A low-pH virus inactivation method based on membrane chromatography
By integrating low-pH virus inactivation with cation exchange membrane chromatography, the problem of uniformity and efficiency in large-scale production of traditional low-pH virus inactivation methods has been solved, achieving efficient and simple virus inactivation and protein purification, and ensuring product quality and safety.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHONGQING HAOYUAN BIOPHARMACEUTICAL CO LTD
- Filing Date
- 2026-05-22
- Publication Date
- 2026-07-24
AI Technical Summary
Traditional low-pH virus inactivation methods are difficult to achieve uniform pH adjustment in large-scale production, leading to target protein aggregation or degradation. They are also time-consuming, require complex equipment, and affect product yield and quality.
A continuous low-pH virus inactivation method based on membrane chromatography was adopted. The virus was inactivated using a cation exchange membrane chromatography device. Through equilibration, loading, washing and elution steps, combined with a specific buffer system, the dynamic integration of virus inactivation and protein purification was achieved.
It improves the uniformity and efficiency of virus inactivation, simplifies operation steps, reduces equipment usage, and enhances the safety and quality of protein drugs, making it suitable for large-scale biopharmaceutical processes.
Smart Images
Figure CN122444809A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of low pH virus inactivation technology, specifically relating to a low pH virus inactivation method based on membrane chromatography. Background Technology
[0002] Recombinant therapeutic proteins (especially therapeutic antibodies) expressed from mammalian cell lines have become mainstream products in the biopharmaceutical field. However, the use of animal cells in production carries an inherent risk of introducing viral contamination, which directly threatens the safety of the final product. Therefore, virus clearance processes are an indispensable and critical step in the downstream production of such drugs.
[0003] To ensure patient safety, guidelines from major global drug regulatory agencies explicitly require that manufacturing processes include at least two orthogonal virus clearance steps based on different mechanisms of action. Low-pH virus inactivation is one of the core inactivation methods for lipid-enveloped viruses. The principle behind this method is that under specific acidic conditions (typically pH 3.0-3.8), the lipid bilayer of the viral envelope becomes unstable, and the conformation of its surface glycoproteins undergoes irreversible changes, thereby destroying the virus's integrity and rendering it incapable of infecting host cells. In conventional antibody production platforms, the low-pH inactivation step is usually performed immediately after Protein A affinity chromatography capture of the target product. The specific procedure typically involves collecting the eluent from the Protein A column in a dedicated inactivation vessel, adjusting the pH to the target range and maintaining it for a period of time (e.g., 30 to 120 minutes), completing the inactivation, neutralizing the solution, and transferring it to subsequent purification steps.
[0004] However, this traditional batch inactivation method has the following drawbacks: On a large production scale, precisely and uniformly adjusting the pH in a large volume of feed solution is cumbersome, and uneven mixing can easily create localized overly acidic areas, which may exacerbate the aggregation or degradation of the target protein, affecting product yield and quality. Furthermore, the entire inactivation process is time-consuming and requires a separate processing container, increasing equipment usage and operational complexity. Summary of the Invention
[0005] To address the problems in the prior art, this invention provides a low-pH virus inactivation method based on membrane chromatography, which improves the process robustness, ease of operation, and production efficiency of the low-pH virus inactivation step, thereby more effectively ensuring the viral safety and product quality of protein drugs.
[0006] The technical problem solved by this invention is achieved by the following technical solution:
[0007] The purpose of this invention is to provide a low-pH virus inactivation method based on membrane chromatography, comprising the following steps:
[0008] S1: Equilibrate the membrane chromatography apparatus with equilibration buffer;
[0009] S2: Load the sample solution containing the target protein into the equilibrated membrane chromatography apparatus;
[0010] S3: Rinse the membrane chromatography apparatus with equilibration buffer;
[0011] S4: Rinse the membrane chromatography apparatus with virus inactivation buffer and keep the virus inactivation buffer in contact with the target protein in the membrane chromatography apparatus to complete virus inactivation;
[0012] S5: Elute and collect the virus-inactivated target protein from the membrane chromatography apparatus using elution buffer.
[0013] Furthermore, in S1, the equilibration buffer is 50 mM NaAC-HAC with a pH of 5.0-6.0.
[0014] Furthermore, in S1, the cation exchange membrane chromatography apparatus is equilibrated with 5 to 10 membrane volumes using equilibration buffer.
[0015] Furthermore, in S2, the target protein is a monoclonal antibody, a bispecific antibody, or an antibody fusion protein.
[0016] Furthermore, in S2, the pH of the loading solution containing the target protein is 5.0~6.0, and the loading volume is 5~25 g / L membrane volume.
[0017] Furthermore, the equilibration buffer in S3 and the virus inactivation buffer in S4 are both 20-50mM NaAC-HAC buffers.
[0018] Furthermore, in S3, the membrane chromatography apparatus is rinsed with equilibration buffer for 5 to 10 membrane volumes.
[0019] Furthermore, in S4, the membrane chromatography apparatus is rinsed with a virus inactivation buffer at pH 3.4-3.8 for 5-10 membrane volumes, and the virus inactivation buffer is allowed to contact the target protein in the membrane chromatography apparatus for 60-90 minutes.
[0020] Furthermore, in S5, the elution buffer is a NaAC-HAC buffer or phosphate system with pH 5.0~7.0 containing 200~500mM NaCl.
[0021] Furthermore, isocratic or gradient elution methods are employed.
[0022] A low-pH virus inactivation method based on membrane chromatography includes the following steps:
[0023] S1: Equilibrate 5-10 membrane volumes of the cation exchange membrane chromatography apparatus using 50mM NaAC-HAC equilibration buffer with a pH of 5.0-6.0;
[0024] The preferred cation exchange membrane chromatography apparatus is a strong cation exchange membrane chromatography apparatus.
[0025] S2: Load the sample solution containing the target protein at pH 5.0~6.0 into the equilibrated membrane chromatography apparatus. The sample volume of the sample solution containing the target protein is 5~25 g / L membrane volume.
[0026] Preferably, the target protein is selected from monoclonal antibodies, bispecific antibodies, or antibody fusion proteins;
[0027] S3: Rinse the membrane chromatography apparatus with 20-50mM NaAC-HAC equilibration buffer for 5-10 membrane volumes to remove unbound impurities;
[0028] S4: Rinse the membrane chromatography apparatus with 20-50mM NaAC-HAC inactivation buffer (pH 3.4-3.8) for 5-10 membrane volumes, and allow the inactivation buffer to contact the target protein in the membrane chromatography apparatus for 60-90 minutes to complete virus inactivation.
[0029] S5: Use an elution buffer containing 200-500 mM NaCl in a NaAc-HAC buffer system or a phosphate system to elute the virus-inactivated target protein from the membrane chromatography device and collect the sample. The concentration of the elution buffer should be 20-50 mM and the pH should be 5.0-7.0.
[0030] Preferably, the elution is performed using an isocratic or gradient elution method.
[0031] Compared with the prior art, the beneficial technical effects of the present invention are as follows:
[0032] 1. This invention provides a continuous low-pH virus inactivation method based on membrane chromatography, dynamically integrating low-pH virus inactivation with cation exchange membrane chromatography to achieve efficient and uniform inactivation of target proteins. This method not only effectively avoids the local over-acidity phenomenon caused by uneven mixing in traditional batch inactivation, but also simplifies the process steps, improves equipment utilization and production robustness, and is suitable for the safe and efficient removal of lipid-enveloped viruses in large-scale biopharmaceutical processes.
[0033] 2. This invention discloses a continuous low-pH virus inactivation method based on membrane chromatography technology. Utilizing the high mass transfer efficiency, low operating pressure, and ease of continuous flow operation of cation exchange membrane chromatography media, the low-pH inactivation step is directly integrated into the chromatography process. This method improves the process robustness, ease of operation, and production efficiency of the low-pH virus inactivation step, thereby more effectively ensuring the viral safety and product quality of protein drugs.
[0034] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the contents of the specification. Furthermore, in order to make the above contents, objectives, features and advantages of the present invention more obvious and understandable, specific embodiments of the present invention are described below. Attached Figure Description
[0035] Figure 1 This is the SEC spectrum of the sample after low-pH inactivation by membrane chromatography in Example 1.
[0036] Figure 2 This is the SEC spectrum of the sample after low-pH inactivation by membrane chromatography in Example 2.
[0037] Figure 3 This is the SEC spectrum of the sample after low-pH inactivation by membrane chromatography in Example 3. Detailed Implementation
[0038] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.
[0039] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or prepared by existing methods.
[0040] Example 1
[0041] A low-pH virus inactivation method based on membrane chromatography: Take 3 mL of an 8 mm Sartobind S nano membrane chromatography buffer. Before use, purge the buffer with 50 mM NaAC-HAC equilibration buffer (pH 5.5) and sterilize with 1 M NaOH. Then, wash the membrane chromatography buffer for 5 volumes with 50 mM NaAC-HAC equilibration buffer (pH 5.5). Take a sample solution containing 45 mg of penicillin-dextrin antibody and adjust the pH to 5.5 with NaAC or HAC to obtain a protein loading solution. Load the protein loading solution onto the membrane chromatography buffer at a volume of 15 g / L. Wash the membrane chromatography buffer for 5 volumes with 50 mM NaAC-HAC equilibration buffer (pH 5.5), and then wash the membrane chromatography buffer for 8 volumes with 50 mM NaAC-HAC inactivation buffer (pH 3.7). Pause for 60 min. The product was eluted and recovered using a 50mM NaAC-HAC elution buffer containing 500mM NaCl at pH 5.5. The eluted fraction was collected to obtain a low-pH virus-inactivated bispecific antibody product with a purity of 94.6% as determined by SEC analysis.
[0042]
[0043] Example 2
[0044] A low-pH virus inactivation method based on membrane chromatography: Take 3 mL of an 8 mm Sartobind S nano membrane chromatography buffer. Before use, purge the buffer with 50 mM NaAC-HAC equilibration buffer (pH 5.0) and sterilize with 1 M NaOH. Then, rinse the membrane chromatography buffer for 5 volumes with 50 mM NaAC-HAC equilibration buffer (pH 5.0). Take a sample containing 24 mg of fusion protein and adjust the pH to 5.0 with NaAC or HAC to obtain the protein loading solution. Load the protein loading solution onto the membrane chromatography buffer at a volume of 8 g / L. Rinse the membrane chromatography buffer for 5 volumes with 50 mM NaAC-HAC equilibration buffer (pH 5.0), and then rinse the membrane chromatography buffer for 8 volumes with 50 mM NaAC-HAC inactivation buffer (pH 3.6). Pause for 60 min. The product was recovered by gradient elution using a 20mM citrate-disodium hydrogen phosphate elution buffer at pH 5.0 and a 20mM sodium citrate-disodium hydrogen phosphate elution buffer at pH 7.0 containing 200mM NaCl. The eluted fractions were collected to obtain the low-pH virus-inactivated fusion protein product, with a purity of 94.04% according to SEC analysis.
[0045] Example 3
[0046] A low-pH virus inactivation method based on membrane chromatography: Take 3 mL of an 8 mm Sartobind S nano membrane chromatography buffer. Before use, purge the buffer with 50 mM NaAC-HAC equilibration buffer (pH 5.5) and sterilize with 1 M NaOH. Then, wash the membrane chromatography buffer for 5 volumes with 50 mM NaAC-HAC equilibration buffer (pH 5.5). Take a sample solution containing 30 mg of monoclonal antibody and adjust the pH to 5.5 with NaAC or HAC to obtain a protein loading solution. Load the protein loading solution onto the membrane chromatography buffer at a volume of 10 g / L. Wash the membrane chromatography buffer for 5 volumes with 50 mM NaAC-HAC equilibration buffer (pH 5.5), and then wash the membrane chromatography buffer for 8 volumes with 50 mM NaAC-HAC inactivation buffer (pH 3.6), pausing for 90 min. The product was recovered by gradient elution using 50 mM NaAC-HAC elution buffer at pH 5.5 and 50 mM NaAC-HAC elution buffer at pH 5.5 containing 500 mM NaCl. The eluted fractions were collected to obtain the low-pH virus-inactivated monoclonal antibody product, with a purity of 98.38% as determined by SEC analysis.
[0047] This invention discloses a continuous low-pH virus inactivation method based on membrane chromatography, primarily applied to the downstream purification process of recombinant therapeutic proteins (especially antibodies) expressed in mammalian cells. This method dynamically integrates low-pH virus inactivation with cation exchange membrane chromatography. By continuously passing the low-pH virus inactivation solution into the membrane chromatography medium, its high mass transfer efficiency and uniform fluid distribution achieve efficient and uniform inactivation of the target protein. This not only effectively avoids the localized over-acidity phenomenon caused by uneven mixing in traditional batch inactivation but also supports continuous flow operation, eliminating the need for a separate inactivation tank, simplifying process steps, improving equipment utilization and production robustness. It is suitable for the safe and efficient removal of lipid-enveloped viruses in large-scale biopharmaceutical processes.
[0048] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.
[0049] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.
Claims
1. A low-pH virus inactivation method based on membrane chromatography, characterized in that, Includes the following steps: S1: Equilibrate the membrane chromatography apparatus with equilibration buffer; S2: Load the sample solution containing the target protein into the equilibrated membrane chromatography apparatus; S3: Rinse the membrane chromatography apparatus with equilibration buffer; S4: Rinse the membrane chromatography apparatus with virus inactivation buffer and keep the virus inactivation buffer in contact with the target protein in the membrane chromatography apparatus to complete virus inactivation; S5: Elute and collect the virus-inactivated target protein from the membrane chromatography apparatus using elution buffer.
2. The low-pH virus inactivation method based on membrane chromatography as described in claim 1, characterized in that: In S1, The equilibration buffer used was 50 mM NaAC-HAC with a pH of 5.0-6.
0.
3. The low-pH virus inactivation method based on membrane chromatography as described in claim 2, characterized in that: In S1, the cation exchange membrane chromatography apparatus is equilibrated with 5 to 10 membrane volumes using equilibration buffer.
4. The low-pH virus inactivation method based on membrane chromatography as described in claim 1, characterized in that: In S2, the target protein is a monoclonal antibody, a bispecific antibody, or an antibody fusion protein.
5. The low-pH virus inactivation method based on membrane chromatography as described in claim 1, characterized in that: In S2, the pH of the loading solution containing the target protein is 5.0~6.0, and the loading amount is 5~25 g / L membrane volume.
6. The low-pH virus inactivation method based on membrane chromatography as described in claim 1, characterized in that: The equilibration buffer in S3 and the virus inactivation buffer in S4 are both 20-50mM NaAC-HAC buffers.
7. The low-pH virus inactivation method based on membrane chromatography as described in claim 1, characterized in that: In S3, rinse the membrane chromatography apparatus with equilibration buffer for 5 to 10 membrane volumes.
8. The low-pH virus inactivation method based on membrane chromatography as described in claim 1, characterized in that: In S4, the membrane chromatography apparatus is rinsed with a virus inactivation buffer at pH 3.4-3.8 for 5-10 membrane volumes, and the virus inactivation buffer is kept in contact with the target protein in the membrane chromatography apparatus for 60-90 minutes.
9. The low-pH virus inactivation method based on membrane chromatography as described in claim 1, characterized in that: In S5, the elution buffer is a NaAC-HAC buffer or phosphate system with pH 5.0~7.0 containing 200~500mM NaCl.
10. The low-pH virus inactivation method based on membrane chromatography as described in claim 1, characterized in that: In S5, isocratic or gradient elution methods are used.