Using Metabolic and Genetic Engineering to Develop Novel Cell Platforms for the Production of Recombinant Glycoproteins with Enhanced Pharmacokinetic Properties
Embargo until
2020-05-01
Date
2019-05-02
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Publisher
Johns Hopkins University
Abstract
Biopharmaceuticals are a burgeoning class of therapeutics that are revolutionizing the way disease is treated. Despite their considerable success, the ability to control glycosylation—a key design parameter that ensures safety, optimizes biological response, and influences the pharmacokinetic properties of biopharmaceuticals—has been elusive, which has slowed the development of this class of drugs. One specific glycan, sialic acid, has received considerable attention due to its role in the efficacy, pharmacodynamic, and pharmacokinetic properties of biopharmaceuticals. The first part of this project used a combined genetic and metabolic engineering strategy to gain enhanced exogenous control over the magnitude and type of sialyation produced by Chinese hamster ovary (CHO) cells. In order to significantly reduce endogenous flux of ManNAc into the sialic acid biosynthetic pathway (SABP) two key enzymes in this pathway, UDP-GlcNAc 2- epimerase/ManNAc kinase (Gne) and renin binding protein (Renbp), were knocked out. This was followed by exogenous supplementation with high flux tri-butyrated mannosamine analogs (1,3,4-O-Bu3ManNAc and 1,3,4-O-Bu3ManNAz) in order to modulate CHO sialylation and install non-natural chemical moieties. Elimination of Gne and Renbp resulted in lower sialylation relative to wild type cells, however analog supplementation could still improve sialylation in a concentration dependent manner. Despite lower basal levels of sialic acid the Gne and Renbp knockout CHO cells showed a higher percent incorporation of the azide modified analog. Overall, these results provide evidence that this strategy could be used to develop biopharmaceuticals with a more homogenous glycoprofile and with increased incorporation of functional groups for bioconjugation. The second portion of this project aimed to develop a CHO cell line producing the coagulation factor IX (FIX) with “humanized” sialylation and an augmented serum half-life. A CHO cell line expressing ST6GAL1, a sialyltransferase found in humans but naturally silent in CHO cells, and producing FIX was successfully created. FIX will provide an excellent test protein to demonstrate that the Yarema Lab’s metabolic analogs can be used to increase sialylation to ultimately improve FIX serum half-life.
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Keywords
metabolic glycoengineering, CRISPR, factor IX, hemophilia, sialic acid, sialylation, ST6GAL1, Chinese hamster ovary, UDP-GlcNAc 2-epimerase/ManNAc kinase, renin binding protein