Method for determining reduction sensitivity of proteins

Abstract

Provided herein are methods for determining the reduction sensitivity of a protein and for adjusting the culture conditions of the protein based on the reduction sensitivity.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 406,994, filed September 15, 2022, and U.S. Provisional Patent Application No. 63 / 497,629, filed April 21, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to methods for determining the reduction sensitivity of a protein and for adjusting the culture conditions of the protein based on the reduction sensitivity. [Background technology]

[0003] Antibody reduction is an area of ​​growing interest and research in the field of cell culture process development. Antibody reduction refers to the process in which disulfide bonds in monoclonal antibodies are broken in a reducing environment, resulting in the degradation of antibody monomers into inactive by-products such as half antibodies and separate heavy and / or light chains. Although antibody reduction is a risk for all processes, different antibodies have different susceptibility to reduction due to the number and structure of disulfide bonds.

[0004] As the cell culture process progresses and cells die, enzymes from the thioredoxin and glutathione reductase pathways are released, creating an increasingly reducing environment for the cell culture medium, leading to antibody reduction. This reduction can occur at multiple stages of the cell culture process: in the bioreactor itself, during clarification, in the harvest holding vessel, and even during Protein A processing. The harvest holding vessel represents the greatest risk of reduction, especially if cells are lysed during clarification. While antibody reduction is a risk for all monoclonal antibody processes, it has been shown that different antibodies can exhibit very different susceptibilities to reduction due to the number and structure of disulfide bonds present. These susceptibilities can be broadly characterized by antibody subtype (e.g., IgG1, IgG2, etc.) and light chain subtype (kappa and lambda), although differences in reduction susceptibility exist even within these broad classes. Some methods, such as chemical reduction assays, are useful for predicting which antibodies will have problems in production and process development, but cannot be used to evaluate different culture conditions because they only take purified antibodies and subject them to chemical reduction reagents, which is very different from the enzyme-induced reduction and complex environment present in cell culture processes. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is a need in the art for a method to determine the reduction sensitivity of proteins that mimics the actual culture environment to obtain rapid and accurate results. [Means for solving the problem]

[0006] Provided herein are small-scale assays, including bench-scale assays, that can be used to accurately predict the risk of antibody reduction of different proteins (e.g., monoclonal antibodies) during cell culture processes. The provided assays can be used to modify manufacturing processes to prevent or reduce the level of protein reduction. Applicants unexpectedly discovered that assay performance is optimized by using glass vials with an internal volume of about 1 mL to about 3 mL (e.g., about 2.5 mL) with a headspace of less than 100 microliters.

[0007]

[0013] Accordingly, in one aspect, provided herein is a method for determining the reduction sensitivity of a protein, the method comprising: (a) providing a first cell culture medium and a second cell culture medium, wherein the first cell culture medium and the second cell culture medium are identical except that the first cell culture medium comprises a cell lysate and the second cell culture medium is essentially free of the cell lysate; (b) mixing the first cell culture medium and the second cell culture medium in a series of ratios, thereby preparing a set of samples; (c) adding a protein to each sample of the set of samples and incubating each sample of the set of samples in an airtight container (i) having an internal volume of about 1 mL to about 3 mL and (ii) having a headspace of less than 100 microliters; (d) determining a level of protein reduction and / or redox potential in each sample of the set of samples; and (e) determining the reduction sensitivity of the protein based on the level of protein reduction and / or redox potential determined for each sample of the set of samples in step (d), thereby determining the reduction sensitivity of the protein.

[0008] In some embodiments, the cell lysate was obtained by cell homogenization.

[0009] In some embodiments, the cell lysate is centrifuged to remove cell debris prior to step (b).

[0010] In some embodiments, the second cell culture medium is obtained by removing essentially all of the cells from the cell culture medium.

[0011] In some embodiments, the protein is a recombinant protein.

[0012] In some embodiments, the recombinant protein is an antibody, or an antigen-binding fragment thereof.

[0013] In some embodiments, the antibody is a monoclonal antibody.

[0014] In some embodiments, the level of protein reduction is determined by assessing the oxidation state of disulfide bonds in the protein.

[0015] In some embodiments, the redox potential is determined by measuring one or more of lactate dehydrogenase (LDH), nicotinamide adenine dinucleotide (NAD+) / reduced NAD+ (NADH) redox pair, NADP+ / reduced NADP+ (NADPH) redox pair, NADPH, thioredoxin (Trx), and / or glutathione (Grx).

[0016] In some embodiments, the redox potential is determined by using a redox potential probe.

[0017] In some embodiments, the level of protein reduction is determined by the amount of intact and / or reduced protein after incubation with each sample in a set of samples.

[0018] In some embodiments, the amount of intact and / or reduced protein is determined using a native SDS-PAGE gel.

[0019] In some embodiments, the amount of intact and / or reduced protein is determined using a microfluidic device.

[0020] In some embodiments, the methods described herein include determining a level of protein reduction and a redox potential in each sample of the set of samples, and further generating a graph of protein reduction susceptibility based on the determined redox potential and the determined level of intact or reduced protein for each sample of the set of samples.

[0021] In some embodiments, the airtight container is an airtight glass vial.

[0022] In some embodiments, the airtight container has an internal volume of about 2 mL to about 2.5 mL.

[0023] In some embodiments, the airtight container has a headspace of less than 50 microliters, less than 10 microliters, or 0 microliters.

[0024] In some embodiments, the protein is incubated with each sample of the set of samples for about 0.5 to about 4 hours prior to step (c).

[0025] In some embodiments, the methods described herein further comprise comparing the reduction susceptibility of the protein to a reference standard.

[0026] In some embodiments, the reference standard is a standard curve of the reduction sensitivity of a reference protein.

[0027] In some embodiments, the methods described herein further comprise selecting proteins for further development based on the reduction sensitivity of the proteins.

[0028] In another aspect, a method of modifying culture conditions of a cell culture medium or bioprocessing conditions of a cell culture fluid used to produce a protein includes: (a) providing a first cell culture fluid and a second cell culture fluid, wherein the first cell culture fluid and the second cell culture fluid are identical except that the first cell culture fluid comprises a cell lysate and the second cell culture fluid is essentially free of the cell lysate; (b) mixing the first cell culture fluid and the second cell culture fluid in a series of ratios, thereby preparing a set of samples; and (c) adding protein to each sample of the set of samples, wherein each sample of the set of samples (i) has an internal volume of between about 1 mL and about 3 mL, and (ii) has an internal volume of between about 1 mL and about 3 mL. (d) determining a level of protein reduction and / or redox potential in each sample of the set of samples; (e) determining the reduction sensitivity of the protein based on the level of protein reduction and / or redox potential determined for each sample of the set of samples in step (d); and (f) modifying one or more culture conditions of the cell culture medium or cell culture bioprocessing conditions used to produce the protein based on the reduction sensitivity of the protein.

[0029] In some embodiments, the cell lysate was obtained by cell homogenization.

[0030] In some embodiments, the cell lysate is centrifuged to remove cell debris prior to step (b).

[0031] In some embodiments, the second cell culture medium is obtained by removing essentially all of the cells from the cell culture medium.

[0032] In some embodiments, the protein is a recombinant protein.

[0033] In some embodiments, the recombinant protein is an antibody, or an antigen-binding fragment thereof.

[0034] In some embodiments, the antibody is a monoclonal antibody.

[0035] In some embodiments, the level of protein reduction is determined by assessing the oxidation state of disulfide bonds in the protein.

[0036] In some embodiments, the redox potential is determined by measuring one or more of lactate dehydrogenase (LDH), nicotinamide adenine dinucleotide (NAD+) / reduced NAD+ (NADH) redox pair, NADP+ / reduced NADP+ (NADPH) redox pair, NADPH, thioredoxin (Trx), and / or glutathione (Grx).

[0037] In some embodiments, the redox potential is determined by using a redox potential probe.

[0038] In some embodiments, the level of protein reduction is determined by the amount of intact and / or reduced protein after incubation with each sample in a set of samples.

[0039] In some embodiments, the amount of intact and / or reduced protein is determined using a native SDS-PAGE gel.

[0040] In some embodiments, the amount of intact and / or reduced protein is determined using a microfluidic device.

[0041] In some embodiments, the airtight container is an airtight glass vial.

[0042] In some embodiments, the airtight container has an internal volume of about 2 mL to about 2.5 mL.

[0043] In some embodiments, the airtight container has a headspace of less than 50 microliters, less than 10 microliters, or 0 microliters.

[0044] In some embodiments, the protein is incubated with each sample of the set of samples for about 0.5 to about 4 hours prior to step (c).

[0045] In some embodiments, modifying one or more culture conditions of the cell culture medium or cell culture bioprocessing conditions comprises adjusting one or more of the components of the liquid culture medium composition, pH, temperature, and dissolved oxygen during production of the protein.

[0046] In another aspect, provided herein is a method for generating a standard curve for protein reduction sensitivity, the method including: (a) providing a first cell culture medium and a second cell culture medium, wherein the first cell culture medium and the second cell culture medium are identical except that the first cell culture medium comprises a cell lysate and the second cell culture medium is essentially free of the cell lysate; (b) mixing the first cell culture medium and the second cell culture medium in a series of ratios, thereby preparing a set of samples; (c) adding a protein to each sample of the set of samples and incubating each sample of the set of samples in an airtight container (i) having an internal volume of about 1 mL to about 3 mL and (ii) having a headspace of less than 100 microliters; (d) determining a level of protein reduction and a redox potential in each sample of the set of samples; and (e) generating a standard curve for protein reduction sensitivity based on the determined level of redox potential and the determined level of protein reduction for each sample of the set of samples, thereby generating the standard curve for protein reduction sensitivity.

[0047] In some embodiments, the cell lysate was obtained by cell homogenization.

[0048] In some embodiments, the cell lysate is centrifuged to remove cell debris prior to step (b).

[0049] In some embodiments, the second cell culture medium is obtained by removing essentially all of the cells from the cell culture medium.

[0050] In some embodiments, the protein is a recombinant protein.

[0051] In some embodiments, the recombinant protein is an antibody, or an antigen-binding fragment thereof.

[0052] In some embodiments, the antibody is a monoclonal antibody.

[0053] In some embodiments, the level of protein reduction is determined by assessing the oxidation state of disulfide bonds in the protein.

[0054] In some embodiments, the redox potential is determined by measuring one or more of lactate dehydrogenase (LDH), nicotinamide adenine dinucleotide (NAD+) / reduced NAD+ (NADH) redox pair, NADP+ / reduced NADP+ (NADPH) redox pair, NADPH, thioredoxin (Trx), and / or glutathione (Grx).

[0055] In some embodiments, the redox potential is determined by using a redox potential probe.

[0056] In some embodiments, the level of protein reduction is determined by the amount of intact and / or reduced protein after incubation with each sample in a set of samples.

[0057] In some embodiments, the amount of intact and / or reduced protein is determined using a native SDS-PAGE gel.

[0058] In some embodiments, the amount of intact and / or reduced protein is determined using a microfluidic device.

[0059] In some embodiments, the airtight container is an airtight glass vial.

[0060] In some embodiments, the airtight container has an internal volume of about 2 mL to about 2.5 mL.

[0061] In some embodiments, the airtight container has a headspace of less than 50 microliters, less than 10 microliters, or 0 microliters.

[0062] In some embodiments, the protein is incubated with each sample of the set of samples for about 0.5 to about 4 hours prior to step (c).

[0063] In another aspect, provided herein is a method for determining the reduction sensitivity of a protein, the method comprising: (a) adding a protein to each sample of a set of samples prepared by mixing a first cell culture medium and a second cell culture medium in a series of ratios, wherein the first cell culture medium and the second cell culture medium are identical except that the first cell culture medium comprises a cell lysate and the second cell culture medium is essentially free of cell lysate; (b) incubating each sample of the set of samples in an airtight container (i) having an internal volume of about 1 mL to about 3 mL and (ii) having a headspace of less than 100 microliters; and (c) determining a level of protein reduction and / or redox potential in each sample of the set of samples and determining the reduction sensitivity of the protein based on the level of protein reduction and / or redox potential, thereby determining the reduction sensitivity of the protein.

[0064] In some embodiments, the cell lysate was obtained by cell homogenization.

[0065] In some embodiments, the cell lysate is centrifuged to remove cell debris prior to step (b).

[0066] In some embodiments, the second cell culture medium is obtained by removing essentially all of the cells from the cell culture medium.

[0067] In some embodiments, the protein is a recombinant protein.

[0068] In some embodiments, the recombinant protein is an antibody, or an antigen-binding fragment thereof.

[0069] In some embodiments, the antibody is a monoclonal antibody.

[0070] In some embodiments, the level of protein reduction is determined by assessing the oxidation state of disulfide bonds in the protein.

[0071] In some embodiments, the redox potential is determined by measuring one or more of lactate dehydrogenase (LDH), nicotinamide adenine dinucleotide (NAD+) / reduced NAD+ (NADH) redox pair, NADP+ / reduced NADP+ (NADPH) redox pair, NADPH, thioredoxin (Trx), and / or glutathione (Grx).

[0072] In some embodiments, the redox potential is determined by using a redox potential probe.

[0073] In some embodiments, the level of protein reduction is determined by the amount of intact and / or reduced protein after incubation with each sample in a set of samples.

[0074] In some embodiments, the amount of intact and / or reduced protein is determined using a native SDS-PAGE gel.

[0075] In some embodiments, the amount of intact and / or reduced protein is determined using a microfluidic device.

[0076] In some embodiments, the methods described herein further comprise determining a level of protein reduction and a redox potential in each sample of the set of samples, and further comprising generating a graph of protein reduction susceptibility based on the determined redox potential and the determined level of intact or reduced protein for each sample of the set of samples.

[0077] In some embodiments, the airtight container is an airtight glass vial.

[0078] In some embodiments, the airtight container has an internal volume of about 2 mL to about 2.5 mL.

[0079] In some embodiments, the airtight container has a headspace of less than 50 microliters, less than 10 microliters, or 0 microliters.

[0080] In some embodiments, the protein is incubated with each sample of the set of samples for about 0.5 to about 4 hours prior to step (c).

[0081] In some embodiments, the methods described herein further comprise comparing the reduction susceptibility of the protein to a reference standard.

[0082] In some embodiments, the reference standard is a standard curve of the reduction sensitivity of a reference protein.

[0083] In some embodiments, the methods described herein further comprise selecting proteins for further development based on the reduction sensitivity of the proteins.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0085] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims.

[0086] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0087] [Figure 1] FIG. 1 is a schematic diagram showing reduction of protein disulfide bonds. [Figure 2] FIG. 1 is a schematic diagram of antibody reduction during the cell culture development process. [Figure 3] FIG. 1 is a schematic diagram of an experimental outline of an exemplary method for determining correlations between reduced markers. [Figure 4A-4B] 1 shows the correlation between the levels of LDH and ORP in serial dilutions of cell culture medium. [Figure 5]

[0023] Figure 1 is a schematic diagram of the experimental outline of an exemplary method for determining the reduction sensitivity of proteins described herein. Specifically, 7-day CHO cultures are harvested and partially lysed by pressure equalization. Lysed and unlysed culture supernatants are mixed to achieve specific lactate dehydrogenase (LDH) levels. Antibody is added and incubated at a specific temperature for a specific period of time. Samples are processed for reduction analysis on a non-reducing SDS gel. [Figure 6A]

[0023] Figure 1 shows the effect of using different containers when performing an exemplary method for determining the reduction susceptibility of a protein described herein. Different containers were tested in a time-based protein reduction assay to show the effect of air permeability to the different containers over time. [Figure 6B]

[0023] Figure 1 shows the results of an exemplary method for determining the reduction sensitivity of proteins described herein. Protein reduction assays were performed using complete lysis cultures, sampled at different time intervals, and using different antibody subtypes. [Figure 6C] 1 shows the reduction sensitivity of different types of proteins (IgG subtypes) as measured by the level of LDH in cell culture medium dilutions. [Figure 7] 1 shows the reduction sensitivity of different types of proteins (IgG subtypes) as measured by the levels of ORP in cell culture medium dilutions. DETAILED DESCRIPTION OF THE INVENTION

[0088] The present disclosure relates to methods for determining the reduction sensitivity of a protein and for adjusting the culture conditions or production methods of the protein based on the determined reduction sensitivity.

[0089] Although chemical reduction assays can be used to detect antibody reduction, they do not mimic the process environment and therefore cannot be used to evaluate process conditions to mitigate reduction risk. In this disclosure, a more process-relevant reduction assay in a cell culture environment is described. One advantage of cell culture-based assays is the ability to probe process conditions, such as oxidation-reduction potential (ORP), for their effect on the protein's susceptibility to reduction.

[0090] The small-scale assay described herein uses cell culture fluid in a more relevant environment to assess the effect of varying cell culture parameters on reduction sensitivity. In this assay, cell lysate is mixed with clarified broth at different ratios to mimic different culture environments in small, sealed vessels. In this way, the reducing environments of bioreactors, clarification, and harvest holding vessels in manufacturing processes can be simulated at bench scale.

[0091] This assay was demonstrated to show varying susceptibility to reduction based on changes in cell culture parameters known to affect reduction, including the amount of cell lysis present, temperature, and oxygen content. This assay can accurately determine the reduction susceptibility of proteins. This assay was further coupled with oxidation-reduction potential (ORP) measurements to correlate with larger-scale findings, demonstrating that reduction patterns change as ORP in the culture medium changes. This assay is also adaptable for addition to any process production run and test harvest holding conditions after cell culture production and clarification, as needed. These assays can also be used to study reduction mechanisms and prevention techniques, including modifying process parameters and cell culture additives to prevent protein reduction in large-scale processes.

[0092] definition As used herein, the term "substantially" or "essentially" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more higher than a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "essentially the same" or "substantially the same" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range that is about the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0093] As used herein, the terms "substantially free" and "essentially free" are used interchangeably and, when used to describe a composition such as a cell culture medium, refer to a composition that is free of a particular substance or source thereof, e.g., 95% free, 96% free, 97% free, 98% free, 99% free of a particular substance or source thereof, or undetectable when measured by conventional methods. The term "free" or "essentially free" of a particular component or substance in a composition also means that such component or substance is (1) not present in the composition at any concentration, or (2) functionally inactive in the composition but not present at low concentrations. A similar meaning can be applied to the term "absence of" when referring to the absence of a particular substance or source thereof in a composition.

[0094] Throughout this specification, unless the context otherwise requires, words such as "comprise," "comprises," and "comprising" should be understood to mean the inclusion of the stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. In certain embodiments, "include," "has," "contains," and "comprise" are used interchangeably.

[0095] "Consisting of" is meant to include, and is limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0096] "Consisting essentially of" means including any elements listed after the phrase, limited to other elements that do not interfere with or contribute to the activity or function specified in the disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or essential, but that other elements are not optional and may or may not be present depending on whether they affect the activity or function of the listed elements.

[0097] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout the specification do not necessarily refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0098] Furthermore, as used herein, "and / or" should be understood as specifically disclosing each of the two specified features or components with or without the other. Thus, the term "and / or," as used in phrases such as "A and / or B," is intended herein to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or," when used in phrases such as "A, B, and / or C," is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B, or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0099] As used herein, the term "subject" refers to any animal, preferably a human patient, livestock, or other domestic animal.

[0100] "Culture" or "cell culture" refers to the maintenance, growth, and / or differentiation of cells (e.g., mammalian cells) in an in vitro environment. Cell culture, in some embodiments, can refer to batch cell culture, fed-batch cell culture, or perfusion cell culture. "Cell culture media," "culture media" (in each case singular "medium"), "supplement," and "media supplement" refer to nutritional compositions in which cell cultures are cultivated.

[0101] As used herein, the term "antibody" refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementarity-determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope in an antigen. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, single variable domain (V) antibodies, and antibodies with a single variable domain (V). H H) Antibodies include chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, the antibody may contain the Fc region of a human antibody. The term antibody also includes derivatives such as multispecific antibodies, bispecific antibodies, single-chain antibodies, diabodies, and linear antibodies formed from these antibodies or antibody fragments.

[0102] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody, which portion of an antibody is capable of specifically binding to an antigen. In some embodiments, an antigen-binding fragment contains at least one variable domain (e.g., a heavy chain variable domain, a light chain variable domain, or a V H Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments, scFv, and V H H is one example.

[0103] The term "recombinant protein," as used herein, refers to any protein or biologically active portion thereof (e.g., a portion that retains the biological activity of the intact protein) that is not a reporter gene or marker gene (e.g., green fluorescent protein) that is expressed from recombinant genetic material encoding amino acids, including peptides, polypeptides, proteins, oligoproteins, and / or fusion proteins. Recombinant protein products can include therapeutic, prophylactic, or diagnostic products.

[0104] The term "protein production" includes techniques used to grow cells, e.g., recombinant cells, in culture and obtain a protein of interest produced by the cultured cells in a form suitable for use. The manufacturing process may include, but is not limited to, one or more of the following: inserting a gene of interest into a host cell to create an engineered host cell, culturing the host cell to expand the cell number, inducing expression of the protein of interest by the host cell, screening host cells that express the protein of interest, harvesting the protein of interest, e.g., by separating the protein of interest from the cultured cells and cell culture medium, and / or purifying the protein of interest. The protein of interest can be an endogenous protein expressed by a native cell or a recombinant heterologous protein encoded in an expression vector (either transient or stable) inserted into a cell.

[0105] As used herein, a "cell culture medium" is a fluid obtained from cell culture, e.g., fed-batch culture, batch culture, or perfusion culture. The cell culture medium can contain cell culture medium, supplements added to the medium during cell culture, cellular components, and / or metabolic products released from the cells during cell culture. In some embodiments, the cell culture medium contains lysed cells or cell lysate. In some embodiments, the cell culture medium is essentially free of lysed cells or cell lysate.

[0106] As used herein, the terms "disulfide bond" or "disulfide bridge" or "SS bond" refer to a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group. The second thiol group can be found in the side chain of a residue on the same polypeptide or protein (intradisulfide bond) or on a different polypeptide or protein (interdisulfide bond). Such bonds are created during protein biosynthesis and / or by oxidation of sulfhydryl groups, a process called oxidative protein folding. Exemplary methods for detecting the disruption of disulfide bonds include non-reducing SDS-PAGE and native two-dimensional electrophoresis. Additional methods for detecting the disruption of disulfide bonds are known in the art.

[0107] The terms "disulfide bond-containing protein" and "disulfide bond-containing protein" are used interchangeably herein and refer to proteins that contain one or more disulfide bonds in their properly folded state. Disulfide bond-containing proteins can have intermolecular or intramolecular disulfide bonds. Such intermolecular or intramolecular disulfide bonds are present in properly folded disulfide bond-containing proteins. The activity of disulfide bond-containing proteins can depend on the presence and reduction state of disulfide bonds. Many molecules, such as immunoglobulins and immunoglobulin domain-containing cell surface receptors, ribonucleases, lactalbumin, insulin, keratin, hemagglutinin, viral membrane proteins, neuroendocrine protein 7B2, epidermal growth factor (EGF), androgenic gland hormones, sulfide dehydrogenase, and lysozyme, function best with the formation of stable intermolecular and intramolecular disulfide bonds that contribute to proper folding. Many therapeutic proteins, such as antibodies, EGF, and insulin, contain disulfide bonds. Producing a therapeutic protein of interest under conditions that increase disulfide bond reduction can result in a low yield of intact protein of interest. As used herein, the term "yield" refers to the amount of protein of interest that is intact, active, properly folded, and obtained with correct disulfide bond pairing. Although a process may produce a large overall amount of protein of interest, if significant disulfide bond reduction occurs in the protein of interest, the yield of intact protein may be low.

[0108] The term "reduced protein" refers to a protein that is exposed to reducing conditions sufficient to reduce reducible residues in the protein structure, such as cysteine. If the reduced protein contains a thiol group or a sulfur-containing residue, the thiol group in the reduced protein exists in its reduced state. For example, a reduced protein containing a cysteine ​​residue exists in a state in which the sulfur atom of the cysteine ​​residue is in a reduced state, often designated as "-SH." The reduced protein may be a disulfide bond-containing protein. A disulfide bond-containing protein can be reduced by exposure to reducing conditions that cleave one or more disulfide bonds (disulfide bridges) in the disulfide bond-containing protein, which may contribute to destabilization of the disulfide bond-containing protein and potential loss of activity or function of the disulfide bond-containing protein.

[0109] The term "redox potential" or "oxidation reduction potential (ORP)," also known as REDOX, is a measurement that reflects the ability of a molecule to oxidize or reduce another molecule. Oxidation is the loss of electrons, so an oxidizing agent accepts electrons from another molecule. Reduction is the gain of electrons, so a reducing agent donates electrons to another molecule. Redox potential is measured as a single voltage in millivolts (mV). Oxidizing agents have positive ORP values, while reducing agents have negative ORP values. Redox potential characterizes the free energy cost and direction of reactions involving electron transfer, which are the most common and important of biochemical reactions. Such reduction-oxidation reactions are characterized by a free energy change that shares some conceptual features with that used to describe pKa in acid-base reactions involving proton transfer rather than electron transfer. Redox potential can be used as a measure of the driving force for a given redox reaction of interest. In some embodiments, redox potential characterizes the oxidation / reduction power of a sample, e.g., a cell culture mixture. Exemplary methods that can be used to determine redox potential are described herein. Additional methods for determining redox potential are known in the art.

[0110] The term "thioredoxin system" refers to the enzymes thioredoxin reductase-1 (TrxR1) and thioredoxin-1 (Trx-1), and the cofactor NADPH. These three components comprise the thioredoxin system, which supports several processes necessary for eukaryotic cell function, including cell proliferation, antioxidant defense, and redox signaling (Lu et al., 2014, Free Radic. Biol. Med., 66:75-87).

[0111] The term "glutathione system" refers to the components glutathione, glutathione reductase (GR), glutaredoxin (Grx), and the cofactor NADPH (Lillig et al., 2008, Biochim. Biophys. Acta-Gen. Subj., 1780:1304-1317).

[0112] The glutathione system and the thioredoxin system are collectively and alternatively referred to herein as the "reductase system" or "reductase systems," i.e., the term "reductase system" encompasses both the glutathione system and / or the thioredoxin system.

[0113] As used herein, "developability" refers to the likelihood that proteins, e.g., disulfide bond-containing proteins, will successfully progress from discovery to development through evaluation of their physicochemical properties, e.g., their reduction sensitivity. Other properties include the tendency for self-interaction and aggregation, thermal stability, colloidal stability, and optimization of those properties through sequence engineering. Selecting proteins with desired properties based on biological function, efficacy, safety, and developability allows for streamlined and successful downstream production. In some embodiments, the methods described herein include assessing the developability of a protein based on the reduction sensitivity of the protein. In some embodiments, the methods described herein include selecting proteins for further development based on the developability of the protein.

[0114] Methods for determining the reduction sensitivity of proteins

[0010] In one aspect, provided herein is a method for determining the reduction sensitivity of a protein, the method comprising: (a) providing a first cell culture medium and a second cell culture medium, wherein the first cell culture medium and the second cell culture medium are identical except that the first cell culture medium comprises a cell lysate and the second cell culture medium is essentially free of the cell lysate; (b) mixing the first cell culture medium and the second cell culture medium in a series of ratios, thereby preparing a set of samples; (c) adding a protein to each sample of the set of samples and incubating each sample of the set of samples in an airtight container (i) having an internal volume of about 1 mL to about 3 mL and (ii) having a headspace of less than 100 microliters; (d) determining a level of protein reduction and / or redox potential in each sample of the set of samples; and (e) determining the reduction sensitivity of the protein based on the level of protein reduction and redox potential determined for each sample of the set of samples in step (d), thereby determining the reduction sensitivity of the protein.

[0115] In another aspect, provided herein is a method for determining the reduction sensitivity of a protein, the method comprising: (a) adding a protein to each sample of a set of samples prepared by mixing a first cell culture medium and a second cell culture medium in a series of ratios, wherein the first cell culture medium and the second cell culture medium are identical except that the first cell culture medium comprises a cell lysate and the second cell culture medium is essentially free of cell lysate; (b) incubating each sample of the set of samples in an airtight container (i) having an internal volume of about 1 mL to about 3 mL and (ii) having a headspace of less than 100 microliters; and (c) determining a level of protein reduction and / or redox potential in each sample of the set of samples, and determining the reduction sensitivity of the protein based on the level of protein reduction and redox potential, thereby determining the reduction sensitivity of the protein.

[0116] The cell lysate in the first cell culture medium can be obtained by any suitable method known in the art. Cell lysis can be achieved using physical or reagent-based methods. In some embodiments, the cell lysate is obtained by cell homogenization. In some embodiments, the cell lysate is obtained by enzymatic treatment. In some embodiments, the cell lysate is centrifuged to remove cellular debris, and then the proteins in each sample of the set of samples are incubated in an airtight container.

[0117] In some embodiments, the cell lysate in the first cell culture comprises one or more enzymes, organelles and / or metabolites.

[0118] In some embodiments, the cell lysate comprises one or more enzymes that contribute to the reduction of proteins, hi some embodiments, the cell lysate comprises one or more enzymes from the thioredoxin and glutathione reductase pathways.

[0119] In some embodiments, the second cell culture medium is obtained by removing essentially all of the cells from the cell culture medium.

[0120] The first and second cell culture media are mixed in a series of ratios to create a set of samples for determining the reduction sensitivity of the protein. In some embodiments, the ratio between the first cell culture media and the second cell culture media in the set of samples increases or decreases by 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 33-fold, or 100-fold intervals. In some embodiments, the first cell culture media and the second cell culture media are mixed in a ratio of about 1:10. 5 , 1:10 4 , 1:1000, 1:100, 1:10, 1:1, 10:1, 100:1, 1000:1, 10 4The first and second cell culture media are mixed in a ratio of 1:1 or any other suitable ratio. The set of samples created by mixing the first and second cell culture media provides a reducing environment ladder for testing the reduction sensitivity of proteins. In some embodiments, the set of samples further includes one or more reference samples having only the first cell culture media or only the second cell culture media.

[0121] The protein in the methods described herein can be any suitable protein. In some embodiments, the protein is a recombinant protein.

[0122] In some embodiments, the recombinant protein is an antibody, or an antigen-binding fragment thereof. In some embodiments, the antibody is a monoclonal antibody.

[0123] In some embodiments, the protein is an immunoglobulin. As used herein, the term "immunoglobulin" encompasses a wide variety of biochemically distinguishable polypeptides or proteins. Those skilled in the art will understand that immunoglobulin heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses within them (e.g., γ1-γ4 or α1-α2). It is the nature of this chain that determines the "isotype" of an antibody as IgG, IgM, IgA IgG, or IgE, respectively. Immunoglobulin subclasses (subtypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc., are well characterized and are known to confer functional specialization. Modified versions of each of these immunoglobulins are readily discernible to those skilled in the art in light of the present disclosure and, accordingly, are within the scope of the present disclosure.

[0124] In some embodiments, reduction sensitivity is determined for a disulfide bond-containing protein. In some embodiments, the level of protein reduction is determined by assessing the oxidation state of disulfide bonds in the protein. The oxidation state of the disulfide bonds.

[0125] In some embodiments, the redox potential is determined by measuring the levels of one or more molecules involved in redox reactions in cells and during protein reduction. These molecules include, but are not limited to, lactate dehydrogenase (LDH), nicotinamide adenine dinucleotide (NAD) / reduced NAD (NADH) redox couple, NADP / reduced NADP (NADPH) redox couple, NADPH, thioredoxin (Trx), and glutathione (Grx). In some embodiments, the redox potential is determined by measuring the levels of one or more molecules involved in the glutathione system. In some embodiments, the redox potential is determined by measuring the levels of one or more molecules involved in the thioredoxin system.

[0126] Methods for detecting and / or determining the level of molecules with respect to redox potential are known in the art. For example, LDH levels can be measured using a CEDEX bioanalyzer (Roche Diagnostics CEDEX BIO LDH, Roche Diagnostics 06343767001). Thioredoxin levels can be measured using a Thioredoxin Reductase Colorimetric Assay Kit (Cayman Chemical, product number 10007892). Glutathione levels can be measured using a Glutathione Colorimetric Detection Kit (Thermo Fisher, catalog number: EIAGSHC). NADPH levels can be measured using an NADPH Assay Kit (colorimetric) (Abcam, ab186031). Levels of the nicotinamide adenine dinucleotide (NAD+) / reduced NAD+ (NADH) redox pair and / or the NADP+ / reduced NADP+ (NADPH) redox pair can be measured by the NAD / NADH-Glo™ and NADP / NADPH-Glo™ Assays (Promega). Other commercially available methods and kits for determining redox potential are known in the art.

[0127] In some embodiments, the redox potential is determined by using a redox potential probe. The redox potential probe measures the ability of a solution to act as an oxidizing or reducing agent. The ionic potential information obtained by the ORP probe is thereby used to indicate the redox potential of a sample, such as a mixture of the first and second cell culture solutions in the methods described herein. Probes used to determine and detect redox potential are commercially available and known in the art. For example, the redox potential (ORP level) can be determined using a laboratory ORP sensor (METTLER TOLEDO).

[0128] In some embodiments, the level of protein reduction is determined by the amount of intact and / or reduced protein after incubation with each sample in the set of samples. Methods for quantifying reduced and intact protein levels are known in the art.

[0129] As used herein, "intact protein" refers to a properly folded protein without reduction of any disulfide bonds. "Reduced protein" refers to a protein that has at least 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30% or more reduction of disulfide bonds.

[0130] In some embodiments, the amount of intact and / or reduced protein is determined using a native, non-reducing SDS-PAGE gel. In some embodiments, the amount of intact and / or reduced protein is determined using a microfluidic device. Any other suitable method for determining the amount of intact and / or reduced protein can be used herein.

[0131] In some embodiments, the methods described herein further comprise generating, for each sample in the set of samples, a graph of protein reduction susceptibility based on the determined redox potential and the determined level of intact or reduced protein.

[0132] In some embodiments, the graph establishes a correlation between the amount of intact and / or reduced protein and the redox potential of each sample in the set of samples, hi some embodiments, the graph establishes a correlation between the reduction susceptibility of the protein and the redox potential of each sample in the set of samples.

[0133] The container used in the methods described herein is important for determining the reduction sensitivity of a protein. Specifically, the present disclosure provides a small-scale assay that can be performed in a high-throughput manner. Therefore, the internal volume of the container in the methods described herein should be within a certain range. In some embodiments, the internal volume of the container is about 0.1 mL to about 100 mL, about 1 mL to about 100 mL, about 1 mL to about 10 mL, or 1 mL to about 3 mL. In some embodiments, the internal volume of the container is about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, or about 10 mL. In some embodiments, the internal volume of the container is about 2 mL. In some embodiments, the internal volume of the container is about 2 mL to about 2.5 mL. In some embodiments, the internal volume of the container is about 2.5 mL.

[0134] The container material is also important for achieving the desired results of the methods described herein. Specifically, the container is airtight to prevent protein oxidation and accurately assess protein reduction. Therefore, the container material should have minimal air permeability. In some embodiments, the airtight container is an airtight glass vial. In some embodiments, the airtight container is an airtight metal container. In some embodiments, the airtight container is an airtight plastic container.

[0135] In the methods described herein, a first cell culture medium and a second cell culture medium are first mixed in different ratios, and the protein to be tested is added to each mixture and incubated. To prevent protein oxidation and accurately assess protein reduction, there should be minimal headspace between the fluid mixture and the sealer, e.g., the cap of a glass vial. Mixing the first cell culture medium and the second cell culture medium and / or mixing the protein with the cell culture medium sample can be performed using any suitable method. For example, mixing can be performed by inverting the container (e.g., glass vial) several times. Mixing can also be performed by gently pipetting the liquid in the container. In some embodiments, mixing is performed using a method that does not disrupt the protein structure in the mixture.

[0136] As used herein, the term "headspace" refers to the distance or volume from the top of a container to the top of a liquid (e.g., a sample containing a mixture of a first cell culture medium and a second cell culture medium). In some embodiments, any extension of a cover or lid over the body of the container is ignored when performing this operation. In some embodiments, the airtight container has a headspace of less than 200 microliters, less than 150 microliters, less than 100 microliters, less than 50 microliters, less than 10 microliters, or 0 microliters.

[0137] In some embodiments, the protein is incubated with each sample in the set of samples for several minutes, several hours, or several days. In some embodiments, the protein is incubated with each sample in the set of samples for about 0.5 to about 24 hours, about 0.5 to about 12 hours, about 0.5 to about 6 hours, about 0.5 to about 5 hours, about 0.5 to about 4 hours, about 0.5 to about 3 hours, about 0.5 to about 2 hours, or about 0.5 to about 1 hour, after which the protein is added to each sample in the set of samples, and each sample in the set of samples is incubated in an airtight container.

[0138] In some embodiments, the protein is incubated with each sample of the set of samples for about 24 hours to about 36 hours, about 24 hours to about 48 hours, about 24 hours to about 60 hours, about 24 hours to about 72 hours, about 1 day to about 4 days, about 1 day to about 5 days, about 1 day to about 6 days, about 1 day to about 7 days, about 1 day to about 8 days, about 1 day to about 9 days, about 1 day to about 10 days or more, after which the protein is added to each sample of the set of samples and each sample of the set of samples is incubated in an airtight container.

[0139] Incubation of each sample of the protein sample set can be carried out under any suitable conditions. In some embodiments, incubation is carried out at a temperature of about 37°C. In some embodiments, incubation is carried out at room temperature. In some embodiments, incubation is carried out at a temperature of about 4°C.

[0140] In some embodiments, incubation is carried out at a CO2 level of about 5%. In some embodiments, incubation is carried out at 95% relative humidity. In some embodiments, incubation is carried out under physiological pH conditions (e.g., a pH value of about 7.3 to about 7.45). In some embodiments, incubation is carried out under conditions that mimic cell culture and holding tank conditions. In some embodiments, the temperature of the holding tank is lower than the temperature of the reactor. In some embodiments, the temperature of the holding tank is room temperature. In some embodiments, the temperature of the holding tank is about 4°C to about 10°C. In some embodiments, the temperature of the holding tank is about 4°C.

[0141] In some embodiments, the methods described herein further comprise comparing the reduction susceptibility of the protein to a reference standard.

[0142] In some embodiments, the reference standard is a standard curve of the reduction sensitivity of a reference protein. The reference protein can be, for example, a standard protein whose reduction sensitivity has been previously determined to be acceptable for further development.

[0143] In some embodiments, the methods described herein further comprise selecting proteins for further development based on the reduction sensitivity of the protein. In some embodiments, the methods described herein comprise evaluating the developability of the protein based on the reduction sensitivity of the protein. In some embodiments, the methods described herein comprise selecting proteins for further development based on the developability of the protein.

[0144] Method for modifying bioprocessing conditions of cell culture media In another aspect, a method of modifying culture conditions of a cell culture medium or bioprocessing conditions of a cell culture fluid used to produce a protein includes: (a) providing a first cell culture fluid and a second cell culture fluid, wherein the first cell culture fluid and the second cell culture fluid are identical except that the first cell culture fluid comprises a cell lysate and the second cell culture fluid is essentially free of the cell lysate; (b) mixing the first cell culture fluid and the second cell culture fluid in a series of ratios, thereby preparing a set of samples; and (c) adding protein to each sample of the set of samples, wherein each sample of the set of samples (i) has an internal volume of between about 1 mL and about 3 mL, and (ii) is 1 mL. (d) determining a level of protein reduction and / or redox potential in each sample of the set of samples; (e) determining the reduction sensitivity of the protein based on the level of protein reduction and redox potential determined for each sample of the set of samples in step (d); and (f) modifying one or more culture conditions or cell culture medium bioprocessing conditions used to produce the protein based on the reduction sensitivity of the protein.

[0145] In another aspect, a method of modifying culture conditions of a cell culture medium or bioprocessing conditions of a cell culture fluid used to produce a protein comprises: (a) adding a protein to each sample of a set of samples prepared by mixing a first cell culture fluid and a second cell culture fluid in a series of ratios, wherein the first cell culture fluid and the second cell culture fluid are identical except that the first cell culture fluid contains a cell lysate and the second cell culture fluid is essentially free of cell lysate; and (b) adding each sample of the set of samples to a cell culture medium (i) having an internal volume of about 1 mL to about 3 mL, and (ii) 1 (c) determining a level of protein reduction and / or redox potential in each sample of the set of samples to determine the reduction sensitivity of the protein based on the level of protein reduction and redox potential; and (d) modifying one or more culture conditions or cell culture medium bioprocessing conditions used to produce the protein based on the reduction sensitivity of the protein.

[0146] In some embodiments, modifying one or more culture conditions of the cell culture medium or cell culture bioprocessing conditions comprises adjusting one or more of the components of the liquid culture medium composition, pH, temperature, and dissolved oxygen during production of the protein.

[0147] In some embodiments, modifying one or more culture conditions of the cell culture medium or cell culture bioprocessing conditions comprises adjusting one or more threshold parameters in the cell culture medium, including, but not limited to, optical density (OD), dissolved oxygen (DO), pH, the concentration of a nutrient in the culture medium, the total concentration of a first carbon source added to the culture medium, or any combination thereof.

[0148] In some embodiments, modifying one or more culture conditions of the cell culture medium or cell culture bioprocessing conditions comprises removing a reducing agent (a by-product of the reduction reaction) from the cell culture medium.

[0149] How to generate a standard curve In another aspect, provided herein is a method for generating a standard curve for protein reduction sensitivity, the method including: (a) providing a first cell culture medium and a second cell culture medium, wherein the first cell culture medium and the second cell culture medium are identical except that the first cell culture medium comprises a cell lysate and the second cell culture medium is essentially free of the cell lysate; (b) mixing the first cell culture medium and the second cell culture medium in a series of ratios, thereby preparing a set of samples; (c) adding a protein to each sample of the set of samples and incubating each sample of the set of samples in an airtight container (i) having an internal volume of about 1 mL to about 3 mL and (ii) having a headspace of less than 100 microliters; (d) determining a level of protein reduction and a redox potential in each sample of the set of samples; and (e) generating a standard curve for protein reduction sensitivity based on the determined level of redox potential and the determined level of protein reduction for each sample of the set of samples, thereby generating the standard curve for protein reduction sensitivity.

[0150] In another aspect, provided herein is a method for generating a standard curve for the reduction sensitivity of a protein, the method comprising: (a) adding a protein to each sample of a set of samples prepared by mixing a first cell culture medium and a second cell culture medium in a series of ratios, wherein the first cell culture medium and the second cell culture medium are identical except that the first cell culture medium comprises a cell lysate and the second cell culture medium is essentially free of cell lysate; (b) incubating each sample of the set of samples in an airtight container (i) having an internal volume of about 1 mL to about 3 mL and (ii) having a headspace of less than 100 microliters; (c) determining a level of protein reduction and / or redox potential in each sample of the set of samples, thereby determining the reduction sensitivity of the protein based on the level of protein reduction and the redox potential; and (d) generating a standard curve for the reduction sensitivity of the protein based on the determined level of redox potential and the determined level of protein reduction for each sample of the set of samples, thereby generating the standard curve for the reduction sensitivity of the protein.

[0151] In some embodiments, standard curves generated using the methods described herein are used to select proteins for further development.

[0152] In some embodiments, the methods described herein further comprise generating a standard curve of reduction sensitivity of a reference protein. The reference protein can be, for example, a standard protein whose reduction sensitivity has been previously determined to be acceptable for further development.

[0153] In some embodiments, the methods described herein further comprise comparing a standard curve of reduction sensitivity of the tested protein to that of a reference protein.

[0154] In some embodiments, the methods described herein further comprise selecting proteins for further development based on comparison of a standard curve of reduction sensitivity of the proteins. In some embodiments, the methods described herein comprise evaluating the developability of the proteins based on the reduction sensitivity of the proteins. In some embodiments, the methods described herein comprise selecting proteins for further development based on the developability of the proteins. [Example]

[0155] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0156] Example 1: Method for determining the reduction sensitivity of a protein As shown in Figure 1, reduction of disulfide bonds disrupts the bonds, resulting in loss of bulk harvest and failure to meet drug substance specifications.

[0157] As shown in Figure 2, reduction occurs primarily in the clarified harvest. Lysed cells resulting from apoptosis at the end of the culture or caused by shear stress in the harvest releasing enzymes from the GSH and / or Trx systems leads to a reducing environment in the clarified harvest. Oxygen consumption leads to low DO and a reducing environment in the clarified harvest. Longer holding times at elevated temperatures in sealed harvests result in more GSH / Trx activity, DO consumption, and a greater risk of Ab reduction.

[0158] Figure 3 is a schematic diagram of the experimental outline of an exemplary method for determining the correlation of protein reduction markers, e.g., LDH and ORP levels. Specifically, cells were cultured to optimal viable cell density (VCD) while maintaining cell viability. The cells were then harvested and homogenized at 8000 psi to rapidly lyse the cells. The homogenized cell lysate was then clarified by centrifugation and passed through a low-protein-binding filter. The clarified lysed cell culture medium was then mixed at different ratios with culture medium essentially free of cell lysate to create serial dilutions.

[0159] Specifically, serial dilutions of cell culture medium were performed in 2 mL minibottles. The dissolved cell culture medium was mixed with undissolved culture medium in a series of ratios. LDH levels were measured via CEDEX (Roche Diagnostics CEDEX BIO LDH, Roche Diagnostics 06343767001). ORP levels were determined using a laboratory ORP sensor (METTLER TOLEDO).

[0160] The data in Figures 4A-4B establish a correlation between LDH and ORP. Cell culture-based assays allow for the study of the effect of different process-related parameters in the culture supernatant, such as ORP, on reduction. Specifically, ORP was monitored at different LDH values ​​and time points in the cell culture assay. As the amount of dissolved material present increases, the environment becomes more reducing. As shown in Figure 4B, ORP decreased with increasing LDH and time. This demonstrates that the experimental data are reproducible and consistent, which is consistent with the theory underlying the experimental design.

[0161] Figure 5 describes the method used to determine the reduction sensitivity of the proteins described herein. Specifically, cells were cultured to optimal viable cell density (VCD) while maintaining cell viability. The cells were then harvested and homogenized at 8000 psi to rapidly lyse the cells. The homogenized cell lysate was then clarified by centrifugation and passed through a low-protein binding filter. The clarified lysed cell culture medium was then mixed at different ratios with culture medium essentially free of cell lysate to create serial dilutions.

[0162] Specifically, serial dilutions of cell culture medium were performed in 2 mL minibottles. Dissolved cell culture medium was mixed with undissolved culture medium in a series of ratios. Each test protein was added to each mixture in the dilution series and incubated. The liquid was filled to the top of the minibottle before sealing, creating an airtight environment. After incubation, the relative level of intact protein was measured relative to the level of LDH in each minibottle, for example, at 4 hours. LDH levels were measured via CEDEX (Roche Diagnostics CEDEX BIO LDH, Roche Diagnostics 06343767001). Protein reduction (relative to intact mAb levels) was measured via Caliper's LabChip GXII system (Caliper Life Sciences).

[0163] The container for culture retention is important. Figure 6A shows the effect of using different types of containers when performing an exemplary cell culture-based reduction assay. Specifically, different containers were tested in a time-based reduction assay to ensure performance was not affected by air permeability over time. As shown in Figure 6A, glass containers are superior to plastic containers for cell culture-based assays in preventing reoxidation.

[0164] Furthermore, using a kinetic approach, reduction in lysed culture supernatants was tested at LDH = 4000 U / L (Figure 6B). Specifically, 7-day CHO cultures were harvested and partially lysed by pressure homogenization. The lysed and unlysed culture supernatants were mixed to achieve the desired lactate dehydrogenase (LDH) value. Each test antibody was added and incubated at the desired temperature and duration. Samples were processed for reduction analysis (measured by the percentage of intact mAb) on a non-reducing SDS gel. Four antibodies were tested: IgG4 (Ab1), IgG1κ (Ab2), IgG1λ (Ab3), and IgG2 (Ab4). These results demonstrate that the assay described herein can provide an accurate assessment of the sensitivity of different antibodies to reduction.

[0165] An LDH-based incubation assay (Figure 6C) was also performed. As shown in Figure 6C, the amount of intact mAb decreases as the amount of lysed material present increases. Specifically, the reduction sensitivity of IgG1λ (Ab3) is greater than that of IgG1κ (Ab2), which in turn is greater than that of IgG4 (Ab1). These results demonstrate improved resolution and differentiation of the reduction sensitivity of κ and λ immunoglobulin subtypes, despite the short incubation period required (4 h incubation).

[0166] As shown in Figures 4A, 4B, and 6C, correlations between LDH and ORP and between LDH and protein reduction were established. Thus, using LDH as an intermediate, a relationship between ORP and protein reduction was established. As shown in Figure 7, as the cell culture environment becomes increasingly reducing (as measured by decreasing ORP levels), the relative amount of intact mAb decreases. Specifically, the reduction sensitivity of IgG1λ (Ab3) is greater than that of IgG1κ (Ab2), which in turn is greater than that of IgG4 (Ab1).

[0167] Additionally, cell culture-based assays can be used to study the effects of different process parameters, such as dissolved oxygen and temperature shifts, on protein reduction, which is not possible with chemical assays. Culture-based assays can also be used to study novel process controls, such as mitigation for reduction by inhibitors or control of ORP beyond the limits identified for reduction in Figures 6A-6C.

[0168] Cell culture assays are more process-relevant than chemical reduction assays and can be used to study the effect of different culture parameters on reduction, as was done with ORP. Going forward, these findings can be directly translated into cell culture processes to control reduction, for example, by using oxygenation or chemical additives to control ORP above the risk limit found for a given antibody in small-scale assays, or by setting LDH-based harvest criteria.

[0169] Other embodiments While the present invention has been described with reference to its detailed description, it will be understood that the foregoing description is for illustrative purposes only and is not intended to limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method for determining the reduction sensitivity of a protein, comprising: (a) providing a first cell culture medium and a second cell culture medium, wherein the first cell culture medium and the second cell culture medium are identical except that the first cell culture medium comprises a cell lysate and the second cell culture medium is essentially free of the cell lysate; (b) mixing the first cell culture medium and the second cell culture medium in a series of ratios, thereby preparing a set of samples; (c) adding the protein to each sample of the set of samples and incubating each sample of the set of samples in an airtight container (i) having an internal volume of about 1 mL to about 3 mL and (ii) having a headspace of less than 100 microliters; (d) determining the level of protein reduction and / or redox potential in each sample of said set of samples; (e) determining the reduction sensitivity of the protein based on the level of protein reduction and / or the redox potential determined for each sample of the set of samples in step (d), thereby determining the reduction sensitivity of the protein; A method comprising:

2. The method of claim 1 , wherein the cell lysate is obtained by homogenization of cells.

3. 3. The method of claim 1 or 2, wherein the cell lysate is centrifuged to remove cell debris prior to step (b).

4. 4. The method of any one of claims 1 to 3, wherein the second cell culture medium is obtained by removing essentially all of the cells from a cell culture medium.

5. The method of any one of claims 1 to 4, wherein the protein is a recombinant protein.

6. The method of claim 5 , wherein the recombinant protein is an antibody or an antigen-binding fragment thereof.

7. The method of claim 6 , wherein the antibody is a monoclonal antibody.

8. The method of any one of claims 1 to 7, wherein the level of protein reduction is determined by assessing the oxidation state of disulfide bonds in the protein.

9. 9. The method of any one of claims 1 to 8, wherein the redox potential is determined by measuring one or more of lactate dehydrogenase (LDH), nicotinamide adenine dinucleotide (NAD+) / reduced NAD+ (NADH) redox pair, NADP+ / reduced NADP+ (NADPH) redox pair, NADPH, thioredoxin (Trx), and / or glutathione (Grx).

10. The method of any one of claims 1 to 8, wherein the redox potential is determined by using a redox potential probe.

11. 11. The method of any one of claims 1 to 10, wherein the level of protein reduction is determined by the amount of intact and / or reduced protein after incubation with each sample of the set of samples.

12. The method of claim 11, wherein the amount of intact and / or reduced protein is determined using a native SDS-PAGE gel.

13. 12. The method of claim 11, wherein the amount of intact and / or reduced protein is determined using a microfluidic device.

14. 14. The method of any one of claims 1 to 13, comprising determining the level of protein reduction and the redox potential in each sample of the set of samples, and further comprising generating a graph of the reduction sensitivity of the protein based on the determined redox potential and the determined level of intact or reduced protein for each sample of the set of samples.

15. The method according to any one of claims 1 to 14, wherein the airtight container is an airtight glass vial.

16. The method of any one of claims 1 to 15, wherein the airtight container has an internal volume of about 2 mL to about 2.5 mL.

17. The method of any one of claims 1 to 16, wherein the airtight container has a headspace of less than 50 microliters, less than 10 microliters, or 0 microliters.

18. 18. The method of any one of claims 1 to 17, wherein the protein is incubated with each sample of the set of samples for about 0.5 to about 4 hours prior to step (c).

19. The method of any one of claims 1 to 18, further comprising comparing the reduction sensitivity of the protein with a reference standard.

20. 20. The method of claim 19, wherein the reference standard is a standard curve of reduction sensitivity of a reference protein.

21. 21. The method of any one of claims 1 to 20, further comprising selecting said protein for further development based on said reduction sensitivity of said protein.

22. 1. A method of modifying culture conditions or cell culture bioprocessing conditions of a cell culture medium used for the production of a protein, comprising: (a) providing a first cell culture medium and a second cell culture medium, wherein the first cell culture medium and the second cell culture medium are identical except that the first cell culture medium comprises a cell lysate and the second cell culture medium is essentially free of the cell lysate; (b) mixing the first cell culture medium and the second cell culture medium in a series of ratios, thereby preparing a set of samples; (c) adding the protein to each sample of the set of samples and incubating each sample of the set of samples in an airtight container (i) having an internal volume of about 1 mL to about 3 mL and (ii) having a headspace of less than 100 microliters; (d) determining the level of protein reduction and / or redox potential in each sample of said set of samples; (e) determining the reduction susceptibility of the protein based on the level of protein reduction and / or the redox potential determined for each sample of the set of samples in step (d); (f) modifying one or more culture conditions of the cell culture medium or cell culture bioprocessing conditions based on the reduction sensitivity of the protein; thereby modifying the culture conditions or the cell culture bioprocessing conditions used for the production of the protein; A method comprising:

23. 23. The method of claim 22, wherein the cell lysate is obtained by cell homogenization.

24. 24. The method of claim 22 or 23, wherein the cell lysate is centrifuged to remove cell debris prior to step (b).

25. 25. The method of any one of claims 22 to 24, wherein the second cell culture medium is obtained by removing essentially all of the cells from a cell culture medium.

26. The method of any one of claims 22 to 25, wherein the protein is a recombinant protein.

27. 27. The method of claim 26, wherein the recombinant protein is an antibody, or an antigen-binding fragment thereof.

28. 28. The method of claim 27, wherein the antibody is a monoclonal antibody.

29. 29. The method of any one of claims 22 to 28, wherein the level of protein reduction is determined by assessing the oxidation state of disulfide bonds in the protein.

30. 30. The method of any one of claims 1 to 29, wherein the redox potential is determined by measuring one or more of lactate dehydrogenase (LDH), nicotinamide adenine dinucleotide (NAD+) / reduced NAD+ (NADH) redox pair, NADP+ / reduced NADP+ (NADPH) redox pair, NADPH, thioredoxin (Trx), and / or glutathione (Grx).

31. 30. The method of any one of claims 22 to 29, wherein the redox potential is determined by using a redox potential probe.

32. 32. The method of any one of claims 22 to 31, wherein the level of protein reduction is determined by the amount of intact and / or reduced protein after incubation with each sample of the set of samples.

33. 33. The method of claim 32, wherein the amount of intact and / or reduced protein is determined using a native SDS-PAGE gel.

34. 33. The method of claim 32, wherein the amount of intact and / or reduced protein is determined using a microfluidic device.

35. The method according to any one of claims 22 to 34, wherein the airtight container is an airtight glass vial.

36. 36. The method of any one of claims 22 to 35, wherein the airtight container has an internal volume of about 2 mL to about 2.5 mL.

37. 37. The method of any one of claims 22 to 36, wherein the airtight container has a headspace of less than 50 microliters, less than 10 microliters, or 0 microliters.

38. 38. The method of any one of claims 22 to 37, wherein the protein is incubated with each sample of the set of samples for about 0.5 to about 4 hours prior to step (c).

39. 39. The method of any one of claims 22-38, wherein the alteration of the one or more culture conditions of the cell culture medium or the cell culture bioprocessing conditions comprises adjusting one or more of a liquid culture medium composition component, pH, temperature, and dissolved oxygen during production of the protein.

40. 1. A method for generating a standard curve of reduction sensitivity of a protein, comprising: (a) providing a first cell culture medium and a second cell culture medium, wherein the first cell culture medium and the second cell culture medium are identical except that the first cell culture medium comprises a cell lysate and the second cell culture medium is essentially free of the cell lysate; (b) mixing the first cell culture medium and the second cell culture medium in a series of ratios, thereby preparing a set of samples; (c) adding the protein to each sample of the set of samples and incubating each sample of the set of samples in an airtight container (i) having an internal volume of about 1 mL to about 3 mL and (ii) having a headspace of less than 100 microliters; (d) determining the level of protein reduction and redox potential in each sample of said set of samples; (e) generating a standard curve of reduction sensitivity of the protein based on the determined level of redox potential and the determined level of protein reduction for each sample of the set of samples; thereby generating said standard curve of reduction sensitivity of said protein; A method comprising:

41. 41. The method of claim 40, wherein the cell lysate is obtained by cell homogenization.

42. 42. The method of claim 40 or 41, wherein the cell lysate is centrifuged to remove cell debris prior to step (b).

43. 43. The method of any one of claims 40 to 42, wherein the second cell culture medium is obtained by removing essentially all of the cells from a cell culture medium.

44. The method of any one of claims 40 to 43, wherein the protein is a recombinant protein.

45. The method of any one of claims 40 to 44, wherein the recombinant protein is an antibody or an antigen-binding fragment thereof.

46. 46. ​​The method of claim 45, wherein the antibody is a monoclonal antibody.

47. 47. The method of any one of claims 40 to 46, wherein the level of protein reduction is determined by assessing the oxidation state of disulfide bonds in the protein.

48. 48. The method of any one of claims 40 to 47, wherein the redox potential is determined by measuring one or more of lactate dehydrogenase (LDH), nicotinamide adenine dinucleotide (NAD+) / reduced NAD+ (NADH) redox couple, NADP+ / reduced NADP+ (NADPH) redox couple, NADPH, thioredoxin (Trx), and / or glutathione (Grx).

49. 48. The method of any one of claims 40 to 47, wherein the redox potential is determined by using a redox potential probe.

50. 50. The method of any one of claims 40 to 49, wherein the level of protein reduction is determined by the amount of intact and / or reduced protein after incubation with each sample of the set of samples.

51. 51. The method of claim 50, wherein the amount of intact and / or reduced protein is determined using a native SDS-PAGE gel.

52. 51. The method of claim 50, wherein the amount of intact and / or reduced protein is determined using a microfluidic device.

53. 53. The method of any one of claims 40 to 52, wherein the airtight container is an airtight glass vial.

54. 54. The method of any one of claims 40 to 53, wherein the airtight container has an internal volume of about 2 mL to about 2.5 mL.

55. 55. The method of any one of claims 40 to 54, wherein the airtight container has a headspace of less than 50 microliters, less than 10 microliters, or 0 microliters.

56. 56. The method of any one of claims 40 to 55, wherein the protein is incubated with each sample of the set of samples for about 0.5 to about 4 hours prior to step (c).

57. 1. A method for determining the reduction sensitivity of a protein, comprising: (a) adding the protein to each sample of a set of samples prepared by mixing a first cell culture medium and a second cell culture medium in a series of ratios, wherein the first cell culture medium and the second cell culture medium are identical except that the first cell culture medium comprises a cell lysate and the second cell culture medium is essentially free of the cell lysate; (b) incubating each sample of said set of samples in an airtight container (i) having an internal volume of about 1 mL to about 3 mL and (ii) having a headspace of less than 100 microliters; (c) determining a level of protein reduction and / or redox potential in each sample of the set of samples and determining the reduction susceptibility of the protein based on the level of protein reduction and / or the redox potential; thereby determining the reduction sensitivity of the protein; A method comprising:

58. 58. The method of claim 57, wherein the cell lysate is obtained by cell homogenization.

59. 59. The method of claim 57 or 58, wherein the cell lysate is centrifuged to remove cellular debris prior to step (b).

60. 60. The method of any one of claims 57 to 59, wherein the second cell culture medium is obtained by removing essentially all of the cells from a cell culture medium.

61. 61. The method of any one of claims 57 to 60, wherein the protein is a recombinant protein.

62. 62. The method of claim 61, wherein the recombinant protein is an antibody, or an antigen-binding fragment thereof.

63. 63. The method of claim 62, wherein the antibody is a monoclonal antibody.

64. 63. The method of any one of claims 57 to 62, wherein the level of protein reduction is determined by assessing the oxidation state of disulfide bonds in the protein.

65. 65. The method of any one of claims 1 to 64, wherein the redox potential is determined by measuring 57 or more of lactate dehydrogenase (LDH), nicotinamide adenine dinucleotide (NAD+) / reduced NAD+ (NADH) redox couple, NADP+ / reduced NADP+ (NADPH) redox couple, NADPH, thioredoxin (Trx), and / or glutathione (Grx).

66. 65. The method of any one of claims 57 to 64, wherein the redox potential is determined by using a redox potential probe.

67. 67. The method of any one of claims 57 to 66, wherein the level of protein reduction is determined by the amount of intact and / or reduced protein after incubation with each sample of the set of samples.

68. 68. The method of claim 67, wherein the amount of intact and / or reduced protein is determined using a native SDS-PAGE gel.

69. 68. The method of claim 67, wherein the amount of intact and / or reduced protein is determined using a microfluidic device.

70. 70. The method of any one of claims 57 to 69, comprising determining the level of protein reduction and the redox potential in each sample of the set of samples, and further comprising generating a graph of the reduction sensitivity of the protein based on the determined redox potential and the determined level of intact or reduced protein for each sample of the set of samples.

71. 71. The method of any one of claims 57 to 70, wherein the airtight container is an airtight glass vial.

72. 72. The method of any one of claims 57 to 71, wherein the airtight container has an internal volume of about 2 mL to about 2.5 mL.

73. 73. The method of any one of claims 57 to 72, wherein the airtight container has a headspace of less than 50 microliters, less than 10 microliters, or 0 microliters.

74. 74. The method of any one of claims 57 to 73, wherein the protein is incubated with each sample of the set of samples for about 0.5 to about 4 hours prior to step (c).

75. 75. The method of any one of claims 57 to 74, further comprising comparing the reduction sensitivity of the protein to a reference standard.

76. 76. The method of claim 75, wherein the reference standard is a standard curve of reduction sensitivity of a reference protein.

77. 77. The method of any one of claims 57 to 76, further comprising selecting said protein for further development based on said reduction sensitivity of said protein.

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