Profiling analysis of oligosaccharides in antibody pharmaceuticals by capillary electrophoresis

Abstract

Carbohydrate chains in glycoprotein pharmaceuticals have important roles for the expression of their biological activities. Therefore, development of an assessment method for the carbohydrate chains is an important parameter for quality control of glycoprotein pharmaceuticals such as newly developed therapeutic antibodies. In this report, we applied capillary electrophoresis with laser-induced fluorescence detection to the analysis of carbohydrate chains after releasing with glycoamidase followed by derivatization with 3-aminobenzoic acid. We found that four major oligosaccharides present in antibody pharmaceuticals were successfully separated with good resolution. The present method showed good precision in both migration times and relative peak areas, and gave comparable accuracy with that using a derivatization method with 8-aminopyrene-1,3,6-trisulfonate.

Quality Control and Stability Studies with the Monoclonal Antibody, Trastuzumab: Application of 1D- vs. 2D-Gel Electrophoresis

“…As we reported previously [2], the high value of gel-to-gel variation in the spot intensity does not allow the detection of these small changes. These data were expected, since in the trastuzumab molecule, N -linked, asialo, biantennary complex oligosaccharides are dominant and, according to the literature data, only a minor percentage of terminal sialidated sugars is present (1%–3%) [8,40]. Furthermore, HC terminal lysine truncation can be a source of charge heterogeneity; however, it can bring about only small pI difference between the “normal” and “truncated” form of the antibody, and this difference could not be detected with 2D-electrophoresis.…”

“…Enzymatic deglycosylation of trastuzumab was carried out according to the published procedures [2,8]. Solutions of the pharmaceutical preparation, trastuzumab (Herceptin ® , F. Hoffmann-La Roche, Basel, Switzerland) 21 mg/mL collected from the vials immediately after clinical use in Vienna Hospital Rudolfstiftung, were dialyzed against Milli-Q water (3 days, 4 °C) using a cellulose dialysis tube (cut-off: 12,000–14,000 Da, Japan Medical Science, Tokyo, Japan).…”

“…The respective theoretical molecular masses (Mr) and pI values of HC and LC are 49,284.65 Da and 8.49 for HC and 23,443.1 Da and 7.76 for LC. The theoretical Mr of pI nonglycosylated trastuzumab is 145,455.5 Da [6,7]; however, the apparent Mr of trastuzumab is higher (~148 kDa), due to the presence of N -linked oligosaccharides [8]. Like other IgG1 molecules, trastuzumab has one N -linked biantennary oligosaccharide on the conserved asparagine residue at position 300, buried between the CH2 domains [5].…”

Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection

“…There are many analytical methods that are commonly used to analyze glycosylation such as NMR, MS, HPLC and CE. The most commonly used quantitative tools to analyze glycosylation are HPLC either with pulsed amperometric detection (PAD) [16,17] or with fluorescence detection employing fluorescently-labeled glycans [18,19,20] and CE with a LIF detector for various fluorescently-labeled glycans [21,22,23]. CE-LIF technique with APTS-labeled glycans is routinely employed in biopharmaceutical industries to analyze the glycosylation heterogeneities in a mAb.…”

Modern Technologies for Creating Synthetic Antibodies for Clinical Application

“…almost all of them have fucosylated carbohydrate components and, as a result, non-optimal aCDC [66]. The fucosylation of the IgG carbohydrate component is carried out in mammal cells with α-1,6-fucosyl transferase (FUT8).…”