Method for reducing host cell proteins in protein sample

Abstract

The invention discloses a method for reducing host cell protein in a protein sample. The method comprises the following steps: carrying out affinity chromatography on a cell supernatant containing a target protein to obtain an eluent; and neutralizing the eluent, and the like. The method at least has the advantages of simplicity, convenience, rapidness, no need of additional expensive instruments, equipment and reagent consumables and the like.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for reducing host cell proteins in a protein sample. Background Art

[0002] Host cell protein (HCP) refers to endogenous proteins extracted from host cells. It is a major type of process-related impurities. Due to its own immunogenicity and proteolytic activity, it seriously affects the stability and efficacy of drugs and is considered to be a key quality attribute of antibody drugs.

[0003] Currently, sandwich ELISA kits are the gold standard for detecting HCP residues. However, this method is highly dependent on the total host cell protein coverage of the anti-HCP antibodies in the kit. Insufficient antibody coverage in the ELISA assay may result in the misdetection of some residual HCPs in the final product, potentially leading to immune reactions and other drug safety issues in patients. Furthermore, residual HCPs in injectable formulations may trigger adverse reactions in patients. Most HCPs have an isoelectric point between 3 and 7, making them amenable to removal through appropriate processes and parameters. However, there are currently no methods specifically designed for the removal of HCP residues in biological products. Most existing reports focus on the removal of HCP residues in animal cells, such as CHO cells, primarily through affinity and ion exchange chromatography, which are complex and costly. HCP residues are ubiquitous in fermentation broths, particularly in cells. While most of these residues are gradually removed during downstream processing, some remain difficult to remove, requiring specialized process routes and parameters.

[0004] Therefore, there is an urgent need in the art to develop new purification methods to reduce host cell proteins in protein samples. Summary of the Invention

[0005] Based on this, it is necessary to provide at least a method for reducing host cell proteins in a protein sample.

[0006] In a first aspect of the present application, a method for reducing host cell protein in a protein sample is provided, the method comprising:

[0007] The cell supernatant containing the target protein is subjected to affinity chromatography to obtain an eluate;

[0008] neutralizing the eluate to obtain a neutralized solution; and

[0009] filtering the neutralized treatment liquid;

[0010] The neutralization treatment of the eluate comprises the following steps a, b, c and d:

[0011] a using an acid solution to adjust the pH of the eluent to 3.0 ~ 4.5, acid treatment to obtain an acid-treated solution;

[0012] b using an alkaline solution 1 to adjust the pH of the acid treatment solution to 4.5 ~ 5.5, the first alkali treatment to obtain an alkaline treatment solution 1;

[0013] c using an alkaline solution 2 to adjust the pH of the alkaline treatment solution 1 is 5.5 ~ 6.5, the second alkali treatment to give an alkaline treatment solution 2; and

[0014] d. Using an alkaline solution 3 to adjust the pH of the alkaline treatment solution 2 to 6.5~7.5 to obtain the neutralization treatment solution.

[0015] In some embodiments, the affinity chromatography comprises the following steps performed sequentially:

[0016] The eluate is obtained by balancing, loading, first elution, second elution, elution and collection.

[0017] In some embodiments, the affinity chromatography satisfies one or more of the following conditions 1) to 7):

[0018] 1) Equilibration buffer includes: 10 mM–50 mM phosphate and 100 mM–500 mM NaCl, pH 6.5–8.5;

[0019] 2) the eluent 1 used for the first elution comprises: 10 mM to 50 mM phosphate and 50 M to 800 M sodium chloride, pH 6.5 to 10.5;

[0020] 3) the eluent 2 used for the second eluent comprises: 10 mM to 50 mM phosphate, pH 6.5 to 10.5;

[0021] 4) Elution buffers include: 50 mM to 200 mM citric acid-sodium citrate, pH 3.0 to 5.5;

[0022] 5) The volume of the equilibration buffer used for the equilibration is 3 to 8 column volumes;

[0023] 6) Rinse the affinity chromatography column with equilibration buffer for 5-10 column volumes before loading the sample; and,

[0024] 7) The chromatography system used for affinity chromatography is AKTA pure 150; optionally, the chromatography column is BXP26 / 30, and the affinity filler is selected from the group consisting of MabSelect PrismA, NMab Pro and UniMab 50HC.

[0025] In some embodiments, the flow rate of the eluent 1 is 120 cm / h to 240 cm / h;

[0026] The flow rate of the eluent 2 is 180 cm / h to 300 cm / h; and

[0027] The flow rate of the eluent is 120 cm / h~240 cm / h.

[0028] In some embodiments, the acid solution comprises 0.5 M to 2 M citric acid.

[0029] In some embodiments, the alkaline solution 1 comprises 0.5 M to 2 M phosphate, pH 7.0 to 9.0;

[0030] The alkaline solution 2 comprises 0.5 M to 2 M Tris, pH 8.0 to 11.0; and

[0031] The alkaline solution 3 includes 0.5 M to 2 M sodium carbonate and has a pH of 9.0 to 11.0.

[0032] In some embodiments, during the neutralization process, one or more of the acid solution, alkaline solution 1, alkaline solution 2, and alkaline solution 3 are added dropwise under stirring.

[0033] In some embodiments, the stirring speed is independently 50 rpm to 250 rpm.

[0034] In some embodiments, the affinity chromatography column is sterilized prior to affinity chromatography.

[0035] In some embodiments, 0.2 M to 1 M NaOH solution is used for disinfection.

[0036] In some embodiments, in the step of filtering the neutralized treatment liquid, the filtering conditions are:

[0037] The filtration rate is 50L / (h·m 2 )~150 L / (h·m 2 ), the filter pore size is 0.02μm~3μm.

[0038] In some embodiments, the host cell is a mammalian cell, a bacterium, or a fungus.

[0039] In some embodiments, the mammalian cell is a CHO cell, a NS0 cell, a HEK293 cell, or a Vero cell.

[0040] In some embodiments, the bacteria is Escherichia coli.

[0041] In some embodiments, the fungus may optionally be Pichia pastoris.

[0042] The method described above has at least the following advantages: it is simple and fast, and does not require the purchase of expensive additional instruments, equipment, reagents and consumables; it is suitable for laboratory milligram to kilogram-scale production and can be easily scaled up; by treating the protein with acid and alkaline solutions, it has a good removal effect on HCP residues that are difficult to remove by conventional processes, and the effect is significant, making it suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the implementation methods and examples of this application and to more completely understand the application and its beneficial effects, the following briefly introduces the drawings required for the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0044] Figure 1 This is a bar chart showing the HCP removal effects of the two process routes of Example 1 and Comparative Example 1 of the present application. DETAILED DESCRIPTION

[0045] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0047] In this application, unless otherwise specified, "one or more" refers to any one of the listed items or any combination of the listed items. Similarly, "one or more" and other similar expressions that refer to "one or more" are also understood in the same way unless otherwise specified.

[0048] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0049] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0050] In this application, the terms "further," "further," "particularly," "for example," "such as," "example," and "for example" are used for descriptive purposes to indicate that the preceding and following technical solutions are related in terms of the content covered, but should not be construed as limiting the preceding technical solution or the scope of protection of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it is understood that A is not limited to B.

[0051] In this application, the terms "optionally," "optional," and "optional" mean optional, that is, they refer to either option selected from the two parallel options of "yes" or "no." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "optional" is independent. Unless otherwise specified, the descriptions "optionally include," "optionally include," etc. in this application, taking "optionally include" as an example, mean "may include or not include."

[0052] As used herein, the terms "comprising," "including," and "include" are synonymous and are inclusive or open-ended, not excluding additional, unrecited members or features. Examples of members or features include materials or components, structures, elements, and instruments. Non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, and states.

[0053] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0054] In this application, exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0055] In this application, the term "first" is used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, "first" serves only as a non-exhaustive enumeration and should not constitute a closed-ended limitation on quantity.

[0056] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval only refers to an integer in the numerical interval, including the two endpoint integers of the numerical range and each integer between the two endpoints, is equivalent to directly enumerating each integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0057] In this application, if a method flow involves multiple steps, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in an order other than the order described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages do not necessarily need to be completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating, or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0058] This application mainly removes secreted HCP residues from CHO cells, especially extracellularly expressed proteins, by adjusting the process route and buffer system pH parameters. It can address the current problem that many HCPs and target proteins are difficult to remove due to similar chemical properties such as isoelectric point and molecular weight. This application develops a solution to this problem, which can remove the residues to meet relevant standards.

[0059] The first aspect of the present application provides a method for reducing host cell protein in a protein sample, the method comprising:

[0060] The cell supernatant containing the target protein is subjected to affinity chromatography to obtain an eluate;

[0061] neutralizing the eluate to obtain a neutralized solution; and

[0062] filtering the neutralized treatment liquid;

[0063] The neutralization treatment of the eluate comprises the following steps a, b, c and d:

[0064] a using an acid solution to adjust the pH of the eluent to 3.0 ~ 4.5, acid treatment to obtain an acid-treated solution;

[0065] b using an alkaline solution 1 to adjust the pH of the acid treatment solution to 4.5 ~ 5.5, the first alkali treatment to obtain an alkaline treatment solution 1;

[0066] c using an alkaline solution 2 to adjust the pH of the alkaline treatment solution 1 is 5.5 ~ 6.5, the second alkali treatment to give an alkaline treatment solution 2; and

[0067] d. Using an alkaline solution 3 to adjust the pH of the alkaline treatment solution 2 to 6.5~7.5 to obtain the neutralization treatment solution.

[0068] In some embodiments, after the acid treatment or the base treatment, the mixture is allowed to stand for 30 min to 60 min.

[0069] Illustratively, the mixture can be left to stand for 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, or 60 min, or a range or value between any two values.

[0070] In some embodiments, the affinity chromatography comprises the following steps performed sequentially:

[0071] The eluate is obtained by balancing, loading, first elution, second elution, elution and collection.

[0072] In some embodiments, in affinity chromatography, the equilibration buffer used for equilibration comprises: 10 mM to 50 mM phosphate and 100 mM to 500 mM NaCl, pH 6.5 to 8.5.

[0073] In some embodiments, the concentration of phosphate in the equilibration buffer is 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or a value or range between any two values.

[0074] In some embodiments, the concentration of NaCl in the equilibration buffer is 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, or a value or range between any two values.

[0075] In some embodiments, the pH of the equilibration buffer is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, or a value or range between any two values.

[0076] In some embodiments, in affinity chromatography, the eluent 1 used for the first wash comprises: 10 mM to 50 mM phosphate and 50 M to 800 M sodium chloride, pH 6.5 to 10.5.

[0077] Without wishing to be bound by any theory, it was found that when the phosphate concentration in eluent 1 was less than 10 mM, the buffering capacity was too weak and the pH was unstable. In addition, if the sodium chloride concentration was higher than 800 M, the sample would easily form aggregates under high salt concentration, and if it was lower than 50 M, the removal of impurities would not be achieved.

[0078] In some embodiments, the concentration of phosphate in eluent 1 is 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or a value or range between any two values.

[0079] In some embodiments, the concentration of NaCl in eluent 1 is 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, or a value or range between any two values.

[0080] In some embodiments, the pH of eluent 1 is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, or a value or range between any two values.

[0081] In some embodiments, in affinity chromatography, the eluent 2 used for the second eluent comprises: 10 mM to 50 mM phosphate, pH 6.5 to 10.5.

[0082] Without wishing to be bound by any theory, it was found that when the concentration of phosphate in eluent 2 was below 10 mM, the buffering capacity was too weak and the pH was unstable.

[0083] In some embodiments, the concentration of phosphate in eluent 2 is 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or a value or range between any two values.

[0084] In some embodiments, the pH of eluent 2 is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, or a value or range between any two values.

[0085] In some embodiments, in affinity chromatography, the eluent used for elution comprises: 50 mM to 200 mM citric acid-sodium citrate, pH 3.0 to 5.5.

[0086] In some embodiments, the concentration of citric acid-sodium citrate in the eluent is 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, or a value or range between any two values.

[0087] In some embodiments, the pH of the eluent is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, or a value or range between any two values.

[0088] In some embodiments, in affinity chromatography, the volume of the equilibration buffer used for equilibration is 3 to 8 column volumes, for example, 3, 4, 5, 6, 7 or 8 column volumes.

[0089] In some embodiments, during affinity chromatography, the affinity chromatography column is washed with equilibration buffer for 5 to 10 column volumes, illustratively, for example, 5, 6, 7, 8, 9, or 10 column volumes, before loading the sample.

[0090] In some embodiments, in affinity chromatography, the chromatography system used for affinity chromatography is AKTA pure 150. For example, the affinity chromatography column is BXP16 / 30, and the affinity filler can be MabSelect PrismA, NMab Pro, or UniMab50HC.

[0091] In some embodiments, the flow rate of eluent 1 is 120 cm / h to 240 cm / h. In some embodiments, the flow rate of eluent 1 is 120 cm / h, 130 cm / h, 140 cm / h, 150 cm / h, 160 cm / h, 170 cm / h, 180 cm / h, 190 cm / h, 200 cm / h, 210 cm / h, 220 cm / h, 230 cm / h, 240 cm / h, or a value or range between any two values.

[0092] The unit "cm / h" indicates the linear flow rate. Without wishing to be bound by any theory, it is assumed that the linear flow rate is the same for chromatography columns of different sizes.

[0093] In some embodiments, the flow rate of eluent 2 is 180 cm / h to 300 cm / h. Exemplarily, the flow rate of eluent 2 is 180 cm / h, 190 cm / h, 200 cm / h, 210 cm / h, 220 cm / h, 230 cm / h, 240 cm / h, 250 cm / h, 260 cm / h, 270 cm / h, 280 cm / h, 290 cm / h, 300 cm / h, or a value or range between any two values.

[0094] Without wishing to be bound by any theory, it has been found that if the flow rates of eluent 1 and eluent 2 are outside the above numerical ranges, the impurities cannot be eluted effectively.

[0095] In some embodiments, the flow rate of the eluent is 120 cm / h to 240 cm / h. Exemplarily, the flow rate of the eluent is 120 cm / h, 130 cm / h, 140 cm / h, 150 cm / h, 160 cm / h, 170 cm / h, 180 cm / h, 190 cm / h, 200 cm / h, 210 cm / h, 220 cm / h, 230 cm / h, 240 cm / h, or a value or range between any two values.

[0096] In some embodiments, the acid solution comprises 0.5 M to 2 M citric acid. Exemplarily, the concentration of citric acid in the acid solution is 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2 M, or a value or range between any two values.

[0097] In some embodiments, the alkaline solution 1 comprises 0.5 M to 2 M phosphate, pH 7.0 to 9.0;

[0098] The alkaline solution 2 includes 0.5 M to 2 M Tris, pH 8.0 to 11.0; and

[0099] The alkaline solution 3 includes 0.5 M to 2 M sodium carbonate, with a pH of 9.0 to 11.0.

[0100] In some embodiments, the concentration of phosphate in the alkaline solution 1 is 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M, or a value or range between any two values.

[0101] In some embodiments, the pH of the alkaline solution 1 is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or a value or range between any two values.

[0102] In some embodiments, the concentration of Tris in the alkaline solution 2 is 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M, or a value or range between any two values.

[0103] In some embodiments, the pH of the alkaline solution 2 is 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, or a value or range between any two values.

[0104] In some embodiments, the concentration of sodium carbonate in the alkaline solution 3 is 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M, or a value or range between any two values.

[0105] In some embodiments, the pH of the alkaline solution 3 is 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, or a value or range between any two values.

[0106] In some embodiments, one or more of the acid solution, alkaline solution 1, alkaline solution 2, and alkaline solution 3 are added dropwise under stirring.

[0107] The stirring speed can be illustratively 50 rpm to 250 rpm. In some embodiments, the stirring speed is, for example, 50 rpm, 60 rpm, 70 rpm, 80 rpm, 90 rpm, 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm, 250 rpm, or a value or range between any two values.

[0108] In some embodiments, the affinity chromatography column is sterilized prior to affinity chromatography. Exemplarily, a 0.2 M to 1 M NaOH solution is used for sterilization. In some embodiments, the concentration of the NaOH solution is 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, or a value or range between any two values.

[0109] In some embodiments, in the step of filtering the neutralized treatment liquid, the filtering conditions are:

[0110] The filtration rate is 50 L / (h·m 2 )~150 L / (h·m 2 ), the filter pore size is 0.02 μm~3 μm.

[0111] In some embodiments, the filtration rate is 50 L / (h·m 2 )、60 L / (h·m 2 )、70 L / (h·m 2 )、80L / (h·m 2 )、90 L / (h·m 2 )、100 L(h·m 2 )、110 L / (h·m 2 )、120 L / (h·m 2 )、130 L / (h·m 2 )、140 L / (h·m 2 )、150 L / (h·m 2 ), or a value or range between any two values.

[0112] In some embodiments, the host cell expressing the host cell protein is a mammalian cell, a bacterium, or a fungus.

[0113] The mammalian cells may be CHO cells, NS0 cells, HEK293 cells or Vero cells.

[0114] In some embodiments, the bacteria may be Escherichia coli.

[0115] In some embodiments, the fungus may be Pichia pastoris.

[0116] In some embodiments, the host cell protein is an extracellularly expressed protein.

[0117] In one aspect, the present application provides a method for reducing host cell protein in a protein sample, which can be applied to the preparation of protein drugs.

[0118] In another aspect of the present application, a method for preparing a protein drug is provided, comprising:

[0119] During the purification of the protein drug, the method for reducing host cell proteins in a protein sample provided in the first aspect is used to remove the host cell proteins in the protein drug.

[0120] There is no limitation on the protein drugs, and illustratively they may be protein drugs prepared by biosynthesis methods, such as antibody drugs.

[0121] Some examples are provided below.

[0122] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and may also be based on the experimental manuals or conventional conditions in this area, or on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0123] Unless otherwise specified, all buffer solutions should be filtered through a 0.2 μm filter and containers should be cleaned and sterilized to avoid contamination of the sample.

[0124] Example 1

[0125] Before preparation, the chromatography system and affinity chromatography column are thoroughly disinfected with 0.2-1M NaOH, and affinity chromatography equilibration solution (or equilibration buffer), affinity chromatography eluent (or elution buffer), affinity chromatography eluent 1 (or eluent 1), affinity chromatography eluent 2 (or eluent 2), pH-adjusting acid solution (or acid solution), pH-adjusting alkaline solution 1 (or alkaline solution 1), pH-adjusting alkaline solution 2 (or alkaline solution 2), and pH-adjusting alkaline solution 3 (or alkaline solution 3) are prepared. Among them:

[0126] The equilibration buffer consisted of: 10 mM phosphate and 200 mM NaCl, pH 7.5 ± 0.5;

[0127] Eluent 1 consisted of: 10 mM phosphate and 500 M sodium chloride, pH 6.5 ± 05;

[0128] Eluent 2 included: 10 mM phosphate, pH 6.5 ± 0.5;

[0129] Elution buffer: 100 mM citric acid-sodium citrate, pH 3.5 ± 0.5.

[0130] 1. The CHO cell supernatant was subjected to affinity chromatography with an initial HCP residual level of ≥30,000 ppm using an AKTApure 150 chromatography system.

[0131] (1) First, equilibrate the chromatography column with 5 column volumes of equilibration buffer.

[0132] (2) During the loading phase, continue flushing with equilibration buffer for 10 column volumes.

[0133] (3) Use eluent 1 and eluent 2 at flow rates of 240 cm / h and 240 cm / h, respectively, to elute impurities.

[0134] (4) The target protein was eluted with an eluent at a flow rate of 240 cm / h, detected at a wavelength of 280 nm, and the elution peak was collected to obtain the eluent. The protein yield was 94%, and the HCP residual content was 3084 ppm.

[0135] The chromatography process conditions are as follows: chromatography column: BXP16 / 30, affinity filler: MabSelect PrismA.

[0136] The function of this step is at least to capture the target protein and remove HCP for the first time. The principle is to use the specific binding of protein and affinity filler to bind the protein to the antibody affinity, while other cell secretion impurities and host proteins flow through. Some HCP bound to the protein is bound to the chromatography column through the antibody protein instrument. After elution with a solution, the HCP is separated from the antibody protein and the residual HCP in the protein is removed again. Elution 1 and elution 2 are one of the key technical points in this step.

[0137] 2. Neutralize the eluate obtained above with acid and alkaline solutions.

[0138] Instruments: magnetic stirrer, pH meter; Containers: 200ml, 500ml glass beakers, graduated cylinder.

[0139] About the solution:

[0140] Acid solution: 1 M citric acid; Alkali solution 1: 1 M phosphate pH 7.0; Alkali solution 2: 1 M tris pH 9.0; Alkali solution 3: 1.5 M sodium carbonate pH 11.0.

[0141] (1) Place the eluate on a magnetic stirrer and start stirring at 150 rpm. Slowly add the acid solution to the eluate and monitor the protein pH. When the pH reaches 3.5 ± 0.2, stop adding the acid solution and let it stand for 60 minutes.

[0142] (2) Stir the eluate treated with the acid solution in step (1) at a stirring speed of 150 rpm, slowly add alkaline solution 1, and monitor the pH value of the protein. When the pH reaches 5.0±0.5, stop adding alkaline solution 1 and let it stand for 30 minutes.

[0143] (3) Stir the eluate after alkali neutralization 1 in step (2) at a stirring speed of 150 rpm, slowly add alkaline solution 2, and monitor the pH value of the protein. When the pH reaches 6.0±0.5, stop adding alkaline solution 2 and let it stand for 30 minutes.

[0144] (4) Stir the eluate after alkali neutralization 2 in step (3) at a stirring speed of 150 rpm, slowly add alkaline solution 3, and monitor the pH value of the protein. When the pH reaches 7.0±0.5, stop adding alkaline solution 3 and let it stand for 30 minutes.

[0145] (5) The eluate from step (4) was clarified and filtered using a deep filter with a filtration pressure of no more than 2 bar and a filtration flow rate of 100 L / h·m 2 The turbidity of the sample after filtration is no more than 10 NTU (Nephelometric Turbidity Units), the HCP content after filtration is 395ppm, and the pore size of the deep filter is: 0.02 μm~3 μm.

[0146] The acid treatment of the eluted solution inactivates viruses in the antibody protein. Neutralization with alkaline solutions of varying pH values ​​utilizes the varying isoelectric points of residual CHP proteins in the antibody protein to precipitate and remove them. The theory that proteins are more easily precipitated near their isoelectric points allows for the stratified removal of HCPs at different isoelectric points. This step precipitates the majority of residual HCPs in the antibody protein, allowing HCPs to be removed from the antibody protein through clarification and filtration, resulting in a target protein with a lower HCP content.

[0147] 3. Clarification and Filtration: Instruments: Peristaltic Pump, Magnetic Stirrer, Turbidimeter, Pressure Sensor; Consumables: Depth Filter. The depth filter used in this experiment is a product recommended by PALL, model: SC050PDE2.

[0148] (1) Connect the deep filter, connect the filter to the peristaltic pump and pressure gauge, turn on the peristaltic pump, open the filter plate exhaust valve, and tilt the filter so that the exhaust port is at a higher position, and pump in purified water for exhaust.

[0149] (2) Water flushing: Use purified water to flush the filter plate at a flow rate of 150-300 L / (m²·h). The flushing volume of a single filter plate is 50 L / m 2 (L / m2), double-layer filter plate flushing volume is 100 L / m 2 .

[0150] (3) Rinse the filter plate with buffer at a volume of 10 L / m 2 ~20 L / m 2 .

[0151] (4) Use a magnetic stirrer to keep the liquid in a mixed state, start the peristaltic pump, adjust the peristaltic pump filtration rate to 50-150LHM to pump the liquid, perform constant flow filtration, collect the filtrate, and record the filtrate volume, filtration time and pressure difference, and test the filtrate turbidity. These parameters can be monitored to determine acceptable contaminant removal.

[0152] Comparative Example 1

[0153] A method for removing HCP residues in CHO cells according to traditional technology (HCP detection kit CygnusF550-1 detects HCP residues in each step) includes the following steps:

[0154] (1) Affinity chromatography was performed on the CHO cell supernatant (initial protein concentration was 8.5 mg / mL) with an initial HCP residual of ≥30,000 ppm using an AKTA pure 150 chromatography system. The column was first equilibrated with equilibration buffer. During the sample loading phase, the column was flushed with equilibration buffer for 5 column volumes. The elution peak was collected and the protein yield was determined. The chromatography process conditions were as follows:

[0155] Chromatography column: BXP26 / 30; flow rate: 20.0 mL / min; detection wavelength: UV 280 nm;

[0156] Chromatography column packing: any one of MabSelect PrismA, NMab Pro, and UniMab 50HC;

[0157] Equilibration buffer: 10 mM phosphate and 200 mM NaCl, pH 7.5 ± 0.5.

[0158] Elution buffer: 100 mM citric acid-sodium citrate, pH 3.0 ± 0.5.

[0159] The sample protein concentration was 11.6 (mg / mL), the protein yield was 95%, and the HCP residual content was 7369 ppm.

[0160] (2) The eluate obtained in step (1) is subjected to alkali neutralization and deep filtration:

[0161] Instruments: magnetic stirrer, pH meter; Containers: 200ml, 500ml glass beakers, graduated cylinder.

[0162] Related solutions: Alkaline solution: 1M tris pH 8.0-11.0;

[0163] 1) Place the eluate on a magnetic stirrer and stir at 150 rpm. Slowly add the alkaline solution to the eluate while monitoring the protein pH. Stop adding when the pH reaches 7.2 ± 0.5 and let it stand for 30-60 minutes.

[0164] 2) The eluate from step 1) was clarified and filtered using a depth filter at a filtration pressure not exceeding 2 bar. The turbidity of the filtered sample was not greater than 10 NTU (Nephelometric Turbidity Units). The HCP content after filtration was 3941 ppm. The pore size of the depth filter was 0.02 μm to 3 μm.

[0165] The comparison of the HCP removal effects of the two process routes of Example 1 and Comparative Example 1 can be found in Figure 1 .

[0166] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0167] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be determined by the appended claims, and the specification and drawings shall serve to interpret the claims.

Claims

1. A method for reducing host cell protein in a protein sample, characterized in that: The method includes: The cell supernatant containing the target protein is subjected to affinity chromatography to obtain an eluate; neutralizing the eluate to obtain a neutralized solution; and filtering the neutralized treatment liquid; The neutralization treatment of the eluate comprises the following steps a, b, c and d: a using an acid solution to adjust the pH of the eluent to 3.0 ~ 4.5, acid treatment to obtain an acid-treated solution; b using an alkaline solution 1 to adjust the pH of the acid treatment solution to 4.5 ~ 5.5, the first alkali treatment to obtain an alkaline treatment solution 1; c using an alkaline solution 2 to adjust the pH of the alkaline treatment solution 1 is 5.5 ~ 6.5, the second alkali treatment to give an alkaline treatment solution 2; and d. Using an alkaline solution 3 to adjust the pH of the alkaline treatment solution 2 to 6.5~7.5 to obtain the neutralization treatment solution.

2. The method according to claim 1, wherein The affinity chromatography comprises the following steps performed in sequence: The eluate is obtained by balancing, loading, first elution, second elution, elution and collection.

3. The method according to claim 2, wherein The affinity chromatography satisfies one or more of the following conditions 1) to 7): 1) The equilibration buffer used for the equilibration comprises: 10 mM to 50 mM phosphate and 100 mM to 500 mM NaCl, pH 6.5 to 8.5; 2) the eluent 1 used for the first elution comprises: 10 mM to 50 mM phosphate and 50 M to 800 M sodium chloride, pH 6.5 to 10.5; 3) the eluent 2 used for the second eluent comprises: 10 mM to 50 mM phosphate, pH 6.5 to 10.5; 4) The elution buffer used for the elution comprises: 50 mM to 200 mM citric acid-sodium citrate, pH 3.0 to 5.5; 5) The volume of the equilibration buffer used for the equilibration is 3 to 8 column volumes; 6) Wash the affinity column with equilibration buffer for 5 to 10 column volumes before loading the sample; and, 7) The chromatography system used for the affinity chromatography is AKTA pure 150; optionally, the affinity chromatography column is BXP16 / 30, and the affinity filler is selected from the group consisting of MabSelect PrismA, NMab Pro and UniMab 50HC.

4. The method according to claim 3, wherein The flow rate of the eluent 1 is 120 cm / h~240 cm / h; The flow rate of the eluent 2 is 180 cm / h to 300 cm / h; and The flow rate of the eluent is 120 cm / h~240 cm / h.

5. The method according to any one of claims 1 to 4, wherein The acid solution includes 0.5 M to 2 M citric acid.

6. The method according to any one of claims 1 to 4, wherein The alkaline solution 1 includes 0.5 M to 2 M phosphate, pH 7.0 to 9.0; The alkaline solution 2 comprises 0.5 M to 2 M Tris, pH 8.0 to 11.0; and The alkaline solution 3 includes 0.5 M to 2 M sodium carbonate and has a pH of 9.0 to 11.

0.

7. The method according to any one of claims 1 to 4, wherein During the neutralization treatment, one or more of the acid solution, alkaline solution 1, alkaline solution 2, and alkaline solution 3 are added dropwise under stirring; Optionally, the stirring speed is independently 50 rpm to 250 rpm.

8. The method according to any one of claims 1 to 4, wherein Sterilize the affinity chromatography column before affinity chromatography; Alternatively, sterilize with 0.2 M to 1 M NaOH solution.

9. The method according to any one of claims 1 to 4, wherein In the step of filtering the neutralized treatment liquid, the filtering conditions are: The filtration rate is 50L / (h·m 2 )~150 L / (h·m 2 ), the filter pore size is 0.02 μm~3 μm.

10. The method according to any one of claims 1 to 4, wherein The host cell expressing the host cell protein is a mammalian cell, a bacterium or a fungus; Wherein, the mammalian cell may be CHO cell, NS0 cell, HEK293 cell or Vero cell; the bacterium may be Escherichia coli; the fungus may be Pichia pastoris; Optionally, the host cell protein is an extracellularly expressed protein.

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