NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods

Abstract

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

A Simple Strategy to Eliminate Hexosylation Bias in the Relative Quantification of N‐Glycosylation in Biopharmaceuticals

“…To assess glycation induced under controlled conditions, we performed a forced glycation experiment using NISTmAb, a widely used reference material whose glycosylation profile has been extensively characterized in a comprehensive interlaboratory study . In agreement with the results of this study, the mass spectrum of intact, untreated NISTmAb displayed a characteristic series of peaks differing by 162 Da, respectively (Figure a).…”

Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclonal antibody product quality attributes

“…All four laboratories returned similar N-glycosylation data with minimal variability for most of the most abundant glycoforms (intra-lab precision < 10% and inter-lab precision from 9.9 to 67.2%). Reported N-glycans were over 1% of relative levels, and no sialylated glycan structure is shown as they were detected at low levels (< 1%), which is in accordance with reported values for NISTmAb [32].…”

“…Modifications from the analysis are shown in Table 1 with the values of each PTM monitored from NIST mAb. Reported PTM values showed lower amounts of sample preparation-induced modifications as well as lower variability than other inter-laboratory studies where NIST mAb was also used [29,30,32].…”

How and why plants and human N-glycans are different: Insight from molecular dynamics into the “glycoblocks” architecture of complex carbohydrates

“…As an additive layer of difficulty, glycosylation is only indirectly dependent on the genome, which often results in a micro-(or macro-)heterogeneity of glycan sequences at specific sites [7]. These complexities are very difficult to resolve, requiring high levels of expertise and multi-layered orthogonal approaches [8][9][10]7]. Within this framework, the contribution of glycoinformatics tools and databases represents an essential resource to advance glycomics [11][12][13][14][15], while molecular simulations fit in very well as complementary and orthogonal techniques to support and advance structural glycobiology research.…”