Kastilan, R.R.KastilanBoes, A.A.BoesSpiegel, H.H.SpiegelVoepel, N.N.VoepelChudobova, I.I.ChudobovaHellwig, S.S.HellwigBuyel, J.F.J.F.BuyelReimann, A.A.ReimannFischer, R.R.Fischer2022-03-052022-03-052017https://publica.fraunhofer.de/handle/publica/25066010.1038/s41598-017-11819-4Pichia 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.enjournal article