Method for evaluating reduction risk of biomolecule and use thereof

Abstract

The present invention belongs to the field of bioengineering, and specifically relates to a method for evaluating the reduction risk of a biomolecule and the use thereof. In the method of the present invention, by means of evaluating the reduction sensitivity of a biomolecule (such as an antibody), and determining the level of a reduction indicator in a cell culture medium, risk points for reduction of different types of biomolecules can be accurately identified, so as to utilize a targeted prevention strategy. The method is suitable for accurately evaluating the reduction risk of a biomolecule such as an antibody in the development stage of preparation processes thereof, and taking a targeted anti-reduction measure on the basis of the identified reduction risk point, so as to significantly improve the success rate of process scale-up and large-scale production, and reduce the costs of process development and production failure caused by reduction issues.

Description

A method for assessing biomolecule reduction risk and its application

[0001] Cross-reference information

[0002] This application claims priority to Chinese patent application No. 2024103963264 filed on April 2, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of bioengineering, and in particular relates to a method for assessing the reduction risk of biomolecules and its application. Background Art

[0004] Abnormal reduction reactions (or uncontrolled reduction reactions) that occur during the production process of biological molecules may destroy their normal structure and function, or even cause them to have the opposite effect, so they need to be avoided as much as possible.

[0005] As a typical example of this phenomenon, antibody disulfide bond reduction has always been a challenging issue in the small-scale, pilot, and scale-up production of monoclonal antibodies. This can reduce product purity, affect the stability of intermediates, and impact the safety, efficacy, and stability of the product, ultimately leading to product release failure.

[0006] The antibody reduction phenomenon is mainly caused by the release of intracellular components of antibody-producing cells (such as CHO cells), including the thioredoxin (Trx / TrxR) / glutathione reductase (GR) system and enzymes related to the generation of reduction reaction intermediates (such as glucose-6-phosphate dehydrogenase, hexokinase) and energy sources (such as NADPH). In addition to external factors, the properties of the antibody itself are intrinsic factors in the occurrence of antibody reduction. For example, antibodies with different light and heavy chain types have different sensitivities to reducing substances.

[0007] Common measures to prevent antibody reduction, depending on the cause of antibody reduction, include continuous aeration of the harvest supernatant, addition of metal ions, inhibition of reductase activity, lowering the antibody storage temperature, reducing sample storage time, adding oxidants, and controlling deep filtration pressure to reduce the release of reducing components. However, blindly adding anti-reduction measures will increase process complexity and production costs, and may also affect product quality. For example, relevant studies have reported that continuous aeration increases the proportion of acidic peaks in antibody molecules, and the addition of metal ions adds additional steps to downstream purification and may affect product quality.

[0008] Currently, methods for assessing antibody reduction risk have been reported, primarily listing a range of assessment methods, such as NADPH, DCPIP, and LDH activity, for assessing reduction risk in cell culture medium supernatants. It is also noted that the primary factor leading to antibody reduction is NADPH release, not TrxR enzyme activity. However, applying the same standard to assess reduction risk for different antibody molecules can lead to misjudgments. Furthermore, establishing a separate standard for each antibody molecule requires significant time and labor costs.

[0009] In summary, it is necessary to develop a more systematic and efficient method to judge the reduction risk in the antibody production process. Summary of the Invention

[0010] The present invention discovered that the reduction risk of biomolecules with different reduction sensitivities varies significantly. By pre-classifying biomolecules (such as antibodies) based on their reduction sensitivity, relatively similar reduction risk assessment criteria exist among biomolecules within different categories (e.g., between reduction-sensitive molecules or between non-reduction-sensitive molecules). By first assessing reduction sensitivity and then applying different reduction risk assessment criteria to different types of biomolecules, a more systematic and efficient reduction risk detection method can be established while ensuring accuracy.

[0011] Based on the above findings, the present invention first provides a method for assessing the reduction risk of biomolecules, wherein the method comprises:

[0012] Obtain the percentage decrease in purity of the main peak of CE-SDS of biomolecules under reducing conditions Δ MS and compare it with the reduction sensitivity threshold S of the molecule th For comparison, when Δ MS ≥S th When the biomolecule is a reduction-sensitive molecule, MS th When the biomolecule is a non-reduction sensitive molecule;

[0013] The reduction indicator level C of the cell culture medium containing reduction-sensitive molecules or non-reduction-sensitive molecules is d comparing with a reduction level threshold to assess whether the biomolecule has a reduction risk;

[0014] Wherein, the reduction sensitivity threshold S th The biomolecule is a protein containing a disulfide bond.

[0015] ​As a preferred embodiment, the biomolecule includes a disulfide bond-containing protein or a complex thereof; more preferably, the biomolecule is selected from one or more of an antibody or a functional fragment thereof, and an antibody complex.

[0016] As a preferred embodiment, when the reduction indicator level of the reduction sensitive molecule in the cell culture medium is C d Greater than or equal to the reduction level threshold C of the reduction-sensitive molecule s When the reduction-sensitive molecule has a reduction risk in the cell culture medium, otherwise, there is no reduction risk; or when the reduction indicator level C of the non-reduction-sensitive molecule in the cell culture medium is d Greater than or equal to the reduction level threshold C of the non-reduction sensitive molecule ns When the non-reduction sensitive molecule is reduced in the cell culture medium, there is a risk of reduction; otherwise, there is no risk of reduction.

[0017] As a preferred embodiment, the reducing condition includes a reducing agent, and the reducing agent is selected from one or more of DTT, Cys, and MESNA; preferably, the reducing condition includes MESNA at a concentration of 0.5 to 2 mM.

[0018] As a preferred embodiment, the reducing conditions also include at least one of the following: 1) the reaction temperature is 15-26°C; 2) the volume percentage of the biomolecule in the reaction system does not exceed 20%; 3) the pH value of the reaction system is 6.5-7.5; 4) the reaction time is 0.25-24h.

[0019] As a preferred solution, the biomolecule reduction level threshold C is determined s and C ns The methods include:

[0020] The cell culture medium is diluted in series to have different reduction levels before being mixed with the biomolecule to be tested and a reaction is established;

[0021] Identify the lowest concentration of cell culture medium at which one or more biomolecules to be tested begin to be reduced, and detect the level of the reduction indicator in the cell culture medium, which is determined as the reduction level threshold C of the corresponding biomolecule. s or C ns .

[0022] As a preferred embodiment, the reduction indicator includes total free sulfhydryl or NADPH.

[0023] As a preferred embodiment, when the biomolecule is a reduction-sensitive molecule, the reduction level threshold C s Including total free thiol threshold C s(总自由巯基) or NADPH threshold C s(NADPH), wherein the C s(总自由巯基) 100±10μM / L, preferably C s(NADPH) It is 0.33±0.10μM / L.

[0024] As a preferred embodiment, when the biomolecule is a non-reduction sensitive molecule, the reduction level threshold C ns Including total free thiol threshold C ns(总自由巯基) or NADPH threshold C ns(NADPH) , the C ns(总自由巯基) 200±10μM / L, preferably C ns(NADPH) It is 0.67±0.10μM / L.

[0025] As a preferred embodiment, the cell culture medium is selected from cell culture supernatant, clarified cell culture fluid, and affinity flow-through fluid.

[0026] Furthermore, the present invention also provides a method for producing a biomolecule, comprising:

[0027] The reduction risk of the biomolecule is assessed using the method described in any one of the above schemes. When the assessment result shows that the biomolecule has a reduction risk in the cell culture medium, anti-reduction measures are applied to the cell culture medium.

[0028] As a preferred option, the reduction risk in the cell culture medium at each link in the biomolecule production process is evaluated separately, and anti-reduction measures are applied to the cell culture medium in the links where reduction risk exists; preferably, the reduction risk assessment is performed on the cell supernatant harvesting and biomolecule purification links in the production process.

[0029] The present invention also provides a kit for evaluating the reduction risk of a biomolecule, comprising: one or more first reagents for determining whether a biomolecule is a reduction-sensitive molecule or a non-reduction-sensitive molecule; and one or more reduction indicator levels C for measuring a cell culture medium. d The second reagent.

[0030] As a preferred embodiment, the reduction indicator includes total free sulfhydryl or NADPH agent.

[0031] As a preferred embodiment, the first reagent includes one or more selected from DTT, Cys, and MESNA; more preferably, the first reagent includes MESNA; further preferably, the final concentration of MESNA when used to determine whether a biomolecule is a reduction-sensitive molecule or a non-reduction-sensitive molecule is 0.5-2 mM.

[0032] Furthermore, the present invention also provides a non-transitory computer-readable storage medium having computer-readable instructions stored thereon. When the computer-readable instructions are executed by a processor, the steps of the method described in any of the previous schemes or the steps of the production method described in any of the previous schemes are implemented.

[0033] By assessing the reduction sensitivity of biomolecules (such as antibodies) and measuring the levels of reduction indicators in cell culture media, the method of the present invention can accurately identify the risk points for reduction of different types of biomolecules, facilitating the implementation of targeted preventive strategies. The method is suitable for accurately assessing the reduction risk of biomolecules such as antibodies during the development phase of their production processes. Based on the identified reduction risk points, targeted anti-reduction measures can be implemented, significantly improving the success rate of process scale-up and large-scale production, and reducing the costs of process development and production failures caused by reduction issues. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] FIG1 shows the antibody reduction system mentioned in the present invention.

[0036] FIG2 is a CE-SDS-NR spectrum of antibody molecule g after reduction in an embodiment of the present invention.

[0037] FIG3 shows the incubation experimental results of reduction-sensitive molecules f and g in an embodiment of the present invention.

[0038] FIG4 shows the incubation experiment results of non-reduction sensitive molecules bd and h in an embodiment of the present invention.

[0039] FIG5 shows the NADPH and total free sulfhydryl contents during the cell culture and harvest stages in an embodiment of the present invention.

[0040] FIG6 shows the total enzyme activity during the cell culture and harvesting stages in an embodiment of the present invention.

[0041] FIG. 7 shows the TrxR enzyme activity during the cell culture and harvest stages in an embodiment of the present invention. DETAILED DESCRIPTION

[0042] The following describes specific embodiments of the present invention in detail. It should be understood that the specific embodiments described herein are intended only to illustrate and explain the present invention and are not intended to limit the present invention. Those skilled in the art may make various modifications and variations to the present invention without departing from the scope or spirit of the present invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0043] Unless otherwise indicated, all terms (including technical and scientific terms) used to disclose the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are provided to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0044] Explanation of terms

[0045] As used herein, the terms "comprising," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0046] The recitation of numerical ranges herein by endpoints includes all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0047] Concentration values ​​used in this invention include fluctuations within a certain range. For example, fluctuations within a certain precision range are permitted. For example, for 2%, fluctuations within ±0.1% are permitted. For larger values ​​or values ​​that do not require overly precise control, greater fluctuations are permitted. For example, for 100 mM, fluctuations within ±1%, ±2%, ±5%, etc. are permitted. Regarding molecular weight, fluctuations within ±10% are permitted.

[0048] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0049] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0050] In the present invention, “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0051] In the present invention, the terms "optionally," "optional," "optionally," "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If multiple "optional" or "optional" terms appear in a technical solution, unless otherwise specified and there are no contradictions or constraints, each "optional" or "optional" term is independent of the others.

[0052] In the present invention, the term "biomolecule" refers to the molecular unit that constitutes an organism, including various biomacromolecules such as carbohydrates, lipids, proteins, and nucleic acids.

[0053] As used herein, the term "antibody" encompasses both monospecific and multispecific antibodies. Functional fragments of antibodies refer to fragments that possess one or more of the following functions: antigen recognition, binding, and neutralization, particularly those containing disulfide bonds. In some embodiments, the functional fragments may be Fab fragments (antigen-binding fragments), scFv (single-chain antibody fragment), and the like.

[0054] As used herein, the term "antibody complex" refers to a complex formed by the binding of one or more antibody molecules to other molecules (e.g., antigens, drugs, etc.). These complexes can be immune complexes formed by the binding of antigens and antibodies, or complexes formed by the interaction of antibodies with other molecules. Antibody complexes play an important role in immunology, pathogen clearance, and inflammatory processes. In some embodiments, the antibody complexes include antibody-drug conjugates (ADCs).

[0055] In the present invention, the term "cell culture medium" refers to the extracellular medium of cells that produce antibody molecules, the main components of which are cell culture medium and cell metabolites. In some embodiments, the cell culture medium can be a cell culture supernatant. In some embodiments, the cell culture medium can be a clarified cell culture fluid. In some embodiments, the cell culture medium can be an affinity flow-through. As used herein, cell culture medium supernatant and clarified cell culture medium refer to cell culture fluid from which cells and cell debris have been removed. In some embodiments, the cell culture medium supernatant and clarified cell culture medium have a turbidity of ≤30 NTU.

[0056] The term "cell culture supernatant" herein refers to the extracellular medium obtained by centrifuging a portion of the cell culture fluid after the cell culture is completed to remove the cells by low-speed centrifugation (e.g., ≤600g), the main components of which are cell culture medium and cell metabolites.

[0057] The term "clarified cell culture fluid" as used herein refers to the cell culture fluid obtained by removing cells and cell debris by centrifugation or deep filtration after the cell culture is completed. The main components of the clarified cell culture fluid are cell culture medium and cell metabolites.

[0058] "Affinity flow-through" in this context refers to the supernatant obtained from a depth-filtered sample subjected to affinity chromatography to remove the target antibody molecules secreted from the medium. During the affinity chromatography process, the storage containers for the depth-filtered sample and flow-through were blanketed with nitrogen to remove air.

[0059] "Reduction" as used herein refers to the phenomenon in which disulfide bonds of a biomolecule (e.g., an antibody or fragment thereof) are cleaved under reducing conditions (e.g., TrxR and glutathione reductase (GR) systems). After disulfide bond cleavage, different antibody fragments can be formed, such as two heavy chains and one light chain, a haptic, two heavy chains, a single heavy chain and a single light chain, etc. The more disulfide bonds in an antibody are cleaved, the more reduced the antibody is.

[0060] "Reduction sensitivity" as used herein refers to the ability of an antibody to tolerate reducing substances (e.g., MESNA, protein sulfhydryl groups, free sulfhydryl groups). Different antibodies have varying degrees of tolerance to reducing substances. Under the same reducing conditions (e.g., the same concentration of MESNA or affinity flowthrough), a higher degree of antibody reduction indicates a weaker tolerance to reducing substances and a more sensitive antibody.

[0061] "Reduction indicator" herein refers to a substance that can be used to assess reduction risk. In some embodiments, the reduction indicator includes total free sulfhydryls and / or NADPH.

[0062] The term "total free sulfhydryls" as used herein primarily refers to protein sulfhydryls (e.g., thioredoxin sulfhydryls and glutathione protein sulfhydryls) and free sulfhydryls in cell culture media (e.g., cell culture supernatant, clarified cell culture fluid, and affinity flowthrough).

[0063] "Total enzyme activity" herein refers to the sum of TrxR and GR enzyme activities. Since the enzyme activity detection of both reductases is calculated based on the increase rate of 2-nitro-5-mercaptobenzoic acid (TNB) at a wavelength of 412 nm, the total enzyme activity of the two enzymes is measured when using the GR kit.

[0064] Methods for detecting restoration risks

[0065] The present invention relates to a method for assessing the reduction risk of biomolecules, wherein the method comprises:

[0066] Obtain the percentage decrease in purity of the main peak of CE-SDS of biomolecules under reducing conditions Δ MS and compare it with the reduction sensitivity threshold S of the moleculeth For comparison, when Δ MS ≥S th When the biomolecule is a reduction-sensitive molecule, MS th When the biomolecule is a non-reduction sensitive molecule;

[0067] The reduction indicator level C of the cell culture medium containing reduction-sensitive molecules or non-reduction-sensitive molecules is d comparing with a reduction level threshold to assess whether the biomolecule has a reduction risk;

[0068] Wherein, the reduction sensitivity threshold S th The biomolecule is a protein containing a disulfide bond.

[0069] The present invention has found that by first evaluating the reduction sensitivity and then applying different reduction risk assessment criteria to different types of biomolecules, the reduction risk of biomolecules can be detected more systematically and accurately.

[0070] In some embodiments, the biomolecule comprises a disulfide-bonded protein or a complex thereof.

[0071] In some embodiments, the biomolecule is selected from one or more of an antibody or a functional fragment thereof (such as a Fab fragment, scFv, etc.), and an antibody complex (such as ADCs, etc.).

[0072] In some embodiments, when the reduction indicator level of the reduction-sensitive molecule in the cell culture medium is C d Greater than or equal to the reduction level threshold C of the reduction-sensitive molecule s When the reduction-sensitive molecule has a reduction risk in the cell culture medium, otherwise, there is no reduction risk; or when the reduction indicator level C of the non-reduction-sensitive molecule in the cell culture medium is d Greater than or equal to the reduction level threshold C of the non-reduction sensitive molecule ns When the non-reduction sensitive molecule is reduced in the cell culture medium, there is a risk of reduction; otherwise, there is no risk of reduction.

[0073] In some embodiments, the reducing conditions include a reducing agent, and the reducing agent is selected from one or more of DTT (dithiothreitol), Cys (cysteine), and MESNA (mercaptoethanesulfonate sodium).

[0074] ​In some preferred embodiments, the reducing conditions include MESNA at a concentration of 0.5 to 2 mM, preferably 0.5 mM / L, 1.0 mM / L, or 2.0 mM / L. This reducing agent can effectively distinguish antibodies, functional fragments, or complexes thereof with different reduction sensitivities.

[0075] In some embodiments, the reducing conditions further include at least one of the following: 1) a reaction temperature of 15-26°C; 2) a volume percentage of the biomolecule in the reaction system of no more than 20%, more preferably no more than 15%, and even more preferably no more than 10%; 3) a pH of 6.5-7.5; and 4) a reaction time of 0.25-24 hours. In some specific examples, the reaction vessel is sealed by purging with nitrogen to remove air (e.g., a 7 mL vial with an aluminum cap and rubber stopper).

[0076] In some embodiments, N-ethylmaleimide (NEM) is added to the reduced reaction system to terminate the reaction. In some embodiments, the termination reaction time is 5 to 30 minutes, preferably, such as 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 30 minutes.

[0077] The above-mentioned given reducing conditions can make antibodies or their functional fragments or their complexes with different reducing sensitivities form different Δ MS Numeric value.

[0078] In some specific embodiments, Δ MS The calculation method is:

[0079] Δ MS / %=Intact control -Intact MESNA

[0080] Among them, “Δ MS " is the percentage of reduction in the purity of the main peak of the antibody determined by non-reducing CE-SDS and other detection methods; "Intact control " indicates the percentage of the purity of the main peak of the antibody without adding MESNA; " MESNA ” indicates the percentage of the purity of the main peak of the antibody after MESNA reduction.

[0081] In some embodiments, determining the biomolecule reduction level threshold C s and C ns The methods include:

[0082] The cell culture medium is diluted in series to have different reduction levels before being mixed with the biomolecule to be tested and a reaction is established;

[0083] Identify the lowest concentration of cell culture medium at which one or more biomolecules to be tested begin to be reduced, and detect the level of the reduction indicator in the cell culture medium, which is determined as the reduction level threshold C of the corresponding biomolecule. s or C ns .

[0084] In the present invention, the reduction level threshold C s or C ns It can be a threshold value of one or any combination of the reduction indicator levels. In some embodiments, the reduction level threshold C s or C ns is a threshold value for one or more reduction indicator levels. In some embodiments, the reduction level threshold C s or C ns It is the weighted sum threshold of multiple reduction indicator levels.

[0085] In some embodiments, the reduction level after the gradient dilution can be a total free thiol gradient dilution level set at 20 μM / L, 40 μM / L, 60 μM / L, 80 μM / L, 100 μM / L, 200 μM / L, 300 μM / L, 500 μM / L, and 1000 μM / L. In some embodiments, the reduction level after the gradient dilution can be a NADPH gradient dilution level set at 0.1 μM / L, 0.2 μM / L, 0.3 μM / L, 0.4 μM / L, 0.5 μM / L, 0.6 μM / L, 0.7 μM / L, 0.8 μM / L, 0.9 μM / L, and 1.0 μM / L; in some embodiments, it can be set at 1.0 μM / L, 2.0 μM / L, 3.0 μM / L, 4.0 μM / L, and 5.0 μM / L. In some preferred embodiments, it can be set to 0.33 μM / L, 0.67 μM / L, 1 μM / L, 1.67 μM / L, or 3.33 μM / L.

[0086] In some embodiments, establishing the reaction comprises reacting the cell culture medium and the biomolecule to be tested in a reaction system at a reaction temperature of 15-26° C. and a pH of 6.5-7.5 (preferably, such as pH 7.2 or pH 7.5).

[0087] In some embodiments, the reaction system further contains one or more selected from Tris-HAc buffer, Tris-acetate buffer, and ultrapure water.

[0088] In some embodiments, biomolecules (such as antibody molecules) need to be ultrafiltration concentrated and exchanged, and the concentration is ≥15 g / L, preferably, such as 15 g / L, 20 g / L, 30 g / L, 60 g / L; the exchange buffer is acetic acid or acetate buffer, preferably, such as 50 mM Tris-HAc pH 7.5, 50 mM Tris-HAc pH 7.2, 110 mM NaCl pH 7.2.

[0089] In some embodiments, the reaction time is 0.25 to 24 hours, more preferably 0.25 to 2 hours. As an example, the reaction time can be 0.25 hours, 0.5 hours, 1.0 hours or 2.0 hours.

[0090] In some specific embodiments, the volume of the reaction system is 1 mL, 3 mL, 9 mL, preferably, 3 mL.

[0091] In some embodiments, N-ethylmaleimide (NEM) needs to be added to the reduced reaction system to terminate the reaction.

[0092] In some embodiments, the termination reaction time is 5 to 30 minutes, preferably, such as 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 30 minutes.

[0093] In some embodiments, the reduction level threshold C s and C ns In the determination method, the onset of reduction refers to a decrease in the purity of the biomolecule in the cell culture medium by more than 1%.

[0094] In some embodiments, the level of the reducing marker is detected using non-reducing CE-SDS or SDS-PAGE, or any combination thereof.

[0095] In some embodiments, the reduction level threshold C s and C ns In the determination method, a cell culture medium with a high reducing level (containing a high level of a reducing indicator) is used. For example, the affinity flow-through or a mixture containing a Tris-HAc saline buffer (e.g., 50mM Tris-HAc, 110mM NaCl, pH 7.2) is used. This provides the reducing conditions of the incubation reaction while mimicking the actual state of the biomolecule in cell culture medium.

[0096] Based on the above method, those skilled in the art can determine the reduction level threshold C corresponding to different types of biomolecules. s and C ns .

[0097] In some embodiments, the reduction indicator comprises total free sulfhydryls or NADPH.

[0098] In some embodiments, the total free sulfhydryl groups include reduced protein sulfhydryl groups and free sulfhydryl groups.

[0099] In some embodiments, the protein thiol group comprises a thioredoxin thiol group and a glutathione protein thiol group.

[0100] Existing reports have not explored the relationship between free thiol levels and the reduction risk of antibody molecules. However, the reduced protein thiol groups in the TrxR / GR reduction system and the free thiol groups in the cell medium are the direct driving forces for antibody reduction (as shown in Figure 1). Therefore, the levels of these two thiol groups can be used as indicators to judge the reduction risk.

[0101] In some specific embodiments, reduction indicators of cell culture media can be determined using commercially available reagents (such as reagents for determining the total free sulfhydryl content and / or reagents for determining the NADPH content) and known methods.

[0102] In some embodiments, when the biomolecule is a reduction-sensitive molecule, the reduction level threshold C s Including total free thiol threshold C s(总自由巯基) or NADPH threshold C s(NADPH) .

[0103] In some preferred embodiments, C s(总自由巯基) The concentration of the aqueous solution is 100±10 μM / L. Specifically, it can be 90 μM / L, 92 μM / L, 95 μM / L, 97 μM / L, 98 μM / L, 99 μM / L, 100 μM / L, 101 μM / L, 102 μM / L, 103 μM / L, 105 μM / L, 107 μM / L or 110 μM / L.

[0104] In some preferred embodiments, C s(NADPH) The concentration of the ethanol is 0.33±0.10 μM / L. Specifically, the concentration of the ethanol may be 0.23 μM / L, 0.28 μM / L, 0.30 μM / L, 0.31 μM / L, 0.32 μM / L, 0.33 μM / L, 0.34 μM / L, 0.35 μM / L, 0.38 μM / L or 0.43 μM / L.

[0105] In some embodiments, when the biomolecule is a non-reduction sensitive molecule, the reduction level threshold C ns Including total free thiol threshold C ns(总自由巯基) or NADPH threshold C ns(NADPH) .

[0106] In some preferred embodiments, Cns(总自由巯基) The concentration of the HCl in the aqueous solution is 200±10 μM / L. Specifically, the concentration of the HCl in the aqueous solution may be 190 μM / L, 195 μM / L, 197 μM / L, 198 μM / L, 199 μM / L, 200 μM / L, 201 μM / L, 202 μM / L, 203 μM / L, 205 μM / L or 210 μM / L.

[0107] In some preferred embodiments, C ns(NADPH) The concentration of the ethanol is 0.67±0.10 μM / L. Specifically, the concentration of the ethanol may be 0.57 μM / L, 0.62 μM / L, 0.65 μM / L, 0.66 μM / L, 0.67 μM / L, 0.68 μM / L, 0.69 μM / L, 0.70 μM / L, 0.73 μM / L or 0.77 μM / L.

[0108] The above thresholds can be widely used in determining the reduction risk of antibodies or their functional fragments, or antibody complexes.

[0109] In some embodiments, the cell culture medium is selected from the group consisting of cell culture supernatant, clarified cell culture fluid, and affinity flow-through.

[0110] In some specific embodiments, the cell culture medium includes one of D09 (cell culture supernatant on the ninth day of cell culture, Control (cell culture supernatant harvested by low-speed centrifugation at the end of cell culture), P1 max (cell culture supernatant when the instantaneous pressure of the first-level membrane package in deep filtration reaches the maximum), DF Pool (mixed cell culture supernatant after deep filtration), and affinity flow-through.

[0111] In some specific embodiments, the method for obtaining the cell culture supernatant may include centrifugation, deep filtration or other solid-liquid separation methods.

[0112] In some specific embodiments, for example, when performing comparative analysis on a control sample, the cell culture supernatant should be harvested by low-speed centrifugation (eg, ≤600 g) to avoid the release of reducing substances in the periplasm.

[0113] In some specific embodiments, during the storage and reaction of the cell culture medium (e.g., D09, Control, P1 max, DF Pool and affinity flow-through), the container needs to be pre-filled with nitrogen and covered with nitrogen again after the addition of the mixed solution. The nitrogen covering time is 10s, 30s, 60s, 1h, 2h, preferably, such as 10s and 60s.

[0114] Those skilled in the art can combine the above embodiments with common sense to obtain a preferred embodiment of the method for assessing the reduction risk of biomolecules in the present invention.

[0115] Production method

[0116] The present invention also provides a method for producing a biomolecule, comprising:

[0117] The reduction risk of the biomolecule is assessed using the method described in any one of the above schemes. When the assessment result shows that the biomolecule has a reduction risk in the cell culture medium, anti-reduction measures are applied to the cell culture medium.

[0118] In some embodiments, the reduction risk of cell culture media at each stage of the biomolecule production process is assessed, and anti-reduction measures are applied to the cell culture media at stages where reduction risk exists. In some preferred embodiments, reduction risk assessments are performed at the cell supernatant harvest and biomolecule purification stages of the production process.

[0119] In some specific implementations, multiple restoration level thresholds may be set to obtain restoration risk results at multiple levels, and different anti-restoration measures may be applied to different levels of restoration risks.

[0120] In some specific embodiments, the anti-reduction measures can be selected from the following groups: ventilation (air, oxygen, etc.), adding reductase inhibitors (such as gold complexes, such as gold thioglucose, gold thiomalate, etc.), inhibiting reductase activity, lowering the antibody storage temperature, reducing the sample storage time, adding oxidants (such as copper sulfate) and controlling the deep filtration pressure to reduce the release of reducing components, etc.

[0121] The present invention does not impose any particular limitations on the subject performing the methods (including methods for assessing biomolecule reduction risk and methods for producing biomolecules). In some embodiments, the method is performed by a human; in some embodiments, the method is performed by a terminal device or a server; and in some embodiments, some steps of the method are performed by a human, while others are performed by a terminal device or a server. Those skilled in the art will determine the specific subject based on actual circumstances.

[0122] Related products

[0123] The present invention also provides a kit for evaluating the reduction risk of a biomolecule, comprising: one or more first reagents for determining whether a biomolecule is a reduction-sensitive molecule or a non-reduction-sensitive molecule; and one or more reduction indicator levels C for determining the reduction risk of a cell culture medium. d The second reagent.

[0124] In specific implementation, the purpose of the first reagent or the second reagent can be achieved by using multiple different reagents, or by using one reagent.

[0125] In some embodiments, the reduction indicator comprises total free sulfhydryls or an NADPH agent.

[0126] In some embodiments, the first reagent comprises one or more selected from DTT, Cys, and MESNA.

[0127] In some embodiments, the first reagent comprises MESNA.

[0128] In some embodiments, it is further preferred that the final concentration of the MESNA used to determine whether a biomolecule is a reduction-sensitive molecule or a non-reduction-sensitive molecule is 0.5-2 mM. In a specific embodiment, this can be achieved by setting the reagent concentration and providing instructions for use.

[0129] The present invention also provides a non-transitory computer-readable storage medium having computer-readable instructions stored thereon. When the computer-readable instructions are executed by a processor, the steps of the method described in any of the previous schemes or the steps of the production method described in any of the previous schemes are implemented.

[0130] The present invention further provides an electronic device, which includes a memory, a processor, and a computer program stored in the memory and run on the processor, wherein when the processor executes the computer program, it implements: the steps of the method described in any of the previous schemes, or the steps of the production method described in any of the previous schemes.

[0131] The present invention further provides a computer program product, which includes a computer program. When the computer program is read and executed by a computing device, the computing device executes: the steps of the method described in any of the previous schemes, or the steps of the production method described in any of the previous schemes.

[0132] Example

[0133] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made to experimental manuals or conventional conditions in the art, other experimental methods known in the art, or conditions recommended by the manufacturer.

[0134] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0135] Example 1: Determination of the main peak purity reduction value Δ of the antibody molecule under given conditionsMS , and according to the reduction sensitivity threshold S th Determining the reduction sensitivity of a molecule

[0136] In this example, under given conditions, the value of the decrease in the purity of the non-reduced CE-SDS main peak of the antibody molecule ah is calculated. MS , by comparing Δ MS and the reduction sensitivity threshold S th The size determines the reduction sensitivity of the molecule.

[0137] 1. Prepare the sample: Take an appropriate volume of sample according to the sample concentration. If the sample is turbid, centrifuge at 12000g for 1 min at room temperature to obtain the supernatant.

[0138] 2. Set up the reaction: Set up the reaction in a 7mL vial. Before the reaction, purge the container with nitrogen to remove air. Add a certain volume of 50mM Tris-Hac pH 7.5 buffer (recommended pH range 6.5-7.5), antibody molecule ah at a final concentration of 2mg / mL, and 600μL of 10mM MESNA (final concentration of 2mM). The control contains only working solution and antibody molecule, with a MESNA concentration of 0, so that the final reaction volume is 3mL. Purge the container with nitrogen again, seal, mix, and incubate at room temperature (15-26°C) for 30 minutes.

[0139] 3. Termination of reaction: After the reaction is completed, use a syringe to take 900 μL of the reaction solution and add 100 μL of 250 mM NEM (final concentration is 25 mM). Incubate at room temperature for 10 min, terminate the reaction, and store at -70 ± 10 ° C. Perform non-reducing CE-SDS detection and calculate Δ MS value.

[0140] The reduction sensitivity of the antibody molecule ah is summarized as shown in Table 1:

[0141] Table 1 Molecular reduction sensitivity

[0142] According to Δ MS ≥S th That is Δ MS ≥5%, the antibody molecule is a reduction-sensitive molecule; Δ MS th That is Δ MS <5%, the antibody molecule is a non-reduction sensitive molecule. As shown in Table 1, the Δ MS Greater than 5%, the molecule has a high reduction sensitivity and is a reduction sensitive molecule; the Δ MS ​Less than 5%, the reduction sensitivity is low, and it is a non-reduction sensitive molecule. Figure 2 shows the CE-SDS-NR spectrum of antibody molecule g after MESNA reduction.

[0143] Example 2: Determination of reduction risk of reduction-sensitive molecules;

[0144] In this example, NADPH and total free sulfhydryl gradient reduction dilutions were set to incubate reduction-sensitive antibody molecules f and g, and the NADPH and total free sulfhydryl contents at the time when the molecules began to be reduced were determined to verify the reduction thresholds, which were C s(NADPH) =0.33 μM / L or C s(总自由巯基) =100 μM / L or a combination of both.

[0145] 1. First, cell culture and deep filtration operations are performed according to the process method to obtain the clarified cell culture medium supernatant containing antibody molecules a, d, e, and f. The deep filtration process is as follows:

[0146] 1) Connect the pipeline, measure the dead volume of the pipeline, and calibrate the flow rate.

[0147] 2) Exhaust and flush: After flushing the pipeline with MilliQ water, connect two primary filter membrane packages D0HC (2*23cm 2 Clamp the outlet, open the exhaust valve, remove bubbles, and when liquid continuously flows out of the exhaust valve, close the exhaust valve and open the outlet. 2 MilliQ water is used to rinse the membrane package at ≤600LMH. Connect the secondary filter membrane package A1HC (23cm 2 ), exhaust and flush, use ≥100L / m 2 Rinse the membrane cassette with MilliQ water at ≤600LMH.

[0148] 3) Balance (≥100L / m 2 ) : Clamp the inlet port and slowly place the membrane into the container containing Buffer A to prevent air from entering. Open the tube clamp and flush the membrane cassette at ≤ 600 LMH until the pH and conductivity of the liquid at the outlet are close to those of Buffer A.

[0149] 4) Filtration: Take out the cell culture medium after secondary centrifugation and weigh it; clamp the feed port and slowly put it into the culture medium to avoid air intake. Open the tube clamp and perform deep filtration at ≤100LMH, and discard ≥5L / m at the outlet. 2 , start collecting.

[0150] 5) Top wash: use ≥40L / m 2 The remaining liquid in the Buffer A top-wash filter should be collected separately in a new bottle and should not be mixed with the liquid collected during the filtration stage.

[0151] 2. The supernatant obtained after deep filtration is subjected to affinity chromatography to remove the antibody molecules d therein to obtain affinity flow-through.

[0152] 3. Gradient reduction dilution setting: Determine the content of NADPH and total free thiol groups in the affinity flow-through as the gradient reduction mother solution. According to the preliminary experiment, the total free thiol reduction gradient was set to Control, 100μM / L, 200μM / L, 300μM / L, 500μM / L; correspondingly, the NADPH reduction gradient was Control, 0.33μM / L, 0.67μM / L, 1.00μM / L, 1.67μM / L. The control group added a reduction dilution containing total free thiol groups (500μM / L) & NADPH (1.67μM / L), and then added NEM and incubated at room temperature for 30min (NEM reacts with reduced thiol groups to prevent them from reducing antibody molecules), and then added antibody molecules for incubation reaction.

[0153] 4. Set up the reaction: Set up the reaction in a 7 mL vial. Purge the container with nitrogen to remove air before the reaction. Add a certain volume of 50 mM Tris-Hac 110 mM NaCl pH 7.2 buffer (recommended pH range 6.5-7.5) in sequence. Add different volumes of affinity flow-through masterbatch to obtain the above-mentioned gradient reduction dilution solution, with a final concentration of 2 mg / mL for antibodies f and g. Incubate the control sample for 30 minutes before adding the antibody molecules, bringing the final reaction volume to 3 mL. Purge the container with nitrogen again, seal, mix, and incubate at room temperature (15-26°C) for 24 hours.

[0154] 5. Termination of reaction: After the reaction is completed, use a syringe to take 900 μL of the reaction solution and add 100 μL of 250 mM NEM (final concentration is 25 mM). Incubate at room temperature for 10 min, terminate the reaction, and store at -70 ± 10 ° C. Perform non-reducing CE-SDS detection and calculate Δ MS value.

[0155] As shown in Figure 3, with the increase of NADPH and total free sulfhydryl concentrations, the percentage of the main peak of antibody molecules f and g decreased more significantly, and the reduction became more intense. It can also be seen from the figure that when the NADPH and total free sulfhydryl concentrations were 0.33μM / L and 100μM / L, Δ MS (As shown in Figure 3, the difference between the main peak value of the control group and the main peak value of the reduction group is Δ MS ) is significantly greater than 1%, and the antibody molecules begin to be reduced.

[0156] Example 3: Determination of reduction risk of non-reduction sensitive molecules;

[0157] In this example, NADPH and total free sulfhydryl gradient reduction dilutions were set to incubate non-reduction sensitive antibody molecules bd and h, and the NADPH and total free sulfhydryl contents at the time when the molecules began to be reduced were determined to verify the reduction thresholds, which were C ns(NADPH) =0.67 μM / L or C ns(总自由巯基) =200 μM / L or a combination of both.

[0158] 1. Gradient reduction dilution setting: Use the affinity flow-through mother solution described in Example 2. According to the preliminary experiment, the total free thiol reduction gradient was set to Control, 200μM / L, 300μM / L, 500μM / L, 1000μM / L; correspondingly, the NADPH reduction gradient was Control, 0.67μM / L, 1.00μM / L, 1.67μM / L, 3.33μM / L. The control group added total free thiol (1000μM / L) & NADPH (3.33μM / L) reduction dilution, then added NEM, and incubated at room temperature for 30min (NEM reacts with reduced thiol groups to prevent them from reducing antibody molecules), and then added antibody molecules for incubation reaction.

[0159] 2. Set up the reaction: Set up the reaction in a 7 mL vial. Purge the container with nitrogen before the reaction to remove air. Add a certain volume of 50 mM Tris-Hac 110 mM NaCl pH 7.2 buffer (recommended pH range 6.5-7.5) in sequence. Add different volumes of affinity flow-through masterbatch to obtain the above-mentioned gradient reduction dilution solution, with a final concentration of 2 mg / mL antibody molecules bd and h. Incubate the control sample for 30 minutes before adding the antibody molecules, finally bringing the final reaction volume to 3 mL. Purge the container with nitrogen again, seal, mix, and incubate at room temperature (15-26°C) for 24 hours.

[0160] 3. Termination of reaction: After the reaction is completed, use a syringe to take 900 μL of the reaction solution and add 100 μL of 250 mM NEM (final concentration is 25 mM) to terminate the reaction. Store in a freezer at -70 ± 10 °C and perform non-reducing CE-SDS detection. Calculate Δ MS value.

[0161] As shown in Figure 4, with the increase of NADPH and total free thiol concentrations, the percentage of the main peak of antibody molecules bd and h decreased more significantly, and the reduction became more intense. It can also be seen from the figure that when the concentrations of NADPH and total free thiol were 0.67μM / L and 200μM / L, Δ MS (The difference between the main peak value of the control group and the main peak value of the reduction group is Δ MS ) is greater than 1%, the antibody molecules begin to be reduced.

[0162] Example 4: Evaluation of the risk of reduction of antibody molecules in cell culture supernatants and clarified cell culture fluids.

[0163] In this example, the reduction index substance level C in the cell culture supernatant and the clarified cell culture fluid of antibody molecules a, d, e, and f was measured. d and with the reduction level threshold C s and C ns Comparative determination of reduction risk of target antibody molecules during cell culture and harvest stages.

[0164] Cell culture and deep filtration were performed according to the platform cell culture process and deep filtration process. Appropriate volumes of corresponding samples were obtained during the process, including D09 (cell culture supernatant from the ninth day of cell culture), Control (cell culture supernatant harvested by low-speed centrifugation at the end of cell culture), P1 max (cell culture supernatant at the time when the instantaneous pressure of the primary membrane package during deep filtration reached maximum), and DF Pool (cell culture supernatant pooled after deep filtration). The levels of reducing indicators and enzyme activities in the samples, including total free sulfhydryl levels, NADPH levels, total enzyme activity, and TrxR enzyme activity, were measured using commercially available kits. The results are shown in Figures 5-7.

[0165] For antibody molecules a(Δ MS <5%), the NADPH and total free sulfhydryl contents of the fermentation broth itself were low, that is, C d(NADPH) <C ns(NADPH) And C d(总自由巯基) <C ns(总自由巯 基) Therefore, there is no risk of reduction of molecule a during the cell culture stage; however, during harvest, cells were more disrupted (ΔP of the first membrane package of the P1 max sample was 5.9 psi), resulting in a significant increase in the content of NADPH and total free sulfhydryl groups in the clarified harvest fluid, that is, C in P1 max and DF Pool samples was d(NADPH) ≥C ns(NADPH) And C d(总自由巯基) ≥C ns(总自由巯基) Therefore, there is a risk of reduction of molecule a during the cell harvest stage. As shown in Table 2, the main peak content of the sample immediately purified after deep filtration was only 84.50%, showing obvious reduction. After one day of storage, the main peak content continued to decrease to 68.22%. This indicates that molecule a does have a high reduction risk during the cell harvest stage and requires anti-reduction measures, such as controlling the pressure of the deep filtration primary membrane package. The smaller the ΔP, the less cell disruption and the less reducing substances that appear during the culture process.

[0166] Table 2 Stability evaluation of molecule a deep filtration samples

[0167] For antibody molecules d(Δ MS <5%), the fermentation broth itself has high NADPH and total free sulfhydryl content, that is, C d(NADPH) ≥C ns(NADPH) And C d(总自由巯基) ≥C ns(总自由巯基) Therefore, there is a risk of reduction of molecule d during the cell culture stage; even if the cell disruption is small (ΔP of the first membrane package of the P1 max sample is 3.6psi), the NADPH and total free sulfhydryl content in the clarified harvest liquid are still high, and C in the sample d(NADPH) ≥C ns(NADPH) And C d(总自由巯基) ≥C ns(总自由巯基) Therefore, molecule d also presents a reduction risk during the cell harvest stage. As shown in Table 3, the main peak content detected in the sample stored at 2-8°C for 2 days during purification was only 57.67%, indicating significant reduction. However, the main peak content of the sample stored at -80°C was still 97.17%, showing no obvious reduction. This indicates that molecule d does have a high reduction risk during the cell culture stage. Furthermore, after deep filtration, the reduced substances are still present in the clarified harvest liquid, so the reduction risk remains high, and anti-reduction measures, such as ventilation, are required.

[0168] Table 3 Stability evaluation of molecule d centrifugation supernatant samples

[0169] For antibody molecules e(Δ MS <5%), the NADPH and total free sulfhydryl content of the fermentation broth itself is low, that is, C d(NADPH) <C ns(NADPH) And C d(总自由巯基) <C ns(总自由巯基) Therefore, there is no risk of reduction of molecule e during the cell culture stage; although there is more cell disruption during harvest (ΔP of the first membrane package of the P1 max sample is 5.5psi), the NADPH and -SH contents in the DF Pool sample are still low, that is, C d(NADPH) <C ns(NADPH) And C d(总自由巯基) <C ns(总自由巯基) Therefore, there is no reduction risk in the DF Pool mixed sample of Molecule e. Table 4 shows that compared with the Control group, the main peak content of samples stored at room temperature for 1 and 2 days did not change significantly. This indicates that Molecule e does not have a reduction risk during the cell culture and harvesting stages, and anti-reduction measures are not required. However, attention should be paid to the deep filtration pressure.

[0170] Table 4 Stability evaluation of molecular e depth filtration samples

[0171] For antibody molecules f(Δ MS≥5%), the NADPH and total free sulfhydryl content in the cell culture and harvest stages were low, that is, C d(NADPH) <C s(NADPH) And C d(总自由巯基) <C s(总自由巯基) Therefore, there is no reduction risk for molecule f during the cell culture and harvesting stages. As shown in Table 5, compared with the Control group, there is no significant change in the main peak content of samples stored at room temperature and 2-8°C for 3 days, which indicates that there is indeed no reduction risk for molecule f during the cell culture and harvesting stages, and no anti-reduction measures are needed.

[0172] Table 5 Stability evaluation of depth filtration samples of molecule f

[0173] According to the kit protocol, the unit enzyme activity for total enzyme activity is defined as the conversion of 1 μM TNB per minute (1U); the unit enzyme activity for TrxR activity is defined as the conversion of 1 nM TNB per minute (1U). Figures 6 and 7 show that the minimum total enzyme activity is approximately 20 mU / mL, corresponding to a daily catalytic reduction of 4.32 g / mL of antibody molecules (calculated based on a MW of 150 kDa), approximately 1,000-fold higher than the typical antibody expression level. The minimum TrxR activity is approximately 10 mU / mL, corresponding to a daily catalytic reduction of 2.16 g / mL of antibody molecules (calculated based on a MW of 150 kDa), also approximately 1,000-fold higher than the typical antibody expression level. Therefore, both total and TrxR enzyme activities are very high during the cell culture and harvest stages, making them unsuitable indicators for measuring the reduction risk of cell culture medium supernatants.

[0174] The measured reduction index substance data are summarized as shown in Table 6:

[0175] Table 6

[0176] As shown in Table 6, the system reducing power level (NADPH and total free sulfhydryl content level) is directly related to whether the antibody molecule is reduced. However, the total enzyme activity and TrxR enzyme activity in the cell culture and harvest stages are both very high and cannot be used as evaluation criteria.

[0177] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A method for assessing the reduction risk of a biomolecule, wherein: The method comprises: Obtain the percentage decrease in purity of the main peak of CE-SDS of biomolecules under reducing conditions Δ MS and compare it with the reduction sensitivity threshold S of the molecule th For comparison, when Δ MS ≥S th When the biomolecule is a reduction-sensitive molecule, MS th When the biomolecule is a non-reduction sensitive molecule;​ The reduction indicator level C of the cell culture medium containing reduction-sensitive molecules or non-reduction-sensitive molecules is d comparing with a reduction level threshold to assess whether the biomolecule has a reduction risk; Wherein, the reduction sensitivity threshold S th The biomolecule is a protein containing a disulfide bond.

2. The method according to claim 1, wherein When the reduction indicator level of reduction-sensitive molecules in cell culture medium is C d Greater than or equal to the reduction level threshold C of the reduction-sensitive molecule s When the reduction-sensitive molecule has a reduction risk in the cell culture medium, otherwise, there is no reduction risk; or when the reduction indicator level C of the non-reduction-sensitive molecule in the cell culture medium is d Greater than or equal to the reduction level threshold C of the non-reduction sensitive molecule ns When the non-reduction sensitive molecule is reduced in the cell culture medium, there is a risk of reduction; otherwise, there is no risk of reduction.

3. The method according to claim 1, wherein The reducing conditions include a reducing agent, and the reducing agent is selected from one or more of DTT, Cys, and MESNA; preferably, the reducing conditions include MESNA at a concentration of 0.5 to 2 mM.

4. The method according to claim 3, wherein the reducing conditions further include at least one of the following: 1) the reaction temperature is 15-26°C; 2) the volume percentage of the biomolecule in the reaction system does not exceed 20%; 3) the pH value of the reaction system is 6.5-7.5; 4) the reaction time is 0.25-24 hours.

5. The method according to claim 1, wherein Determine the biomolecule reduction level threshold C s or C ns The methods include: The cell culture medium is diluted in series to have different reduction levels before being mixed with the biomolecule to be tested and a reaction is established; Identify the lowest concentration of cell culture medium at which one or more biomolecules to be tested begin to be reduced, and detect the level of the reduction indicator in the cell culture medium, which is determined as the reduction level threshold C of the corresponding biomolecule. s or C ns .

6. The method according to any one of claims 1 to 5, wherein: The reduction indicators include total free sulfhydryls or NADPH.

7. The method according to claim 6, wherein: When the biomolecule is a reduction-sensitive molecule, the reduction level threshold C s Including total free thiol threshold C s(总自由巯基) or NADPH threshold C s(NADPH) , wherein the C s(总自由巯基) is 100±10μM / L, the C s(NADPH) It is 0.33±0.10μM / L.

8. The method according to claim 6, wherein: When the biomolecule is a non-reduction sensitive molecule, the reduction level threshold C ns Including total free thiol threshold C ns(总自由巯基) or NADPH threshold C ns(NADPH) , the C ns(总自由巯基) 200±10μM / L, the C ns(NADPH) It is 0.67±0.10μM / L.

9. The method according to claim 1, wherein The cell culture medium is selected from cell culture supernatant, clarified cell culture fluid or affinity flow-through fluid.

10. A method for producing a biomolecule, comprising: The method according to any one of claims 1 to 9 is used to assess the reduction risk of a biomolecule, and when the assessment result shows that the biomolecule has a reduction risk in a cell culture medium, anti-reduction measures are applied to the cell culture medium.

11. The method for producing a biomolecule according to claim 10, wherein: The reduction risk in the cell culture medium at each link in the biomolecule production process is evaluated separately, and anti-reduction measures are applied to the cell culture medium in the links where reduction risk exists; preferably, the reduction risk assessment is performed on the cell supernatant harvesting and biomolecule purification links in the production process.

12. A kit for evaluating the reduction risk of a biomolecule, comprising: one or more first reagents for determining whether a biomolecule is a reduction-sensitive molecule or a non-reduction-sensitive molecule; and, One or more reduction indicators for determining the level of cell culture medium C d The second reagent.

13. The kit according to claim 12, wherein The reduction indicators include total free sulfhydryls or NADPH.

14. The kit according to claim 12, wherein The first reagent includes one or more selected from DTT, Cys, and MESNA; preferably, the first reagent includes MESNA; further preferably, the final concentration of MESNA when used to determine whether a biomolecule is a reduction-sensitive molecule or a non-reduction-sensitive molecule is 0.5-2 mM.

15. A non-transitory computer-readable storage medium having computer-readable instructions stored thereon, which, when executed by a processor, implement the steps of the method according to any one of claims 1 to 9, or the steps of the production method according to claim 10 or 11.

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