Pichia pastoris is an alternative yeast system that has gained rapid acceptance for recombinant protein production. This is driven largely by high product yields when using a promoter derived from the alcohol oxidase I (AOX1) gene, the similarity of molecular genetic manipulation techniques to those of Saccharomyces cerevisiae, and the strong preference for respiratory growth leading to high cell densities. However, relatively few products from Pichia pastoris have gained FDA approval so far, compared to baker’s yeast, despite the decision in 1993 by Phillips Petroleum Company in Bartlesville (continued by Research Corporation Technologies) to release the P. pastoris expression system to academic research laboratories. The high cell densities obtainable using Pichia pastoris provide high product concentrations in the culture supernatant.
High titre headlines do not equal net yield realities
However, high cell densities mean there is less supernatant per litre of whole culture, so g/L supernatant values are not representative of g/L fermentation capacity. Additionally, the benefits of Pichia systems using the AOX1 promoter require the use of both oxygen and methanol, which present significant safety hazards during large-scale manufacture. While methanol-free promoters are becoming more widely available, yields may be lower, lacking a competitive advantage over other systems, such as baker’s yeast, with a more substantial track record for regulatory approvals. Importantly, undesirable post-translational modifications, including protein folding issues, have been documented for some Pichia-derived products, which will likely complicate downstream processing Frahm et al. 2014. In 2009, it was discovered that Mitsubishi Tanabe Pharma Corporation (MTPC) had violated Good Manufacturing Practice (GMP) guidelines by falsifying data for Medway Injection 5%, containing rHSA derived from Pichia pastoris. As a result of this discovery, Mitsubishi Tanabe Pharma withdrew its marketing approval for the product.
Suitability for non-therapeutic proteins
Nevertheless, Pichia pastoris appears highly attractive for producing non-therapeutic products with lower regulatory barriers, such as selected industrial proteins and/or cell culture components. While technical solutions in downstream processing may be possible for removing undesirable product-related impurities, this may substantially reduce total plant productivity, increasing the CoGs and negating perceived advantages from high g/L supernatant yields.
Requirement to integrate protein sequence hampers optimisation
Furthermore, the genetic sequence for the protein to be manufactured using Pichia has to be directly integrated into the genome. Because it is difficult to subsequently remove the expression construct from the genome, a strain optimised by step-by-step engineering for one biologic cannot easily be repurposed for another. In contrast, baker’s yeast uses episomal plasmids, which can easily be removed in laboratory conditions to allow repurposing of the strain while being stable for many generations during industrial fermentation campaigns. The integrated plasmids Pichia systems make rapid strain optimisation by breeding impractical compared to baker’s yeast.
Toxic markets and residual antibiotic risks
Additionally, the selectable markers commonly used for Pichia pastoris tend to be highly toxic and expensive for large-scale manufacture, e.g. zeocin, hygromycin B and geneticin (G418), compared to the safe auxotrophic markers used with baker’s yeast. Therefore, the regulatory authorities require evidence for removal of residual antibiotics from the final product. The disposal of biomass from Pichia fermentation also presents environmental risks due to the antibiotic-resistant genes inserted into the genome. In contrast, baker’s yeast strains for biologics manufacture typically comprise only yeast DNA plus the synthetic gene of interest, presenting no theoretical antimicrobial resistance risks during disposal.
Patent Restrictions
Importantly, for commercial manufacture of biologics, the more widespread use of Pichia pastoris for recombinant protein production in recent years has led to a larger number of patent filings for this expression system compared to other yeasts. This significantly complicates the IP landscape for biologics manufacture, e.g. compared to baker’s yeast, where the essential patents protecting first-generation expression systems have already lapsed.In summary, while Pichia pastoris is generally the most widely adopted yeast system for recombinant protein production, it is not as well established for FDA-approved biologics as baker’s yeast and has significant disadvantages for large-scale manufacturing compared to second-generation baker’s yeast systems.
