Lectin microarray profiles glycans on intact therapeutic antibodies, matching mass spectrometry

Researchers evaluated a second-generation lectin microarray for glycan profiling directly on intact therapeutic monoclonal antibodies, according to a paper in AAPS Open. Glycan profiles from the array were highly concordant with mass spectrometry of released glycans, the authors report biomedcentral.comAAPS Openbiomedcentral.comOpen source ↗. N-linked glycosylation is a critical quality attribute of therapeutic antibodies and influences efficacy, safety and pharmacokinetics.

How the lectin microarray works

The platform, LecChip-IgG-mAb, carries nine tailored lectins printed in triplicate across 14 wells. It targets nine glycan epitopes common in approved antibodies: core fucose, terminal N-acetylglucosamine, terminal beta-galactose, high mannose, terminal alpha-galactose, terminal alpha-2,3-linked N-acetylneuraminic acid, terminal alpha-2,6-linked N-glycolylneuraminic acid, triantennary structures and bisecting N-acetylglucosamine. One glycan can display several epitopes, and one epitope occurs on several coexisting glycoforms, so the signal reflects the overall presence of an epitope. This makes the readout semi-quantitative.

The array analyzes the intact protein without chemical or enzymatic release of glycans and needs one microgram of antibody. The conventional workflow releases glycans with PNGase F, tags them with fluorescent labels, separates them by hydrophilic interaction chromatography and identifies them by mass spectrometry; even rapid protocols consume up to 15 micrograms of antibody, and single injections can last up to an hour. Evanescent-field fluorescence detection excites only fluorophores bound to lectins at the chip surface, so no washing step is needed after incubation.

Validation on anti-HER2 antibodies and adalimumab

For validation, the team produced anti-HER2 antibodies with the trastuzumab sequence in HEK293T cells. Chemoenzymatic glycoengineering yielded eight glycoform variants: Ab-G0, Ab-G0F, two Ab-G1F forms (3-arm and 6-arm galactose), Ab-G2, Ab-G2F, Ab-S2G2 and Ab-S2G2F biomedcentral.comAAPS Openbiomedcentral.comOpen source ↗. MALDI-TOF analysis of released glycans showed Ab-G0, Ab-G0F, Ab-G2 and Ab-G2F to be fully homogeneous. The G1F preparations contained 12.2 percent and 0.1 percent residual G0F. The sialylated variants were partly desialylated after transport and storage, leaving 70.4 percent intact S2G2 and 64.4 percent intact S2G2F.

Intact Cy3-labeled antibodies were diluted to 125 nanograms per milliliter, incubated overnight on the array and imaged on a GlycoStation Reader 2300. The authors report that both approaches agreed closely, and that further studies confirmed the dynamic range and intermediate precision of the platform. The array was also tested on three lots of the adalimumab reference product Humira and eight biosimilars. According to the authors, this confirmed its use as a complementary tool for characterizing therapeutic antibodies.