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.

Figure 1

Time course of the three-phase fermentation strategy for the production of VAMAX1 (straight lines) and VAMAX2 (dotted lines) at the 15-L scale. The strategy included a typical glycerol fed-batch phase to increase the cell density before induction. An oxygen-limited induction mode with a constant methanol concentration of 2.5 mL L⁻¹ was used for product expression. (a) Course of dry cell weight (DCW, •) and dissolved oxygen tension (DOT). (b) Course of added glycerol (Gly) and methanol (Me), methanol concentration (MC), and product concentration (▴). The x-axis is normalized to the time point of induction. Error bars indicate the standard deviation of technical triplicates.

Figure 2

Time course of the two-phase fermentation strategy for the production of VAMAX1 (straight lines) and VAMAX2 (dotted lines) at the 15-L scale. The typical glycerol fed-batch phase was omitted in this strategy, allowing the direct transition to an induction phase after the depletion of the batch glycerol. The induction time was expanded to incorporate the time usually allocated for the glycerol fed-batch phase, resulting in almost the same total process duration as the three-phase strategy. An oxygen-limited induction mode with a constant methanol concentration of 2.5 mL L⁻¹ was used for product expression. (a) Course of dry cell weight (DCW, •) and dissolved oxygen tension (DOT). (b) Course of added methanol (Me), methanol concentration (MC), and product concentration (▴). The x-axis is normalized to the time point of induction. Error bars indicate the standard deviation of technical triplicates.

Figure 3

Analysis of fermentation supernatants during the three-phase fermentation process for the production of VAMAX1. (a) Proteins in 15 µL supernatant were separated by LDS-PAGE and stained with SimplyBlue SafeStain. Numbered arrows indicate two impurities that were more abundant in the supernatant when the three-phase strategy was used instead of the two-phase strategy. (b) The proteins separated above were further analyzed by immunoblotting using the reduction-sensitive mAb 4G2. Times above the lanes indicate the elapsed hours of each process phase. M: protein marker. EoB: end of batch-phase. GlyFeed: glycerol fed-batch phase. Numbered arrows: samples analyzed by mass spectrometry. V1: target protein VAMAX1.

Figure 4

Analysis of fermentation supernatants during the two-phase fermentation process for the production of VAMAX1. (a) Proteins in 15 µL supernatant were separated by LDS-PAGE and stained with SimplyBlue SafeStain. (b) The proteins separated above were further analyzed by immunoblotting using the reduction-sensitive mAb 4G2. Times above the lanes indicate the elapsed hours of each process phase. M: protein marker. EoB: end of batch-phase. V1: target protein VAMAX1.

Figure 5

The purity of VAMAX1 in the fermentation supernatant. (a) Proteins in 15 µL supernatant taken at the end of the three-phase (3 P) and two-phase (2 P) fermentation strategies were separated by LDS-PAGE and visualized with SimplyBlue SafeStain. (b) Densitometric analysis of the separated proteins using AIDA Image Analyzer. Arrows represent the target protein VAMAX1. Numbered bands/peaks represent impurities that were cut from the gel for analysis by mass spectrometry.

Figure 6

Analysis of fermentation supernatants during the three-phase fermentation process for the production of VAMAX2. (a) Proteins in 15 µL supernatant were separated by LDS-PAGE and stained with SimplyBlue SafeStain. Numbered arrows indicate two impurities that were more abundant in the supernatant when the three-phase strategy was used instead of the two-phase strategy. (b) The proteins separated above were further analyzed by immunoblotting using the reduction-sensitive mAb 4G2. Times above the lanes indicate the elapsed hours of each process phase. M: protein marker. EoB: end of batch-phase. GlyFeed: glycerol fed-batch phase. Numbered arrows: samples analyzed by mass spectrometry. V2: target protein VAMAX2.

Figure 7

Analysis of fermentation supernatants during the two-phase fermentation process for the production of VAMAX2. (a) Proteins in 15 µL supernatant were separated by LDS-PAGE and stained with SimplyBlue SafeStain. (b) The proteins separated above were further analyzed by immunoblotting using the reduction-sensitive mAb 4G2. Times above the lanes indicate the elapsed hours of each process phase. M: protein marker. EoB: end of batch-phase. V2: target protein VAMAX2.

Figure 8

The purity of VAMAX2 in the fermentation supernatant. (a) Proteins in 15 µL supernatant taken at the end of the three-phase (3 P) and two-phase (2 P) fermentation strategies were separated by LDS-PAGE and visualized with SimplyBlue SafeStain. (b) Densitometric analysis of the separated proteins using AIDA Image Analyzer. Arrows represent the target protein VAMAX2. Numbered bands/peaks represent impurities that were cut from the gel for analysis by mass spectrometry.