Profiling the Glycoforms of the Intact α Subunit of Recombinant Human Chorionic Gonadotropin by High-Resolution Capillary Electrophoresis−Mass Spectrometry

Abstract

With the rapid growth of complex heterogeneous biological molecules, effective techniques that are capable of rapid, characterization of biologics are essential to ensure the desired product characteristics. To address this need, we have developed a method for analysis of intact glycoproteins based on high resolution capillary electrophoretic separation coupled to an LTQ-FT mass spectrometer. We evaluated the performance of this method on the alpha subunit of mouse cell line-derived recombinant human chorionic gonadotrophin (r-αhCG), a protein that is glycosylated at two sites and is part of the clinically-relevant gonadotrophin family. Analysis of r-αhCG, using capillary electrophoresis (CE) with a separation time under 20 minutes, resulted in the identification of over 60 different glycoforms with up to nine sialic acids. High resolution CE/FT-MS allowed separation and analysis of not only intact glycoforms with different numbers of sialic acids but also intact glycoforms that differed by the number and extent of neutral monosaccharides. The high mass resolution of the FT-MS enabled a limited mass range to be targeted for the examination of the protein glycoforms, simplifying the analysis without sacrificing accuracy. In addition, the limited mass range resulted in a fast scan speed that enhanced the reproducibility of the relative quantitation of individual glycoforms. The intact glycoprotein analysis was complemented with the analysis of the tryptic glycopeptides and glycans of r-αhCG to enable the assignment of glycan structures to individual sites, resulting in a detailed characterization of the protein. Samples of r-αhCG obtained from a CHO cell line were also analyzed and briefly shown to be significantly different from the murine cell line product. Taken together, the results suggest that the CE coupled to high resolution FT-MS can be one of the effective tools for in-process monitoring, as well as for final product characterization.

Identification and characterization of protein glycosylation using specific endo- and exoglycosidases

“…The lower band on lane 2 (arrow) is faint on the Emerald Green image, while the upper band on lane 2 is bright, indicating that many glycans groups are still present. These data support the conclusion that recombinant hCGβ expressed in mouse cells contains multiple glycoforms 9 . These different glycoforms are due to the inherent heterogeneity of glycosylation where some polypeptides do not receive a glycan in each consensus site and/or some glycans are extended while others even on the same protein are not.…”

The glycosylation aspects of biosimilarity

“…N-glycosylation analysis of therapeutic proteins can be performed at three different levels (intact protein, glycopeptide, and released glycans), as depicted in Figure 3 [64]. Intact glycoproteins can be analyzed by mass spectrometry (MS) via direct infusion or coupling with separation techniques, such as liquid chromatography (LC)-MS or capillary electrophoresis (CE)-MS [65,66]. Such top-level analysis also offers information about glycan pairing on the two heavy chains of an IgG molecule.…”

Protein glycosylation analysis with capillary-based electromigrative separation techniques

“…A favorable neutral coating, which is very hydrophilic and thus strongly reduces adsorption problems within a large pH range, is polyvinylalcohol, either commercially available or selfmade by in-capillary crystallization of the polymer. It was applied for the characterization of the alpha subunit of recombinant human chorionic gonadotropin by CE-MS [125]. Neutral coatings may give rise to problems with spray stability of electrospray ionization mass spectrometry, sheathless interfacing is not feasible [182,[188][189][190].…”

“…For a complex-type N-glycan with a high branching, up to four sialic acids can be attached (in some cases polysialic acid structures (see Fig. 1)) which may lead to a higher number of sialic acids attached to the glycan, e.g., [48,125]). The different degree of sialylation of glycoproteins, glycopeptides, and glycans gives rise to large charge heterogeneity.…”