08/23/2021
By Susan Pryputniewicz

The Biomedical Engineering and Biotechnology program invites you to attend a doctoral proposal defense by Saani Yakubu on “Modulation of the N-glycan of Recombinant Glycoproteins in Human and Mammalian Cell Lines using Molecular and Media Engineering Strategies.”

Name: Saani Yakubu
Date: Tuesday, Aug. 31, 2021
Time: 2 to 4 p.m.
Location: This will be a virtual defense via Zoom. Those interested in attending should contact the student (saani_yakubu@student.uml.edu) and committee advisor (Peter_Gaines@uml.edu) at least 24 hours prior to the defense to request access to the meeting.

Committee Chair (Advisor): Peter Gaines, Ph.D., Professor, Department of Biological Science, University of Massachusetts Lowell

Committee Members:

  • Jin Xu, Ph.D., Professor, Department of Chemistry, University of Massachusetts Lowell
  • Sanjeev Manohar, Ph.D., Professor/Chair, Department of Chemical Engineering, University of Massachusetts Lowell
  • Paul Mclean, Ph.D., Principal Scientist, Analytical Development, Takeda Pharmaceuticals

Abstract:

Glycans are sugar moieties that are covalently linked to specific amino acid residues in critical functional domains of glycoproteins. Glycan formation is initiated in the endoplasmic reticulum (ER) as mannose core structures (14- sugar residues anchored to dolichol pyrophosphate), which are transferred ‘en bloc’ to an asparagine residue of newly translated proteins undergoing post-translational modifications (PTMs). The primary mannose core structure is further modified in the Golgi apparatus with the addition of sugar residues, such as mannose-6-phosphate, galactose, and sialic acid, which provide for complex glycan forms. The fully formed glycans attached to the protein domain confers functional attributes to the glycoprotein, including receptor binding capacities and half-life extension in blood serum. The asparagine-linked glycan (N-linked glycan) is an aggregated monosaccharide composed of fucose (Fuc), N-Acetylglucosamine (GlcNAc), mannose (Man), galactose (gal), and terminal N-acetylneuraminic acid (Ac), with each sugar moiety formed through complex enzymatic reactions involving specific substrates and enzymes.

The N-linked glycans play a crucial role in defining the efficacy of recombinant protein therapeutics by mediating their pharmacokinetics (half-life extension and biodistribution) through terminal sialylation with N-acetyl neuraminic acid. The N-glycans also mediate cellular internalization of glycoproteins through IGF-II/cation-dependent mannose-6-phosphate receptor interactions. A glycoprotein with incomplete N-glycans produces less therapeutic efficacy due to reduced receptor binding and lower half-life.

Due to intrinsic complexity of human and mammalian type glycoproteins, the post-translational modifications (glycosylation) of recombinant glycoproteins could result in incomplete glycoforms with lesser complex residues of mannose-6-phosphate, galactose, and terminal sialic acid. The incomplete glycoforms produced have lesser efficacy, and hence introduce variability in therapeutic efficacy from batch to batch which impacts accurate dosing of therapeutic protein to patients. The batch variability is also problematic for drug approval by the Food and Drug Administration (FDA) which requires the Pharma companies to define the range of glycan variability for new drugs filed.

In previous studies, several factors have been identified to potentially impact the glycan map formation during recombinant proteins expression in a cell line. These factors include cell line the protein is being expressed, the cell culture medium, condition of the cell environment (such as metabolites, temperature, pH, CO2, O2 etc.), availability and spatial distribution of substrates and enzymes in the ER and Golgi, and residence or transit time of glycoproteins in the ER and Golgi.

In this study, we have hypothesized that the terminal sialic acid along with other residues of N-glycans can be enriched through:

  • genetic engineering of the cell line the recombinant protein is expressed
  • enrichment of cell culture media with specific substrates (nucleotide-sugars)

The sialic acid enrichment may include enrichment of other sugar residues preceding the sialic acid residue in the N-glycan structure. Using molecular techniques, we engineered model CHO and HEK293 cell lines with vector constructs containing target glycan modifying genes and then stably expressed proteins in the engineered cell lines cultured in a substrate-enriched medium. The produced proteins were then purified and then subjected to glycan analyses.

Our results indicate the glycan maps of the proteins expressed in the engineered cell lines, which showed significant modifications consistent with the glycan gene mRNA profiles. Also, the terminal sialic acid content, which is indicative of enrichment of other N-glycan sugar residues, showed increased complexity versus the native protein’s N-glycan. The four sialic acid peak groups increased as follows: di-sialylated species (di-SA) (3-4% increase), tri-sialylated species (tri-SA) (2-3% increase), and tetra-sialylated species (tetra-SA) (1-2% increase). A proportional decrease in mono-sialylated species (mono-SA) (4-5%) was also observed and attributed to formation of the complex glycan species. Together these data indicate that specific modifications of the most commonly used producer cells for protein engineering, CHO and HEK293, either via genetic insertions of glycan genes or enriched media, can significantly improve 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.

All interested students and faculty members are invited to attend the online defense via remote access.