Wenger Marc D, Dephillips Peter, Price Colleen E, Bracewell Daniel G
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London, UK.
Biotechnol Appl Biochem. 2007 Jun;47(Pt 2):131-9. doi: 10.1042/BA20060240.
The development of fermentation processes for recombinant vaccines requires optimizing expression while maintaining high product quality. Changes to cell fermentation conditions are typically evaluated following cell disruption, with expression levels quantified by immunoassay, liquid chromatography or enzyme activity. However, assay titres do not always predict the effects that intracellular aggregation, proteolysis, post-translational modifications and differences in relative impurity levels can have on purification yield and product purity. Furthermore, heterogeneity in the size and surface properties inherent in viral particles makes unit operations such as chromatography less predictable. In these cases, the purification procedure (or a mimic thereof) must be carried out to give accurate information on the impact of changes in fermentation conditions on purification process performance. This was demonstrated for the development of a recombinant vaccine against human papillomavirus produced in Saccharomyces cerevisiae, where the most informative feedback on fermentation variables was obtained by completing a multistep chromatographic purification to evaluate process yield and product purity. To increase the purification throughput and reduce labour, the chromatography was miniaturized 1000-fold from the laboratory scale using microlitre volumes of adsorbent in a pipette tip and automated on a robotic workstation. The microscale purification is shown to be predictive of the laboratory-scale purification in terms of yield and purity, while providing over a 10-fold increase in throughput and allowing for increased monitoring of fermentation processes. In addition, by reducing the volume of cells needed for this assessment, the fermentation can be correspondingly reduced in scale and carried out in parallel for additional throughput gains.
重组疫苗发酵工艺的开发需要在保持高产品质量的同时优化表达。细胞发酵条件的改变通常在细胞破碎后进行评估，表达水平通过免疫测定、液相色谱或酶活性进行定量。然而，测定效价并不总能预测细胞内聚集、蛋白水解、翻译后修饰以及相对杂质水平差异对纯化收率和产品纯度的影响。此外，病毒颗粒固有的大小和表面性质的异质性使得诸如色谱等单元操作的可预测性降低。在这些情况下，必须进行纯化程序（或其模拟）以提供关于发酵条件变化对纯化工艺性能影响的准确信息。这在酿酒酵母中生产的抗人乳头瘤病毒重组疫苗的开发中得到了证明，通过完成多步色谱纯化以评估工艺收率和产品纯度，获得了关于发酵变量的最有用反馈。为了提高纯化通量并减少劳动力，使用移液器吸头中的微升体积吸附剂将色谱从实验室规模缩小了1000倍，并在机器人工作站上实现自动化。微尺度纯化在收率和纯度方面被证明可以预测实验室规模的纯化，同时通量提高了10倍以上，并允许增加对发酵过程的监测。此外，通过减少该评估所需的细胞体积，可以相应地缩小发酵规模并并行进行以获得额外的通量提升。
