Analysis of Three Epoetin Alpha Products by LC and LC-MS Indicates Differences in Glycosylation Critical Quality Attributes, Including Sialic Acid Content

Abstract

Erythropoietin (EPO) is one of the main therapeutics used to treat anemic patients, greatly improving their quality of life. In this study, biosimilars Binocrit and a development product, called here CIGB-EPO, were compared to the originator product, Eprex. All three are epoetin alpha products, reputed to have similar glycosylation profiles. The quality, safety, and efficacy of this biotherapeutic depend on the following glycosylation critical quality attributes (GCQAs): sialylation, N-glycolyl-neuraminic acid (Neu5Gc) content, branching, N-acetyl-lactosamine (LacNAc) extensions, and O-acetylation pattern. Reverse-phase ultra-high-pressure liquid chromatography (RP-UHPLC) analysis of acid-released, 1,2-diamino-4,5-methylenedioxybenzene (DMB) labeled sialic acid derivatives and hydrophilic interaction liquid chromatography (HILIC) in combination with mass spectrometry (HILIC-UHPLC-MS) of procainamide (PROC) labeled N-glycans were the analytical tools used. An automated method for enzymatic release and PROC labeling was applied for the first time to the erythropoiesis stimulating agent (ESA) products, which facilitated novel, in-depth characterization, and allowed identification of precise structural features including the location of O-acetyl groups on sialic acid (SA) moieties. Samples were digested by a sialate-O-acetylesterase (NanS) to confirm the presence of O-acetyl groups. It was found that Eprex contained the greatest relative abundance of O-acetylated derivatives, Binocrit expressed the least Neu5Gc, and CIGB-EPO showed the greatest variety of high-mannose-phosphate structures. The sialylation and LacNAc extension patterns of the three ESAs were similar, with a maximum of four N-acetyl-neuraminic acid (Neu5Ac) moieties detected per glycan. Such differences in SA derivatization, particularly O-acetylation, could have consequences for the quality and safety of a biotherapeutic, as well as its efficacy.

Physicochemical and biological evaluation of JR-131 as a biosimilar to a long-acting erythropoiesis-stimulating agent darbepoetin alfa

“…Our data showed that the degree of O-acetylation of sialic acid in JR-131 differs to some extent from that of darbepoetin alfa, which may affect in vivo clearance [16]. However, the pharmacokinetic parameters in humans including plasma t 1/2 are similar for the two drugs PLOS ONE [20], and the erythropoiesis-stimulating activity of these products in the model rats was not different (Fig 9), indicating that this glycan variation does not affect the pharmacokinetics and in vivo efficacy of the two drugs.…”

“…The dominant glycan compositions contain sialylated tetra-antennary structures (Gal 4 GlcNAc 4 Fuc 1 NeuAc 4 Man 3 GlcNAc 2 ) at all glycosylation sites. Although the major peaks were not different between JR-131 and the originator, the signal intensities of the O-acetylated sialic acid ion-derived subpeaks were somewhat different (S2A- S2C Fig), which indicates that JR-131 contains less O-acetylated sialic acid than darbepoetin alfa and this may affect the therapeutic half-life of these drugs [16].…”

The sweet spot for biologics: recent advances in characterization of biotherapeutic glycoproteins

“…Glycoprotein digestion with neuraminidase and subsequent UPLC or MS analysis could provide information about presence and amount of sialic acids [64,65], and other exoglycosidase digestions are used for characterization of other glycan moieties [67,68]. Additionally, level of sialic acids (Neu5Ac, Neu5Gc, and O-acetylated sialic acids) could be determined by mild hydrolysis and subsequent labeling with 1,2-diamino-4,5-methylenedioxybenzene using a kit developed by Ludger Ltd, which has recently been used in analysis of three epoetin alpha products [69].…”

“…On the other hand, O-glycans released by other methods may be fluorescently labeled. Several fluorescent labels have been described for glycan labeling for glycoproteins: 2-aminopyridine (PA) [78], 2-aminobenzoic acid (2-AA) [79,80], 2-aminobenzamide (2-AB) [56,81], procainamide (4amino-N-(2-diethylaminoethyl) benzamide) [69,82] and more recently aminoquinoline carbamate (AQC) [44] and RapiFluor-MS (Waters) for the UPLC and MS techniques. For CGE-LIF, 8-aminopyrene-1,3,6-trisulfonate (APTS) and 2-amino-1-naphthalenesulfonic acid (2-ANSA) can be used.…”

Assays for the identification and quantification of sialic acids: Challenges, opportunities and future perspectives

“…Most recently chromatography has been employed to provide a means for the separation, identification and subsequent quantification of sialic acids in biological [64][65][66] and other samples such as infant formula 67 and milk products. 68 For the analysis of sialic acids, derivatisation is required using fluorescent labels and reverse phase chromatography is employed to separate the sialic acids from the bulk material and from each other, allowing closer scrutiny of a variety of sialic acid derivatives in one sample.…”