The Effect of Sample Glucose Content on PNGase F-Mediated N-Glycan Release Analyzed by Capillary Electrophoresis

• Published in: Molecules (Basel, Switzerland) Vol. 27; no. 23; p. 8192
• Main Authors: Torok, Rebeka, Auer, Felicia, Farsang, Robert, Jona, Eszter, Jarvas, Gabor, Guttman, Andras
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 24.11.2022; MDPI
• Subjects: Antibodies; Asparagine; Biological products; Capillary electrophoresis; capillary gel electrophoresis; Carbohydrates; Deglycosylation; Dextrose; Digestion; Efficiency; Electrophoresis; Electrophoresis, Capillary - methods; Enzymatic activity; Enzyme activity; Enzyme kinetics; Enzymes; Gel electrophoresis; Glucose; Glycan; Glycoproteins; Glycoproteins - metabolism; Glycoside Hydrolases; Health aspects; Heterogeneity; Immunogenicity; immunoglobulin G; Isoelectric focusing; Kinetics; Michaelis-Menten kinetics; Mode of action; N-glycans; N-glycome; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Pharmacokinetics; PNGase F; Polysaccharides; Polysaccharides - metabolism; Proteins; Quality management; Sodium dodecyl sulfate; Structure-function relationships; Hungary; PNGase F; Michaelis-Menten kinetics; immunoglobulin G; N-glycome; enzyme activity; capillary gel electrophoresis
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules27238192

Protein therapeutics have recently gained high importance in general health care along with applied clinical research. Therefore, it is important to understand the structure-function relationship of these new generation drugs. Asparagine-bound carbohydrates represent an important critical quality attribute of therapeutic glycoproteins, reportedly impacting the efficacy, immunogenicity, clearance rate, stability, solubility, pharmacokinetics and mode of action of the product. In most instances, these linked N-glycans are analyzed in their unconjugated form after endoglycosidase-mediated release, e.g., PNGase F-mediated liberation. In this paper, first, N-glycan release kinetics were evaluated using our previously reported in-house produced 6His-PNGase F enzyme. The resulting deglycosylation products were quantified by sodium dodecyl sulfate capillary gel electrophoresis to determine the optimal digestion time. Next, the effect of sample glucose content was investigated as a potential endoglycosidase activity modifier. A comparative Michaelis-Menten kinetics study was performed between the 6His-PNGase F and a frequently employed commercial PNGase F product with and without the presence of glucose in the digestion reaction mixture. It was found that 1 mg/mL glucose in the sample activated the 6His-PNGase F enzyme, while did not affect the release efficiency of the commercial PNGase F. Capillary isoelectric focusing revealed subtle charge heterogeneity differences between the two endoglycosidases, manifested by the lack of extra acidic charge variants in the cIEF trace of the 6His-PNGase F enzyme, which might have possibly influenced the glucose-mediated enzyme activity differences.