) a variable domain including high-frequency variable regions CDR1', CDR2', and CDR3' and the constant region of a human light chain or a fragment thereof (the CDR1' has amino acid sequence number 4, the CDR2' has amino acid sequence number 5, and the CDR3' has amino acid sequence number 6).

[0075] In one embodiment, the IL-17 antibody or its antigen-binding fragment is selected from a single-chain antibody or its antigen-binding fragment that includes, in order: a) a first domain comprising high-frequency variable regions CDR1, CDR2, and CDR3 (CDR1 having amino acid sequence number 1, CDR2 having amino acid sequence number 2, and CDR3 having amino acid sequence number 3); b) a second domain comprising high-frequency variable regions CDR1', CDR2', and CDR3' (CDR1' having amino acid sequence number 4, CDR2' having amino acid sequence number 5, and CDR3' having amino acid sequence number 6); and c) an antigen-binding site comprising a peptide linker that binds to the N-terminus of the first domain and the C-terminus of the second domain, or to the C-terminus of the first domain and the N-terminus of the second domain.

[0076] Alternatively, the IL-17 antibody or its antigen-binding fragment may, when used in the method disclosed herein, include a derivative of the IL-17 antibody described herein by sequence (e.g., a PEGylated version of secukinumab). Alternatively, the IL-17 antibody or its antigen-binding fragment used in the method disclosed herein may include a V H or V L The domain is V as described herein. H or V L V is substantially identical to the domain (for example, those described in sequence numbers 8 and 10). H or V LIt may have a domain. The human IL-17 antibody disclosed herein may include a heavy chain substantially identical to that described as SEQ ID NO: 15 (in the presence or absence of C-terminal lysine) and / or a light chain substantially identical to that described as SEQ ID NO: 14. The human IL-17 antibody disclosed herein may include 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.

[0077] Alternatively, the IL-17 antibody or its antigen-binding fragment used in the disclosed method may be an amino acid sequence variant of the standard IL-17 antibody shown herein, insofar as it contains CysL97. This disclosure includes secukinumab V H or V L This also includes IL-17 antibodies or their antigen-binding fragments (e.g., secukinumab) in which one or more amino acid residues (other than CysL97), typically only a very small number (e.g., 1 to 10), of the domain are altered, for example, by mutations in the corresponding DNA sequence, such as site-directed mutagenesis. In all such cases of derivatives and variants, the IL-17 antibody or its antigen-binding fragment can inhibit the activity of human IL-17 by 50% at concentrations of the molecule 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, and the inhibitory activity is measured against hu-IL-17-induced IL-6 production in human dermal fibroblasts, as described in Example 1 of International Publication No. 2006 / 013107.

[0078] In some embodiments, an IL-17 antibody or its antigen-binding fragment, such as secukinumab, binds to mature human IL-17 epitopes including Leu74, Tyr85, His86, Met87, Asn88, Val124, Thr125, Pro126, Ile127, Val128, and His129. In some embodiments, an IL-17 antibody, such as secukinumab, binds to mature human IL-17 epitopes including Tyr43, Tyr44, Arg46, Ala79, and Asp80. In some embodiments, an IL-17 antibody, such as secukinumab, binds to an epitope of an IL-17 homodimer having two mature human IL-17 chains (the epitope includes 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 fact that residue 1 is the first amino acid of the mature protein (i.e., IL-17A lacks a 23-amino acid N-terminal signal peptide and starts with glycine). The sequence of immature IL-17A is described in Swiss-Prot entry Q16552. In some embodiments, the IL-17 antibody is present at approximately 100-200 pM K D In some embodiments, the IL-17 antibody has an IC50 of approximately 0.4 nM for the in vitro neutralization of the biological activity of human IL-17A, which is approximately 0.67 nM. 50 In some embodiments, the absolute bioavailability of the IL-17 antibody administered subcutaneously (sc) is in the range of 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 T of about 7 to 8 days max It has.

[0079] A particularly preferred IL-17 antibody or its antigen-binding fragment for use in the method disclosed herein is a human antibody, in particular secukinumab as described in Examples 1 and 2 of International Publication No. 2006 / 013107. Secukinumab is an IgG1 / kappa isotype recombinant high-affinity fully human monoclonal anti-human interleukin-17A (IL-17A, IL-17) antibody currently in clinical trials for the treatment of immune-mediated inflammatory conditions. Secukinumab (see, e.g., International Publication No. 2006 / 013107 and International Publication No. 2007 / 117749) has a very high affinity for IL-17, i.e., about 100-200 pM K D , and IC25 in vitro neutralization of the biological activity of human IL-17A at approximately 0.4 nM and approximately 0.67 nM. 50 It possesses this property. Therefore, secukinumab inhibits the antigen in a molar ratio of approximately 1:1. This high binding affinity makes secukinumab antibodies particularly suitable for therapeutic use. Furthermore, secukinumab has a very long half-life of approximately 4 weeks, which has been determined to allow for extended intervals between administrations, an exceptional characteristic when treating lifelong chronic disorders such as rheumatoid arthritis.

[0080] Disclosed herein are methods for formulations of the IL-17 antibody and its antigen-binding fragment (e.g., secukinumab). For convenience and to reduce costs, the disclosed methods may be applied to antibody formulations (e.g., IL-17 antibody, e.g., secukinumab). The “formulation” of the antibody refers to a composition (e.g., a solution) having multiple antibody molecules. The “formulation” includes any liquid composition containing the IL-17 antibody or its antigen-binding fragment. Thus, the formulation may contain, for example, the IL-17 antibody or its antigen-binding fragment, e.g., secukinumab, in water or a buffer, in a column eluate, in a dialysis buffer, etc. In some embodiments, the initial antibody formulation contains a pool of the IL-17 antibody or its antigen-binding fragment, e.g., secukinumab, in a buffer (e.g., Tris, e.g., 1 mM to 1 M Tris, pH 6.0 to 8.0) or in WFI.

[0081] In some embodiments of the above method, the IL-17 antibody or its antigen-binding fragment is an immunoglobulin heavy chain variable domain (V) comprising an amino acid sequence described as SEQ ID NO: 8. H );ii) an immunoglobulin light chain variable domain (V) containing the amino acid sequence described as sequence number 10 L );iii) Immunoglobulin V containing the amino acid sequence described as SEQ ID NO: 8 H Immunoglobulin V containing the domain and the amino acid sequence described as SEQ ID NO: 10 L Domain; iv) Immunoglobulin V containing a hypervariable region described as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 within its sequence. H Domain;v) Immunoglobulin V containing a hypervariable region described as SEQ ID NOs: 4, 5, and 6 within the sequence. L Domain; vi) Immunoglobulin V containing a hypervariable region described as SEQ ID NOs: 11, 12, and 13 within its sequence. H Domain; vii) Immunoglobulin V containing a hypervariable region described as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 within its sequence. H Immunoglobulin V containing a hypervariable region within its domain and sequence, described as SEQ ID NOs: 4, 5, and 6. L Domain; and viiii) Immunoglobulin V containing a hypervariable region described as SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 within the sequence. H Immunoglobulin V containing a hypervariable region within its domain and sequence, described as SEQ ID NOs: 4, 5, and 6. L The domain is included. In some embodiments of the methods disclosed herein, the IL-17 antibody or its antigen-binding fragment is a human antibody of the IgG1 isotype. In some embodiments of the methods disclosed herein, the antibody is secukinumab.

[0082] Recombinant antibody production The preparation of recombinant polypeptides is traditionally divided into two main steps: upstream (cell culture and synthesis of the target polypeptide) and downstream (purification of the polypeptide and preparation into a viable product or formulation).

[0083] More specifically, preparations of monoclonal antibodies or their antigen-binding fragments may be recombinantly produced by any mammalian cells using any mammalian cell line, such as Chinese hamster ovary cells (CHO) cells, mouse myeloma NS0 cells, baby hamster kidney (BHK) cells, human embryonic kidney cell line HEK-293, human retinal cell line Per.C6 (Crucell,NL), and HKB11 cell clones (derived from a hybrid cell fusion of HEK293S and Burkitt lymphoma cell line 2B8). "Recombinantly produced by mammalian cells" means that antibody production within mammalian cells is achieved using recombinant DNA technology.

[0084] CHO cells are currently the most widely used mammalian host in biological and medical research, particularly for the expression of human therapeutic proteins, with approximately 70% of recombinant therapeutic proteins reported to be produced in CHO cell lines. While CHO cells require expensive culture media and grow relatively slowly compared to E. coli and yeast expression systems, they allow for more precise protein glycation, assembly, and folding, similar to human cells. Therefore, for some proteins whose activity is closely related to post-translational modifications, CHO cells are a preferred production host. CHO cells also efficiently synthesize extremely large molecules that may not 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 Pro 3-, CHO DG44, CHO P12 or dhfr-CHO cell line DUK-BII (Urlaub G & Chasin LA (1980) PNAS 77(7):4216-4220), DUXBI 1 (Simonsen CC & Levinson AD (1983) PNAS 80(9):2495-2499), or CHO-K1SV (Lonza, Basel, Switzerland). Many CHO cell-derived therapies 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).

[0085] In the industrial production of recombinant proteins, the most common culture modes used in biomanufacturing are fed-batch and perfusion. The use of one or the other technique depends on different factors related to the protein or host, where cells are cultured attached to a carrier or in suspension (Kadouri & Spier, (1997) Cytotechnology 24:89-98). One of the most common processes is the batch bioreactor, where cells proliferate and produce until, after inoculation, a limit is reached due to medium consumption and the cell density begins to decrease. A second very common process is the fed-batch, where nutrient limitation is prevented by adding a highly concentrated feed at different points in time during culture. Thus, the culture duration is longer than in the batch mode, and the final productivity is increased. In continuous processes where culture medium is continuously supplied and collected material is continuously removed, one of the simplest is the chemostat process, where medium is added at a constant flow rate, bioreactor contents are removed at the same flow rate, and there is no cell retention (Henry O et al., (2008) Biotechnol. Prog., 921-931). An alternative continuous process is perfusion, where inflow and outflow are constant, but the cells are retained inside the bioreactor. The current industry standard for the production of stable proteins such as monoclonal antibodies is the fed-boil process in agitated tank bioreactors up to 20 kL. These culture vessels offer very high mixing and material transport rates, can also offer high flexibility in terms of working volume, and can be used for different cell types and operating modes (Rodrigues ME et al., (2010) Biorechnol. Prog. 26:332-51).

[0086] Since the beginning of biomanufacturing, culture medium development has been the most important aspect of cell culture development and optimization, primarily aimed at process performance, but more importantly, at safety. The first cell media were prepared using animal-derived products (Yao & Asayama, (2017) Reprod. Med. Biol., 16:99-117). The consequences in patients were exposure to many harmful factors such as viruses and prions, and the risk of infection in patients was significant, especially in cases of chronic disease, due to continuous exposure to the drugs (Grillberger L et al., (2009) Biotechnol. J., 4:186-201). Process inconsistencies due to batch variability were also a driver of the reduction of animal-derived or even plant-derived media components, but even today, considerable effort is being spent on optimizing synthetic media that can enhance cell proliferation.

[0087] Synthetic culture media are now commercially available, and most large-scale biomanufacturing companies are developing their own formulations. For example, highly concentrated feeds can be challenging because the physical properties of some compounds can limit solubility or stability. Specifically, further optimization of CHO cell media and process parameters for the commercial production of monoclonal antibodies and other recombinant polypeptides has resulted in dramatic increases in cell density and protein expression, in some cases reaching titers exceeding 10 g / L (LiF et al., (2010) MAbs, 2(5):466-479; Lu F et al., (2013) Biotechnol Bioeng., 110(1):191-205; Xing Z et al., (2011) Process Biochem., 46(7):1432-9). Several companies specializing in cell culture media are developing and optimizing combinations of base and feed media, particularly for recombinant CHO manufacturing processes (ThermoFisher, Waltham, MA, USA; GE Healthcare, Waukesha, WI, USA; MilliporeSigma, St. Louis, MO, USA; Lonza, Basel, Switzerland; Irvine Scientific, Santa Ana, CA, USA).

[0088] While these commercially available culture medium formulations are typically proprietary, all cell media require similar basic nutrients essential to support cell survival and proliferation. Water (with sources of carbon, nitrogen, and phosphate), specific amino acids, fatty acids, vitamins, trace elements, and salts are all supplied at concentrations based on knowledge of nutrient depletion rates that can maintain and extend cell viability by replenishing the cell's chemical structure, the calculated amounts required to reach the desired cell density, and the major components. In particular, amino acids are major components in CHO cell media, especially synthetic media, and studies have shown that small changes in the amino acid composition of cell media can not only alter growth characteristics and titer but also significantly affect the glycosylation pattern of the product (Fan Y et al., (2015) Biotechnol. Bioeng., 112(3):521-35).

[0089] Generally, amino acids can be classified into non-essential amino acids, which can be synthesized by mammalian cells, and essential amino acids, which cells cannot synthesize and therefore must be supplied as components of the cell medium. Both non-essential and essential amino acids can have a significant effect on CHO cell proliferation, and it has been shown that optimizing the relative concentrations of non-essential and essential amino acids in the culture medium improves the productivity of recombinant monoclonal antibodies (Parampalli A et al., (2007) Cytotechnology, 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 medium.

[0090] Non-essential amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine. Despite the fact that non-essential amino acids can be synthesized by mammalian cells under culture, most cell media further contain most or all of these amino acids to support cell proliferation and polypeptide production. Most non-essential amino acids can have a significant effect on cell culture processes.

[0091] In particular, cysteine, a simple thiol-containing amino acid, is a special non-essential amino acid in monoclonal antibody production. The formation of interhydroxyl disulfide crosslinks on cysteine ​​residues supports the folding of the tertiary and quaternary structures of both the structural proteins and recombinant antibody products of CHO cells. Cysteine ​​restriction can be lethal and irreversible in the case of CHO cell proliferation, potentially leading to decreased cell viability. A study by Ghaffari et al. (2020) investigated the effects of restricting glutamine, asparagine, and cysteine ​​on cell proliferation, metabolism, antibody production, and product glycosylation in three Chinese hamster ovary (CHO) cell lines (CHO-DXB11, CHO-K1SV, and CHO-S). Cysteine ​​restriction was detrimental to both cell proliferation and productivity in all three CHO cell lines. Of the three amino acid restrictions tested, cysteine ​​restriction had the greatest detrimental effect on culture growth and productivity, as well as mAb glycosylation. Cysteine ​​has low solubility and can be limiting in discontinuous supply protocols commonly used on an industrial scale, especially at high cell concentrations. Ghaffari et al. investigated the duration for which CHO-DXB11 cells, initially grown in cysteine-free BIOGRO medium, could tolerate cysteine ​​restriction. Then, on either day 1 or day 2 of culture, the cysteine ​​concentration was restored to 0.4 mM by adding a concentrated cysteine ​​solution. When cysteine ​​levels were restored on day 1, cells remained in the induction phase for the remainder of the days, then growth resumed on day 2; by day 5, these cultures reached concentrations similar to the control. Restoring cysteine ​​levels two days after cysteine ​​restriction proved ineffective, and cells did not grow (Ghaffari N et al., (2020) Biotech.Prog., 36:e2946). In contrast, cysteine ​​concentrations >1 mM can be toxic to mammalian cells, likely due to lipid peroxidation and hydroxyl radical formation, which may be further accelerated in the presence of copper (Ritacco FV et al (2018) Biotechnol. Prog., 34(6):1407-26).In mammals, the cysteine ​​pool is regulated by the liver, and if such a regulatory mechanism is not present in CHO cells, the cysteine ​​concentration in the culture medium needs to be carefully designed and controlled for the cell culture process (Stipanuk MH et al., (2006) J. Nutr., 136(6):1652S-59S).

[0092] Recombinant polypeptide formulations for use in the methods described herein, for example, IL-17 antibodies or their antigen-binding fragments, may be recombinantly produced by any mammalian cells using any mammalian cell line. Preferably, the mammalian cell line is CHO cells. The recombinant polypeptide, for example, anti-IL-17 antibody or its antigen-binding fragment, may be produced in a continuous production system or using a fed-batch system with the addition of a feed to the culture medium. As described above, the anti-IL-17 antibody secukinumab contains a free unpaired cysteine ​​involved in antigen recognition and binding. This free cysteine ​​residue is found after amino acid 8 of L-CDR3 described as SEQ ID NO: 6, which corresponds to cis-proline in the light chain complementarity-determining region (CDR) 3 loop, i.e., amino acid 97 of the light chain variable region described as SEQ ID NO: 10, and is referred to as "CysL97". To maintain overall activity, this free cysteine ​​residue cannot be shielded by oxidative disulfide pairing with other cysteine ​​residues or oxidation by exogenous compounds. Furthermore, this modification of free cysteine ​​can have a negative effect on antibody activity and stability, potentially leading to increased immunogenicity. Therefore, processing secukinumab can be challenging, as the final product may contain a significant amount of inactive antibody material. However, since secukinumab is produced using mammalian cells, particularly CHO cells, cell-based modification of CysL97 certainly occurs, which can affect yield and antibody activity.

[0093] As discussed above, the term "cys equivalent" refers to cysteine ​​and cystine available to cells in the culture medium within the culture vessel, regardless of whether they originate from the basic medium or / or feed medium. Surprisingly, it has been found that reducing the amount of cys equivalent in the cell culture growth medium for secukinumab leads to a decrease in the modification of free cysteine ​​CysL97. This, in turn, leads to an increase in the active antibodies produced by the production process, i.e., an improvement in product quality. Contrary to established studies (e.g., Ghaffari et al., above), a decrease in cysteine ​​in the production medium and medium feed did not have a negative effect on the yield of secukinumab.

[0094] Purification of recombinant polypeptides A purification step is necessary to obtain a substantially homogeneous formulation of recombinant polypeptides produced according to the cell culture process described herein. As a first step, particulate cell debris is removed, usually by centrifugation of the culture medium or lysate. The produced polypeptides can be purified by hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography for convenience. Other techniques for protein purification are also available, including fractionation with ion-exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography with silica, chromatography with heparin Sepharose, chromatography with anion or cation exchange resins (such as polyaspartate columns), isoelectric focusing, SDS-PAGE, and ammonium sulfate precipitation.

[0095] Pharmaceutical compositions, administrations, and kits Provided herein are pharmaceutical compositions comprising recombinant polypeptides, as described herein, in combination with one or more pharmaceutically acceptable excipients, diluents, or carriers. To prepare a pharmaceutical composition or sterile composition comprising the molecules of this disclosure, the molecules are mixed with pharmaceutically acceptable carriers or excipients. The expression “pharmaceutically acceptable” means approved by a federal or state regulatory authority for use in animals, and more specifically in humans, or listed in the United States Pharmacopeia or other generally accepted pharmacopoeias. The term “pharmaceutical composition” means a mixture of at least one active ingredient (e.g., the antibody or fragment of this disclosure) and at least one pharmaceutically acceptable excipient, diluent, or carrier. “Pharmaceutical” means a substance used for medical treatment.

[0096] Pharmaceutical compositions for therapeutic and diagnostic agents can be prepared by mixing them with physiologically acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (e.g., 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: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al., (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. See also: al., (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner & Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0097] The selection of a dosage regimen for a therapeutic agent depends on several factors, including the metabolic turnover rate of the entity's serum or tissue, the level of symptoms, the entity's immunogenicity, and the reachability of target cells in the biological matrix. In certain embodiments, the dosage regimen maximizes the amount of therapeutic agent delivered to the patient to match an acceptable level of side effects. Therefore, the amount of bioagent 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 (e.g., 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 Engl. J. Med. 348:601-608; Milgrom et al., (1999) New Engl. J. Med. 341:1966-1973; Slamon, et al. (2001) New Engl.J.Med.344:783-792;Beniaminovitz et al.,(2000)New Engl.J.Med.342:613-619;Ghosh et al.,(2003)New Engl.J.Med.348:24-32;Lipsky et al.,(2000)New (See Engl.J.Med.343:1594-1602).

[0098] This disclosure further encompasses kits for treating patients with IL-17-mediated pathological disorders, such as autoimmune diseases or inflammatory disorders or conditions. Such kits include a therapeutically effective dose of an antibody produced according to the method described herein and a package leaflet, the package leaflet indicating a recommended administration plan of the anti-IL-17 antibody to the patient. Preferably, the antibody is an anti-IL-17 antibody such as secukinumab. In addition, such kits may include means for administering the antibody (e.g., an automated injector, syringes and vials, pre-filled syringes, pre-filled pens) and instructions for use. The kit may also include instructions for administering the anti-IL-17 antibody to treat the patient. Such instructions may provide dosage, route of administration, regimen, and total duration of treatment in the use of the inclusion antibody. The expression “means for administration” refers to any available devices for systemic administration of a drug to a patient, including, but not limited to, pre-filled syringes, vials and syringes, injection pens, auto-injectors, IV drip bags, infusion pumps, patches, infusion bags and needles. Using such products, the patient may self-administer the drug (i.e., administer the drug without the assistance of a physician), or a physician may administer the drug.

[0099] Details of one or more embodiments of this disclosure are described in the accompanying description above. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, but preferred methods and materials are described herein. Other features, purposes and advantages of this disclosure will be apparent from this description and the claims. In this specification and the appended claims, the singular form includes multiple references unless otherwise clearly indicated by the context. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. All patents and publications referenced herein are incorporated by reference. The following examples are provided to better illustrate preferred embodiments of this disclosure. These examples should not be construed as limiting the scope of the disclosed patient matter as defined by the appended claims. [Examples]

[0100] The following experiments are intended to further illustrate the present invention as defined herein.

[0101] Example 1 The experimental setup was designed to observe whether or not cys97 oxidation of secukinumab occurs extracellularly.

[0102] The purified secukinumab was diafiltration with cell medium containing standard amounts of cysteine / cystine (Amicon Ultracel tubes). The solution was then diluted with medium to a concentration of 1.5 g / L to match the bioreactor titer. The solution was then incubated at 37°C. Finally, samples were collected at 24 and 48 hours to obtain the percentage of free cys97 in cystamine-CEX.

[0103] Table 2 shows that the percentage of free cys97 significantly decreased after incubation of the culture medium, suggesting that cys97 may be oxidized in the extracellular environment containing cysteine / cystine after secukinumab secretion. This understanding led to the hypothesis that the oxidation of cys97 may occur due to the presence of cysteine / cystine in the cell culture medium.

[0104] [Table 2]

[0105] The results confirmed that cysteine / cystine in the cell culture medium oxidizes cys97.

[0106] The secukinumab raw material was incubated with media containing different amounts of cysteine / cystine. Figure 1 shows that lower amounts of cysteine / cystine in the media reduced the oxidation of cys97. This incubation test of the media suggests that cysteine / cystine does indeed oxidize cys97 on secukinumab.

[0107] Example 2 Previous observations have shown that incubation of secukinumab at 37°C in media such as perfusion medium leads to a decrease in antibody activity over time. To determine whether the level of cysteine / cystine (cys equivalent) in the medium could reduce the decrease in activity, secukinumab eluate was incubated in different variations of media based on standard perfusion medium, and the effects of the media components were investigated.

[0108] To minimize dilution when adding the sample to the culture medium, the starting solution was diafiltration with 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, reproducing a typical antibody bioreactor titer. The solution was incubated at 37°C (standard bioreactor temperature) for 2 days. After 24 and 48 hours, 25 ml samples were taken and the antibody was captured. All samples were analyzed for activity by Cystamine-CEX. Cysteine ​​was supplied to standard perfusion medium in the form of cysteine ​​hydrochloride monohydrate, and cystine was supplied from a stock solution containing tyrosine and cystine.

[0109] Secukinumab was incubated in three different culture media to investigate the effects of the culture medium composition.

[0110] [Table 3]

[0111] As shown in Figure 2, the activity of secukinumab significantly decreased from approximately 98% to 25% within 2 days of incubation in baseline medium. Reducing the amount of cys equivalent by 75% reduced the activity decrease to 60%. With no cys equivalent present, the activity decrease was further reduced to just 83%. In addition, when the activity decreased from 96% to 84%, no significant improvement was observed by removing trace elements.

[0112] Example 3 In a fed-batch reactor, experiments were designed based on the standard principle of secukinumab expression in CHO cells to determine the effect of changes in cysteine / cystine, i.e., the cys equivalent, in the fed-batch reactor method. The cysteine ​​concentration was varied in both the basal and feed media. The cystine concentration was changed relative to that added from the tyrosine / cystine stock solution. The variations of media tested are shown in Table 4 below, where fed-batch cell culture was performed for 10 days. Even in cases of reduction or removal of cysteine ​​from the basal or feed media, cystine was still present in the media from the tyrosine / cystine stock solution. Therefore, the total cys equivalent in baseline and media variations is also shown in Table 4.

[0113] [Table 4]

[0114] As shown in Figure 3, changing the content of the cys equivalent in the basal and feed media resulted in secukinumab cell proliferation and expression titers (mg / ml) similar to all atypia, including baseline. As shown in Figure 4, only small changes in the final antibody titer (mg / ml) were detected in the atypia (from approximately 2.4 mg / ml in atypia 1 to approximately 2.6 mg / ml in atypia 3).

[0115] Figure 5 is a graph showing the activity (%) of secukinumab expressed in different culture media variants. The activity of secukinumab expressed in cell media with reduced cys equivalent was over 80%. In contrast, the activity of secukinumab expressed in baseline cell media was approximately 60%.

[0116] As demonstrated in this experiment, reducing and / or removing the cys equivalent from the basal medium and / or feed medium had no effect on the yield of secukinumab in the fed-batch process and further resulted in antibody products with higher activity. Therefore, these results suggest that secukinumab expressed at reduced concentrations of the cys equivalent in the cell medium reduces undesirable cell-based modifications of CysL97 and improves product quality.

[0117] Example 4 An integrated sequester production process utilizing a 1000L high cell density perfused batch (HDPB) culture with approximately 1 reactor volume of perfused medium per day is adapted for secukinumab production from CHO cells. The peak viable cell density (VCD) of the HDPB production process is approximately 16 million cells / mL, and the process duration is approximately 19 days. Compared to fed-boil production medium, the HDPB production medium ensures robust growth and desired product quality with minimal adjustment (concentrations of components including manganese, Pluronic® F68, glucose, glutamine, cysteine, and NaCl). No new components were introduced. The volumetric productivity of the HDPB bioreactor is approximately 1.2 g / L / day (or a cumulative titer of 22.8 g / L).

[0118] During the evaluation of candidate culture medium formulations, it was revealed that reducing the cysteine ​​concentration increased the biological activity of secukinumab, as measured by cystamine CEX. As shown in Tables 5 and 6 and Figure 6, biological activity increased by approximately 6–8% in a laboratory-scale experimental recovery pool by reducing the cysteine ​​concentration in the perfusion medium by 50%. This was also evaluated from a downstream perspective due to the direct impact of the reduction step, in addition to the effect revealed in the upstream process, confirming the presence of a cysteine ​​effect.

[0119] [Table 5]

[0120] [Table 6]

[0121] Lower cysteine ​​concentrations resulted in a lower acidity profile compared to other conditions, which was also considered advantageous.

[0122] Table 7

[0123] Table 8

Claims

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 culture medium does not contain added cysteine, and the feed culture 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 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 culture 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 group of recombinant polypeptides recovered from the medium contains at least 10% higher levels of reduced free cysteine ​​compared to the group 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 comprising 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 medium with fresh cell 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 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 polypeptide in mg units of active recombinant polypeptide per liter of medium compared to a control method which comprises 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.

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