Abstrato

Development of liquid and solid-phase extraction sample treatment methods for quantitation and purity assessment of recombinant Kunitz domain proteins in Pichia fermentation culture by RP-HPLC

Jie Chen, Mike DiLeo, Yanyu Zhang, Jill Settles, Mark Stochl, Craig Bodycombe, Andrew E Nixon and Arthur Ley

Background: Pichia pastoris is a well-established cell system for the expression of recombinant proteins. However, the complex pigments and native proteins concurrently produced by this organism have presented significant challenges when attempting to determine protein product titer and purity in fermentation samples. Results: A methodology of treating P. pastoris fermentation samples using either liquid extraction or solid-phase extraction is presented, which consistently yields over 90% recovery for recombinant Kunitz domain proteins produced via high cell density P. pastoris fermentation. The developed sample treatment method allows subsequent HPLC assay to not only provide similar specificity and accuracy to that offered by a validated ELISA assay for titer analysis, but also to deliver product purity information and the benefit of lower assay variability. Conclusion: The use of the novel sample pretreatment methods described in this work can significantly improve the viability and outcome of subsequent analyses. This includes enabling the use of reliable and easily automated HPLC assays to perform quantitative and qualitative analysis of recombinant protein products produced by P. pastoris cultures during fermentation process development and manufacturing control.