Improvement of a fermentation process for the production of two PfAMA1-DiCo-based malaria vaccine candidates in Pichia pastoris

Robin Kastilan,Andreas Reimann,Johannes Felix Buyel,Alexander Boes,Holger Spiegel,Ivana Chudobová,Stephan Hellwig,Nadja Voepel,Rainer Fischer

doi:10.1038/s41598-017-11819-4

Abstract

Pichia pastoris is a simple and powerful expression platform that has the ability to produce a wide variety of recombinant proteins, ranging from simple peptides to complex membrane proteins. A well-established fermentation strategy is available comprising three main phases: a batch phase, followed by a glycerol fed-batch phase that increases cell density, and finally an induction phase for product expression using methanol as the inducer. We previously used this three-phase strategy at the 15-L scale to express three different AMA1-DiCo-based malaria vaccine candidates to develop a vaccine cocktail. For two candidates, we switched to a two-phase strategy lacking the intermediate glycerol fed-batch phase. The new strategy not only provided a more convenient process flow but also achieved 1.5-fold and 2.5-fold higher space-time yields for the two candidates, respectively, and simultaneously reduced the final cell mass by a factor of 1.3, thus simplifying solid–liquid separation. This strategy also reduced the quantity of host cell proteins that remained to be separated from the two vaccine candidates (by 34% and 13%, respectively), thus reducing the effort required in the subsequent purification steps. Taken together, our new fermentation strategy increased the overall fermentation performance for the production of two different AMA1-DiCo-based vaccine candidates.

Highlights

The yeast Pichia pastoris was initially used for the production of single-cell protein, with the C1 compound methanol as a carbon source[1], but is widely exploited as an expression host for a broad range of recombinant proteins[2,3]
Our previously reported expression of the three malaria vaccine candidates VAMAX1, VAMAX2 and VAMAX420 was based on the fermentation strategy in the “Pichia fermentation process guidelines”[13] modified by us as described above
We decided to omit the glycerol fed-batch phase in favor of a two-phase fermentation strategy comprising a batch phase directly followed by an induction phase

Summary

Introduction

The yeast Pichia pastoris was initially used for the production of single-cell protein, with the C1 compound methanol as a carbon source[1], but is widely exploited as an expression host for a broad range of recombinant proteins[2,3]. Unlike E. coli, it provides the eukaryotic machinery for posttranslational modification, and allows the efficient secretion of correctly folded proteins[5] This is a key advantage of highly purified proteins because P. pastoris secretes only a few host cell proteins into the fermentation broth. The recommended fermentation process uses the strong inducible AOX1 promoter to control recombinant protein expression[14,15]. The process-scale addition of pure oxygen is expensive and special precautions are required because the use of compressed oxygen is accompanied by safety issues

Methods

Results

Conclusion