Synthetic thiazolidines as cysteine delivery methods in cell culture feed

Abstract

In some aspects, this disclosure relates to methods for preparing cysteine ​​source solutions containing thiazolidinediones. In some other aspects, this disclosure relates to methods for using these cysteine ​​source solutions (such as in the preparation of cell culture media, cell culture, and / or peptide production), and systems and kits comprising these cysteine ​​source solutions.

Description

Cross-reference to related applications

[0001] This application claims priority and benefit to French Patent Application No. 2313030, filed on November 24, 2023, and European Patent Application No. 24305405.3, filed on March 18, 2024, the contents of each of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0002] In some aspects, this disclosure relates to methods for preparing cysteine ​​source solutions containing thiazolidinediones. In some other aspects, this disclosure relates to methods for using these cysteine ​​source solutions (such as in the preparation of cell culture media, cell culture, and / or the production of cell products, such as peptides), and systems and kits comprising these cysteine ​​source solutions. Background Technology

[0003] For many cell types, cysteine ​​is an important amino acid involved in, for example, cell proliferation, metabolism, and stability, and has been associated with the production of cells desiring high titers of recombinant proteins (e.g., antibodies). In culture, certain cell types require the provision of exogenous cysteine. For example, in mammals, the liver regulates the free cysteine ​​pool, and therefore, for cells that cannot synthesize cysteine ​​de novo, a cysteine ​​source exists in vivo. However, for certain mammalian cells cultured alone, adequate cell growth and maintenance require a source of exogenous cysteine. Exogenous cysteine ​​is conventionally provided via cell culture medium. However, when used in cell culture media, cysteine ​​has unfavorable properties, as demonstrated by its low solubility at neutral pH. Due to this property, cysteine ​​is known to frequently precipitate from cell culture media, which greatly hinders the use of such media for adequately supporting cell growth and maintenance. Summary of the Invention

[0004] In some respects, this article provides a method for preparing a cysteine ​​source solution suitable for cell culture, the method comprising: reacting cysteine ​​with an α-keto acid compound having formula (A). (A), Alternatively, the corresponding carboxylate anion reacts in aqueous solution to produce a cysteine ​​source solution containing a thiazoline compound having formula (B). (B), Wherein R is an optionally substituted C1-C6 alkyl group, wherein the reaction is carried out under the following conditions: the concentration of cysteine ​​is at least about 30 mM, and / or the concentration of the α-keto acid compound having formula (A) or its corresponding carboxylate anion is at least about 30 mM, and / or the pH is about 1.5 to about 12.5, and / or the ratio of cysteine ​​to the α-keto acid compound having formula (A) is about 2:1 to about 1:2.

[0005] In some embodiments, the reaction is not carried out in a cell culture medium. In some embodiments, the reaction is carried out for 5 hours or less.

[0006] In some embodiments, the residual concentration of cysteine ​​or cystine in the cysteine ​​source solution is about 0.5 mM or less. In some embodiments, the residual concentration of the α-keto acid compound having formula (A) is about 0.5 mM or less.

[0007] In some embodiments, the purity of the thiazoline compound of formula (B) in the resulting cysteine ​​source solution is at least about 95% of the original cysteine ​​concentration.

[0008] In some embodiments, the concentration of cysteine ​​reacting with an α-keto acid compound having formula (A) is from about 30 mM to about 5000 mM. In some embodiments, the concentration of cysteine ​​reacting with an α-keto acid compound having formula (A) is at least about 150 mM. In some embodiments, the concentration of cysteine ​​reacting with an α-keto acid compound having formula (A) is at least about 1800 mM.

[0009] In some embodiments, the α-keto acid compound having formula (A) is pyruvate (Pyr) or pyruvate salt. In some embodiments, the α-keto acid compound having formula (A) is α-ketoglutarate (AKG) or α-ketoglutarate salt. In some embodiments, the α-keto acid compound having formula (A) reacting with cysteine ​​has a concentration of about 30 mM to about 5000 mM. In some embodiments, the concentration of the α-keto acid compound having formula (A) reacting with cysteine ​​is at least about 150 mM. In some embodiments, the concentration of the α-keto acid compound having formula (A) reacting with cysteine ​​is at least about 1800 mM.

[0010] In some embodiments, the reaction of cysteine ​​with an α-keto acid compound having formula (A) is carried out at a pH from about 3 to about 12. In some embodiments, the reaction of cysteine ​​with an α-keto acid compound having formula (A) is carried out at a pH of about 4.1. In some embodiments, the reaction of cysteine ​​with an α-keto acid compound having formula (A) is carried out at a pH of about 7. In some embodiments, the reaction of cysteine ​​with an α-keto acid compound having formula (A) is carried out at a pH of about 12.

[0011] In some embodiments, cysteine ​​and an α-keto acid compound having formula (A) react at a cysteine:α-keto acid concentration ratio from 1:1 to 1:2. In some embodiments, cysteine ​​and an α-keto acid compound having formula (A) react at a cysteine:α-keto acid concentration ratio of 1:1.

[0012] In some embodiments, cysteine ​​is L-cysteine.

[0013] In other respects, this document provides a method for preparing a cysteine ​​source solution suitable for cell culture, the method comprising: reacting at least about 1800 mM of cysteine ​​with at least about 1800 mM of pyruvate or a corresponding carboxylate anion in an aqueous solution to produce a cysteine ​​source solution comprising 2-methyl-1,3-thiazolidin-2,4-dicarboxylic acid, wherein the reaction is initiated at a pH of about 3.5 to about 12. In some embodiments, the reaction is initiated at a pH of about 4.1. In some embodiments, the reaction is initiated at a pH of about 7.

[0014] In other respects, this document provides a method for preparing a cysteine ​​source solution suitable for cell culture, the method comprising: reacting at least about 1800 mM of cysteine ​​with at least about 1800 mM of α-ketoglutarate or the corresponding carboxylate anion in an aqueous solution to produce a cysteine ​​source solution comprising 2-(2-carboxyethyl)thiazolidin-2,4-dicarboxylic acid, wherein the reaction is initiated at a pH of about 4 to about 8. In some embodiments, the reaction is carried out at a pH of about 7.

[0015] In other respects, this document provides a method for preparing a cysteine ​​source solution suitable for cell culture, the method comprising: reacting at least about 1800 mM of cysteine ​​with at least about 1800 mM of oxaloacetic acid or a corresponding carboxylate anion in an aqueous solution to produce a cysteine ​​source solution comprising 2-(carboxymethyl)thiazolidin-2,4-dicarboxylic acid, wherein the reaction is initiated at a pH of about 4 to about 8. In some embodiments, the reaction is carried out at a pH of about 7.

[0016] In some embodiments, the reaction is carried out for 2 hours or less. In some embodiments, the residual concentration of cysteine ​​or cystine in the cysteine ​​source solution is about 0.5 mM or less. In some embodiments, the residual concentration of the corresponding carboxylate anion of pyruvate, α-ketoglutarate, or oxaloacetic acid, or any one thereof, is about 0.5 mM or less. In some embodiments, the purity of thiazolidinediones in the resulting cysteine ​​source solution is at least about 95%.

[0017] In other respects, this document provides a cysteine ​​source solution produced according to the method described herein.

[0018] In other aspects, this document provides a method for preparing a cell culture medium, the method comprising mixing a basal cell culture medium with a cysteine ​​source solution to prepare the cell culture medium, wherein the cysteine ​​source solution is produced according to the method described herein. In some embodiments, the cell culture medium is suitable for cysteine-dependent cells. In some embodiments, the basal cell culture medium is not suitable for cysteine-dependent cells without further modification. In some embodiments, the basal cell culture medium has a pH of about 5 to about 8. In some embodiments, the method further comprises preparing a thiazolidinyl side solution according to the method described herein. In some embodiments, the method further comprises adjusting the pH of the basal cell culture medium. In some embodiments, the method further comprises adding cell culture medium to an appropriate volume after mixing the basal cell culture medium and the cysteine ​​side solution. In some embodiments, the basal cell culture medium is substantially free of cysteine ​​and cystine.

[0019] In other respects, this article provides a method for culturing cysteine-dependent cells, the method comprising culturing the cysteine-dependent cells in a cell culture medium prepared according to the method described herein.

[0020] In other respects, this document provides a method for generating a polypeptide, the method comprising culturing cysteine-dependent cells in a cell culture medium prepared according to the method described herein, and obtaining the polypeptide therefrom. In some embodiments, the polypeptide is a therapeutic polypeptide or a precursor thereof. In some embodiments, the therapeutic polypeptide or its precursor is an antibody or a fragment thereof. In some embodiments, the antibody or its fragment is an antibody-drug conjugate.

[0021] In other respects, this document provides a cell culture system comprising: cells; and a basal cell culture medium mixed with the cysteine ​​source solution described herein or in a dry or semi-dry form thereof. In some embodiments, the basal cell culture medium is substantially free of cysteine ​​and cystine.

[0022] In other respects, this document provides a method for culturing cells, the method comprising providing the cells with a cell culture medium comprising a basal medium and a cysteine ​​source solution containing thiazolidinediones, and wherein the cell culture medium contains cysteine ​​at a concentration not exceeding about 20% relative to the amount of thiazolidinediones in the cell culture medium; and culturing the cells. In some embodiments, the cells are cysteine-dependent cells. In some embodiments, the cell culture medium is unsuitable for culturing cells without further containing cysteine ​​or a source thereof. In some embodiments, the cysteine ​​source solution is the sole cysteine ​​source for the cells. In some embodiments, the cell culture medium does not contain cysteine. In some embodiments, the cysteine ​​source solution is produced according to the method described herein. Attached Figure Description

[0023] Figure 1A The Cys / Pyr, Cys / AKG, and Cys / Oxa reaction curves obtained by DTNB determination in Optimization Test 1 are shown. Figure 1B Showing from Figure 1A The selected DTNB measurement data.

[0024] Figure 2 The Cys / Pyr, Cys / AKG, and Cys / Oxa reaction curves obtained by DTNB determination in Optimization Test 2 are shown.

[0025] Figure 3A The Cys / Pyr, Cys / AKG, and Cys / Oxa reaction curves obtained by DTNB determination in optimization test 3 are shown. Figure 3B Showing from Figure 3A The selected DTNB measurement data.

[0026] Figure 4A A representative chromatogram obtained by applying the method described herein is shown. Figure 4B A representative chromatogram obtained by applying the method described herein is shown.

[0027] Figure 5 shows the chemical structures of three complexes: (A) Cys-Pyr, (B) Cys-AKG, and (C) Cys-Oxa.

[0028] Figure 6 The Cys-Pyr molecule corresponding to peak RT = 6.04 is shown, with theoretical fragment weight and experimental data.

[0029] Figure 7A and Figure 7B MS-MS of Cys-Pyr molecular fragments and daughter ions are shown, with data obtained via Mass Frontier. TM(SR1 version 8.0) Predicted fragment structure.

[0030] Figure 8 The Cys-AKG molecule corresponding to peak RT = 11.18 is shown, with theoretical fragment weight and experimental data.

[0031] Figure 9A and Figure 9B MS-MS of Cys-AKG molecular fragments and daughter ions are shown, with data obtained via Mass Frontier. TM (SR1 version 8.0) Predicted fragment structure.

[0032] Figure 10 The Cys-Oxa molecule corresponding to peak RT = 9.20 is shown, with theoretical fragment weight and experimental data.

[0033] Figure 11A and Figure 11B MS-MS of Cys-Oxa molecular fragments and daughter ions are shown, with data obtained via MassFrontier. TM (SR1 version 8.0) Predicted fragment structure.

[0034] Figure 12 VCD curves for four different cell lines in an ambr15 fed-batch bioreactor with six Cys delivery methods are shown.

[0035] Figure 13 Viability curves for four different cell lines in an ambr15 fed-batch bioreactor with six Cys delivery methods are shown.

[0036] Figure 14 The titer profiles for four different cell lines in an ambr15 fed-batch bioreactor with six Cys delivery methods are shown.

[0037] Figure 15A-15F The graph shows the normalized free cysteine ​​concentration over time under certain cell culture conditions.

[0038] Figures 16A-16E The diagram shows the cell culture performance and product quality of certain cell cultures.

[0039] Figures 17A-17B The graph shows the changes of certain metabolites over time in studies of certain cell culture conditions. Detailed Implementation

[0040] In some aspects, this application provides a method for preparing a cysteine ​​source solution suitable for providing a cysteine ​​source for cell culture, wherein the cysteine ​​source solution comprises a thiazolidinyl compound. As described herein, the cysteine ​​source solution can be used as an alternative to the need to directly provide cysteine ​​to the cell culture medium. Therefore, in some embodiments, the cysteine ​​source solution does not contain cysteine ​​(or does not contain a large amount of cysteine, such as containing no more than about 20% relative to the amount of thiazolidinyl, such as 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less). The disclosure of this application is based, at least in part, on the inventors' discovery of an efficient method for producing cysteine ​​source solutions with high yields and purity of thiazolidinyl compounds suitable as alternatives to other cysteine ​​sources routinely used in cell culture. It has been found that the synthesized thiazolidinyl compounds are highly soluble and stable in the cysteine ​​source solutions and cell culture media, overcoming a significant drawback of directly using cysteine, namely the known cysteine ​​solubility problem in cell culture media that leads to cysteine ​​precipitation. Precipitated cysteine ​​cannot be used by cells and, in addition, introduces manufacturing quality control problems. Furthermore, it has been found that cells can use the synthesized thiazolidinyl compounds as a cysteine ​​source. As demonstrated in the examples, the cysteine ​​source solutions taught herein can be combined with basal cell culture media lacking other cysteine ​​sources, and the resulting cell culture media provides an environment suitable for cell maintenance, growth, and the production of recombinant peptides at desired titers. Moreover, the thiazolidinyl compounds in the taught cysteine ​​source solutions (or compositions containing them, such as cell culture media) are highly soluble and do not result in the presence of precipitated cysteine. The thiazolidinedones in the cysteine ​​source solutions described herein are highly bioavailable and non-toxic, readily produced, highly soluble, suitable for single-feed cell culture techniques, protect the thiol groups on the cysteine ​​components from redox reactions, and represent a cost-effective method for providing cysteine ​​sources for cell culture. The advantages provided by the cysteine ​​source solutions described herein, including the advantages of the methods for producing said cysteine ​​source solutions, represent a significant advancement in the field of cell culture and the products that can be generated therefrom.

[0041] Therefore, in some respects, this document provides a method for preparing a cysteine ​​source solution suitable for cell culture, the method comprising reacting cysteine ​​with an α-keto acid compound in an aqueous solution to produce a cysteine ​​source solution containing a thiazolidinyl compound, wherein the reaction is carried out under the following conditions: the concentration of cysteine ​​is at least about 30 mM, and / or the concentration of an α-keto acid compound having formula (A) or its corresponding carboxylate anion is at least about 30 mM, and / or the pH is about 1.5 to about 12.5, and / or the ratio of cysteine ​​to an α-keto acid compound having formula (A) is about 2:1 to about 1:2.

[0042] In other respects, this article provides a method for preparing a cysteine ​​source solution suitable for cell culture, the method comprising: reacting cysteine ​​with an α-keto acid compound having formula (A). (A), Alternatively, the corresponding carboxylate anion reacts in aqueous solution to produce a cysteine ​​source solution containing a thiazoline compound having formula (B). (B), Where R is an optionally substituted C1-C6 alkyl group, The reaction takes place under the following conditions: The concentration of cysteine ​​is at least about 30 mM, and / or The concentration of an α-keto acid compound having formula (A) or its corresponding carboxylate anion is at least about 30 mM, and / or pH is from about 1.5 to about 12.5, and / or The ratio of cysteine ​​to an α-keto acid compound having formula (A) is about 2:1 to about 1:2.

[0043] In other aspects, a method is provided for preparing a cysteine ​​source solution suitable for cell culture, the method comprising: reacting at least about 1800 mM of cysteine, comprising about 1800 mM of pyruvate or a corresponding carboxylate anion in an aqueous solution to produce a cysteine ​​source solution comprising 2-methyl-1,3-thiazolidin-2,4-dicarboxylic acid, wherein the reaction is initiated at a pH of about 3.5 to about 12. In some embodiments, the reaction is initiated at a pH of about 4.1. In some embodiments, the reaction is initiated at a pH of about 7.

[0044] In other aspects, a method for preparing a cysteine ​​source solution suitable for cell culture is provided, the method comprising: reacting at least about 1800 mM of cysteine, comprising about 1800 mM, with at least about 1800 mM of α-ketoglutarate or a corresponding carboxylate anion in an aqueous solution to produce a cysteine ​​source solution comprising 2-(2-carboxyethyl)thiazolidin-2,4-dicarboxylic acid, wherein the reaction is initiated at a pH of about 4 to about 8. In some embodiments, the reaction is carried out at a pH of about 7.

[0045] In other aspects, a method for preparing a cysteine ​​source solution suitable for cell culture is provided, the method comprising: reacting at least about 1800 mM of cysteine, comprising about 1800 mM of cysteine, with at least about 1800 mM of oxaloacetic acid or a corresponding carboxylate anion in an aqueous solution to produce a cysteine ​​source solution comprising 2-(carboxymethyl)thiazolidin-2,4-dicarboxylic acid, wherein the reaction is initiated at a pH of about 4 to about 8. In some embodiments, the reaction is carried out at a pH of about 7.

[0046] In other respects, a cysteine ​​source solution produced according to any of the methods described herein is provided.

[0047] In other respects, a method for preparing a cell culture medium is provided, the method comprising mixing a basal cell culture medium with a cysteine ​​source solution to prepare the cell culture medium, wherein the cysteine ​​source solution is produced according to any of the methods described herein.

[0048] In other respects, a method for culturing cysteine-dependent cells is provided, the method comprising culturing the cysteine-dependent cells in a cell culture medium prepared according to the description provided herein.

[0049] In other respects, a method for producing a polypeptide is provided, the method comprising culturing cysteine-dependent cells in a cell culture medium prepared according to the description provided herein, and obtaining the polypeptide therefrom.

[0050] In other respects, a cell culture system is provided comprising: cells; and a basic cell culture medium mixed with the cysteine ​​source solution described herein. A. Definition

[0051] Unless otherwise defined, all industry terms, symbols, and other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some instances, for clarity and / or for ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of these definitions herein should not be construed as representing a material difference from the commonly understood meaning in the art.

[0052] As used herein, the terms “peptide” and “protein” are used interchangeably and refer to polymers containing amino acid residues, and are not limited to a minimum length. Such polymers may contain native or non-native amino acid residues or combinations thereof, and include, but are not limited to, peptides, polypeptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. Full-length polypeptides or proteins and fragments thereof are covered in this definition. These terms also include the types of modifications they may have, such as post-translational modifications of one or more residues, such as methylation, phosphorylation, glycosylation, sialylation, or acetylation.

[0053] The term “antibody” is used in the broadest sense in this article and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they contain an Fc domain.

[0054] The term “full-length antibody” is used herein to refer to an antibody having a structure substantially similar to that of a natural antibody or having a heavy chain containing an Fc region as defined herein.

[0055] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. Each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3). Similarly, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, from the N-terminus to the C-terminus, followed by a constant light (CL) domain. Based on the amino acid sequence of its constant domains, the light chains of an antibody can be assigned to one of two types (called kappa (κ) and lambda (λ)).

[0056] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five main antibody classes: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ.

[0057] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a specific source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0058] "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of a complete antibody that binds to an antigen. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); immunoglobulin monovariable domains, such as VHH; and multispecific antibodies formed from antibody fragments.

[0059] "Human antibody" is an antibody whose amino acid sequence corresponds to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human antibody encoding sequence using a human antibody library or other human antibodies. This definition of human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0060] A “humanized” antibody is a chimeric antibody comprising amino acid residues from a nonhuman hypervariable region (HVR) and amino acid residues from a human frame region (FR). In some embodiments, the humanized antibody will comprise at least one, and typically substantially all, of the two variable domains, wherein all or substantially all of the HVR (e.g., CDR) corresponds to the HVR of the nonhuman antibody, and all or substantially all of the FR corresponds to the FR of the human antibody. Optionally, the humanized antibody may comprise at least a portion of the antibody constant region derived from the human antibody. The “humanized form” of an antibody (e.g., a nonhuman antibody) refers to an antibody that has undergone humanization.

[0061] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homologous group of antibodies, meaning that the individual antibodies constituting this group are identical and / or bind to the same epitopes, typically present in trace amounts except for possible variant antibodies (e.g., containing naturally occurring mutations or arising during the production of the monoclonal antibody formulation). In contrast to polyclonal antibody formulations, which typically contain different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates the characteristic of an antibody derived from a substantially homologous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method.

[0062] The term "therapeutic antibody" refers to an antibody used to treat a disease. Therapeutic antibodies can have various mechanisms of action. Therapeutic antibodies can bind to a target associated with an antigen and neutralize its normal function. For example, a monoclonal antibody that blocks the activity of a protein required for cancer cell survival leads to cell death. Another type of therapeutic monoclonal antibody can bind to a target associated with an antigen and activate its normal function. For example, a monoclonal antibody can bind to a protein on a cell and trigger apoptosis signals. Yet another type of monoclonal antibody can bind to a target antigen expressed only in diseased tissue; conjugation of a toxic payload (effective agent) (such as a chemotherapeutic agent or radiopharmaceutical) with a monoclonal antibody can produce an agent for specifically delivering the toxic payload to diseased tissue, thereby reducing damage to healthy tissue. The "biological functional fragment" of a therapeutic antibody will exhibit at least one (if not some or all) biological function attributable to the complete antibody, which includes at least specific binding to a target antigen. In some embodiments, the therapeutic antibody is an antibody conjugate, such as an antibody-drug conjugate. In some embodiments, this characteristic is performed covalently (including via a linker) or non-covalently with the antibody conjugate.

[0063] Throughout this disclosure, all aspects of the claimed subject matter are presented in range form. It should be understood that this range form is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Therefore, the description of a range should be considered to include all possible subranges of the exact disclosure, as well as individual numerical values ​​within that range. For example, where a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of the range (unless the context explicitly states otherwise, to one-tenth of the lower limit unit), and any other stated or intermediate value within the range, is encompassed within this disclosure, subject to any particular exclusions of the range. Where the range includes one or both of the included limits, ranges excluding any or both of these included limits are also included in this disclosure. In some embodiments, two opposing and open ranges are provided for a feature, and in such a description, it is contemplated that a combination of these two ranges is provided herein. For example, in some embodiments, a feature greater than about 10 units is described, and a feature less than about 20 units is described (as in another sentence), and thus a range of about 10 units to about 20 units is described herein.

[0064] As used herein, the term "about" refers to a typical range of error readily known in the art for a given value. The "about" value or parameter mentioned herein includes (and describes) variations relating to that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0065] Unless the context clearly indicates otherwise, as used herein (including in the appended claims), the singular forms “a,” “or,” and “the” include multiple referents. For example, “a / an” means “at least one” or “one or more”. It should be understood that aspects and variations described herein include embodiments that “consist of such aspects and variations” and / or “consist substantially of such aspects and variations.”

[0066] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this disclosure. The following description illustrates the disclosure and should not, of course, be construed in any way as limiting the scope of the invention described herein. B. Method for preparing cysteine ​​source solution

[0067] This document provides methods for preparing cysteine ​​source solutions suitable for cell culture and methods for using cysteine ​​source solutions. In some embodiments, the method for preparing a cysteine ​​source solution includes reacting cysteine ​​with an α-keto acid compound to produce a cysteine ​​source solution containing a thiazolidinyl compound. As described herein, in some embodiments, the reaction is carried out under the following conditions: a cysteine ​​concentration of at least about 30 mM, and / or a concentration of the α-keto acid compound or its corresponding carboxylate anion of at least about 30 mM, and / or a pH of about 1.5 to about 12.5, and / or a cysteine ​​to α-keto acid compound ratio of about 2:1 to about 1:2.

[0068] In some embodiments, this document provides a method for preparing a cysteine ​​source solution suitable for cell culture and / or using the cysteine ​​source solution. In some such embodiments, the method includes: reacting cysteine ​​with an α-keto acid compound having formula (A): (A), Or corresponding carboxylate anionic compounds with formula (AI): (AI), The reaction is carried out in an aqueous solution to produce a cysteine ​​source solution containing a thiazolidinyl compound having formula (B): (B), Where R is an optionally substituted aliphatic, optionally substituted unsaturated alkyl, or C(O)R 1 C(O)OR 2 、or -OR 1 ; Where R 1 Is it arbitrarily R 2 or C(O)R 2 Substituted alkyl groups; and R 2 It is an alkyl group or H that is optionally substituted; The reaction takes place under the following conditions: The concentration of cysteine ​​is at least about 30 mM, and / or The concentration of the α-keto acid compound having formula (A) or the corresponding carboxylate anionic compound having formula (AI) is at least about 30 mM, and / or pH is from about 1.5 to about 12.5, and / or The ratio of cysteine ​​to an α-keto acid compound having formula (A) or a corresponding carboxylate anionic compound having formula (AI) is about 4:1 to about 1:4.

[0069] Some of the embodiments provided herein describe the pH of the reaction. It should be understood that, unless otherwise stated, this pH is the pH at the start of the reaction (e.g., mixing two or more reactants). It is understood in providing this instruction that the pH of the reaction mixture can change during the reaction. In some embodiments, unless specifically indicated in such embodiments provided herein, the pH of the reaction will change to a pH outside the defined range of the reaction during the process, and such embodiments are not outside the instruction of this specification.

[0070] As disclosed herein, aspects of the methods provided herein can be implemented in a variety of ways and forms. Further discussion of aspects of the provided methods is included in the following sections. This modular discussion of such aspects does not limit the scope of the invention, and those skilled in the art will readily understand how certain features from the following sections and this document can be combined to carry out the methods taught herein. i. Cysteine

[0071] The method provided herein for preparing cysteine ​​source solutions suitable for cell culture includes cysteine ​​as a reactant. As described herein, unless otherwise instructed, cysteine ​​can be present in various forms, and this disclosure envisions that such forms are readily substituted (and may be present in certain mixtures).

[0072] In some embodiments, cysteine ​​is L-cysteine.

[0073] In some embodiments, cysteine ​​is present with an acid (e.g., cysteine ​​hydrochloride). In some embodiments, cysteine ​​is hydrated, such as a monohydrate. In some embodiments, cysteine ​​is formulated as an ester, such as cysteine ​​methyl ester.

[0074] In some embodiments, cysteine ​​is in the form of a solution. In some embodiments, cysteine ​​is in the form of a powder, such as a lyophilized powder.

[0075] In some embodiments, the reaction described herein is carried out under the following conditions: the concentration of cysteine ​​is from about 30 mM to about 5000 mM, such as any one of about 500 mM to about 2000 mM, about 500 mM to about 1250 mM, about 750 mM to about 1500 mM, about 1000 mM to about 2000 mM, about 1500 mM to about 2000 mM, about 1700 mM to about 1900 mM, about 1500 mM to about 5000 mM, or about 2500 mM to about 5000 mM. In some embodiments, the reaction described herein is carried out under the following conditions: the concentration of cysteine ​​is at least about 30 mM, such as at least about 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM. 1700 mM 2700 mM 4600 mM Any of 4700 mM, 4800 mM, 4900 mM, or 5000 mM.In some embodiments, the reactions described herein are carried out under the following conditions: cysteine ​​concentrations of approximately 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM. 1750 mM 2700 mM The concentrations of cysteine ​​reactants are any one of 2800 mM, 2900 mM, 3000 mM, 3100 mM, 3200 mM, 3300 mM, 3400 mM, 3500 mM, 3600 mM, 3700 mM, 3800 mM, 3900 mM, 4000 mM, 4100 mM, 4200 mM, 4300 mM, 4400 mM, 4500 mM, 4600 mM, 4700 mM, 4800 mM, 4900 mM, or 5000 mM. Those skilled in the art will readily understand the form (including its working variations) of the cysteine ​​reactants necessary to achieve the desired cysteine ​​concentrations described herein. Furthermore, the amount of cysteine ​​indicates the initial amount of cysteine ​​present at the start of the reaction with the α-keto acid, and it is well understood that such initial reactants will decrease in a successful reaction. ii. α-Keto acids

[0076] The methods provided herein for preparing cysteine ​​source solutions suitable for cell culture include α-keto acids or corresponding carboxylate anions as reactants. As described herein, unless otherwise instructed, α-keto acids or corresponding carboxylate anions may be present in various forms, and this disclosure envisions that such forms are readily substituted (and may be present in certain mixtures).

[0077] In some embodiments, the α-keto acid is a compound having formula (A): (A), Where R is an optionally substituted aliphatic, optionally substituted unsaturated alkyl, or C(O)R 1 C(O)OR 2 、or -OR 1 ;where R 1 Is it arbitrarily R 2 or C(O)R 2 Substituted alkyl groups; and R 2 R is an optionally substituted alkyl group or H. In some such embodiments, R is an optionally substituted mono- to 6-membered aliphatic group. In some such embodiments, R is an optionally substituted C1-C6 alkyl group. In some such embodiments, R is optionally halogenated, OH, SH, 6- to 10-membered aryl, or C(O)OR. 2 Substituted C1-C6 alkyl groups, wherein the 6- to 10-membered aryl group is optionally selected from OH, C1-C6 alkyl, or C(O)OR. 2 One or more groups are substituted for R. In some such embodiments, R is optionally replaced by one or more C(O)OR groups. 2 Substituted C1-C6 alkyl groups, wherein R 2 It is H or a C1-C6 alkyl group. In some such embodiments, R is a C1-C6 alkyl group optionally substituted with COOH. In some such embodiments, R is a C1-C6 alkyl group substituted with one or more COOH groups. In some such embodiments, R is optionally selected from halogenated groups, OH, SH, or C(O)OR. 2 One or more groups substituted for a C2-C6 alkenyl group. In some such embodiments, R is C(O)R. 1 , where R 1 It is a C1-C6 alkyl group. In some such embodiments, R is C(O)OR. 2 , where R 2 It is a C1-C6 alkyl group or H. In some such embodiments, R is -OR 1 , where R 1 Is it optional to be R 2 Substituted C1-C6 alkyl groups. In some such embodiments, the α-keto acid is the corresponding carboxylate anion having the formula (AI). (AI), The variable group R is as previously defined for equation (A).

[0078] In some embodiments, the α-keto acid having formula (A) is pyruvate, α-ketoglutarate, or oxaloacetate, or the corresponding carboxylate anion having formula (AI) is pyruvate, α-ketoglutarate, or oxaloacetate. In some such embodiments, the α-keto acid is pyruvate. In some such embodiments, the corresponding carboxylate anion is pyruvate. In some such embodiments, the α-keto acid is α-ketoglutarate. In some such embodiments, the corresponding carboxylate anion is α-ketoglutarate. In some such embodiments, the α-keto acid is oxaloacetate. In some such embodiments, the corresponding carboxylate anion is oxaloacetate.

[0079] In some embodiments, the reaction described herein is carried out under the following conditions: the concentration of the α-keto acid or the corresponding carboxylate anion (e.g., pyruvate, α-ketoglutarate, or oxaloacetic acid, or the corresponding carboxylate anion of any one thereof) is from about 30 mM to about 5000 mM, such as from about 500 mM to about 2000 mM, from about 500 mM to about 1250 mM, from about 750 mM to about 1500 mM, from about 1000 mM to about 2000 mM, from about 1500 mM to about 2000 mM, from about 1700 mM to about 1900 mM, from about 1500 mM to about 5000 mM, or from about 2500 mM to about 5000 mM.In some embodiments, the reactions described herein are carried out under the following conditions: the concentration of the α-keto acid or the corresponding carboxylate anion (e.g., pyruvate, α-ketoglutarate, or oxaloacetic acid, or the corresponding carboxylate anion of any of these) is at least about 30 mM, such as at least about 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, etc. 1400 mM mM, 1500mM, 1550mM, 1600mM, 1650mM, 1700mM, 1750mM, 1800mM, 1850mM, 1900mM, 1950mM, 2000mM, 2050mM, 2100mM, 2150mM, 2200mM, 2300 2400mM Any of 4300 mM, 4400 mM, 4500 mM, 4600 mM, 4700 mM, 4800 mM, 4900 mM, or 5000 mM.In some embodiments, the reactions described herein are carried out under the following conditions: the concentration of the α-keto acid or the corresponding carboxylate anion (e.g., pyruvate, α-ketoglutarate, or oxaloacetic acid, or the corresponding carboxylate anion of any of these) is about 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, etc. 1400 mM 2200 mM mM, 2300mM, 2400mM, 2500mM, 2600mM, 2700mM, 2800mM, 2900mM, 3000mM, 3100mM, 3200mM, 3300mM, 3400mM, 3500mM, 3600mM, 3700mM, 3800mM, 3900mM, 4000mM, 4100 Any of 4200 mM, 4300mM, 4400mM, 4500mM, 4600mM, 4700mM, 4800mM, 4900mM, or 5000mM. Those skilled in the art will readily understand that the form (including variations thereof) of the α-keto acid reactant or the corresponding carboxylate anion (e.g., pyruvate, α-ketoglutarate, or oxaloacetic acid, or the corresponding carboxylate anion of any of these) is necessary to achieve the desired concentration of the α-keto acid or the corresponding carboxylate anion for the reactions described herein.Furthermore, the amount of the α-keto acid or the corresponding carboxylate anion (e.g., pyruvate, α-ketoglutarate, or oxaloacetic acid, or the corresponding carboxylate anion of any of these) indicates the initial amount of the α-keto acid or the corresponding carboxylate anion present at the start of the reaction with cysteine, and it is well understood that such initial reactants will decrease in a successful reaction. iii. Ratio of reactants

[0080] The methods provided herein for preparing cysteine ​​source solutions suitable for cell culture cover examples at least in part based on the ratio between reactants (i.e., cysteine ​​to α-keto acids or corresponding carboxylate anions). In some embodiments, the reaction of cysteine ​​and α-keto acids or corresponding carboxylate anions is carried out in a 1:1 ratio to produce thiazolidinediones. In some embodiments, the reactions described herein are configured based on the desired composition of the cysteine ​​source solution after the reactions described herein. For example, in some embodiments, the cysteine ​​source solution is substantially free of (including not containing) cysteine ​​after the reaction, and in such reactions, the ratio of cysteine ​​to α-keto acids or corresponding carboxylate anions is 1:1. In other embodiments, it may be desirable to have residual amounts of cysteine ​​and / or α-keto acids or corresponding carboxylate anions in the cysteine ​​source solution after the reactions described herein. In such embodiments, the ratio of reactants can be established accordingly.

[0081] In some embodiments, at the start of the reaction described herein, the molar ratio of cysteine ​​to α-keto acid or the corresponding carboxylate anion is about 4:1 to about 1:4, such as about any one of about 3:1 to about 1:3, about 2.5:1 to about 1:2.5, about 2:1 to about 1:2, or about 1.5:1 to about 1:1.5. In some embodiments, at the start of the reaction described herein, the molar ratio of cysteine ​​to α-keto acid or the corresponding carboxylate anion is about 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, or 1:4. In some embodiments, at the start of the reaction described herein, the molar ratio of cysteine ​​to α-keto acid or the corresponding carboxylate anion is about 1:1. iv. Reaction conditions and characteristics

[0082] The method for preparing cysteine ​​source solutions provided in this article can be carried out under different conditions.

[0083] As described herein, the reactions described herein may exhibit pH changes as the reaction progresses, and therefore the description of the pH of the reaction (including the initial pH) describes when these reactants are brought together for the reaction steps. In some embodiments, the pH, such as during and after the production of thiazolidin, is outside the prescribed pH of the reaction as the reaction progresses. Such embodiments are still covered within the scope of this application. In some embodiments, the reaction is carried out at a pH of about 1 to about 13 (including the initial pH), such as any one of about 1.5 to about 12.5, about 3 to about 12, about 3 to about 5, about 6 to about 8, or about 11 to about 13. In some embodiments, the reaction is carried out at a pH of at least about 3 (including the initial pH), such as any one of at least about 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the reaction is carried out at a pH of about 12 or less (including the initial pH), such as any one of about 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the reaction is carried out at any one of the following pH values ​​(including the initial pH): about 3, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 5, 6, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 8, 9, 9, 10, 11, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, or 12.5. In some embodiments, the reaction is carried out at a pH of about 4.1 (including the initial pH). In some embodiments, the reaction is carried out at a pH of about 7 (including the initial pH). In some embodiments, the reaction is carried out at a pH of about 12 (including the initial pH).

[0084] In some embodiments, the reaction takes about 5 hours or less, such as about 4.5 hours or less, 4 hours or less, 3.5 hours or less, 3 hours or less, 2.5 hours or less, 2 hours or less, 1.5 hours or less, or 1 hour or less.

[0085] The reactions described herein can be carried out under a variety of other conditions. For example, in some embodiments, the reaction is carried out at about 10°C to about 50°C (including room temperature or 37°C). In some embodiments, the reaction is carried out under light. In some embodiments, the reaction is carried out in the dark. In some embodiments, the reaction is carried out under an inert atmosphere.

[0086] In some embodiments, the reaction is not carried out in the cell culture medium. v. The form of the resulting cysteine ​​source solution

[0087] The reactions described herein provide cysteine ​​source solutions suitable for cell culture. The cysteine ​​source solutions provided herein can be arrayed in various forms, such as having different concentrations of thiazolidinediones, and in some embodiments, can be further modified. In some embodiments, the cysteine ​​source solution does not contain cysteine ​​(or does not contain a significant amount of cysteine, such as containing no more than about 2% relative to the amount of thiazolidinediones, such as 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, or 3% or less, or 2% or less). As discussed herein, in some embodiments, the cysteine ​​source solution can be in a dry or semi-dry form.

[0088] In some embodiments, the residual concentration of cysteine ​​or cystine (including the sum thereof) in the cysteine ​​source solution is about 1 mM or less, such as about 0.95 mM or less, 0.9 mM or less, 0.85 mM or less, 0.8 mM or less, 0.75 mM or less, 0.7 mM or less, 0.65 mM or less, 0.6 mM or less, 0.55 mM or less, 0.5 mM or less, 0.45 mM or less, 0.4 mM or less, 0.35 mM or less, 0.3 mM or less, 0.25 mM or less, 0.2 mM or less, 0.15 mM or less, 0.1 mM or less, or 0.05 mM or less. In some embodiments, the cysteine ​​source solution contains a certain amount of residual cysteine, including none or substantially none, such that the resulting cysteine ​​source solution does not form (or substantially does not form) cysteine ​​precipitates (such as compared to solutions containing an equivalent amount of cysteine ​​based on the amount of thiazolidin in the cysteine ​​source solution or a composition containing the cysteine ​​source solution).

[0089] In some embodiments, the residual concentration of α-keto acid or corresponding carboxylate anions (including their sum) in the cysteine ​​source solution is about 1 mM or less, such as any one of about 0.95 mM or less, 0.9 mM or less, 0.85 mM or less, 0.8 mM or less, 0.75 mM or less, 0.7 mM or less, 0.65 mM or less, 0.6 mM or less, 0.55 mM or less, 0.5 mM or less, 0.45 mM or less, 0.4 mM or less, 0.35 mM or less, 0.3 mM or less, 0.25 mM or less, 0.2 mM or less, 0.15 mM or less, 0.1 mM or less, or 0.05 mM or less.

[0090] In some embodiments, the yield of thiazolidinediones in the cysteine ​​source solution is at least about 90% (compared to the expected amount of thiazolidinediones from complete reaction or reactants), such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the concentration of thiazolidinediones produced by the reaction is at least about 90% of the concentration of one or more limiting reagents, such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the purity of the thiazolidinedione compound in the resulting cysteine ​​source solution is at least about 90% relative to any residual cysteine ​​or α-keto acid or corresponding carboxylate anion, such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0091] In some embodiments, the cysteine ​​source solution comprises a thiazolidinedion at a concentration of about 30 mM to about 5000 mM, such as any one of about 500 mM to about 2000 mM, about 500 mM to about 1250 mM, about 750 mM to about 1500 mM, about 1000 mM to about 2000 mM, about 1500 mM to about 2000 mM, about 1700 mM to about 1900 mM, about 1500 mM to about 5000 mM, or about 2500 mM to about 5000 mM. In some embodiments, the cysteine ​​source solution comprises a concentration of at least about 30 mM of thiazolidin, such as at least about 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM. 1750 mM 2800 mM 4700 mM Any of 4800 mM, 4900 mM, or 5000 mM.In some embodiments, the cysteine ​​source solution comprises the following concentrations of thiazolidin: approximately 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM, 900 mM. 1750 mM 2900 mM 3000mM, 3100mM, 3200mM, 3300mM, 3400mM, 3500mM, 3600mM, 3700mM, 3800mM, 3900mM, 4000mM, 4100mM, 4200mM, 4300mM, 4400mM, 4500mM, 4600mM, 4700mM, 4800mM Either mM, 4900 mM, or 5000 mM.

[0092] In some embodiments, the cysteine ​​source solution is a liquid, such as that obtained directly from the reaction described herein. In some embodiments, the cysteine ​​source solution is added in sufficient quantities (QS) to the final desired volume and / or measurement. In some embodiments, the cysteine ​​source solution comprises water and thiazolidinyl.

[0093] In some embodiments, the cysteine ​​source solution may be processed into a dry form after the reaction, such as via lyophilization, crystallization, and / or another drying technique. For the sake of brevity, the embodiments herein refer to cysteine ​​source solutions; however, those skilled in the art will readily understand when a cysteine ​​source solution can be a derivative thereof, such as a dry form of the cysteine ​​source solution. For example, in some embodiments, mixing the cysteine ​​source solution with a basal culture medium is provided. In some embodiments, the cysteine ​​source solution is in liquid form and mixed with a basal culture medium. In some embodiments, the cysteine ​​source solution is in dry or semi-dry form and mixed with a basal culture medium.

[0094] As described herein, the cysteine ​​source solutions provided are suitable for cell culture. Those skilled in the art will readily understand that the cysteine ​​source solutions covered by this description, including how to assess suitability, are not intended to be suitable for all cell cultures. In some embodiments, suitability for cell culture is based on suitability for single-cell types. Further descriptions of cell culture are provided in other parts of this document and are well known in the art. vi. Exemplary Model

[0095] In some aspects, this document provides a method for preparing a cysteine ​​source solution suitable for cell culture, the method comprising: reacting about 1000 mM to about 2500 mM of cysteine ​​with about 1000 mM to about 2500 mM of pyruvate or a corresponding carboxylate anion in an aqueous solution to produce a cysteine ​​source solution comprising 2-methyl-1,3-thiazolidin-2,4-dicarboxylic acid, wherein the reaction is initiated at a pH of about 3.5 to about 12. In some embodiments, the reaction comprises reacting at least about 1500 mM of cysteine, such as at least about 1600 mM cysteine, 1700 mM cysteine, 1800 mM cysteine, 1900 mM cysteine, or 2000 mM cysteine. In some embodiments, the reaction comprises reacting about 1800 mM of cysteine. In some embodiments, the reaction comprises reacting at least about 1500 mM pyruvate or a corresponding carboxylate anion, such as at least about 1600 mM pyruvate or a corresponding carboxylate anion, 1700 mM pyruvate or a corresponding carboxylate anion, 1800 mM pyruvate or a corresponding carboxylate anion, 1900 mM pyruvate or a corresponding carboxylate anion, or 2000 mM pyruvate or a corresponding carboxylate anion. In some embodiments, the reaction comprises reacting about 1800 mM pyruvate or a corresponding carboxylate anion. In some embodiments, the reaction is initiated at a pH of about 3.5 to about 4.5 (inclusive, about 4.1). In some embodiments, the reaction is initiated at a pH of about 6.5 to about 7.5 (inclusive, about 7). As described herein, the reactions described herein may exhibit pH changes as the reaction progresses, and therefore the description of the starting pH describes when these reactants are brought together for the reaction step. In some embodiments, the reaction comprises reacting cysteine ​​and pyruvate or the corresponding carboxylate anion at a ratio of about 2:1 to about 1:2 (including a ratio of about 1:1). In some embodiments, the reaction is carried out for about 5 hours or less, including any one of about 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less. In some embodiments, the residual concentration of cysteine ​​or cystine in the cysteine ​​source solution is about 0.5 mM or less. In some embodiments, the residual concentration of pyruvate or the corresponding carboxylate anion of either is about 0.5 mM or less. In some embodiments, the purity of the thiazolidinedion in the resulting cysteine ​​source solution is at least about 95%, such as any one of at least about 96%, 97%, 98%, 99%, or 100%. In some embodiments, the cysteine ​​is L-cysteine.

[0096] In some aspects, this document provides a method for preparing a cysteine ​​source solution suitable for cell culture, the method comprising: reacting about 1000 mM to about 2500 mM of cysteine ​​with about 1000 mM to about 2500 mM of α-ketoglutarate or a corresponding carboxylate anion in an aqueous solution to produce a cysteine ​​source solution comprising 2-(2-carboxyethyl)thiazolidin-2,4-dicarboxylic acid, wherein the reaction is initiated at a pH of about 4 to about 8. In some embodiments, the reaction comprises reacting at least about 1500 mM of cysteine, such as at least about 1600 mM cysteine, 1700 mM cysteine, 1800 mM cysteine, 1900 mM cysteine, or 2000 mM cysteine. In some embodiments, the reaction comprises reacting about 1800 mM of cysteine. In some embodiments, the reaction comprises reacting at least about 1500 mM α-ketoglutarate or a corresponding carboxylate anion, such as at least about 1600 mM α-ketoglutarate or a corresponding carboxylate anion, 1700 mM α-ketoglutarate or a corresponding carboxylate anion, 1800 mM α-ketoglutarate or a corresponding carboxylate anion, 1900 mM α-ketoglutarate or a corresponding carboxylate anion, or 2000 mM α-ketoglutarate or a corresponding carboxylate anion. In some embodiments, the reaction comprises reacting about 1800 mM α-ketoglutarate or a corresponding carboxylate anion. In some embodiments, the reaction is initiated at a pH of about 6.5 to about 7.5 (inclusive). As described herein, the reactions described herein can exhibit pH changes as the reaction progresses, and therefore the description of the initial pH describes when these reactants are brought together for the reaction step. In some embodiments, the reaction comprises reacting cysteine ​​and α-ketoglutarate or the corresponding carboxylate anion at a ratio of about 2:1 to about 1:2 (including a ratio of about 1:1). In some embodiments, the reaction is carried out for about 5 hours or less, including any one of about 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less. In some embodiments, the residual concentration of cysteine ​​or cystine in the cysteine ​​source solution is about 0.5 mM or less. In some embodiments, the residual concentration of the corresponding carboxylate anion of α-ketoglutarate or any of them is about 0.5 mM or less. In some embodiments, the purity of the thiazolidinedion in the resulting cysteine ​​source solution is at least about 95%, such as any one of at least about 96%, 97%, 98%, 99%, or 100%. In some embodiments, the cysteine ​​is L-cysteine.

[0097] In some aspects, this document provides a method for preparing a cysteine ​​source solution suitable for cell culture, the method comprising: reacting about 1000 mM to about 2500 mM of cysteine ​​with about 1000 mM to about 2500 mM of oxaloacetic acid or a corresponding carboxylate anion in an aqueous solution to produce a cysteine ​​source solution comprising 2-(carboxymethyl)thiazolidin-2,4-dicarboxylic acid, wherein the reaction is initiated at a pH of about 4 to about 8. In some embodiments, the reaction comprises reacting at least about 1500 mM of cysteine, such as any one of at least about 1600 mM cysteine, 1700 mM cysteine, 1800 mM cysteine, 1900 mM cysteine, or 2000 mM cysteine. In some embodiments, the reaction comprises reacting about 1800 mM of cysteine. In some embodiments, the reaction comprises reacting at least about 1500 mM oxaloacetate or the corresponding carboxylate anion, such as at least about 1600 mM oxaloacetate or the corresponding carboxylate anion, 1700 mM oxaloacetate or the corresponding carboxylate anion, 1800 mM oxaloacetate or the corresponding carboxylate anion, 1900 mM oxaloacetate or the corresponding carboxylate anion, or 2000 mM oxaloacetate or the corresponding carboxylate anion. In some embodiments, the reaction comprises reacting about 1800 mM oxaloacetate or the corresponding carboxylate anion. In some embodiments, the reaction is initiated at a pH of about 6.5 to about 7.5 (inclusive). As described herein, the reaction can exhibit pH changes as the reaction progresses, and therefore the description of the starting pH describes when the two reactants are brought together for the reaction step. In some embodiments, the reaction comprises reacting cysteine ​​and oxaloacetate or the corresponding carboxylate anion in a ratio of about 2:1 to about 1:2 (inclusive). In some embodiments, the reaction is carried out for about 5 hours or less, including any one of about 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less. In some embodiments, the residual concentration of cysteine ​​or cystine in the cysteine ​​source solution is about 0.5 mM or less. In some embodiments, the residual concentration of the corresponding carboxylate anion of oxaloacetate or any of them is about 0.5 mM or less. In some embodiments, the purity of thiazolidinediol in the resulting cysteine ​​source solution is at least about 95%, such as at least about 96%, 97%, 98%, 99%, or 100%. In some embodiments, the cysteine ​​is L-cysteine.

[0098] In some embodiments, a method is provided comprising preparing a cysteine ​​source solution according to the methods provided herein, preparing (in any order or in parallel) a cell culture medium (such as preparing a cell culture medium in a cell culture vessel), adding a certain amount of the cysteine ​​source solution to the cell culture medium, adding the cell culture medium containing the cysteine ​​source solution to an appropriate amount, and aseptically filtering the cell culture medium containing the cysteine ​​source solution. C. Other compositions containing cysteine ​​source solutions and their uses

[0099] The invention provided herein also covers methods for producing cysteine ​​source solutions (and cysteine ​​source solutions and derivatives thereof) and aspects achieved by using the taught cysteine ​​source solutions.

[0100] In some embodiments, a method for preparing a cell culture medium is provided, the method comprising mixing a basal cell culture medium with a cysteine ​​source solution (including its dried form) to prepare the cell culture medium, wherein the cysteine ​​source solution is produced according to any of the methods described herein. In some embodiments, the cell culture medium is suitable for cysteine-dependent cells. In some embodiments, the basal cell culture medium is not suitable for cysteine-dependent cells without further modification, e.g., without the addition of a sufficient amount of cysteine ​​(or alternatively, the cysteine ​​source solution taught herein). In some embodiments, the basal cell culture medium has a pH of about 5 to about 8. In some embodiments, the method further comprises preparing a thiazolidinyl side solution according to the methods described herein. In some embodiments, the method further comprises adjusting the pH of the basal cell culture medium. In some embodiments, the method further comprises adding cell culture medium to an appropriate volume after mixing the basal cell culture medium and the cysteine ​​side solution. In some embodiments, the basal cell culture medium is substantially free of cysteine ​​and cystine (e.g., free of a certain amount of cysteine ​​and / or cystine that enables the growth and maintenance of cysteine-dependent cells).

[0101] In some embodiments, this document provides a method for culturing cysteine-dependent cells, the method comprising culturing the cysteine-dependent cells in a cell culture medium prepared according to any method provided herein. In some embodiments, the culturing method comprises maintaining the level of thiazolidinediones by further subjecting the cells to a cysteine ​​source solution, such as by adding the cysteine ​​source solution directly to the cell culture medium containing the cells and / or providing the cells with fresh cell culture medium containing the cysteine ​​source solution.

[0102] In some embodiments, this document provides a method for generating a polypeptide, the method comprising culturing cysteine-dependent cells in a cell culture medium prepared according to any of the methods described herein, and obtaining the polypeptide therefrom. In some embodiments, the polypeptide is a therapeutic polypeptide or a precursor thereof. In some embodiments, the therapeutic polypeptide or its precursor is an antibody or a fragment thereof. In some embodiments, the antibody or its fragment is an antibody-drug conjugate or a precursor thereof for post-conjugation. It should be noted that the inventors have further envisioned advantages for preparing drug conjugates using the methods of the present invention, including the absence or low levels of cysteine ​​residues available for binding the polypeptide, thus greatly facilitating the generation of drug conjugates compared to manufacturing techniques using cell culture media containing cysteine.

[0103] The inventions provided herein relate to many cell types, and particularly to the culture of cysteine-dependent cells. Cell culture media are well known in the art and may include or require the addition of a cysteine ​​source. This application provides means for preparing suitable cell culture media using thiazolidinediones, without requiring the use of cysteine ​​or its forms, such as cystine. In some embodiments, a cell culture medium is generated by using a basal cell culture medium lacking a sufficient amount of a cysteine ​​source and adding a cysteine ​​source solution taught herein. Culture media and components required to support cell growth are described in the following: Ham et al., Meth Enz, 58, 1979; Barnes et al., Anal Biochem, 102, 1980; U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; International Patent Application Nos. WO 90 / 03430 or WO 87 / 00195; and U.S. Patent Reissue No. 30,985, which are hereby incorporated herein by reference in their entirety. If necessary, any of these culture media may be further supplemented with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the GENTAMYCIN™ drug), trace elements (defined as inorganic compounds typically present in a final concentration in the micromolar range), and glucose or an equivalent energy source. In some embodiments, the culture medium further contains a glucose source. In some embodiments, the culture medium further contains a mannose source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art.

[0104] Methods of culturing cells in culture media are well known to those skilled in the art. See, for example, Li et al., MAbs, 2, 2010. In some embodiments, the culture technique is a fed-batch culture method. In some embodiments, the culture technique is a batch culture method. In some embodiments, the culture technique is a continuous culture method. In some embodiments, the culture technique is a perfusion culture method.

[0105] In some embodiments, the culture includes performing the reactions described herein to produce a cysteine ​​source solution, mixing the cysteine ​​source solution with a basal cell culture medium lacking sufficient cysteine ​​source to support the growth and maintenance of cysteine-dependent cells, aseptically filtering the resulting cell culture medium, and providing the cell culture medium to the cells as needed during the culture process. In some embodiments, the culture technique is a fed-batch culture method. In some embodiments, the culture technique is a batch culture method. In some embodiments, the culture technique is a continuous culture method. In some embodiments, the culture technique is a perfusion culture method. In some embodiments, the method further includes obtaining a product generated by the cells, such as a peptide.

[0106] Culture conditions (such as temperature, pH, etc.) will be obvious to those skilled in the art. For example, protein production is typically carried out on a large scale (such as commercial scale). To achieve a cell population suitable for commercial-scale production, those skilled in the art will recognize the utility of using a stepwise approach to expand the cell population. For example, this approach involves growing the desired cells at a smaller scale to allow the cell population to increase (such as seed train). To further increase the cell population, the approach typically involves using seed train to inoculate larger culture vessels, such as inoculation tanks or bioreactors. This process will provide a suitable cell population for culture in a production culture vessel. In some embodiments, the production culture vessel is a 1000L culture vessel. The cysteine ​​source solution provided herein can be used in any one or more of the culture steps. In some embodiments, the cell culture is maintained at a specified temperature. In some embodiments, the specified temperature is from about 15°C to about 45°C. In some embodiments, the specified temperature is about 30°C. In some embodiments, the specified temperature is less than about 37°C. In some embodiments, the specified temperature is less than about 35°C. In some embodiments, the specified temperature is less than about 34°C. In some embodiments, the cell culture is maintained at a specified pH. In some embodiments, cell culture is maintained at a specified dissolved oxygen concentration. In some embodiments, cell culture is maintained at a specified nutrient level.

[0107] In some embodiments, this document provides a method for culturing cells, the method comprising providing the cells with a cell culture medium comprising a basal medium and a cysteine ​​source solution containing thiazolidinediones, and wherein the cell culture medium contains cysteine ​​at a concentration not exceeding about 20% relative to the amount of thiazolidinediones in the cell culture medium; and culturing the cells. In some embodiments, the cells are cysteine-dependent cells. In some embodiments, the cell culture medium is unsuitable for culturing cells without further containing cysteine ​​or its source. In some embodiments, the cysteine ​​source solution is the sole cysteine ​​source for the cells. In some embodiments, the cell culture medium does not contain cysteine ​​or cystine. In some embodiments, the method of culturing cells is a fed-batch technique performed in a bioreactor. In some embodiments, cell culture comprises fed-batch cell culture in a bioreactor. In some embodiments, fed-batch cell culture is performed for a duration of 1 to 21 days, such as any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days. In some embodiments, the cysteine ​​source solution has a thiazolidinyl concentration of about 5 mM to about 50 mM, such as any one of about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM. In some embodiments, the cysteine ​​source solution is added to the bioreactor daily. In some embodiments, the cysteine ​​source solution is added throughout the fed-batch cell culture process. In some embodiments, the fed-batch cell culture process is a live cell density-dependent strategy. In some embodiments, the volume of cysteine ​​source solution added to the bioreactor is based on the live cell density. In some embodiments, fed-batch cell culture is performed at approximately 1 × 10⁻⁶ cells / year. 6 To approximately 8 × 10 6 Seeding density is [number] live cells / mL. In some embodiments, the peak density of live cells is 15 × 10⁶ cells / mL. 6 To approximately 50 × 10 6 Cells / mL. In some embodiments, the viable cell density is approximately 15 × 10⁶ cells / mL. 6 To approximately 50 × 10 6Viable cells / mL. In some embodiments, the daily feed volume is about 0.5% to about 8.5% of the daily working volume. Techniques for measuring viable cell density are known in the art and include techniques such as trypan blue exclusion assays and dielectric constant measurements, for example, Riss et al., Cell Viability Assays, The Assay Guidance Manual, 2016 and Rosner et al., Bioengineering, 9, 2022, which are hereby incorporated herein by reference in their entirety. In some embodiments, the method further includes harvesting when cell viability in cell culture decreases to about 70% to about 80%. In some embodiments, the daily feed volume is about 0.5% to about 8.5% of the daily working volume. In some embodiments, the cells are mammalian host cells transformed with genes encoding biomolecules of interest. In some embodiments, the mammalian host cells are CHO cells, HeLa cells, HEK293 cells, Vero cells. In some embodiments, the mammalian host cells are CHO cells. In some embodiments, the biomolecule includes an antibody or an antigen-binding fragment thereof, an enzyme, a viral vector containing a therapeutic transgene, or a recombinant enzyme. In some embodiments, the antibody or an antigen-binding fragment thereof includes a monoclonal antibody (mAb), a bispecific antibody, a trispecific antibody, or an immunoglobulin monovariable domain.

[0108] The cells described herein typically require a cysteine ​​source for culture. This cysteine ​​source is usually provided, for example, by cysteine ​​in the cell culture medium. Generally, the cells in question include eukaryotic cells, such as yeast or higher eukaryotic cells, and such higher eukaryotic cells include established cell lines derived from insects and mammals. Examples of suitable mammalian cells include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell, 23, 1981), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines grown in serum-free medium (Rasmussen et al., Cytotechnology, 28, 1998), HeLa cells, BHK (ATCC CRL10) cell lines, and the CVI / EBNA cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) (as described by McMahan et al., EMBO J, 10, 1991), human embryonic kidney cells (such as 293, 293 EBNA, or MSR). 293) Human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro cultures of primary tissues, primary explants, HL-60, U937, HaK, or Jurkat cells. Optionally, mammalian cell lines such as HepG2 / 3B, KB, NIH 3T3, or S49 can be used as cells for the methods provided herein.

[0109] In some embodiments, the cells are CHO cells. CHO cells are well known in the art. See, for example, Xu et al., Nat Biotechnol, 29, 2011. In some embodiments, the cells are DP12 host cells. In some embodiments, the host cells are DUXB-11-derived DHFR-deficient DP12 cells. In some embodiments, the cells are CHO-K1 host cells. In some embodiments, the cells are DHFR-positive CHO-K1 host cells. In some embodiments, the cells are CHOK1M cells.

[0110] In some embodiments, the cell is a mouse host cell. In some embodiments, the cell is an Sp2 / O host cell. In some embodiments, the cell is an NSO host cell.

[0111] In some embodiments, the cells are hybridomas. In some embodiments, the hybridomas are antibody-producing cells, wherein antibody-producing cells are collected from the host after immunization with an antigen. In some embodiments, antibody-producing cells are fused with myeloma cells. In some embodiments, the cells are mouse myeloma-derived cell lines.

[0112] Alternatively, the cell can be a lower eukaryote, such as yeast. Suitable yeasts include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida species, or any yeast strain capable of expressing heterologous peptides.

[0113] As described herein, the methods provided can be used to produce products, such as peptides, from cells. In some embodiments, this document provides a method for producing a peptide, the method comprising culturing cysteine-dependent cells in a cell culture medium prepared according to any of the embodiments provided herein, and obtaining the peptide therefrom. In some embodiments, the peptide is a therapeutic peptide or a precursor thereof. In some embodiments, the therapeutic peptide or its precursor is an antibody or a fragment thereof. In some embodiments, the antibody or its fragment is an antibody-drug conjugate.

[0114] In some respects, this document provides methods for using cell cultures, including concentrated cell culture media containing a cysteine ​​source solution containing the thiazolidinedane described herein. Exemplary methods for using the fed-batch culture techniques taught herein include fed-batch culture techniques, perfusion culture techniques, and online dilution culture techniques. The culture media described herein can be used at various scales, from small-scale research cell cultures to commercial-scale production cultures. D. Reagent kits, components, compositions, and systems

[0115] In other respects, this document also provides kits, components, compositions, and systems comprising a cysteine ​​source solution (or a component of the reaction). For example, in some embodiments, this document provides kits comprising components for performing the reactions taught herein. In some embodiments, this document provides a kit comprising a cysteine ​​source solution or a precursor thereof produced according to the methods described herein. In some embodiments, the kit includes instructions for use according to the methods described herein. In some embodiments, this document provides a cysteine ​​source solution produced according to the methods described herein. In some embodiments, a cell culture medium is provided comprising a cysteine ​​source solution produced according to the methods described herein. In some embodiments, the cysteine ​​source solution and / or cell culture medium are sterile (e.g., by sterile filtration).

[0116] In some aspects, this document provides a concentrated basal cell culture medium comprising the thiazolidinedonum described herein, for example, synthesized as described herein. In some embodiments, the concentrated basal cell culture medium is about 2x to about 10x, such as any one of about 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x. In some embodiments, the basal cell culture medium is substantially free of cysteine ​​or cystine, such as containing 2% or less of cysteine ​​or cystine.

[0117] In some embodiments, this document provides a cell culture system comprising: cells; and a basal cell culture medium mixed with a cysteine ​​source solution provided herein. In some embodiments, the basal cell culture medium is substantially free of cysteine ​​and cystine. Example Example 1: Materials and Methods Thiazolidine Synthesis

[0118] Cysteine ​​(Cys) (CAS: 52-90-4, Sigma-Aldrich, catalog C7352) and keto acids were mixed in aqueous solutions with different pH values, concentrations, and ratios under ambient conditions (room temperature) to form thiazolidinediones (cysteine ​​source solutions). Three keto acids were used in the optimized synthesis: sodium pyruvate (Pyr) (CAS: 113-24-6, Thermo Fisher Scientific, catalog AAJ6184018), α-ketoglutarate (AKG) (CAS: 328-50-7, Sigma-Aldrich, catalog K1128), and oxaloacetic acid (Oxa) (CAS: 328-42-7, Sigma-Aldrich, catalog O7753). DTNB solution:

[0119] The synthesis of thiazoline was indirectly measured using DTNB assay. The conversion of cysteine ​​(Cys) to thiazoline occurs via a reaction with a keto acid, leading to the formation of a thiocyclic structure. The disappearance of free thiol groups during the reaction was quantified using DTNB assay, with readout obtained by absorbance measurement at 412 nm.

[0120] DTNB solutions are prepared by dissolving 2.5 mM 5,5'-dithio-bis-(2-nitrobenzoic acid) (CAS: 69-78-3, Thermo Fisher Scientific, Catalogue No. 22582) in 0.5 M Tris buffer. A strong buffer is used because DTNB will decompose outside the pH range of 7–8. The solution is used on the day it is prepared and stored in a freezer when not in use.

[0121] The primary objective in preparing the diluent is to achieve the optimal thiol concentration for DTNB determination and to ensure that the pH of the experimental solution falls within the ideal range of 7-8. The thiol solution sample is diluted with Tris buffer to a final concentration of up to 1 mM, and the pH of the solution is measured and maintained within the appropriate range for determination.

[0122] Mix 50 µL of diluted sample with 150 µL of DTNB solution in the wells of a 96-well plate with a clear bottom. Mix the contents by carefully pipetting up and down, ensuring no air bubbles form in the wells. Prepare control wells with 50 µL of deionized water (RODI) and 150 µL of DTNB solution as blank references. Prepare fresh blanks at each time point. This practice ensures that any potential decomposition of the DTNB stock solution is properly accounted for during absorbance measurements and maintains the accuracy of the determination. Measure the absorbance of the resulting well plate at 412 nm using a SpectraMax plate reader. LC-MS:

[0123] The purity of thiazoline was confirmed by direct measurement of thiazoline synthesis using LC-MS analysis. LC-MS analysis was performed using a Thermo Q-Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, USA) equipped with a heated electrospray ionization source (HESI-II) and a Vanquish UPLC system. LC separation was achieved by injecting 10 μL of sample or standard solution into a Hypercarb porous graphite carbon HPLC column with 4.6 mm id × 100 mm dimensions and a 4.6 μm particle size (Thermo Fisher Scientific, San Jose, USA, part number 35003-104630). Mobile phase A consisted of 20 mM ammonium formate, 0.1% FA, and water, while mobile phase B consisted of 20 mM ammonium formate, 0.1% FA, and 90% MeOH: water. The method was set up with a gradient from 10% to 80% B from 0 to 10 min, followed by a gradient from 80% to 100% B from 10 to 12 min, and held isocratic until 18 min, then held isocratic at 0% B from 18 to 20 min. The total run time was 20 min, and the flow rate was 0.5 mL / min. Samples were analyzed by electrospray ionization in positive polarity from 60 to 900 m / z in full-scan MS mode. During the analysis, the resolution was fixed at 70,000 FWHM, with an automatic gain control (AGC) target of 3.0 × 10⁻⁶. 6 The maximum ion implantation time is 100 ms. The software used to operate the LC-HRMS is Xcalibur™ (version 4.1).

[0124] For data-dependent acquisition (DDA), the mass spectrometer was operated in positive ion mode, acquiring survey scans at a resolution of 70,000, with a maximum injection time of 100 ms and a target value of 3.0 × 10⁻⁶ m / s over a mass range of 60 to 900 m / s. 6 The target candidate qualities of 192.0322, 250.0376, and 236.0220 Daltons were added to the inclusion list. Fragments were recorded at a resolution of 35,000 Daltons, with an injection time of 50 ms and a target value of 1 × 10⁻⁶. 5 Set the separation window to 4 m / z and the normalized collision energy (NCE) to 35. Fed-batch cell culture:

[0125] Internal cell culture media and feed were used in cell culture experiments. The process was conducted in an Ambr 15 bioreactor with up to 48 disposable cell culture bioreactor vessels (Sartorius StedimBiotech GmbH) or a 3 L stirred tank bioreactor with a DeltaV controller. Ambr15 culture was performed using a platform strategy: agitation at 1400 rpm, air saturation at 40% DO, pH ≥ 6.9, and temperature variation from 36.5°C to 34°C on day 4. To prevent foaming, 20 μL of FoamAway™ Irradiated AOF (animal-free) antifoaming agent was added every 48 h. Culture in a 3 L bioreactor was performed using an enhanced process: VCD inoculation target of 10 Mvc / mL, agitation at 250 rpm, air saturation at 40% DO, pH ≥ 6.9, and temperature variation from 36.5°C to 34°C on day 2. Example 2: Optimization of thiazolidin production Optimization Test 1

[0126] Fresh Cys solution was mixed with fresh Pyr (Cys / Pyr), AKG (Cys / AKG), and Oxa (Cys / Oxa) solutions at a ratio of 1:3 to Cys, resulting in a final concentration of 11.25 mM. The three mixtures were prepared under acidic pH conditions with initial pH values ​​of 5.5, 2.1, and 2.2, respectively. The initial pH values ​​for the three mixtures were then adjusted to 7.0 and 12.0, respectively, by titration with NaOH solution. DTNB measurements were performed at T = 0.5, 2.5, and 5 h to monitor the rate of free Cys disappearance, which corresponds to the thiazolidinyl ester synthesis rate. See also Figure 1ASee Table 1. The results revealed that the Cys / Pyr mixture had the highest synthesis rate, followed by the Cys / AKG and Cys / Oxa mixtures. Cys / Pyr exhibited the highest synthesis rate at pH 12, while Cys / AKG and Cys / Oxa showed their highest rates at pH 7. Table 1: Cys disappearance determined by DTNB in ​​Test 1 Optimization Test 2

[0127] The substrate concentration and ratio for thiazolidin synthesis were optimized using Cys / Pyr as an example. The assay involved three concentration conditions: 30 mM Cys and Pyr mixed at a 1:1 ratio, 150 mM Cys and Pyr mixed at a 1:3 ratio, and 450 mM Cys and Pyr mixed at a 1:1 ratio. All three conditions were tested at initial pH values ​​of 4.6 and 12. DTNB determinations were performed for each mixture condition at T = 1, 2, 3, and 4 h to monitor the rate of free Cys disappearance, which corresponds to the thiazolidin synthesis rate. See also Figure 2 See Table 2. The results show that higher substrate concentrations of Cys / Pyr resulted in higher synthesis rates, and that higher substrate concentrations helped to balance the Cys / Pyr substrate ratio to 1:1 and achieve appropriate synthesis rates. For the Cys / Pyr mixture, adjusting the pH to the initial pH 12 exhibited a higher synthesis rate than the initial pH 4.6, consistent with the trend observed in Test 1 regarding the effect of pH. Table 2: Cys disappearance determined by DTNB in ​​Test 2 Optimize Test 3

[0128] To develop optimal synthetic methods for all three thiazolidines—Cys / Pyr, Cys / AKG, and Cys / Oxa—an initial pH of 7 (achieved by titration to neutral pH with NaOH) and a high initial concentration were selected for further optimization tests. Based on the pH-related trends observed in optimization test 1, an initial pH of 7 should yield the highest synthetic rates compared to acidic or alkaline pH for Cys / AKG and Cys / Oxa.

[0129] The initial concentration of Cys was increased to 1800 mM and mixed with Pyr (Cys / Pyr), AKG (Cys / AKG), or Oxa (Cys / Oxa) at a 1:1 concentration ratio, with the acidic or neutral pH adjusted by NaOH. DTNB measurements were performed for each mixture condition at T = 1, 2, 3, and 5 h to monitor the rate of free Cys disappearance, which corresponds to the thiazolidinyl ester synthesis rate. See also Figure 3A See Table 3. The results show that for thiazolidinedioides of Cys / Pyr, Cys / AKG, and Cys / Oxa, all three reactions of the substrate at a 1:1 ratio were completed in almost one hour at a high initial concentration of 1800 mM at an initial pH of 7. Table 3: Cys disappearance determined by DTNB in ​​Test 3

[0130] In optimization tests 1, 2, and 3, the effects of pH and concentration on the thiazolidin synthesis rate were consistent. Therefore, a 1:1 Cys:α-keto acid ratio, a concentration range of 900–1800 mM, and pH 7 were determined to be the optimal initial conditions for the conversion of Cys to high-purity thiazolidin in aqueous solution.

[0131] like Figure 1B and Figure 3B The differences shown between the two, optimization of pH, concentration, and ratio resulted in a significant reduction in reaction time, for example, achieving complete reaction in approximately 1 hour after optimization. Figure 3B ), and observed in Figure 3B A substrate in a 1:1 ratio produces a pure product (see reference). Figure 1B An excess of α-keto acid was observed after the reaction. Example 3: Confirmation of high-purity and stable synthetic thiazolidinedonous solutions.

[0132] Thiazolidine solutions (cysteine ​​source solutions) were prepared using optimal methods and added to mammalian cell culture feed during culture medium preparation. The structures and associated purities of the thiazolidinedions in the solutions were confirmed using LC-MS and HPLC.

[0133] The sample solution was diluted 100-fold with mobile phase A, and the LC peaks were eluted with retention times of 6.04, 11.18, and 9.20 min for Cys-Pyr, Cys-AKG, and Cys-Oxa thiazolidinyl samples, respectively. Figure 4A Repeat analysis was performed, and the chromatograms of thiazolidinyl samples of Cys-Pyr, Cys-AKG, and Cys-Oxa were obtained. Figure 4BProvided in [the relevant section]. Relevant mass spectra of Cys-Pyr, Cys-AKG, and Cys-Oxa were obtained (data not provided). In repeat analyses, some amounts of Cys-Oxa were observed to be converted to Cys-Pyr. The chemical structures of the proposed complexes are shown in Figure 5, and the theoretical exact mass of each molecule was calculated using ChemDraw Professional software (version 22.2.0.3300). Protonated molecular weights at 192.0322, 250.0376, and 236.0220 Daltons were identified for the three complexes of interest. For Cys-Pyr, [the relevant data were obtained] in high-resolution mass spectrometry ([data not provided]). Figure 6 The strongest peak at 192.0322 m / z in the C6H spectrum is related to the C6H spectrum. 10 NO4S + The theoretical single isotopic mass of 192.0325 matches, and the remaining peaks also appear to be isotopic matches. However, a detailed structure cannot be specified by this result because it does not provide the atomic arrangement within the molecule. To further elucidate the structure, tandem mass spectrometry was used to provide fragmented patterns to aid interpretation. Therefore, 192.0325 m / z (retention time 6.04 min) was chosen as the parent ion of DDA. The structure of the daughter ion was predicted using ChemDraw. The spectroscopic results are shown in […]. Figure 7A and Figure 7B The diagram shows the predicted fragment structure applied to tandem mass spectrometry.

[0134] Furthermore, for Cys-AKG and Cys-Oxa, 250.0376 was found ( Figure 8 ) and 236.0220 ( Figure 10 The spectral characteristics of ) are respectively related to C8H 12 NO6S + and C7H 10 NO6S + The theoretical mass matching was achieved. Similar to the analysis with Cys-Pyr, the parent ions at 250.0376 at 11.18 min and 236.0220 at 9.20 min were fragmented into daughter ions, such as... Figure 9A and Figure 9B as well as Figure 11A and Figure 11B The diagram shows the predicted fragment structure applied to tandem mass spectrometry. These results are consistent with the proposed chemical structure shown in Figure 5, thus confirming the formation of the complex of interest.

[0135] In addition, the purity was tested by injecting a 10-fold diluted solution onto LC-MS as described above, and the results showed that the resulting thiazolidinedione solution (cysteine ​​source solution) was substantially pure for thiazolidinedione.

[0136] It should be noted that we observed in Cys / Oxa solution that the complex is a thiazolidinyl mixture of Cys-Pyr and Cys-Oxa. Without being bound by theory, the mixture of Cys-Pyr and Cys-Oxa is attributed to the following proposed chemical reaction of Cys-Oxa:

[0137] A complex of mass 236.0220 was used as a chemical marker to quantify the Cys-Oxa concentration in cell culture samples.

[0138] NMR studies were also conducted, which confirmed the production of certain thiazolidinedanes described in this paper (data not shown). Stability of synthetic thiazolidinediones in mammalian cell culture feed

[0139] The internal feed was prepared using a 20x concentrated thiazolidin solution (cysteine ​​source solution; relative to the final desired concentration in the feed matrix). The final feed solution was aged at 4°C for 10 days. As shown in Table 4, the solution exhibited negligible free thiols, indicating the stability of the thiazolidin in the feed matrix, demonstrating their reliability and durability. Table 4: Free thiols in cell culture feed variants detected by DTNB assay Example 4: Performance evaluation of synthetic thiazolidinediones as the sole cysteine ​​source in cell culture

[0140] During fed-batch cell culture in ambr15, the cell culture performance and product quality of feed media with unique Cys variants were evaluated using four different CHO cell lines (CL1, CL2, CL3, and CL4) that produced monoclonal antibodies or fusion proteins as different Cys delivery methods. Three synthetic thiazolidinyl options—Cys-Pyr, Cys-AKG, and Cys-Oxa—were compared with three other Cys delivery methods: a mixture of Cys and n-acetyl-Cys (Cys / NAC), a bis-lysyl-cysteine ​​peptide (KCCK), and a high-pH cysteine ​​side solution.

[0141] In cell culture performance ( Figure 12-14 Regarding the synthesis of thiazolidinedioides using Cys-Pyr, Cys-AKG, and Cys-Oxa, and the product quality properties (not shown), no significant differences were found compared to other known Cys delivery methods. Therefore, the cysteine ​​source solution presented in this paper provides sufficient nutrients for culturing cysteine-dependent cells while avoiding the known drawbacks of directly supplementing cell culture medium with cysteine. Example 5: Metabolite analysis of thiazolidinediones used as the sole cysteine ​​source in cell culture

[0142] Metabolite analysis of daily consumed culture media from a 3 L bioreactor using an enhanced fed-batch method was performed by LC-MS. Specifically, the analysis was conducted under three conditions: cell culture feed paired with the cystine-side solution according to the examples above, cell culture feed containing Cys-Pyr thiazolidinone, and cell culture feed containing Cys-AKG thiazolidinone, wherein the Cys-Pyr and Cys-AKG stock solutions were prepared according to the methods described in the examples above.

[0143] The normalized concentration of cysteine ​​source in the daily consumed culture medium (free cysteine ​​converted to cystine due to oxidation in the measurement environment) Figures 15A-15C As shown in the image. Provided Figure 15D-15E To show Figures 15A-15C A magnified view of some of the data presented. For example... Figures 15A-15C As shown, cells can consume thiazolidinediones as a monocysteine ​​source. (As...) Figures 17A-17B As shown, measurements of free pyruvate and α-ketoglutarate indicate that cells can convert thiazolidinediones into native cysteine ​​and associated α-keto acids. A comparable amount of cysteine ​​source was consumed between the cysteine-side solution and thiazolidinedione conditions. Comparable cell culture performance, as assessed via LDH, viability, VCD, and titer, was observed between the test conditions. Figures 16A-16E Therefore, these data demonstrate that the cysteine ​​source solution containing thiazolidinedioides described in this paper can be used as a single Cys source for cell culture of cells capable of producing products such as recombinant peptides, e.g., antibodies.

Claims

1. A method for preparing a cysteine ​​source solution suitable for cell culture, the method comprising: To react cysteine ​​with an α-keto acid compound having formula (A) (A)， Alternatively, the corresponding carboxylate anion reacts in an aqueous solution to produce a cysteine ​​source solution containing a thiazoline compound having formula (B). (B)， Where R is an optionally substituted C1-C6 alkyl group, The reaction takes place under the following conditions: The concentration of this cysteine ​​is at least about 30 mM, and / or The concentration of the α-keto acid compound having formula (A) or its corresponding carboxylate anion is at least about 30 mM, and / or pH is from about 1.5 to about 12.5, and / or The ratio of the cysteine ​​to the α-keto acid compound having formula (A) is about 2:1 to about 1:

2.

2. The method of claim 1, wherein the reaction is not carried out in a cell culture medium.

3. The method of claim 1 or 2, wherein the reaction is carried out for 5 hours or less.

4. The method according to any one of claims 1-3, wherein the residual concentration of cysteine ​​or cystine in the cysteine ​​source solution is about 0.5 mM or lower.

5. The method according to any one of claims 1-4, wherein the residual concentration of the α-keto acid compound having formula (A) is about 0.5 mM or less.

6. The method according to any one of claims 1-5, wherein the purity of the thiazoline compound having formula (B) in the resulting cysteine ​​source solution is at least about 95% of the original cysteine ​​concentration.

7. The method according to any one of claims 1-6, wherein the concentration of cysteine ​​reacting with the α-keto acid compound having formula (A) is from about 30 mM to about 5000 mM.

8. The method according to any one of claims 1-7, wherein the concentration of cysteine ​​reacting with the α-keto acid compound having formula (A) is at least about 150 mM.

9. The method according to any one of claims 1-8, wherein the concentration of cysteine ​​reacting with the α-keto acid compound having formula (A) is at least about 1800 mM.

10. The method according to any one of claims 1-9, wherein the α-keto acid compound having formula (A) is pyruvate (Pyr) or pyruvate salt.

11. The method according to any one of claims 1-9, wherein the α-keto acid compound having formula (A) is α-ketoglutaric acid (AKG) or α-ketoglutarate.

12. The method according to any one of claims 1-9, wherein the α-keto acid compound having formula (A) is oxaloacetic acid (Oxa) or an oxaloacetic acid salt.

13. The method according to any one of claims 1-12, wherein the concentration of the α-keto acid compound having formula (A) reacting with cysteine ​​is from about 30 mM to about 5000 mM.

14. The method according to any one of claims 1-13, wherein the concentration of the α-keto acid compound having formula (A) reacting with cysteine ​​is at least about 150 mM.

15. The method according to any one of claims 1-14, wherein the concentration of the α-keto acid compound having formula (A) reacting with cysteine ​​is at least about 1800 mM.

16. The method according to any one of claims 1-15, wherein the reaction of cysteine ​​with the α-keto acid compound having formula (A) is carried out at a pH from about 3 to about 12.

17. The method according to any one of claims 1-16, wherein the reaction of cysteine ​​with the α-keto acid compound having formula (A) is carried out at a pH of about 4.

1.

18. The method according to any one of claims 1-16, wherein the reaction of cysteine ​​with the α-keto acid compound having formula (A) is carried out at a pH of about 7.

19. The method according to any one of claims 1-16, wherein the reaction of cysteine ​​with the α-keto acid compound having formula (A) is carried out at a pH of about 12.

20. The method according to any one of claims 1-19, wherein the cysteine ​​and the α-keto acid compound having formula (A) react at a cysteine:α-keto acid concentration ratio from 1:1 to 1:

2.

21. The method according to any one of claims 1-20, wherein the cysteine ​​and the α-keto acid compound having formula (A) react at a cysteine: α-keto acid concentration ratio of 1:

1.

22. The method of any one of claims 1-21, wherein the cysteine ​​is L-cysteine.

23. A method for preparing a cysteine ​​source solution suitable for cell culture, the method comprising: At least about 1800 mM of cysteine ​​is reacted with at least about 1800 mM of pyruvate or the corresponding carboxylate anion in an aqueous solution to produce a cysteine ​​source solution containing 2-methyl-1,3-thiazolidin-2,4-dicarboxylic acid. The reaction begins at a pH of approximately 3.5 to approximately 12.

24. The method of claim 23, wherein the reaction is initiated at a pH of about 4.

1.

25. The method of claim 23, wherein the reaction is initiated at a pH of about 7.

26. A method for preparing a cysteine ​​source solution suitable for cell culture, the method comprising: At least about 1800 mM of cysteine ​​is reacted with at least about 1800 mM of α-ketoglutaric acid or the corresponding carboxylate anion in an aqueous solution to produce a cysteine ​​source solution containing 2-(2-carboxyethyl)thiazolidin-2,4-dicarboxylic acid. The reaction begins at a pH of approximately 4 to approximately 8.

27. The method of claim 26, wherein the reaction is carried out at a pH of about 7.

28. A method for preparing a cysteine ​​source solution suitable for cell culture, the method comprising: At least about 1800 mM of cysteine ​​is reacted with at least about 1800 mM of oxaloacetic acid or the corresponding carboxylate anion in an aqueous solution to produce a cysteine ​​source solution containing 2-(carboxymethyl)thiazolidin-2,4-dicarboxylic acid. The reaction begins at a pH of approximately 4 to approximately 8.

29. The method of claim 27, wherein the reaction is carried out at a pH of about 7.

30. The method of any one of claims 23-29, wherein the reaction is carried out for 2 hours or less.

31. The method of any one of claims 23-30, wherein the residual concentration of cysteine ​​or cystine in the cysteine ​​source solution is about 0.5 mM or lower.

32. The method according to any one of claims 23-31, wherein the residual concentration of the corresponding carboxylic acid anion of pyruvate, α-ketoglutarate, or oxaloacetic acid, or any one thereof, is about 0.5 mM or less.

33. The method according to any one of claims 23-32, wherein the purity of the thiazolidinedion in the resulting cysteine ​​source solution is at least about 95%.

34. A cysteine ​​source solution, wherein the cysteine ​​source solution is produced according to the method according to any one of claims 1-33.

35. A method for preparing a cell culture medium, the method comprising: The cell culture medium is prepared by mixing a basic cell culture medium with a cysteine ​​source solution, wherein the cysteine ​​source solution is produced according to any one of claims 1-33.

36. The method of claim 35, wherein the cell culture medium is suitable for cysteine-dependent cells.

37. The method of claim 35 or 36, wherein the basal cell culture medium is not suitable for cysteine-dependent cells without further modification.

38. The method of any one of claims 35-37, wherein the basal cell culture medium has a pH of about 5 to about 8.

39. The method of any one of claims 35-38, further comprising preparing a thiazolidinedione side solution according to any one of claims 1-29.

40. The method of any one of claims 35-39, further comprising adjusting the pH of the basal cell culture medium.

41. The method of any one of claims 35-40, further comprising adding the cell culture medium to an appropriate amount after mixing the basal cell culture medium and the cysteine-side solution.

42. The method of any one of claims 35-41, wherein the basic cell culture medium is substantially free of cysteine ​​and cystine.

43. A method for culturing cysteine-dependent cells, the method comprising culturing the cysteine-dependent cells in a cell culture medium prepared according to any one of claims 35-42.

44. A method for producing a polypeptide, the method comprising culturing cysteine-dependent cells in a cell culture medium prepared according to any one of claims 35-42, and obtaining the polypeptide therefrom.

45. The method of claim 44, wherein the polypeptide is a therapeutic polypeptide or a precursor thereof.

46. ​​The method of claim 45, wherein the therapeutic polypeptide or its precursor is an antibody or a fragment thereof.

47. The method of claim 46, wherein the antibody or a fragment thereof is an antibody-drug conjugate.

48. A cell culture system comprising: Cells; and A basic cell culture medium mixed with the cysteine ​​source solution as described in claim 34 or its dry or semi-dry form.

49. The cell culture system of claim 48, wherein the basic cell culture medium is substantially free of cysteine ​​and cystine.

50. A method for culturing cells, the method comprising: Provide the cell culture medium, The cell culture medium contains a basal medium and a cysteine ​​source solution, which contains thiazolidinone. The cell culture medium contains cysteine ​​at a concentration not exceeding approximately 20% relative to the amount of thiazolidin in the cell culture medium; and Culture the cell.

51. The method of claim 50, wherein the cell is a cysteine-dependent cell.

52. The method of claim 50 or 51, wherein the cell culture medium is unsuitable for culturing the cells without further containing cysteine ​​or a source thereof.

53. The method of any one of claims 50-52, wherein the cysteine ​​source solution is the sole cysteine ​​source for the cell.

54. The method of any one of claims 50-53, wherein the cell culture medium does not contain cysteine.

55. The method of any one of claims 50-54, wherein the cysteine ​​source solution is produced according to any one of claims 1-33.

56. The method of any one of claims 50-55, wherein the cell culture comprises fed-batch cell culture.

57. The method of claim 56, wherein the fed-batch cell culture lasts for 1 to 21 days.

58. The method of claim 57, wherein the fed-batch cell culture lasts for 14 days.

59. The method of any one of claims 56-58, wherein the cysteine ​​source solution has a thiazolidin concentration of about 5 mM to about 50 mM.

60. The method of claim 59, wherein the cysteine ​​source solution is added to the cell culture daily.

61. The method of claim 59 or 60, wherein the cysteine ​​source solution is added throughout the fed-batch culture process.

62. The method of claim 60 or 61, wherein the volume of the cysteine ​​source solution added to the cell culture is based on the live cell density.

63. The method of any one of claims 56-62, wherein the fed-batch cell culture is inoculated at a cell density of about 1 x 10 6 to about 8 x 10 6 viable cells / mL.

64. The method of any one of claims 56-63, wherein the peak density of viable cells is 15 x 10 6 to about 50 x 10 6 cells / mL.

65. The method of any one of claims 56-64, wherein the cell culture has a viable cell density of about 15 x 10 6 to about 50 x 10 6 viable cells / mL.

66. The method of any one of claims 56-65, further comprising harvesting when the percentage of cell viability in the cell culture decreases to about 70% to about 80%.

67. The method of any one of claims 56-66, wherein the daily feed volume is about 0.5% to about 8.5% of the daily working volume.

68. The method of any one of claims 1-67, wherein the cell is a mammalian host cell transformed with a gene encoding a biomolecule of interest.

69. The method of claim 68, wherein the mammalian host cell is a CHO cell, a HeLa cell, a HEK293 cell, or a Vero cell.

70. The method of claim 68, wherein the mammalian host cell is a CHO cell.

71. The method of any one of claims 68-70, wherein the biomolecule comprises an antibody or an antigen-binding fragment thereof, an enzyme, a viral vector containing a therapeutic transgene, a gene-editing complex, or a recombinase.

72. The method of claim 71, wherein the antibody or its antigen-binding fragment comprises a monoclonal antibody (mAb), a bispecific antibody, a trispecific antibody, or an immunoglobulin monovariable domain.

73. A concentrated basal cell culture medium comprising thiazolidinediones.

74. The concentrated basal cell culture medium of claim 73, wherein the concentrated basal cell culture medium is 2x to 10x.

75. The concentrated basal cell culture medium as described in claim 73 or 74, wherein the basal cell culture medium is substantially free of cysteine ​​or cystine.

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