Analysis of Nonhuman N-Glycans as the Minor Constituents in Recombinant Monoclonal Antibody Pharmaceuticals

Abstract

Minor N-linked glycans containing N-glycolylneuraminic acid residues and/or α-Gal epitopes (i.e., galactose-α1,3-galactose residues) have been reported to be present in recombinant monoclonal antibody (mAb) therapeutics. These contaminations are due to their production processes using nonhuman mammalian cell lines in culture media containing animal-derived materials. In case of the treatment of tumors, we inevitably use such mAbs by careful risk-benefit considerations to prolong patients' lives. However, expanding their clinical applications such as for rheumatism, asthma, and analgesia demands more careful evaluation of the product characteristics. The present work for detailed evaluations of N-glycans demonstrates the methods using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and a combination of high-performance liquid chromatography and electrospray ionization time-of-flight mass spectrometry. The CE-LIF method provides excellent separation of both major and minor N-glycans from six commercial mAb pharmaceuticals within 30 min and clearly indicates that a possible trigger of immunogenicity in humans due to the presence of nonhuman N-glycans is present. We strongly believe that the proposed method will be a powerful tool for the analysis of N-glycans of recombinant mAb products in various development stages, such as clone selection, process control, and routine release testing to ensure safety and efficacy of the products.

Comparative Studies ofN-Glycans and Glycosaminoglycans Present in SIRC (Statens Seruminstitut Rabbit Cornea) Cells and Corneal Epithelial Cells from Rabbit Eyes

“…29 For the analysis of 2AA-labeled N-glycans, electrophoresis was performed with a DB-1 capillary (100 mm i.d. Â 40 cm) in 100 mM Tris-phosphate buffer (pH 8.3).…”

Glycosylation analysis of Chinese hamster ovary produced glycoproteins

“…Detection is typically through laser induced fluorescence (LIF) and elution profiles can be standardized using a dextran ladder, allowing for the conversion of peak elution times to GU values, as in HILIC analysis. Newer methods for labeling with APTS [84], rhodamine 10 [85] and 4fluoro 7nitro 2,1,3benzoxadiazole (NBDF) [86], have increased the sensitivity. CE-LIF has the advantage of very rapid elution times (under 30 min), and also has been shown useful in the detection of the antigenic Neu5Gc and Galα1,3Gal (αGal) sugars [87] which has recently been found to be produced in one CHO biotherapeu tic in very low amounts [46].…”

Capillary Electrophoresis of Glycans: Validation of Antibody Pharmaceuticals Based on Glycan Profiles

“…The fluorescently labeled glycans with negatively charged tags such as 2-aminobenzoic acid (2-AA) or 8-aminopyrene-1,3,6-trisulfonate (APTS) are well resolved by CE in a running buffer solution containing neutral polymer as sieving matrix (e.g., polyethylene glycol, polyethylene oxide) (Kamoda et al 2004(Kamoda et al , 2006Maeda et al 2012). The CE technology has been increasingly used in the field of protein biopharmaceuticals, and it is now one of the important tools to evaluate heterogeneity based on the "glycan profile.…”

“…However, those of tocilizumab, bevacizumab, and adalimumab are NeuAc. Generally, the dominant types (over 93 %) in mAbs product are alternately NeuAc type or NeuGc type (Maeda et al 2012). Therefore, CE-LIF method is quite useful for rapid and extensive evaluation of nonhuman N-glycans in mAb pharmaceuticals.…”