03/28/2022
By Saani Yakubu

You are invited to attend a Ph.D. Dissertation Defense by Saani Yakubu. Topic: "N-Linked Glycan Modulation of Recombinant Glycoproteins in Mammalian and Human Cell Lines using Molecular Engineering and Media Enrichment Strategies."

Date: Tuesday, April 12
Time: 3 to 5 p.m. EST
Location: Virtual

Dissertation Chair: Peter Gaines
Dissertation Committee:

  • Sanjeev Manohar
  • Jin Xu
  • Paul McLean

Abstract:
The goal of this thesis was to determine approaches which would lead to higher levels of sialylation in non-monoclonal antibody recombinant therapeutics in which this attribute was essential for full function and half-life. Glycans are the sugar moieties covalently linked to specific amino acid residues in glycoproteins. Glycans are categorized into N-linked and O-linked. The N-liked glycans (covered in this study), are initiated co-translationally in the endoplasmic reticulum (ER) as a bi-antennary14-sugar residue anchored to dolichol pyrophosphate which is transferred ‘en bloc’ to an asparagine residue in the polypeptide chain of newly translated proteins during post-translational modifications (PTMs). The Asparagine linked 14-sugar residue is processed to primary mannose core structure in the ER, followed by further modifications in the Golgi apparatus to form the full N-glycan.

he N-linked glycan plays a crucial role in the therapeutic functions of recombinant proteins including receptor binding capacities (efficacy) as well as half-life extension. Incomplete N-linked glycan development could lead to glycoproteins with poor therapeutic efficacy, and this has been attributed to several factors including the host cell line the protein is expressed, cell culture media composition, culture conditions, and other factors. This study was intended to evaluate the impact of cell genetic engineering and media enrichment of two commonly used cell lines (CHO and HEK-293) on the glycan modulation of two glycoproteins (X1 and X2). The results showed a significant increase in sialylation of N-glycan of X2 expressed in the engineered CHO cells by 36% compared to the native cell line, and a significant shift in the iso-electric point (pI) of X1 (indicative of increased terminal sialylation) expressed in the engineered HEK293 cells compared to the native cell line. Overall, the study found that engineering of commonly used producer cells either via genetic insertions of glyco-genes and or enriched media, could significantly improve the N-glycan formation and thereby enhance both the efficacy (i.e., via improved receptor binding) and half-life of critical bioengineered proteins important to biomedical applications.