Physiological state as transferable operating criterion to improve recombinant protein production in Pichia pastoris through oxygen limitation

BACKGROUND: The yeast Pichia pastoris is widely used as a production platform for secreted recombinant protein. The application of oxygen-limiting conditions leads to an important increase in protein specific productivity driven by the GAP promoter. RESULTS: The physiological and metabolic adaptation of the host to a wide range of oxygen availability has been systematically studied in glucose-limited chemostat cultivations producing an antibody fragment (Fab). A weighty increase of up to 3-fold of the specific Fab production rate (qFab) and Fab yield (YPX) has been achieved for the optimal conditions. Besides the remarkable increase on both Fab yield and productivity, as a consequence of the metabolic shift from respiratory to respiro-fermentative pathways, a decrease on biomass yield and generation of several secreted by-products have been observed. CONCLUSION: The accurate system characterization achieved throughout the bioprocess specific rates and the monitoring of cell physiology allowed the determination of the optimal conditions to enhance bioprocess efficiency. This work also presents a versatile approach based on the physiological state of the yeast that can be used to implement the desired oxygen-limiting conditions to fermentations set-ups with different oxygen transfer capacities, alternative operating modes, and even for the production of other proteins of interest

The trispecific DARPin ensovibep inhibits diverse SARS-CoV-2 variants

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19)