(630d) A Deep Examination of Carbon Feeding during the Production of Various Sized Vaccine and Therapeutic Candidates in Komagataella Phaffi

Komagataella phaffi (Pichia pastoris) is a widely used host for heterologous protein expression, owing to its fast growth to high cell density and strong methanol-inducible promoters, such as pAOX1. Strong transgene expression and tight inducibility enabled by pAOX1 have led to its widespread use. However, further optimization of other key process parameters, such as central carbon metabolism, is often tricky. Such factors are highly coupled by a mutual dependence on methanol concentration in P. pastoris cultivations. Furthermore, previous studies indicate large protein production requires additional media additives to facilitate proper protein folding, glycosylation, and secretion. In this study, we introduce the use of carbon source metabolism as a pivotal perturbation to investigate such effects P. pastoris cultivations have on the production of various sized proteins ranging from low molecular weight nanobodies and insulin glargine to high molecular weight monoclonal antibodies and endogenous human proteins. In particular, these perturbations’ effects on protein folding, cellular stress, and post-translational modifications will be addressed. We first explored strategies to tune transgene expression levels and found that specific carbon sources increase transgene expression beyond typical induced methanol expression and increase cellular growth. Next, we identified specific metabolic pathways responsible for cellular development, stress, translation, protein folding, and secretion using high-throughput bulk mRNA and ribosomal sequencing. We used these pathways to determine the optimal combination of carbon sources at the cellular level that reduces cellular stress while increasing protein translation and secretion. Finally, secreted product titers were determined, sequenced using mass spectrometry, and post-translational modifications analyzed using high-performance liquid chromatography. The results from this study indicate the need for optimized carbon source feeding strategies centered around the complexity and the size of the desired protein. This strategy may help accelerate advancements of candidate vaccines and therapeutics from alternative hosts, such as P. pastoris, to the clinic.