The use of lectin microarray for assessing glycosylation of therapeutic proteins

Abstract

Glycans or carbohydrates attached to therapeutic glycoproteins can directly affect product quality, safety and efficacy, and therefore must be adequately analyzed and controlled throughout product life cycles. However, the complexity of protein glycosylation poses a daunting analytical challenge. In this study, we evaluated the utility of a lectin microarray for assessing protein glycans. Using commercial lectin chips, which contain 45 lectins toward distinct glycan structures, we were able to determine the lectin binding patterns of a panel of 15 therapeutic proteins, including 8 monoclonal antibodies. Lectin binding signals were analyzed to generate glycan profiles that were generally consistent with the known glycan patterns for these glycoproteins. In particular, the lectin-based microarray was found to be highly sensitive to variations in the terminal carbohydrate structures such as galactose versus sialic acid epitopes. These data suggest that lectin microarray could be used for screening glycan patterns of therapeutic glycoproteins.

Keywords: Glycan analysis; lectin microarray; monoclonal antibodies; therapeutic glycoproteins.

Figure 1.

Schematic view of lectin microarray. (A) Lectin microchips used in this study consist of 45 distinct lectins that selectively bind structural variants of carbohydrates attached onto a protein. Each lectin is printed in triplicate. The lectin-printing layout of lectin chips was provided by the vendor (GlycoTechnica). (B) Protein samples are labeled with a fluorescent dye (e.g., Cy3) and then applied onto the lectin chips. The binding signals at each lectin spots are measured using an evanescent-field fluorescence scanner, detecting the presence or absence of glycan variants in the testing sample based on the known selectivity of lectins toward particular glycan structures.

Figure 2.

Lectin binding profiles of therapeutic IgG monoclonal antibodies. The indicated therapeutic mAbs, including bevacizumab, trastuzumab, adalimumab, infliximab, rituximab, omalizumab, and panitumumab were labeled with Cy3 and applied onto the lectin chips containing 45 distinct lectins with each being printed in triplicate. (A) Lectin binding images of the indicated samples. (B) Relative binding signals at specific lectin spots were derived from the images in A and normalized to protein markers on the same chip (mean ± SD). Shown are representatives of 3 independent experiments. The coefficient of variation (CV) was determined to be < 10% for most lectin-glycan binding signals of the samples tested. (C) Typical glycan structures present in the Fc portion of therapeutic IgG1 mAbs.

Figure 3.

Lectin binding profiles of cetuximab and etanercept. Cy3-labeled samples were applied onto the lectin chips as in Figure 2. Shown are (A) representative lectin binding images, (B) Relative binding signals at specific lectin spots (mean ± SD), and (C) Typical glycan structures present in the Fab portion of cetuximab.

Figure 4.

Glycan profiling of Fabs and Fcs. The Fabs and Fcs of rituximab (A) and cetuximab (B) were prepared as described in Materials and Methods. Purified Fab and Fc were analyzed by reducing SDS-PAGE (left panel) and lectin microarray (right panel). As noted, the dimeric Fcs (˜ 55 kDa) were reduced to monomeric products (˜ 30 kDa) on SDS-PAGE under reducing conditions.

Figure 5.

Glycan profiles of proteins produced by different host cell systems. The proteins tested include therapeutic proteins produced by CHO cells (darbepoetin alfa and dornase alfa), yeast strains (rasburicase), or E. coli (filgrastim), and human transferrin protein expressed by recombinant rice (transferrin-rice) or isolated from human plasma (transferrin-human). (A) Lectin binding images. (B) Relative binding signals at specific lectin spots (mean ± SD). (C) Typical glycan structures present in darbepoetin alfa.

Figure 6.

Assessing glycan variants in glycan-engineered rituximab protein samples. Rituximab was incubated with a reaction buffer alone (rituximab + buffer), β1-4-galactosyltransferase (rituximab + β1-4GalT), and β1-4-galactosyltransferase followed by α2-6-sialyltransferase (rituximab + β1-4GalT + α2-6SiaT) (see detail in Materials and Methods). After affinity purification, the resulting samples were analyzed using mass spectrometry and lectin microarray, respectively. Shown are representatives of deconvoluted mass spectra (A) and corresponding lectin binding profiles (B) for the samples produced under the indicated conditions.