The difficulties in purification of VLP-based recombinant hepatitis B surface antigen (rHBsAg) are mainly emerged from inefficient semi-purification step plus proteins physicochemical properties and these issues make the downstream processing (DSP) very lengthy and expensive. In this study, optimization of rHBsAg (recombinantly-expressed in Pichia pastoris) DSP was performed using selection of buffering conditions in the semi-purification step. In the semi-purification optimization step, up to 73% of the protein impurities were eliminated and the utmost increase in rHBsAg purity (ca. 3.6-fold) was achieved using 20 mM sodium acetate, pH 4.5. By using rHBsAg binding and nonbinding situations obtained from the response surface plot in design of experiments (DOE), additional bind-elute and flow-through purification mode experiments were conducted and rHBsAg with high purity (near 100%) and recovery (> 83%) was achieved. Following assessment of critical quality attributes (i.e., purity, particle size distribution, host cell DNA, host cell protein, secondary structures, specific activity and relative potency), it was indicated that the characteristics of rHBsAg purified by the new DSP were similar or superior to the ones obtained from conventional DSP. The purification performance of the resin was constantly retained (97–100%) and no significant resin damage took place after 10 adsorption–elution–cleaning cycles. The new DSP developed for production of rHBsAg in this study can substitute the conventional one with granting satisfactory target protein quality, long-lasting resin efficacy, shorter and less expensive process. This process may be also employable for purification of both non-VLP- and VLP- based target proteins expressed in the yeast.

基于VLP的重组乙型肝炎表面抗原（rHBsAg）的纯化困难主要源于低效的半纯化步骤以及蛋白质的理化特性，这些问题使得下游处理（DSP）非常漫长且昂贵。在本研究中，rHBsAg（在毕赤酵母中重组表达）的优化) 在半纯化步骤中选择缓冲条件来进行 DSP。在半纯化优化步骤中，使用 20 mM 乙酸钠（pH 4.5）可消除高达 73% 的蛋白质杂质，并最大限度地提高 rHBsAg 纯度（约 3.6 倍）。利用实验设计（DOE）中响应面图获得的rHBsAg结合和非结合情况，进行额外的结合-洗脱和流通纯化模式实验，获得高纯度（接近100%）和回收率（> 83%）的rHBsAg ） 已实现。在评估关键质量属性（即纯度、粒度分布、宿主细胞 DNA、宿主细胞蛋白质、二级结构、比活性和相对效力）后，表明新型DSP纯化的rHBsAg的特性与传统DSP纯化的rHBsAg相似或优于传统DSP。经过10次吸附-洗脱-清洗循环后，树脂的纯化性能始终保持（97%–100%），并且没有发生明显的树脂损坏。本研究中开发的用于生产 rHBsAg 的新型 DSP 可以替代传统的 DSP，具有令人满意的目标蛋白质量、持久的树脂功效、更短且更便宜的工艺。该方法还可用于纯化酵母中表达的基于非VLP和基于VLP的靶蛋白。经过10次吸附-洗脱-清洗循环后，树脂的纯化性能始终保持（97%–100%），并且没有发生明显的树脂损坏。本研究中开发的用于生产 rHBsAg 的新型 DSP 可以替代传统的 DSP，具有令人满意的目标蛋白质量、持久的树脂功效、更短且更便宜的工艺。该方法还可用于纯化酵母中表达的基于非VLP和基于VLP的靶蛋白。经过10次吸附-洗脱-清洗循环后，树脂的纯化性能始终保持（97%–100%），并且没有发生明显的树脂损坏。本研究中开发的用于生产 rHBsAg 的新型 DSP 可以替代传统的 DSP，具有令人满意的目标蛋白质量、持久的树脂功效、更短且更便宜的工艺。该方法还可用于纯化酵母中表达的基于非VLP和基于VLP的靶蛋白。