Expression, purification and activity determination of humanized anti-HER2 monoelonal antibody in CHO / 中国药学杂志

ABSTRACT

OBJECTIVE: To screen for the optimal CHO cell clone which stably expresses recombinant humanized anti-HER2 monoclonal antibody(rhHER2-mAb) and to measure its bioactivity. METHODS: Disposable orbital shaking bioreactor, Tubespin, was utilized for high-throughput screening, and then orbital shaking flask was used for scale-up. Protein A affinity chromatography was utilized to purify the fusion protein. SDS-PAGE and HPLC were utilized to detect its purity, ELISA was utilized to detect its production amount, and MTT was utilized to detect its bioactivity. RESULTS: Through the cell clone selection, it was found that #38 cell clone had the best expression performance, with the productivity in shaking flask of(152±20) mg · L-1. After scale up, enough supernatant was got for the purification. The molecular weight of the purified protein was about 150 × 103, and its purity was higher than 96%. Analysis of bioactivity and binding affinity showed that its activity and bind affinity were close to the commercialized product, Herceptin. CONCLUSION: A cell clone with high productivity was obtained by cell clone screening. The antibody protein was successfully obtained by orbital shaking scale-up cultivation and purification. Its bioactivity and binding affinity were consistent with the commercialized drug. This method has laid a foundation for large-scale fermentation of this recombinant protein.