P15: Upstream optimization of glycoengineered Pichia pastoris expressing a highly sialylated therapeutic glycoprotein

Monday, November 7, 2011
Capri Ballroom (Marriott Marco Island)
Muralidhar Reddy Mallem, Adam C Nylen, Marc d'Anjou, Sujatha Gomathinayagam, Eric Brown, Stephen Hamilton, John Bukowski, Daniel Hopkins, Irina Burnina, Andy Stadheim, Ishaan Shandil and Sehoon Kim, Fermentation, GlycoFi/ Merck, Lebanon, NH
Most therapeutic biologics are glycoproteins in which the carbohydrate moiety plays an important role in its drug properties such as circulatory half-life and efficacy.  Currently, mammalian cell culture is the most clinically validated option for recombinant expression despite its drawbacks such as the presence of non-human glycans and a lack of process robustness.  Highly engineered alternative expression systems, such as GlycoFi’s humanized yeast technology, offer the promise of more uniform and human-like glycosylation.  This work describes the expression of a marketed therapeutic glycoprotein whose drug properties are known to be significantly impacted by the total sialic acid content contributed by its glycan moiety.  Glycofi’s glycoengineering technology platform was optimized to produce a yeast-derived molecule that demonstrated similar efficacy in preclinical models of disease as the marketed version.

 The current study describes the effect of various fermentation process conditions such as pH, temperature, methanol feed rate, oxygen transfer rate, and  several media supplementation strategies (basal salts media, amino acids, vitamins, complex media) on N- and O-linked glycosylation, total sialic acid (TSA) content, cell fitness and productivity It was concluded from this study that the TSA content was very sensitive to the change in process conditions especially methanol feed rate and specific productivity. Improvements to productivity and a 2-fold increase in TSA content was achieved by optimization of these different fermentation process conditions.

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