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为克服血源免疫球蛋白制品的不足 ,开发了抗甲肝病毒基因工程单克隆抗体anti_HAVIgG。用无血清培养基培养rCHO工程细胞株 ,上清液经过rProteinASFF亲和层析→脱盐→离子交换层析→超滤换液纯化后 ,所得anti_HAVIgG纯度达 99%以上 ,比活性约 10 0IU mg ,anti_HAVIgG活性回收率 40 %。所纯化的anti_HAVIgG分子量150kD ,等电点 8 4～ 9 3。免疫印迹实验证实anti_HAVIgG为人源全抗体分子。亲和层析介质rProteinASFF确实存在亲和配基脱落问题 ,但通过后续纯化步骤可有效除去。在亲和层析过程中加入高盐清洗步骤 ,可有效降低宿主DNA残留量水平。对样品中自由巯基含量进行了测定 ,认为非还原电泳图谱中低分子量条带是由于抗体分子内存在自由巯基引起。用该工艺制备的anti_HAVIgG各项纯度检测指标均达到我国对基因工程产品的质量要求
To overcome the lack of blood-derived immunoglobulin products, the anti-HAVIgG genetically engineered monoclonal antibody against Hepatitis A virus has been developed. RCHO engineered cell lines were cultured in serum-free medium, the supernatant was purified by rProteinASFF affinity chromatography → desalination → ion exchange chromatography → ultrafiltration, the purity of anti_HAVIgG was over 99%, the specific activity was about 10 0 mg mg, anti_HAVIgG activity recovery rate of 40%. The purified anti_HAVIgG molecular weight of 150kD, isoelectric point of 84 ~ 93. Western blotting experiments confirmed that anti_HAVIgG is a human full antibody molecule. Affinity chromatography medium rProteinASFF does have affinity ligand shedding problems, but can be effectively removed by subsequent purification steps. In the affinity chromatography process by adding salt cleaning, can effectively reduce the level of host DNA residues. The content of free sulfhydryl in the sample was determined. It is considered that the low molecular weight band in the non-reducing electrophoresis pattern is caused by the presence of free thiol in the antibody molecule. The purity of anti_HAVIgG prepared by the process of detection indicators have reached the quality of our country’s genetic engineering products