Screening for glycosylation changes on recombinant human IgG using lectin methods

Abstract

Two lectin-binding methods were investigated as possible ways of monitoring the glycosylation of human monoclonal antibodies during their development and production. Carbohydrate composition was assessed in various preparations that were produced in different host cell types, cell sublines or batches of the same cells. The lectin binding was measured with ELISA and surface plasmon resonance (SPR). For comparative purposes, the monosaccharide content of many of the preparations was also measured by high-pressure anion exchange chromatography (HPAEC)/pulsed amperometric detection (PAD). Both lectin methods detected modifications in glycosylation when antibodies were produced in different ways; SPR was more sensitive than ELISA for some lectins and vice versa. Generally, the lectin results agreed with those obtained by the monosaccharide analysis; however, the former were much better for assessing N -acetylneuraminic acid changes. The latter were impossible to assess by HPAEC/PAD because of their low levels. The lectin-based methods also had the advantages that they were quicker to perform and required less expertise and could quickly identify structures that monosaccharide analysis might miss. It is suggested that, in the development of therapeutic proteins, monosaccharide analysis and/or oligosaccharide profiling is initially performed but later routine batches of the glycoprotein are screened with a lectin method. Of the two lectin methods used, SPR is much quicker when performing a screen, whereas ELISA is particularly useful for comparing a particular carbohydrate feature on different samples of the same glycoprotein.

N‐linked glycosylation is an important parameter for optimal selection of cell lines producing biopharmaceutical human IgG

“…The employed chromatographic analysis of purified IgG is time consuming. The throughput would be considerably increased in case supernatant could be used for glycan analyses, for instance by using lectin‐based approaches 37–40. The concern here is that such techniques might not be quantitative, specific, and/or sensitive enough to probe small differences.…”

Recent Progress in Plantibody Technology

“…The pattern of N-glycosylation differs qualitatively and quantitatively between kingdoms but also between closely related species, including mammals [99]. Moreover, Nglycan structure can be influenced by culture conditions [100][101][102], developmental stage [103], production levels and subcellular targeting [87,104,105]. This applies not only to the various production systems discussed in this review but also to human serum IgG.…”