A method for reducing the formation of protein multimers in protein L gel chromatography

Abstract

The present invention relates to a method for reducing the formation of protein multimers in protein L gel chromatography, which includes: (1) adding cysteine (Cys) to the protein L elution system and neutralizing the elution system with NaOH solution; (2) treating the sample after protein L gel chromatography purification with Sephadex G25 gel chromatography to remove Cys. The present invention effectively inhibits the formation of multimers caused by low pH elution, and the purified protein does not degrade. The process of the present invention is simple in operation and obvious in effect, providing a reference for the protein L gel chromatography process in laboratory research and industrial production, and also providing a new idea for further research on methods for removing multimers formed by other similar proteins during purification.

Description

Technical Field

[0001] The invention relates to a method for reducing the formation of polymers by proteins in protein L gel chromatography, belonging to the technical field of biotechnology. Background Art

[0002] Protein L is an affinity chromatography medium (resin) suitable for capturing antibodies and antibody fragments. It can bind to immunoglobulins and immunoglobulin Kappa (κ) light chains without affecting antigen binding, and has a wider range of antibody fragment selection, higher purity and yield. However, the optimal elution condition for the target protein is low in pH, generally at pH 3 or even lower. High-concentration proteins are extremely unstable in a low pH environment and are prone to forming polymers, which affects the molecular arrangement of the protein after chromatography and the efficiency of active recovery.

[0003] Recombinant protein is a highly active recombinant fusion protein with human epidermal growth factor receptor (HER2) as the recognition target, and is a new type of targeted anti-breast cancer protein drug. It has the advantages of simple production process, high biological activity, and few product impurities, and has good application value and market prospects. During the purification process using protein L chromatography column, a certain amount of polymers are formed due to the constraints of elution conditions. The formation and content of polymers directly affect the quality of the product, the safety of clinical drug use and the qualified rate of the product. From the perspective of medical safety, reducing or removing the content of polymers as much as possible is one of the very important process links. Current studies have shown that this method can significantly inhibit the formation of recombinant protein polymers on the basis of improving the purity of fusion protein, so that it can meet certain quality index requirements. 。 Summary of the invention

[0004] The purpose of the present invention is to provide a method for reducing the formation of polymers in protein L gel chromatography. The present invention solves the problem of polymer formation during purification by adding reagents such as cysteine ​​to the elution buffer used in protein L chromatography. This improves protein purity and purification efficiency, and provides a reference for studying the removal of polymers generated in other similar protein purifications. The technical solution of the present invention is as follows:

[0005] A method for reducing the formation of polymers by protein L gel chromatography, comprising:

[0006] (1) Add cysteine ​​(Cys) to the protein L elution system and neutralize the elution system with NaOH solution;

[0007] (2) The protein L gel chromatography purified sample was treated with G25 dextran gel chromatography to remove Cys;

[0008] In step (1), the pH of the elution system is 2.0 - 7.0; preferably, the elution system is selected from any one of citrate buffer, acetate buffer, glycine - hydrochloride buffer, phthalic acid - hydrochloride buffer, disodium hydrogen phosphate - citrate buffer, citric acid - sodium citrate buffer, acetic acid - sodium acetate buffer.

[0009] The fusion protein 4D5Fv - PE25 contains the variable region Fv of trastuzumab and Pseudomonas aeruginosa exotoxin PE25 (the invention name is fusion protein 4D5Fv - PE25 and its preparation method and uses, application number is 201510674652.8)

[0010] Further, 0.1M Cys is added to the elution system, and the elution system is neutralized to pH 8.5 ± 0.3 with 0.5M NaOH solution.

[0011] Furthermore, the method for reducing the formation of multimers of the fusion protein 4D5Fv - PE25 in protein L gel chromatography is as follows:

[0012] (1) Equilibrate the protein L gel chromatography column with 5 column volumes of PBS equilibration buffer;

[0013] (2) Load the fusion protein 4D5Fv - PE25 at a loading amount of 20 mg / ml gel;

[0014] (3) Then equilibrate the protein L gel chromatography column with 5 column volumes of PBS equilibration buffer;

[0015] (4) Elute the protein with an elution system containing cysteine and collect the eluate;

[0016] (5) After gently mixing the elution system, immediately neutralize the elution system to pH 8.5 ± 0.3 with NaOH solution;

[0017] (6) Concentrate the neutralized solution to a protein content of 2 mg / ml by ultrafiltration concentration;

[0018] (7) Load the concentrated sample onto a G25 Sephadex gel column equilibrated with PBS, elute with PBS buffer, and collect the eluate to obtain the monomer of the target protein.

[0019] Further, in step (4), the protein is eluted with an elution system containing 0.1M cysteine; preferably, the protein is eluted with a 30 mM citrate elution buffer containing 0.1M cysteine.

[0020] Further, after gently mixing the elution system in step (4), immediately neutralize the elution system to a pH of 8.5 ± 0.3 with 0.5 M NaOH solution.

[0021] The present invention has the following advantages compared with the prior art:

[0022] In the present invention, by adding Cys to the protein L eluent and neutralizing it with NaOH solution, the formation of multimers caused by low-pH elution is effectively inhibited. The addition of Cys to the elution system does not affect the original elution conditions, and Cys can be effectively removed when replacing the buffer with Sephadex G25 gel chromatography. When using NaOH solution to neutralize the eluent, the purified protein does not degrade. Cys in the present invention can not only effectively reduce the formation of multimers during the purification process, but also prevent the degradation of recombinant proteins during the neutralization process, and is an excellent protective agent. The present invention can effectively inhibit the formation of multimers caused by protein purification, with simple process operation and obvious effects, providing a reference for the protein L gel chromatography process in laboratory research and industrial production, and also providing a new idea for further research on methods for removing multimers formed by other similar proteins during purification. Description of the Drawings

[0023] To more clearly illustrate the background art or the technical solution of the present invention, the drawings used in the prior art or specific embodiments are briefly introduced below; obviously, the following drawings in combination with specific embodiments are only for facilitating the understanding of the embodiments of the present invention, and those of ordinary skill in the art can also obtain other drawings based on these drawings without creative efforts;

[0024] Figure 1 It is the elution effect diagram after protein L gel chromatography in Example 1 using different elution systems;

[0025] Figure 2 It is the elution effect diagram after protein L gel chromatography in Example 2 using different elution systems;

[0026] Figure 3 It is the elution effect diagram after protein L gel chromatography in Example 3 using different elution systems;

[0027] Figure 4 It is the elution effect diagram after protein L gel chromatography in Example 4 using different elution systems;

[0028] Figure 5 It is the elution effect diagram after protein L gel chromatography in Example 5 using different elution systems; CIP peak: in-situ cleaning peak;

[0029] Figure 6It is a detection chart of the polymer content; among them, 1 is that the elution system is 30 mM citric acid elution buffer added with 25 mM Cys; 2 is that the elution system is 30 mM citric acid elution buffer added with 50 mM Cys; 3 is that the elution system is 30 mM citric acid elution buffer added with 100 mM Cys. Detailed implementation mode

[0030] The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer as the description progresses. However, the embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0031] Example A method for reducing the formation of polymers of proteins in protein L gel chromatography

[0032] (1) Equilibrate the protein L gel chromatography column with 5 column volumes of PBS equilibration solution at a flow rate of 1.0 ml / min and a detection wavelength of 280 nm;

[0033] (2) Load the fusion protein 4D5Fv-PE25 at a loading amount of 20 mg / ml gel;

[0034] (3) Equilibrate the protein L gel chromatography column with 5 column volumes of PBS equilibration solution;

[0035] (4) Elute the protein with the elution system and collect the eluate;

[0036] (5) After gently mixing the elution system, immediately neutralize the elution system to pH 8.5 ± 0.3 with 0.5 M NaOH solution;

[0037] (6) Concentrate the neutralized solution to a protein content of 2 mg / ml by ultrafiltration concentration;

[0038] (7) Load the concentrated sample onto a G25 Sephadex gel column equilibrated with PBS, and elute with PBS buffer, and collect the eluate to obtain the monomer of the target protein.

[0039] The elution systems are shown in Table 1:

[0040] Table 1

[0041]

[0042] Test Example 1 Protein separation under different elution systems using protein L gel chromatography

[0043] After detection, the separation effect in Example 1 is very good. For example, Figure 1 as shown, only part of the target protein can be eluted in Examples 2, 3 and 5. The target protein can be completely eluted only when the chromatography column needs to be cleaned, as shown in Figure 2 , Figure 3 and Figure 5 respectively. In Example 4, only the baseline of the chromatogram increases, and the elution efficiency is not affected, as shown in Figure 4 shown.

[0044] Test Example 2 Detection of the Content of Polymers

[0045] The content of polymers after treatment in Control Example 1, Control Example 2 and Example 4 neutralized with NaOH was detected by non-reducing SDS-PAGE electrophoresis

[0046] Control Example 1: The elution system was 30 mM citric acid elution buffer added with 25 mM Cys, and other steps were the same as in Example 4;

[0047] Control Example 2: The elution system was 30 mM citric acid elution buffer added with 50 mM Cys, and other steps were the same as in Example 4.

[0048] The detection results are as shown in Figure 6 shown, Figure 6 in which 1 is the elution system of 30 mM citric acid elution buffer added with 25 mM Cys; 2 is the elution system of 30 mM citric acid elution buffer added with 50 mM Cys; 3 is the elution system of 30 mM citric acid elution buffer added with 100 mM Cys (Example 4); thus, it can be seen that as the Cys concentration in the elution system increases, the content of polymers gradually decreases, and when the Cys concentration is increased to 100 mM, the polymers are basically invisible.

Claims

1. A method for reducing the formation of multimers of a protein in protein L gel chromatography, characterized in that, The method includes: (1) Adding cysteine (Cys) to the protein L elution system and neutralizing the elution system with NaOH solution; (2) Treating the sample after protein L gel chromatography purification with Sephadex G25 gel chromatography to remove Cys; In the step (1), the pH of the elution system is 2.0 - 7.0; The specific steps are as follows: (a) Equilibrating the protein L gel chromatography column with 5 column volumes of PBS equilibration buffer; (b) Loading the fusion protein 4D5Fv-PE25 at a loading amount of 20 mg / ml gel; (c) Equilibrating the protein L gel chromatography column again with 5 column volumes of PBS equilibration buffer; (d) Eluting the protein with an elution system containing cysteine and collecting the eluate; (e) After gently mixing the elution system, immediately neutralizing the elution system to pH 8.5 ± 0.3 with 0.5 M NaOH solution; (f) Concentrating the neutralized solution to a protein content of 2 mg / ml by ultrafiltration concentration; (g) Loading the concentrated sample onto a Sephadex G25 gel column equilibrated with PBS, eluting with PBS buffer, and collecting the eluate to obtain the monomer of the target protein; In the step (d), the protein is eluted with a 30 mM citrate elution buffer containing 0.1 M cysteine.

2. The method according to claim 1, wherein Reducing the formation of multimers of the fusion protein 4D5Fv-PE25 in protein L gel chromatography.

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