Effect of N-Glycan Profiles on Binding Affinity of Diagnostic Antibody Produced by Hybridomas in Serum-Free Suspension

Abstract

Serum-free suspension culture for hybridomas is one of the important key steps for efficient diagnostic antibody production while maintaining protein quality and function. Based on the importance of N-glycan profiles in therapeutic antibody production in mammalian cells, the effect of changes in the N-glycan profiles on the function of diagnostic antibody must also be validated. To investigate the influence of diagnostic antibodies with different N-glycan profiles on the binding affinity with target antigens, four glycosylation regulators, tunicamycin, Bis-Tris, galactose, and N-acetylmannosamine, were administered separately to diagnostic antibody-producing hybridomas cultures. Supplementation with these four glycosylation modulators inhibited glycosylation and increased mannosylation, galactosylation, and sialylation in serum-free suspended hybridomas. In particular, the diagnostic antibody produced from a culture with tunicamycin exhibited a significant increase in the aglycosylated form compared with those without tunicamycin or with other glycosylation modulators. Surprisingly, diagnostic antibody with different N-glycan compositions did not significantly affect binding affinity with the target antigen and even aglycosylated antibodies did not affect binding affinity. Taken together, the results indicate that the change in the N-glycan profile of the diagnostic antibody produced in serum-free suspension hybridomas in an altered culture environment did not significantly affect their biological function, which provides valuable insight for the production and quality control of diagnostic antibody.

Keywords: Hybridomas; binding affinity; diagnostic antibody; glycosylation; suspension culture.

Fig. 1. Profiles of cell growth, viability, and diagnostic antibody production from hybridomas (A) in serumcontaining adherent and serum-free suspended culture mode (B) with and without four glycosylation modulators.

The arrow indicates the time of glycosylation modulator addition to the culture media. Error bars represent the standard deviations calculated from technical duplicate experiments.

Fig. 2. Specific consumption rates of amino acids in serum-free suspended hybridomas supplemented with and without four glycosylation modulators.

The specific consumption rates of the amino acids were calculated from 2– 3-day cultures, which were expected before and after the addition of the glycosylation modulators, respectively. Error bars represent the standard deviations calculated from technical triplicate analyses.

Fig. 3. (A) SDS-PAGE analysis of the purified diagnostic antibody produced from hybridomas supplemented with and without four glycosylation modulators, followed by treatment with (+) and without (‒) PNGase F.

The diagnostic antibody was purified by protein G affinity chromatography from the culture supernatants harvested on day 3 with cell viability greater than 80% and treated with PNGase F for the release of N-glycans. Equal amounts of proteins were loaded under reducing conditions and the gel was stained with Coomassie brilliant blue. (B) Hydrophilic interaction HPLC analysis of the major N-glycans obtained from the purified diagnostic antibody from hybridomas on day 3. The relative abundance of six major N-glycans (G0, G0F, Man, G1F, G2F, G2S1F, and G2S2F) was obtained from cultures with and without four glycosylation modulators. Error bars represent standard deviations calculated from triplicate experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Fig. 4. Fold change in intracellular nucleotide sugars in serum-free suspended hybridomas supplemented with and without four glycosylation modulators.

Nucleotide sugars were extracted and quantified from cells on day 3 using ion-pair reversed-phase HPLC. The fold change for each nucleotide sugar was calculated based on each nucleotide sugar in the culture without glycosylation modulators. Error bars represent the standard deviations calculated from technical triplicate experiments. *p ≤ 0.05, **p ≤ 0.01

Fig. 5. The binding affinity assay for the purified diagnostic antibody with a specific target antigen.

The absorbance (OD_450nm) was measured using a conventional ELISA method with a diagnostic antibody produced from hybridomas in the absence and presence of four glycosylation modulators. Error bars represent standard deviations calculated from data obtained in technical triplicate experiments.