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

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.

Comparison of microbial and transient expression (tobacco plants and plant‐cell packs) for the production and purification of the anticancer mistletoe lectin viscumin

“…The complexity also increases the documentation requirements and the likelihood of errors compared with simpler processes. Other plant lectins have been expressed in the yeast Pichia pastoris at levels of 6–20 mg/L (Lannoo, Vervecken, Proost, Rouge, & Van Damme, ; Oliveira, Felix, Moreira, Teixeira, & Domingues, ), which is similar to the ~7 mg/kg that we report here, assuming that 1 kg of plant biomass is approximately equivalent to 1 L of fermentation broth because both contain about 6% (~60 g/kg) dry cell mass (Buyel, Gruchow, Tödter, & Wehner, ; Kastilan et al, ). However, glycoproteins produced in yeasts often carry predominantly high‐mannose rather than complex‐type glycans (Strasser, ), which increase the risk of an immune response.…”

Development of a New High-Cell Density Fermentation Strategy for Enhanced Production of a Fungus β-Glucosidase in Pichia pastoris

“…Several induction strategies have been established, such as DO-stat, μ-stat, m-stat, etc. ( Trinh et al, 2003 ; Fang et al, 2014 ; Kastilan et al, 2017 ). The DO-stat strategy is an indirect feedback mode which automatically controls the substrate feeding rate to efficiently maintain the on-line DO at a near-constant level.…”

Evaluation of two Plasmodium vivax sexual-stage antigens as transmission-blocking vaccine candidates

“…For the development of recombinant protein-based TBVs, expression of the recombinant proteins with properly-folded conformational epitopes can be critical for inducing antibodies with TR activities [22,25]. The yeast protein expression system has been widely used to produce many human vaccines [26,27]. Compared to the prokaryotic expression system, the yeast system has the advantage of higher biomass expression and secretion yields.…”