Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis

In this paper, we report on a novel oriented peptide-N-glycosidase F (PNGase F) immobilization approach onto methacrylate based monolithic support for rapid, reproducible and efficient release of the N-linked carbohydrate moieties from glycoproteins. The glutathione-S-transferase-fusion PNGase F (PNGase F-GST) was expressed in Escherichia coli using regular vector technology. The monolithic pore surface was functionalized with glutathione via a succinimidyl-6-(iodoacetyl-amino)-hexanoate linker and the specific affinity of GST toward glutathione was utilized for the oriented coupling. This novel immobilization procedure was compared with reductive amination technique commonly used for non-oriented enzyme immobilization via primary amine functionalities. Both coupling approaches were compared using enzymatic treatment of several glycoproteins, such as ribonuclease B, fetuin and immunoglobulin G followed by MALDI/MS and CE-LIF analysis of the released glycans. Orientedly immobilized PNGase F via GST-glutathione coupling showed significantly higher activity, remained stable for several months, and allowed rapid release of various types of glycans (high-mannose, core fucosylated, sialylated, etc.) from glycoproteins. Complete protein deglycosylation was obtained as fast as in several seconds when using flow-through immobilized microreactors

Glycosimilarity assessment of biotherapeutics 1: Quantitative comparison of the N -glycosylation of the innovator and a biosimilar version of etanercept

The carbohydrate moieties on the polypeptide chains in most glycoprotein based biotherapeutics andtheir biosimilars play essential roles in such major mechanisms of actions as antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, anti-inflammatory functions and serumclearance. In addition, alteration in glycosylation may influence the safety and efficacy of the product.Glycosylation, therefore, is considered as one of the important critical quality attributes of glycoproteinbiotherapeutics, and consequently for their biosimilar counterparts. Thus, the carbohydrate moieties ofsuch biopharmaceuticals (both innovator and biosimilar products) should be closely scrutinized during allstages of the manufacturing process. In this paper we introduce a rapid, capillary gel electrophoresis basedprocess to quantitatively assess the glycosylation aspect of biosimilarity (referred to as glycosimilarity)between the innovator and a biosimilar version of etanercept (Enbrel®and Benepali®, respectively),based on their N-linked carbohydrate profiles. Differences in sialylated, core fucosylated, galactosylatedand high mannose glycans were all quantified. Since the mechanism of action of etanercept is TNF� bind-ing, only mannosylation was deemed as critical quality attribute for glycosimilarity assessment due toits influence on serum half-life

High-throughput profiling of the serum N-glycome on capillary electrophoresis microfluidics systems: toward clinical implementation of glycoHepatoTest

We developed a 3 h procedure for preparing serum N-glycans and labeling them with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) by sequential addition of reagents to the serum and incubation in a polymerase chain reaction (PCR) thermocycler. Moreover, we succeeded in analyzing these samples by capillary electrophoresis on three commercial microfluidics-based platforms: the MCE-202 MultiNA, the 2100 Bioanalyzer, and a modified prototype of the eGene system which were originally designed for nucleic acid separation and detection. Although these instruments use short separation channels, our technical improvements made it possible to reliably measure the N-glycans constituting GlycoHepatoTest. This test comprises a panel of biomarkers that allows follow-up of liver fibrosis patients starting from the early stage. In this way and for the first time, we demonstrate a clinical glycomics assay on an affordable, robust platform so that clinical chemistry laboratories can exploit glycomics in the diagnosis and monitoring of chronic liver disease. Another potential application is the rapid screening of the N-glycosylation of recombinant glycoproteins produced for pharmaceutical use