Method for improving sample turbidity of three antibodies in virus inactivation and neutralization process

Abstract

The invention belongs to the field of biology, and particularly relates to a method for improving sample turbidity of three antibodies in virus inactivation and neutralization processes. Carrying out virus inactivation on the chromatographic eluent containing the three antibodies, neutralizing the solution after virus inactivation, and filtering after neutralization to obtain a clear filtrate; the neutralization method comprises the following steps: diluting the final concentration of a chromatographic eluent to 1.7 g / L by using 0.1 M Gly-HCl with the pH value of 3.2, and neutralizing and adjusting by using 0.2 M PB with the pH value of 7.0 until the pH value is 7.0-8.0; or 0.2 M NaCl is used for diluting the final concentration of the chromatographic eluent to 1.7 g / L, then 1M Tris-HCl and pH 9.0 are used for neutralization adjustment, and the pH is neutralized to 7.0-8.0. By adopting the neutralization method in the anti-virus inactivation process, the sample is not turbid or precipitated, and the recovery rate is high.

Description

Technical Field

[0001] The present invention belongs to the field of biology, and particularly relates to a method for improving the turbidity of samples during virus inactivation and neutralization of trispecific antibodies. Background Art

[0002] In the field of biomedicine, the research and production of trispecific antibody drugs have attracted increasing attention. The occurrence and development of many diseases often involve multiple targets and signaling pathways. Trispecific antibodies can simultaneously recognize and bind to three different targets, thereby more comprehensively intervening in the pathophysiological process of diseases, playing a more effective therapeutic role, and providing a more precise solution for the treatment of some complex diseases. Trispecific antibodies add two specific antigen-binding sites, which is beneficial for redirecting the drug to diseased sites such as tumors, increasing binding specificity, improving the accuracy of the drug targeting diseased cells, reducing off-target toxicity, and reducing side effects on normal tissues, making the treatment more precise and effective. By acting on multiple targets simultaneously, trispecific antibodies can produce a synergistic effect and enhance the therapeutic effect on diseases. For example, in tumor treatment, some trispecific antibodies can simultaneously activate immune cells, block immunosuppressive signaling pathways, and inhibit tumor angiogenesis, attacking tumor cells from multiple angles and achieving an effect of "1 + 1 + 1 > 3", effectively inhibiting tumor growth and metastasis, and improving the survival rate and quality of life of patients. Moreover, the long-term use of drugs with a single target is prone to the generation of drug resistance. The multi-target action mechanism of trispecific antibodies enables them to overcome drug resistance problems caused by single-target mutations or changes in signaling pathways, providing new treatment options for drug-resistant patients.

[0003] The molecular structure of trispecific antibody drugs is more complex than that of monoclonal antibodies and bispecific antibodies. It needs to simultaneously bind to three different targets, which requires a more refined and unique structural design. This complex structure may lead to unstable internal molecular interactions and is prone to conformational changes under the influence of external conditions, thereby affecting the stability of the drug.

[0004] Therefore, during the virus inactivation and neutralization process, the phenomenon of sample turbidity is often encountered: For example, during the virus inactivation process, the commonly used low-pH treatment step may cause a decrease in the solubility of trispecific antibodies, denaturation, or aggregation, thereby causing precipitation and turbidity.

[0005] For example, during the high-temperature inactivation process, the increase in temperature may cause the proteins in trispecific antibodies to undergo thermal denaturation or aggregation, resulting in their precipitation from the solution and forming turbidity.

[0006] In some virus inactivation processes, organic solvents such as tributyl phosphate (TNBP) are used, which may interact with the proteins in the tertiary antibody, causing protein denaturation or aggregation, thereby causing turbidity. Or the use of detergents such as Tween-80 may destroy the structure of the tertiary antibody during the inactivation process, reducing its solubility, resulting in precipitation and turbidity.

[0007] In addition, in high-concentration third antibody solutions, protein molecules may interact with each other (such as hydrophobic interactions or electrostatic interactions between proteins) to form aggregates, resulting in turbidity of the solution.

[0008] This phenomenon seriously affects the smooth progress of subsequent detection and analysis, purification process, product quality control, recovery rate, etc. For example, if the protein in the tertiary antibody denatures or aggregates, this will destroy the natural conformation of the protein, causing it to lose its original biological activity and function, thereby reducing the effectiveness of the product.

[0009] The mechanisms that lead to the turbidity of triple antibodies are diverse, and there is a lack of targeted and efficient solutions, which has become one of the key technical bottlenecks restricting the development and production of triple antibody drugs. Summary of the invention

[0010] The present invention innovatively proposes a new method, which has remarkable simplicity and effectiveness, and its core purpose is to effectively improve the sample turbidity phenomenon that occurs during the virus inactivation and neutralization process of the three antibodies. In the virus inactivation process, the complex environment in which the three antibodies are located often triggers a series of physical and chemical changes, causing the sample to be turbid, which not only affects the recovery rate of the three antibodies, but also brings great obstacles to subsequent analysis and detection work. The method provided by the present invention can effectively intervene in the key factors that cause turbidity by optimizing and adjusting specific process steps and introducing special treatment reagents and technical means. Whether it is an in-depth exploration of the characteristics of the three antibodies in a laboratory research environment, or a strict control of the quality of the three antibody products in a large-scale industrial production process, this method has shown extremely outstanding application value, and is expected to become a key breakthrough point in solving the problem of sample turbidity in the process of virus inactivation and neutralization of the three antibodies, and open up a smoother and more efficient new path for research and production activities in related fields.

[0011] In order to achieve the above object, the present invention provides the following technical solutions: The present invention discloses a method for improving sample turbidity during virus inactivation and neutralization by three antibodies, comprising the following steps: Inactivating the virus in the chromatography eluate containing the tertiary antibody, neutralizing the virus-inactivated solution, and filtering after neutralization to obtain a clear filtrate; The neutralization method is as follows: Dilute the final concentration of the chromatography eluate to 1.7 g / L with 0.1M Gly-HCl, pH3.2, and then adjust the neutralization with 0.2 M PB, pH7.0 until the pH reaches 7.0 - 8.0; or Dilute the final concentration of the chromatography eluate to 1.7 g / L with 0.2M NaCl, and then adjust the neutralization with 1M Tris-HCl, pH9.0 until the pH reaches 7.0 - 8.0.

[0012] Among them, the virus inactivation method for the trispecific antibody can adopt methods such as pasteurization, dry heat method, organic solvent / detergent (S / D) method, low pH inactivation method, nanofiltration membrane filtration method, chromatographic separation method, etc.

[0013] Preferably, the neutralization method is as follows: Dilute the final concentration of the chromatography eluate to 1.7 g / L with 0.1M Gly-HCl, pH3.2, and then adjust the neutralization with 0.2 M PB, pH7.0 until the pH reaches 7.5; or Dilute the final concentration of the chromatography eluate to 1.7 g / L with 0.2M NaCl, and then adjust the neutralization with 1M Tris-HCl, pH9.0 until the pH reaches 7.0.

[0014] As an embodiment of the present invention, the chromatography eluate is obtained by affinity chromatography of the cell culture solution containing the trispecific antibody. Preferably, Capto L is used for affinity chromatography.

[0015] Among them, the preparation method of the chromatography eluate is as follows: Take the clarified harvest solution, load the sample at a flow rate of 268 cm / h, ensure that the column load of the chromatography column is 15 - 20 g / L. After the sample loading is completed, rinse the chromatography column with 5 column volumes of the eluent at a flow rate of 268 cm / h. The eluent is 20 mM PB, 150 mM NaCl, pH7.4. Rinse the chromatography column with 5 column volumes of the washing buffer at a flow rate of 268 cm / h. The washing buffer is 20 mM Na-Citrate, pH6.0. Elute with the elution buffer at a flow rate of 268 cm / h. The elution buffer is 0.1M Gly-HCl, pH3.2. Measure the protein concentration of the eluted solution by ultraviolet absorption method, and collect the elution peak when the absorbance is greater than or equal to 0.5 AU to obtain the chromatography eluate.

[0016] Preferably, the preparation method of the clarified harvest solution is: Take the cell culture solution containing the trispecific antibody, and obtain the clarified harvest solution after deep filtration. The concentration of the trispecific antibody is: 0.87 g / L.

[0017] Compared with the prior art, the present invention has the following technical effects: In the neutralization method during the process of the present invention, the sample will not become turbid or precipitate, and the recovery rate is high.

[0018] In the neutralization method during the three-antiviral inactivation process of the present invention, the process operation is simple, the required buffer components are few, the cost is saved, there are no additives, and a series of complex and cumbersome problems caused by the need to remove the additive residues subsequently are avoided. Specific embodiments

[0019] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0020] Example 1: Take the cell culture medium containing three antibodies, and obtain a clarified harvest solution (volume: 2.5 L, concentration: 0.87 g / L) after deep filtration. Use Capto L for affinity chromatography to obtain the first chromatographic eluate. Specifically as follows: Take the clarified harvest solution and load it at a flow rate of 268 cm / h. The column load of the chromatographic column is ensured to be 15 - 20 g / L. After loading, use 5 column volumes of the eluent to wash the chromatographic column at a flow rate of 268 cm / h. The eluent is 20 mM PB, 150 mM NaCl, pH 7.4. Use 5 column volumes of the wash buffer 2 to wash the chromatographic column at a flow rate of 268 cm / h. The wash buffer 2 is 20 mM Na-Citrate, pH 6.0. Use the eluent to elute at a flow rate of 268 cm / h. The elution buffer is 0.1M Gly-HCl, pH 3.2. Use the ultraviolet absorption method (280 nm) to measure the protein concentration, and collect the elution peak when the absorbance is greater than or equal to 0.5 AU to obtain the first chromatographic eluate.

[0021] For the first chromatographic eluate obtained in the previous step, perform virus inactivation, and neutralize the solution after virus inactivation. After neutralization, filter to obtain the filtrate. The neutralization method is as follows: Dilute the first chromatographic eluate with the neutralizing solution to a final concentration of 1.7 g / L, and then further perform neutralization adjustment with the adjusting solution until the corresponding pH value. The corresponding reagents and conditions are shown in Table 1 below.

[0022] Table 1 Neutralizing solution Adjusting solution Neutralize to pH value Example 1 0.1M Gly-HCl, pH3.2 0.2 M PB, pH7.0 7.5 Example 2 1M Tris-HCl, pH9.0 NA 6.0-7.0 Example 3 4M NaCl 1M Tris-HCl, pH9.0 6.0-7.0 Example 4 0.1 M Arg NA 5.0 Example 5 0.1 M Arg NA 6.0 Example 6 0.1 M Arg NA 7.0 Example 7 0.2M NaCl 1M Tris-HCl, pH9.0 5.0 Example 8 0.2M NaCl 1M Tris-HCl, pH9.0 5.5 Example 9 0.2M NaCl 1M Tris-HCl, pH9.0 6.0 Example 10 0.2M NaCl 1M Tris-HCl, pH9.0 6.5 Example 11 0.2M NaCl 1M Tris-HCl, pH9.0 7.0 Example 12 0.2M NaCl 1M Tris-HCl, pH9.0 8.0 Example 13 0.05% PS80 1M Tris-HCl, pH9.0 6.0 Example 14 0.05% PS80 1M Tris-HCl, pH9.0 7.0 Example 15 0.05% PS80 1M Tris-HCl, pH9.0 8.0 Example 16 8% Sucrose 1M Tris-HCl, pH9.0 6.0 Example 17 8% Sucrose 1M Tris-HCl, pH9.0 7.0 Example 18 8% Sucrose 1M Tris-HCl, pH9.0 8.0 Example 19 8.5% PEG 1M Tris-HCl, pH9.0 6.0 Example 20 8.5% PEG 1M Tris-HCl, pH9.0 7.0 Example 21 8.5% PEG 1M Tris-HCl, pH9.0 8.0 Example 22 8.5% Trehalose 1M Tris-HCl, pH9.0 6.0 Example 23 8.5% Trehalose 1M Tris-HCl, pH9.0 7.0 Example 24 8.5% Trehalose 1M Tris-HCl, pH9.0 8.0 Comparative Example 1 NA 1M Tris-HCl, pH9.0 7.0 Experimental results: See Table 2 below.

[0023] Table 2 Experiment serial number Yield % Appearance state 1 94.49 Clear 2 64.61 Turbid 3 67.11 Turbid 4 37.53 Turbid 5 59.57 Turbid 6 53.15 Turbid 7 55.46 Turbid 8 55.09 Turbid 9 54.34 Turbid 10 56.96 Turbid 11 94.77 Clear 12 91.60 Clear 13 52.66 Turbid 14 48.59 Turbid 15 42.95 Turbid 16 51.91 Turbid 17 46.03 Turbid 18 59.83 Turbid 19 51.07 Turbid 20 47.62 Turbid 21 63.04 Turbid 22 45.00 Turbid 23 45.00 Turbid 24 93.00 Clear According to the above experimental results, by comparing the results of Example 1 with those of Examples 2-25 and Comparative Example 1, it can be seen that the neutralization method of the trispecific antibody in the virus inactivation step of the present invention (diluting the first chromatographic eluate to a final concentration of 1.7 g / L with 0.1 M Gly-HCl, pH 3.2, and then neutralizing and adjusting with 0.2 M PB, pH 7.0 to a pH of 7.0-8.0) can effectively reduce the turbidity of the sample product, improve the yield, and without adding any additives, avoiding a series of complex and cumbersome problems caused by the subsequent removal of additive residues, greatly simplifying the process flow, improving the production efficiency and the stability of product quality, and providing a highly innovative and practical technical solution for the production practice in related fields.

[0024] The above is only the preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, shall be covered by the protection scope of the present invention.

Claims

1. A method for improving sample turbidity during virus inactivation and neutralization by three antibodies, characterized in that: The steps include: Inactivating the virus in the chromatography eluate containing the tertiary antibody, neutralizing the virus-inactivated solution, and filtering after neutralization to obtain a clear filtrate; The neutralization method is: diluting the final concentration of the chromatography eluate to 1.7 g / L with 0.1 M Gly-HCl, pH 3.2, and then neutralizing and adjusting with 0.2 M PB, pH 7.0 to a pH of 7.0-8.0; or The chromatography eluate was diluted to a final concentration of 1.7 g / L with 0.2 M NaCl, and then neutralized with 1 M Tris-HCl, pH 9.0 to a pH of 7.0-8.

0.

2. A method for improving sample turbidity during virus inactivation and neutralization by three antibodies according to claim 1, characterized in that: The neutralization method is: diluting the final concentration of the chromatography eluate to 1.7 g / L with 0.1 M Gly-HCl, pH 3.2, and then neutralizing and adjusting with 0.2 M PB, pH 7.0 to pH 7.5; or The chromatography eluate was diluted to a final concentration of 1.7 g / L with 0.2 M NaCl, and then neutralized to pH 7.0 with 1 M Tris-HCl, pH 9.

0.

3. A method for improving sample turbidity during virus inactivation and neutralization by three antibodies according to any one of claim 1 or 2, characterized in that: The chromatography eluate is obtained by performing affinity chromatography on a cell culture fluid containing the three antibodies.

4. A method for improving sample turbidity during virus inactivation and neutralization of three antibodies according to claim 3, characterized in that: The preparation method of the chromatography eluent is as follows: The clarified harvest liquid was taken and loaded at a flow rate of 268 cm / h. The loading capacity of the chromatography column was ensured to be 15-20 g / L. After the loading was completed, the chromatography column was rinsed with 5 column volumes of eluent at a flow rate of 268 cm / h. The eluent was 20 mM PB, 150 mM NaCl, pH7.

4. The chromatography column was rinsed with 5 column volumes of elution buffer at a flow rate of 268 cm / h. The elution buffer was 20 mM Na-Citrate, pH6.

0. The elution was eluted with elution buffer at a flow rate of 268 cm / h. The elution buffer was 0.1 M Gly-HCl, pH3.

2. The protein concentration of the eluted solution was determined by ultraviolet absorption method. The elution peak with an absorbance greater than or equal to 0.5 AU was collected to obtain the chromatography eluate.

5. A method for improving sample turbidity during virus inactivation and neutralization of three antibodies according to claim 4, characterized in that: The preparation method of the clarified harvest solution is as follows: taking a cell culture solution containing three antibodies, and obtaining a clarified harvest solution after deep filtration, wherein the concentration of the three antibodies is 0.87 g / L.