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Method for purifying bispecific antibody
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A kind of technology of bispecific antibody, purification method
Pending Publication Date: 2022-01-25
INNOVENT BIOLOGICS (SUZHOU) CO LTD
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[0003] In order to solve the technical problem of high residual HCP after protein purification of the existing bispecific antibody (as described in the present invention), the present invention provides a method for purifying the bispecific antibody of the present invention
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Embodiment 1
[0083] Example 1 Construction of LAG-3 / PD-L1 bispecific antibody
[0084] According to the method described in PCT application number PCT / CN2020 / 073964, construct the following figure 1 The LAG-3 / PD-L1 bispecific antibody with the structure shown, wherein the antigen A is LAG-3, and the antigen B is PD-L1. Antibodies were expressed recombinantly in CHO cells.
[0085] The specific sequence of the bispecific antibody is shown in the table below:
[0086] The protein sequence number of the bispecific antibody of the present invention
[0094] The CHO cellculture fluid expressing the LAG-3 / PD-L1 bispecific antibody as described in Example 1 was filtered with Millistak DOHC and XOHC deep adsorption membranes from Merckmilipore Company. The protein content of the sample was determined to be 3.9 g / L in the clarified harvest liquid.
[0096] A MabSelect SuRe prepacked column (HiTrap) from GE Company was used, with a column height of 5 cm and a column volume of 4.8 mL. After the chromatographic column was equilibrated for 5 column volumes (CV) with an equilibration buffer (20mM Tris-HCl, 150mM NaCl, pH 7.2), the above sample protein was loaded on the AKTA pure M purification system (the optical path of the ultravioletdetector was 2mm). The sample load is 47g / L packing, and the sample retention time is 6min when loading the sample, that is, the flow rate is 0.83ml / min. After sample loading, equili...
[0104] The preparation process was the same as in Example 2, and the protein content of the sample was measured to be 4.3 g / L in the clarified harvest liquid.
[0106]Use GE's MabSelect SuRe LX filler to fill Merck Millipore's LLaboratory Column VL 11×250 chromatography column, column height 7.5cm, column volume 8mL. After the chromatographic column was equilibrated for 5 column volumes (CV) with equilibration buffer (20mM Tris-HCl, 150mM NaCl, pH 7.2), the sample was loaded using the AKTA pure M purification system (the optical path length of the UV detector was 2mm). 40g / L filler, the sample retention time is 6min when the sample is loaded, that is, 1.33ml / min. After loading the samples, equilibrate 3CV with affinity rebalance solution (20mM Tris-HCl, 150mM NaCl, pH 7.2), and then wash 3CV with different affinity wash solutions (see Table 2 for wash solution...
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Abstract
The invention discloses a method for purifying a bispecific antibody. The method comprises the step of purifying the bispecific antibody by using affinity chromatography, wherein after the bispecific antibody is loaded, a washing buffer solution containing CaCl2 is used for washing a chromatographic column. The invention further provides a preparation method of the bispecific antibody and application of the washing buffer solution in purification of the bispecific antibody. When the bispecific antibody disclosed by the invention is purified by using the purification method disclosed by the invention, HCP can be controlled in a process acceptable range, and the content of residual HCP in collected liquid is lower than about 7000 ppm.
Description
technical field [0001] The invention relates to a method for purifying a bispecific antibody. Background technique [0002] Host protein residues (HCP) usually refer to some extracellular proteins secreted by host cells such as CHO cells during the culture process and some intracellular proteins released by cell breakdown during clarification and filtration, which are process-related impurities. Regulatory authorities have strict requirements on HCP residues in biological products, and HCP residues need to be reduced to an acceptable range. Even very low doses of HCP in the final product may cause antibody proteininstability or immunogenicity in patients. Therefore, HCP carryover is often a critical quality attribute in downstream purification processes. In the prior art, HCP is often reduced through multiple process steps, such as flocculation, chromatography and adsorption depth filtration. In the face of purification of protein molecules with different properties, it ...
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