N-glycan analysis of human α1-antitrypsin produced in Chinese hamster ovary cells

Abstract

Human alpha-1-antitrypsin (α1AT) is a glycoprotein with protease inhibitor activity protecting tissues from degradation. Patients with inherited α1AT deficiency are treated with native α1AT (nAT) purified from human plasma. In the present study, recombinant α1AT (rAT) was produced in Chinese hamster ovary (CHO) cells and their glycosylation patterns, inhibitory activity and in vivo half-life were compared with those of nAT. A peptide mapping analysis employing a deglycosylation reaction confirmed full occupancy of all three glycosylation sites and the equivalency of rAT and nAT in terms of the protein level. N-glycan profiles revealed that rAT contained 10 glycan structures ranging from bi-antennary to tetra-antennary complex-type glycans while nAT displayed six peaks comprising majorly bi-antennary glycans and a small portion of tri-antennary glycans. In addition, most of the rAT glycans were shown to have only core α(1 - 6)-fucose without terminal fucosylation, whereas only minor portions of the nAT glycans contained core or Lewis X-type fucose. As expected, all sialylated glycans of rAT were found to have α(2 - 3)-linked sialic acids, which was in sharp contrast to those of nAT, which had mostly α(2 - 6)-linked sialic acids. However, the degree of sialylation of rAT was comparable to that of nAT, which was also supported by an isoelectric focusing gel analysis. Despite the differences in the glycosylation patterns, both α1ATs showed nearly equivalent inhibitory activity in enzyme assays and serum half-lives in a pharmacokinetic experiment. These results suggest that rAT produced in CHO cells would be a good alternative to nAT derived from human plasma.

Proteolytic andN-Glycan Processing of Humanα1-Antitrypsin Expressed inNicotiana benthamiana

“…By coexpressing a2,6-sialyltransferase (ST6), secreted plant-derived A1AT carries exclusively a2,6-linked terminal sialic acid (Castilho et al, 2010) corresponding to the native therapeutic counterpart (Prolastin) and in sharp contrast to A1AT expressed in Chinese hamster ovary cells, where mainly a2,3-linked sialylation is present (Lee et al, 2013). Pharmacokinetic (PK) studies in rats have shown that the mean residence time of A1AT can be modified by varying the sialic acid linkage (Brinkman et al, 2012).…”

“…Concerns over the supply and safety of the products have urged searches for alternative recombinant sources of A1AT. Recombinant A1AT has been produced in human and nonhuman cell production systems with limited success (Blanchard et al, 2011;Brinkman et al, 2012;Ross et al, 2012;Lee et al, 2013). The production suffers from two major drawbacks: low expression levels and/or incorrect glycosylation (Garver et al, 1987;Chang et al, 2003;McDonald et al, 2005;Hasannia et al, 2006;Karnaukhova et al, 2006;Plesha et al, 2007;Agarwal et al, 2008;Nadai et al, 2009;Huang et al, 2010;Arjmand et al, 2011;Jha et al, 2012).…”

Mass spectrometry for glycosylation analysis of biopharmaceuticals

“…For spectrophotometric detection, released glycans need to be derivatized, commonly by attachment of a fluorophore [e.g., 2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), or 1-aminopyrene-3,6,8-trisulfonate] at the reducing end [15,16]. Separation is then commonly achieved by hydrophilic interaction liquid chromatography (HILIC)-HPLC or UPLC [16,17], or by C(G)E with fluorescence detection [18,19].…”

“…Separation is then commonly achieved by hydrophilic interaction liquid chromatography (HILIC)-HPLC or UPLC [16,17], or by C(G)E with fluorescence detection [18,19]. As presented in a multilaboratory study, fluorescent labeling is the critical step in profiling due to possible incomplete derivatization, resulting in greater laboratory variability than MS-based Nglycomic methods [20].…”

“…Likewise, due to its speed and low consumption of sample, MALDI-TOF-MS is suitable for oligosaccharide sequencing via exoglycosidase digestion, providing information on the anomericity of glycosidic linkages [16]. This approach provides an attractive, sensitive alternative to traditional linkage-analysis techniques, such as methylation GC-MS or NMR.…”