Comprehensive Characterization of Biotherapeutics by Selective Capturing of Highly Acidic Glycans Using Stepwise PGC-SPE and LC/MS/MS

Abstract

The glycosylation of biologics is an important factor in pharmacological functions such as efficacy, safety, and biological activity and is easily affected by subtle changes in the cellular environment. Hence, comprehensive and in-depth glycomic characterization of biological products should be performed to ensure product quality and process consistency prior to regulatory approval, but it is still highly challenging due to glycan microheterogeneity produced by enzymatic machinery. In this study, we have developed a systematic methodology for the separation and characterization of various glycans of biotherapeutics using the combination of solid-phase extraction (SPE) and high resolution LC/MS. Neutral and multiple-acidic glycans were selectively fractionated by SPE with a porous graphitized carbon (PGC) cartridge according to their molecule size and polarity (acidity, pK a). Subsequent LC-MS and -MS/MS analyses enabled us to obtain glycan compositions, structures, and quantitative information. Indeed, we have successfully performed glycomic characterization of agalsidase-beta, a representative therapeutic enzyme containing both phosphorylated and sialylated glycans. In addition, a comparative analysis of functional glycans released from different batches of enzymes was performed to verify our method. These results suggest that stepwise PGC-SPE and LC/MS/MS pairwise assays can be used as an efficient tool to detect glycosylation changes of therapeutic glycoproteins including abundant acidic species in biologics or biosimilar development.

Isomer-Specific Monitoring of Sialylated N-Glycans Reveals Association of α2,3-Linked Sialic Acid Epitope With Behcet’s Disease

“…Three sialylated glycans were further separated and analyzed using a PGC column, which is excellent for isomer-specific separation of native glycans ( Hua et al, 2011 ; Seo et al, 2019 ). Figure 3 shows the representative PGC-LC/MRM chromatograms of Hex 5 HexNAc 4 NeuAc 1 , Hex 5 HexNAc 4 Fuc 1 NeuAc 1 , and Hex 5 HexNAc 4 NeuAc 2 , respectively.…”

“…Glycans can have numerous isomers depending on position, linkage, branching, and anomeric structure by various feedback mechanisms of glycosidases and glycosyltransferases (Baum and Cobb, 2017), even if they are identical in composition. In particular, Three sialylated glycans were further separated and analyzed using a PGC column, which is excellent for isomer-specific separation of native glycans (Hua et al, 2011;Seo et al, 2019). Figure 3 shows the representative PGC-LC/MRM chromatograms of Hex 5 HexNAc 4 NeuAc 1 , Hex 5 HexNAc 4 Fuc 1 NeuAc 1 , and Hex 5 HexNAc 4 NeuAc 2 , respectively.…”

Discovery of N-glycan Biomarkers for the Canine Osteoarthritis

“…The search was performed within the mass error of 10 ppm. Biomarkers were selected by the comparison of patients and healthy controls using the normalized absolute peak intensity ( NAPI ) calculated as follows [ 22 , 40 ]; …”

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

“…PGC can be used both in SPE formats to enrich glycopeptides and in online methods, sometimes in combination with reversed-phase separation, for LC-MS/MS. PGC-SPE formats have yet to be widely adopted for enrichment ( 271 , 272 ), likely because complicated retention mechanisms make method optimization challenging and less intuitive. That said, PGC has the attractive potential of chromatographically separating isomeric glycopeptides based on their glycan structures, and several studies have utilized online PGC separations to characterize intact glycopeptides ( 233 , 273 , 274 , 275 , 276 , 277 , 278 , 279 , 280 ).…”