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  • Influence of media composition on recombinant monoclonal IgA1 glycosylation analysed by lectin-based protein microarray and MALDI-MS.

Influence of media composition on recombinant monoclonal IgA1 glycosylation analysed by lectin-based protein microarray and MALDI-MS.

Journal of biotechnology (2020-04-17)
Lucia Pažitná, Marek Nemčovič, Zuzana Pakanová, Peter Baráth, Teimur Aliev, Dmitry Dolgikh, Victoria Argentova, Jaroslav Katrlík
ABSTRACT

Glycosylation of therapeutic glycoproteins significantly affects their physico-chemical properties, bioactivity and immunogenicity. The determination of glycan composition is highly important regarding their development and production. Therefore, there is a demand for analytical techniques enabling rapid and reliable glycoprofiling of therapeutic proteins. For the investigation of changes in glycan structures, we have employed two platforms: lectin-based protein microarray, and MALDI-MS. In lectin-based microarray analysis, the samples of IgA were printed on the microarray slide, incubated with the set of lectins with various specificity and evaluation of changes in glycosylation was based on differences in reactivity of samples with lectins. MALDI-MS was used for N-glycan analysis of IgA1 samples. IgAs are effective as therapeutic agents in defense against viruses that use sialic acid as a receptor. Dimeric IgA1 antibodies were produced by stable cell line IgA1/2G9 on the basal medium at different conditions (different supplementation and feeding) and we also evaluated the effect of different conditions on lactate production, which correlates with IgA productivity. Decrease of lactate levels was observed during supplementation with succinic acid, asparagine, or with mannose feeding. We found by lectin-based microarray analysis that the metabolic shift from glutamine to asparagine or feeding with glucose caused increase of high mannose type glycans what was confirmed also by MALDI-MS. Among other changes in IgA glycosylation determined by lectin-based protein microarray were, for example, reduced galactosylation after supplementation with succinic acid and increase of both sialylation and galactosylation after supplementation with glutamine and feeding with mannose. The elucidation of mechanism of determined changes requires further investigation, but the described analytical approach represent effective platform for determination, screening and evaluation of glycosylation of therapeutic proteins.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
D-(+)-Glucose, powder, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.5%
Sigma-Aldrich
D-(+)-Mannose, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
IgA from human serum, reagent grade, ≥95% (HPLC), buffered aqueous solution
Millipore
D-(+)-Galactose, suitable for microbiology, ≥99.0%