Automatic method for developing affinity chromatography and virus inactivation process by using gravity column

Abstract

The invention discloses an automatic method for developing an affinity chromatography and virus inactivation process by using a gravity column. The method comprises the following steps: S1, preparing a biological macromolecular biological product containing potential viruses; s2, setting a protein A affinity chromatography gravity column layout, wherein the protein A affinity chromatography gravity column layout comprises protein A affinity chromatography elution condition screening and subsequent material preparation which are carried out at the same time; and S3, developing a low-pH virus inactivation process by using an automatic workstation, wherein the low-pH virus inactivation process comprises the following steps: automatically completing addition and uniform mixing of acid and alkali adjusting liquid, automatically completing low-pH adjustment, incubating and neutralizing to neutral pH. According to the method, the yield of the gravity column is increased, the development efficiency of the affinity chromatography process is improved, and the development of the biological medicine downstream affinity chromatography process and the pH virus inactivation process can be quickly and automatically completed.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical analysis, and in particular to an automated method for developing affinity chromatography and virus inactivation processes using a gravity column. Background Art

[0002] The downstream process of monoclonal antibody protein usually includes protein A affinity chromatography, low pH virus inactivation and other purification chromatography steps. The process development of protein A affinity chromatography usually requires first screening for suitable elution conditions, and then using the eluted sample for subsequent low pH virus inactivation and purification process development.

[0003] Affinity chromatography process development usually uses corresponding chromatographic columns, and there are two ways of using them in the prior art: one is to use a gravity column for purification, which does not control the pressure during use, does not require special instruments and equipment, and uses gravity to complete purification, and has a high flux. Therefore, using a gravity column for purification is a more commonly used purification method and is widely used in the pharmaceutical industry. The other is to install the affinity chromatography column on a purification instrument, such as an AKTA purifier or a liquid chromatograph. This method can be automated, but only one sample can be purified at a time, and the flux is low, making it difficult to complete the affinity chromatography elution condition screening and material preparation at the same time.

[0004] Biotechnology products prepared from mammalian CHO cell lines cannot completely eliminate the risk of viral contamination. Regulatory agencies require viral inactivation and clearance studies to demonstrate that downstream processes are effective in inactivating / clearing viruses and to quantitatively estimate the level of viral reduction that can be achieved by the overall downstream process. Low pH viral inactivation is a common step in the downstream process to inactivate enveloped viruses. In general, this step is effective and robust for the inactivation of enveloped viruses, but in order to assess whether it will compromise the quality of the product, the viral inactivation pH needs to be developed. Typically, protein A affinity chromatography process development and sample preparation require 2 to 4 experiments to complete, and low pH adjustment, incubation, and neutralization to neutral pH all require manual operations, which take up a lot of time for developers.

[0005] Therefore, there is an urgent need in the art for an automated method for developing affinity chromatography and virus inactivation processes using gravity columns, improving the yield of gravity columns, improving the efficiency of affinity chromatography, and at the same time realizing the automation of gravity column-based protein A affinity chromatography process development, sample preparation, and low pH virus inactivation process development. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an automated method for developing affinity chromatography and virus inactivation processes using a gravity column, the method comprising the following steps:

[0007] S1. Preparation of biological macromolecules containing potential viruses;

[0008] S2. Setting a protein A affinity chromatography gravity column layout, wherein the protein A affinity chromatography gravity column layout includes simultaneous protein A affinity chromatography elution condition screening and subsequent material preparation;

[0009] S3. Use an automated workstation to develop a low pH virus inactivation process, wherein the low pH virus inactivation process includes automated addition and mixing of acid and alkali adjustment solutions, automated low pH adjustment, incubation, and neutralization to a neutral pH.

[0010] Specifically, in S1, the biomacromolecule is selected from monoclonal antibody protein, bispecific antibody or Fc fusion protein.

[0011] Specifically, in S1, the potential virus is an endogenous virus expressed by a mammalian cell line or a virus introduced during the production process.

[0012] Specifically, in S1, the biological product is a biological product for injection.

[0013] Specifically, in S2, the compression factor of the filler is calculated when the protein A affinity chromatography gravity column is packed, and the filler used is an affinity filler with protein A as a ligand, preferably MabSelect PrismA or MabSelect SuRe LX, and the compression factor is 1.15.

[0014] Specifically, in S2, the liquid dripping speed of the protein A affinity chromatography gravity column is 120-140 seconds for each drop of 1 mL of pure water.

[0015] Specifically, in S2, the sample loading method of the protein A affinity chromatography gravity column is: after the current sample completely flows through the gravity column, the next sample loading is performed.

[0016] Specifically, in S2, the layout of the protein A affinity chromatography gravity column includes: supporting three elution pH screenings, and supporting pH 3.6 elution conditions for sample preparation.

[0017] Specifically, in S2, the volume of each sample loading on the protein A affinity chromatography gravity column is 0.45-0.55 mL.

[0018] Specifically, in S3, the average ratio of the acid and alkali regulating solutions in the automated workstation is:

[0019]

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The experimental method provided by the present invention optimizes the performance and layout of the protein A affinity chromatography gravity column based on the high-throughput advantage of the gravity column, thereby improving the elution efficiency of the protein A affinity chromatography gravity column, achieving the effect of completing sample preparation under the pH 3.6 elution condition while performing three elution pH screenings of pH 4.0, pH 3.8, and pH 3.6, thereby solving the problem that conventional purification methods are difficult to simultaneously complete affinity chromatography elution condition screening and sample preparation.

[0022] 2. The experimental method provided by the present invention optimizes the loading method of the protein A affinity chromatography gravity column, adopts a method of adding 0.45-0.55 mL of sample each time and then adding the next sample after the sample has completely flowed through the gravity column, thereby improving the binding efficiency of the sample and the filler, solving the disadvantage of low yield of traditional gravity columns, and improving the comparability of affinity chromatography gravity column development data with AKTA chromatography development data.

[0023] 3. The detection method provided by the present invention realizes the automation of affinity chromatography process development, sample preparation, low pH adjustment, incubation, and neutralization to neutral pH by designing and optimizing the steps of adding acid-base regulating solution and mixing. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a gravity column layout diagram for high-throughput screening of three elution pHs: pH 4.0, pH 3.8, and pH 3.6, and sample preparation under pH 3.6 elution conditions. The shaded background column position is used for elution pH screening, and the blank background column position is used for sample preparation under pH 3.6 elution conditions;

[0025] Figure 2 This is the layout diagram of the automated workstation for process development using gravity column protein A affinity chromatography and low pH process development. DETAILED DESCRIPTION

[0026] The technical scheme of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The materials, instruments and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0027] Embodiment 1

[0028] In this example, the process of developing a monoclonal antibody protein A affinity chromatography process and a low pH virus inactivation process using the method of the present invention is shown. Using a monoclonal antibody expressed by a CHO cell line, the loading sample information is shown in Table 1 below:

[0029] Table 1 Sample information

[0030]

[0031]

[0032] In this embodiment, unless otherwise specified, the protein A affinity chromatography process development will be performed according to the process parameters in Table 2 below:

[0033] Table 2 Affinity chromatography process development experimental parameters

[0034] step Solution Solution volume (mL) Rinse 1 50mM Tris, HAc, 150mM NaCl, pH 7.4 3 Pre-disinfection 0.5M NaOH 5 balance 50mM Tris, HAc, 150mM NaCl, pH 7.4 5 Loading sample As needed Rinse 1 50mM Tris, HAc, 150mM NaCl, pH 7.4 4 Rinse 2 50mM NaAc-HAc, 1M NaCl, pH 5.5 4 Rinse 3 30 mM NaAc-HAc, pH 5.5 4 Elution 30 mM NaAc, pH 4.0, 3.8, 3.6 5 regeneration 0.12M HAc 4 Rinse 2 50mM Tris, HAc, 150mM NaCl, pH 7.4 3 Post-disinfection 0.5M NaOH 5 Rinse 3 50mM Tris, HAc, 150mM NaCl, pH 7.4 3 Column preservation 20% EtOH 3

[0035] In this example, the gravity column was filled with MabSelect PrismA filler from Cytiva. Please refer to Table 3 below, which shows the yield comparison of the two cases of not calculating the compression factor and calculating the compression factor in the optimization of the performance of the gravity column.

[0036] Table 3 Gravity column performance optimization

[0037]

[0038] It can be seen that the gravity column that is conventionally loaded without calculating the compression factor of the filler has an unstable binding effect between the filler and the protein between different gravity columns, resulting in large differences in the yield of the eluent. In the method provided in the present application, after calculating the compression factor of 1.15 to load the gravity column, a gravity column in which 1 mL of pure water flows through the gravity column for 120-140 seconds is selected, and the loading method of the protein A affinity chromatography gravity column is optimized. The method of adding 0.45-0.55 mL of sample each time and the next loading after the sample completely flows through the gravity column is adopted, which increases the comparability between gravity columns, improves the robustness and binding efficiency of the combination of sample and filler, solves the drawbacks of large yield fluctuations and low yields of traditional gravity columns, and improves the representativeness of gravity columns for protein A affinity process development.

[0039] The optimized protein A affinity chromatography gravity column in the above steps was applied, and the sample loading method of 0.45-0.55 mL was added each time, and the next loading was performed after the sample completely flowed through the gravity column. The protein A affinity process of two monoclonal antibody molecules with different SEC purities was further developed. The comparison results of the affinity chromatography gravity column development data and the AKTA chromatography development data are shown in Table 4 below.

[0040] Table 4 Comparison of affinity chromatography gravity column development data and AKTA chromatography development data

[0041]

[0042] As shown in Table 4, the gravity column and loading method optimized by the present application method successfully completed the screening of elution pH in the protein A affinity chromatography process and obtained results comparable to the AKTA chromatography equipment development data.

[0043] Please refer to Figure 1-Figure 2 This embodiment also shows the optimization of the layout of the protein A affinity chromatography gravity column based on the advantage of the high throughput of the gravity column, and the screening of three elution pHs of pH 4.0, pH 3.8, and pH 3.6 and the sample preparation of the pH 3.6 elution condition. In addition, based on the automated workstation, the automation of the low pH virus inactivation process development is explored. Among them, the addition ratio of the acid and alkali regulating solution is shown in Table 5 below.

[0044] Table 5 Addition ratio of acid and alkali regulating solution

[0045]

[0046] By exploring the addition ratio of acid and alkali adjusting solutions, the optimal ratio is obtained as shown in Table 5. The automatic addition and mixing steps of acid and alkali adjusting solutions can be completed by using an automated workstation, realizing the automation of low pH adjustment, incubation, and neutralization to neutral pH.

[0047] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An automated method for developing affinity chromatography and virus inactivation processes using a gravity column, characterized in that: The method comprises the following steps: S1. Preparation of biological macromolecules containing potential viruses; S2. Setting a protein A affinity chromatography gravity column layout, wherein the protein A affinity chromatography gravity column layout includes simultaneous protein A affinity chromatography elution condition screening and subsequent material preparation; S3. Use an automated workstation to develop a low pH virus inactivation process, wherein the low pH virus inactivation process includes automated addition and mixing of acid and alkali adjustment solutions, automated low pH adjustment, incubation, and neutralization to a neutral pH.

2. The automated method for developing affinity chromatography and virus inactivation process using a gravity column according to claim 1, characterized in that: In S1, the biomacromolecule is selected from monoclonal antibody protein, bispecific antibody or Fc fusion protein.

3. The automated method for developing affinity chromatography and virus inactivation process using a gravity column according to claim 1, characterized in that: In S1, the potential virus is an endogenous virus expressed by a mammalian cell line or a virus introduced during the production process.

4. The automated method for developing affinity chromatography and virus inactivation process using a gravity column according to claim 1, characterized in that: In S1, the biological product is a biological product for injection.

5. The automated method for developing affinity chromatography and virus inactivation process using a gravity column according to claim 1, characterized in that: In S2, the compression factor of the filler is calculated when the protein A affinity chromatography gravity column is packed. The filler used is an affinity filler with protein A as a ligand, and the compression factor is 1.

15.

6. The automated method for developing affinity chromatography and virus inactivation process using a gravity column according to claim 1, characterized in that: In S2, the liquid dripping speed of the protein A affinity chromatography gravity column is 120-140 seconds for each drop of 1 mL of pure water.

7. The automated method for developing affinity chromatography and virus inactivation process using a gravity column according to claim 1, characterized in that: In S2, the sample loading method of the protein A affinity chromatography gravity column is: after the current sample has completely flowed through the gravity column, the next sample loading is performed.

8. The automated method for developing affinity chromatography and virus inactivation process using a gravity column according to claim 1, characterized in that: In S2, the layout of the protein A affinity chromatography gravity column includes: supporting three elution pH screenings and supporting pH 3.6 elution conditions for sample preparation.

9. The automated method for developing affinity chromatography and virus inactivation process using a gravity column according to claim 1, characterized in that: In S2, the sample volume of each loading of the protein A affinity chromatography gravity column is 0.45-0.55 mL.

10. The automated method for developing affinity chromatography and virus inactivation process using a gravity column according to claim 1, characterized in that: In S3, the average ratio of acid and alkali regulating solution in the automated workstation is:

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