Screening for glycosylation changes on recombinant human IgG using lectin methods.
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.
Fotinopoulou
Angeliki
A
Department of Clinical Biochemistry, The Medical School, Newcastle upon Tyne NE2 4HH, U.K.
Meyers
Timothy
T
Varley
Paul
P
Turner
Graham
G
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007074:Immunoglobulin G / Q000032:analysis* / Q000737:chemistry* / Q000145:classification; D037102:Lectins; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000145:classification; D013045:Species Specificity; D020349:Surface Plasmon Resonance / Q000379:methods*
D007074:Immunoglobulin G; D037102:Lectins; D011994:Recombinant Proteins