Combining metabolic and process engineering strategies to improve recombinant glycoprotein production and quality

From National Research Council Canada

DOIResolve DOI: https://doi.org/10.1007/s00253-017-8513-0
AuthorSearch for: ; Search for: 1; Search for: ; Search for:
Name affiliation
  1. National Research Council Canada. Human Health Therapeutics
FormatText
TypeArticle
Journal titleApplied Microbiology and Biotechnology
ISSN0175-7598
1432-0614
Volume101
Issue21
Pages7837–7851
SubjectHEK293 cells; PYC2-overexpression; protein sialylation; nutrient substitution
Abstract
Increasing recombinant protein production while ensuring a high and consistent protein quality remains a challenge in mammalian cell culture process development. In this work, we combined a nutrient substitution approach with a metabolic engineering strategy that improves glucose utilization efficiency. This combination allowed us to tackle both lactate and ammonia accumulation and investigate on potential synergistic effects on protein production and quality. To this end, HEK293 cells overexpressing the pyruvate yeast carboxylase (PYC2) and their parental cells, both stably producing the therapeutic glycoprotein interferon α2b (IFNα2b), were cultured in media deprived of glutamine but containing chosen substitutes. Among the tested substitutes, pyruvate led to the best improvement in growth (integral of viable cell density) for both cell lines in batch cultures, whereas the culture of PYC2 cells without neither glutamine nor any substitute displayed surprisingly enhanced IFNα2b production. The drastic reduction in both lactate and ammonia in the cultures translated into extended high viability conditions and an increase in recombinant protein titer by up to 47% for the parental cells and the PYC2 cells. Product characterization performed by surface plasmon resonance biosensing using Sambucus nigra (SNA) lectin revealed that the increase in yield was however accompanied by a reduction in the degree of sialylation of the product. Supplementing cultures with glycosylation precursors and a cofactor were effective at counterbalancing the lack of glutamine and allowed improvement in IFNα2b quality as evaluated by lectin affinity. Our study provides a strategy to reconcile protein productivity and quality and highlights the advantages of PYC2-overexpressing cells in glutamine-free conditions.
Publication date
PublisherSpringer
LanguageEnglish
Peer reviewedYes
NPARC number23002662
Export citationExport as RIS
Report a correctionReport a correction
Record identifierc5d18592-a153-46e8-82d8-38caaab27a6d
Record created2017-12-15
Record modified2020-03-16
Report a problem on this page
Date modified: