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CN-115960241-B - Protein A purification preservation method for bispecific antibody of epidermal growth factor receptor

CN115960241BCN 115960241 BCN115960241 BCN 115960241BCN-115960241-B

Abstract

The invention discloses a Protein A purification and preservation method for an epidermal growth factor receptor bispecific antibody, which comprises the steps of eluting the epidermal growth factor receptor bispecific antibody by using Protein eluent, and preserving the purified bispecific antibody in a Protein storage solution, wherein Protein eluent and components of the Protein storage solution are specifically disclosed. The invention optimizes the eluent and the storage liquid of the bispecific antibody aEGFR-aPDL BsAb of the EGFR, finds a stable buffer composition, and remarkably improves the recovery rate and the purity of the protein.

Inventors

  • ZHANG YUHAN
  • GUO CHAO
  • WANG FENG
  • ZHANG JIE

Assignees

  • 苏州生物医药转化工程中心

Dates

Publication Date
20260505
Application Date
20211012

Claims (10)

  1. 1. A method for purifying and preserving Protein A of an EGFR bispecific antibody is characterized in that Protein A is taken as an affinity filler, the EGFR bispecific antibody is eluted by Protein eluent, the purified EGFR bispecific antibody is stored in a Protein storage solution in a liquid-exchanging way, wherein, The epidermal growth factor receptor bispecific antibody is formed by fusing an epidermal growth factor receptor antibody and a cell apoptosis-ligand 1 antibody; the composition of the protein eluent is as follows: 0.08-0.15M sodium citrate, 0.1-0.2M sodium chloride and 240-260 mM sucrose, pH3.0; or 0.08-0.15M sodium citrate, 0.1-0.2M sodium chloride and 240-260 mM sucrose, pH3.5; the composition of the protein stock solution is as follows: 0.08-0.15M sodium acetate and 0.1-0.2M sodium chloride, pH 6.0; Or 0.08-0.15M sodium acetate, 0.1-0.2M sodium chloride and 240-260 mM sucrose, pH 6.0; or 0.08-0.15M sodium acetate and 0.1-0.2M sodium chloride, and the pH is 5.0; Or 0.08-0.15M sodium acetate, 0.1-0.2M sodium chloride and 240-260 mM sucrose, pH 5.0.
  2. 2. The method of claim 1, wherein the composition of the protein stock solution is: 0.1M sodium acetate and 0.1M to 0.2M sodium chloride, and the pH value is 6.0; Or 0.1M sodium acetate, 0.1M-0.2M sodium chloride and 250mM sucrose, pH 6.0; or 0.1M sodium acetate and 0.1M to 0.2M sodium chloride, and the pH is 5.0; or 0.1M sodium acetate, 0.1M to 0.2M sodium chloride and 250mM sucrose, pH 5.0.
  3. 3. The method of claim 2, wherein the composition of the protein eluent is: 0.1M sodium citrate, 0.1M to 0.2M sodium chloride and 250mM sucrose, pH 3.0; or 0.1M sodium citrate, 0.1M to 0.2M sodium chloride and 250mM sucrose, pH 3.5.
  4. 4. The method of claim 1, wherein the protein eluent is used for neutralizing after eluting the bispecific antibody of the EGFR, and the neutralizing liquid is 1M Tris-HCl, and the pH value is 8.9.
  5. 5. The method according to claim 1, wherein the protein eluate is added and incubated at 20-30℃for 5-10min.
  6. 6. The method of claim 1, wherein the volume ratio of the EGFR bispecific antibody solution to the protein eluent is 20-40:1 when the protein eluent is used for eluting the EGFR bispecific antibody.
  7. 7. The method of claim 1, wherein the bispecific antibody for EGFR is concentrated by centrifugation at 3500-4500rpm when the replacement fluid is stored in the protein stock fluid.
  8. 8. The method of claim 7, wherein the volume ratio of the epidermal growth factor receptor bispecific antibody concentrate to the protein stock solution obtained after concentration is 1:50-100.
  9. 9. The method according to claim 1, characterized in that it comprises the following steps: (1) Washing the Protein A affinity filler with phosphate buffer; (2) Slowly adding the Protein supernatant to be purified into the chromatographic column, repeating for a plurality of times, and then washing the Protein A affinity filler with phosphate buffer again; (3) Adding protein eluent into the chromatographic column, incubating for 5-10min at 20-30 ℃, eluting the bispecific antibody of the EGFR, and repeating for a plurality of times until the bispecific antibody of the EGFR is completely eluted; (4) Adding a neutralizing solution into the eluted epidermal growth factor receptor bispecific antibody, wherein the neutralizing solution is 1M Tris-HCl, and the pH value is 8.9; (5) Centrifuging at 3500-4500rpm to obtain an EGF receptor bispecific antibody concentrate, adding the protein stock solution, and centrifuging to make the EGF receptor bispecific antibody in the protein stock solution.
  10. 10. The method according to claim 9, wherein in the step (1) and the step (2), the phosphate buffer is DPBS, pH7.5.

Description

Protein A purification preservation method for bispecific antibody of epidermal growth factor receptor Technical Field The invention relates to the field of antibody separation and purification, in particular to a Protein A purification and preservation method for an epidermal growth factor receptor bispecific antibody. Background PD-L1 is called programmed death receptor ligand-1, participates in immune escape, can be combined with PD-1, inhibits T cell proliferation and cytokine secretion, and negatively regulates lymphocyte activation. PD-1, which is a general name of programmed death receptor-1, belongs to a CD28 superfamily member, is a type I transmembrane protein composed of 268 amino acids, and can be expressed on the surface of immune cells such as T cells, B cells and the like. However, when the T cells are not activated, PD-1 is hardly expressed, and only after T cell activation, PD-1 is expressed on the surface of the T cells. As previously described, PD-1 can bind to PD-L1, both as receptors and ligands. The immune system normally responds to foreign antigens that accumulate in the lymph nodes or spleen, triggering antigen-specific cytotoxic T cells (cd8+ Tcell proliferation). And the apoptosis receptor-1 (PD-1) is combined with the apoptosis-ligand 1 (PD-L1) to transmit an inhibitory signal and reduce the proliferation of the lymph node CD8+ T cells. aPD-L1 is a monoclonal antibody that binds to PD-L1 and blocks its interaction with the PD-1 receptor. This releases PD-L1/PD-1 mediated suppression of immune responses, including activation of anti-tumor immune responses without induction of antibody dependent cellular cytotoxicity. In syngeneic mouse tumor models, blocking PD-L1 activity resulted in reduced tumor growth. EGFR (Epidermal Growth Factor Receptor) are receptors for Epithelial Growth Factor (EGF) cell proliferation and signaling. Studies have shown that EGFR mutation rates in many solid tumors, including head and neck, breast, bladder, ovarian, kidney, colon, and non-small cell lung cancers, can reach 50% in asian lung cancer populations. EGFR is involved in the inhibition of proliferation, angiogenesis, tumor invasion, metastasis and apoptosis of tumor cells. EGFR is an important target in clinical treatment of tumors. Bispecific antibodies (bispecific monoclonal antibody, bsAb) are a special antibody that has been engineered to bind two different antigens simultaneously. The research of bispecific antibodies has great significance for the immunotherapy of cancer, and has been developed as a major hotspot for clinical treatment of tumors. The advantages of bispecific antibodies compared with monoclonal antibodies are mainly that they can mediate timing or spatial effects, but the technological threshold and development costs are high, and the construction stage may suffer from low expression level and poor stability. Protein a is a bacterial cell wall Protein isolated from staphylococcus aureus and binds to mammalian IgG mainly through the Fc region. Protein a has five IgG binding domains. In the purification process of the antibody-related Protein, protein A resin is preferred as an affinity chromatography medium for antibody capture. Typically, antibodies or fusion proteins containing an Fc region bind to Protein a under neutral conditions and the Protein is eluted under acidic conditions. Most monoclonal antibodies adopt 0.1mol/L glycine elution solution with pH of 2.5-pH3.0, and under the condition, the monoclonal antibodies have higher purity and recovery rate. However, for some unstable bispecific antibodies, such as an EGFR bispecific antibody formed by fusing an EGFR antibody and a apoptosis-ligand 1 antibody, proteins are easily precipitated after elution with the above solution, resulting in a decrease in recovery and purity of the proteins. Thus, there remains a need to find a purification method for epidermal growth factor receptor bispecific antibodies that can stabilize proteins. Disclosure of Invention In order to solve the technical problems, the invention discloses a Protein A purification and preservation method for an EGFR bispecific antibody, which improves a Protein storage solution and solves the problems of low Protein recovery rate and unstable Protein in the storage process. The invention discloses a Protein A purifying and preserving method for an EGFR bispecific antibody, which uses Protein A as an affinity filler, uses Protein eluent to elute the EGFR bispecific antibody, and stores the purified EGFR bispecific antibody in a Protein storage liquid, The composition of the protein eluent is as follows: 0.08-0.15M sodium citrate, 0.1-0.2M sodium chloride and 240-260 mM sucrose, pH3.0; Or 0.08-0.15M sodium citrate, 0.1-0.2M sodium chloride and 240-260 mM sucrose, pH3.5. The composition of the protein stock solution is: 0.08-0.15M sodium acetate and 0.1-0.2M sodium chloride, pH 6.0; Or 0.08-0.15M sodium acetate, 0.1-0.2M sodium chloride and 240-260 mM sucrose,