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目的 利用国产无血清培养基T22,建立人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白(rhTNFR:Fc)重组CHO细胞大规模生产工艺及质控方法。方法用500 L生物反应器培养重组CHO细胞,通过深层过滤、亲和层析、离子交换层析、疏水层析等方法纯化表达上清中的目的 蛋白,超滤浓缩制成原液,并按照《中国药典》三部(2010版)及现有的质控方法和标准建立相应的质控方法。结果重组CHO细胞在500 L生物反应器中培养15 d左右,细胞密度最高可达8.6×106个/ml,收获时细胞活力为70%左右,rhTNFR:Fc蛋白表达量可达3.3 g/L;纯化的目的 蛋白纯度可达98%以上,总收率超过30%;各项质量指标均符合新药申报标准。结论建立的rhTNFR:Fc重组CHO细胞生产工艺及质控方法稳定可靠,使用国产培养基大大降低了生产成本。
Objective To establish a large scale production process and quality control method of recombinant human Tumor Necrosis Factor Receptor - Antibody Fusion Protein (rhTNFR: Fc) recombinant CHO cells by using domestic serum - free medium T22. Methods Recombinant CHO cells were cultured in a 500 L bioreactor. The recombinant protein was purified by deep filtration, affinity chromatography, ion-exchange chromatography and hydrophobic chromatography. The recombinant protein was concentrated by ultrafiltration to obtain a stock solution. Chinese Pharmacopoeia "three (2010 version) and the existing quality control methods and standards to establish the appropriate quality control methods. Results The recombinant CHO cells were cultured in a 500 L bioreactor for about 15 days with a cell density of 8.6 × 106 cells / ml. The viability of the recombinant CHO cells was about 70% at harvest and the expression level of rhTNFR: Fc was 3.3 g / L. Purified target protein purity up to 98%, the total yield of more than 30%; the quality indicators are in line with the new drug reporting standards. Conclusion The established rhTNFR: Fc recombinant CHO cells production technology and quality control method is stable and reliable, using domestic medium greatly reduces the production cost.