人源性抗HBsAg Fab抗体的发酵生产研究

为了适应工业生产的需要，利用fed—batch方法，重组人源性抗HBsAg Fab抗体酵母工程菌在30L发酵罐中进行了高密度发酵，发酵最适温度30℃，pH值范围5．0～5．3，溶氧范围20％～30％。发酵液OD600值达到300时开始诱导，甲醇最佳诱导浓度为10mL／L。重组人源性抗HBsAg Fab抗体经离子交换层析纯化，纯化产品经SDS-PAGE、Western blot进行分析和ELISA方法进行活性测定。结果显示，重组Fab抗体在Fed-batch发酵系统中可高效表达，经过192h的发酵生产，重组人源性抗HBsAg Fab抗体的表达量可达412mg／L。发酵上清经过离子交换层析纯化，获得纯度为95％的重组Fab抗体，该Fab抗体经ELISA分析具有较高的HBsAg抗原亲和力和特异性。结果证实可以通过高密度发酵毕赤酵母工程菌来高效生产重组人源性抗HBsAg Fab抗体，为后续的工业化生产应用奠定了基础。

Production of Recombinant Humanized anti-HBsAg Fab Antibody by Fermentation

In order to produce recombinant human anti-HBsAg Fab antibody in Pichia pastoris, the recombinant yeast was fermented using fed-batch system in a 30 L bioreactor. The fermentation temperature was 30 degrees C, the pH was 5.0 approximately 5.3, and the DO was 20% approximately 30%. The recombinant Fab antibody was purified from crude culture supernatant by ion exchange and analyzed by SDS-PAGE and western blot and ELISA. When the absorbance (OD600) of broth reach 300 at the end of fed-batch phase, the induced phase was initiated. The results showed that recombinant human anti-HBsAg Fab antibody was high-level expressed in recombinant Pichia pastoris using a fed-batch fermentation system. Both chains of the Fab were successfully expressed upon methanol induction. After 192 h of induction, the expression level of recombinant Fab (soluble) reached 412 mg/L. The recombinant Fab antibody was purified effectively by ion-exchange chromatography from the fermentation supernatant to a purity of 95%. And the affinity activities of the purified recombinant Fab antibdy and fermentation supernatant were detected, and both of them showed high affinity activities. The results demonstrated that recombinant human anti-HBsAg Fab antibody could be high level produced by fed-batch fermentations in Pichia pastoris. Which can be efficiently used in industrial production.