Hardy E, Martínez E, Diago D, Díaz R, González D, Herrera L
Center for Genetic Engineering and Biotechnology, Havana, Cuba.
J Biotechnol. 2000 Feb 17;77(2-3):157-67. doi: 10.1016/s0168-1656(99)00201-1.
The ability of the Pichia pastoris-based technology for large-scale production of recombinant hepatitis B virus surface antigen (HBsAg) and both reproducibly purify HBsAg and remove most of the relevant contaminants was ascertained by evaluating ten industrial production batches, five in 1993 and five in 1998. At an early stage, the clarification of mechanically disrupted yeast cells by acid precipitation renders HBsAg with a purity as low as 3.8 +/- 0.6%. However, by adsorption/desorption from diatomaceous earth matrix, the purity of HBsAg rapidly increases to 18.8 +/- 5%, which is suitable for chromatographic processing. This step also eliminates non-particulated forms of HBsAg, significantly lowers the amount of carbohydrates and lipids, and concentrates the HBsAg 4.8-fold. Finally, a sequential purification procedure that includes large-scale immunoaffinity, ion-exchange, and size-exclusion chromatographies further purifies the preparation, resulting in a product (HBsAg at a concentration of 1.3 +/- 0.2 g l-1) with a purity of 95% or more. Furthermore, each of the other contaminants measured reaches the following low levels per 20 micrograms HBsAg: host deoxyribonucleic acid (< 10 pg), carbohydrates (1.2 +/- 0.02 micrograms), lipids (14 +/- 0.28 micrograms), immunopurification-released immunoglobulin G (less than 100 ppm), and endotoxins (106.7 +/- 19.3 pg). These values are below those specified for recombinant DNA hepatitis B vaccines according to World Health Organization (WHO) guidelines.
通过评估1993年的5个批次和1998年的5个批次共10个工业生产批次，确定了基于毕赤酵母的技术大规模生产重组乙型肝炎病毒表面抗原（HBsAg）以及可重复纯化HBsAg并去除大多数相关污染物的能力。在早期阶段，通过酸沉淀澄清机械破碎的酵母细胞得到的HBsAg纯度低至3.8±0.6%。然而，通过从硅藻土基质上吸附/解吸，HBsAg的纯度迅速提高到18.8±5%，这适用于色谱处理。这一步骤还消除了非颗粒形式的HBsAg，显著降低了碳水化合物和脂质的含量，并使HBsAg浓缩了4.8倍。最后，包括大规模免疫亲和、离子交换和尺寸排阻色谱的连续纯化程序进一步纯化制剂，得到纯度为95%或更高的产品（浓度为1.3±0.2 g l-1的HBsAg）。此外，每20微克HBsAg中测得的其他每种污染物均达到以下低水平：宿主脱氧核糖核酸（<10 pg）、碳水化合物（1.2±0.02微克）、脂质（14±0.28微克）、免疫纯化释放的免疫球蛋白G（<100 ppm）和内毒素（106.7±19.3 pg）。这些值低于世界卫生组织（WHO）指南规定的重组DNA乙型肝炎疫苗的标准值。
