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

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.

Use of PASEF for Accelerated Protein Sequence Confirmation and De Novo Sequencing with High Data Quality.

Biopharmaceutical sequences can be well confirmed by multiple protease digests-e.g., trypsin, elastase, and chymotrypsin-followed by LC-MS/MS data analysis. High quality data can be used for de novo sequencing as well. PASEF (Parallel Accumulation and Serial Fragmentation) on the timsTOF instrument has been used to accelerate proteome and protein sequence studies and increase sequence coverage concomitantly.Here we describe the protein chemical and LC-MS methods in detail to generate high quality samples for sequence characterization from only 3 digests. We applied PASEF to generate exhaustive protein sequence coverage maps by combination of results from the three enzyme digests using a short LC gradient. The data quality obtained was high and adequate for determining antibody sequences de novo.Nivolumab and dulaglutide were digested by 3 enzymes individually. For nivolumab, 94/94/90% sequence coverage and 86/84/85% fragment coverage were obtained from the individual digest analysis with trypsin/chymotrypsin/elastase, respectively. For dulaglutide, 96/100/90% sequence coverage and 92/90/83% fragment coverage were obtained. The merged peptide map from the 3 digests for nivolumab resulted in ∼550 peptides; enough to safely confirm the full sequences and to determine the nivolumab sequence de novo.

Full validation of therapeutic antibody sequences by middle-up mass measurements and middle-down protein sequencing.

The regulatory bodies request full sequence data assessment both for innovator and biosimilar monoclonal antibodies (mAbs). Full sequence coverage is typically used to verify the integrity of the analytical data obtained following the combination of multiple LC-MS/MS datasets from orthogonal protease digests (so called "bottom-up" approaches). Top-down or middle-down mass spectrometric approaches have the potential to minimize artifacts, reduce overall analysis time and provide orthogonality to this traditional approach. In this work we report a new combined approach involving middle-up LC-QTOF and middle-down LC-MALDI in-source decay (ISD) mass spectrometry. This was applied to cetuximab, panitumumab and natalizumab, selected as representative US Food and Drug Administration- and European Medicines Agency-approved mAbs. The goal was to unambiguously confirm their reference sequences and examine the general applicability of this approach. Furthermore, a new measure for assessing the integrity and validity of results from middle-down approaches is introduced - the "Sequence Validation Percentage." Full sequence data assessment of the 3 antibodies was achieved enabling all 3 sequences to be fully validated by a combination of middle-up molecular weight determination and middle-down protein sequencing. Three errors in the reference amino acid sequence of natalizumab, causing a cumulative mass shift of only -2 Da in the natalizumab Fd domain, were corrected as a result of this work.

Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques.

The European Medicines Agency received recently the first marketing authorization application for a biosimilar monoclonal antibody (mAb) and adopted the final guidelines on biosimilar mAbs and Fc-fusion proteins. The agency requires high similarity between biosimilar and reference products for approval. Specifically, the amino acid sequences must be identical. The glycosylation pattern of the antibody is also often considered to be a very important quality attribute due to its strong effect on quality, safety, immunogenicity, pharmacokinetics and potency. Here, we describe a case study of cetuximab, which has been marketed since 2004. Biosimilar versions of the product are now in the pipelines of numerous therapeutic antibody biosimilar developers. We applied a combination of intact, middle-down, middle-up and bottom-up electrospray ionization and matrix assisted laser desorption ionization mass spectrometry techniques to characterize the amino acid sequence and major post-translational modifications of the marketed cetuximab product, with special emphasis on glycosylation. Our results revealed a sequence error in the reported sequence of the light chain in databases and in publications, thus highlighting the potency of mass spectrometry to establish correct antibody sequences. We were also able to achieve a comprehensive identification of cetuximab's glycoforms and glycosylation profile assessment on both Fab and Fc domains. Taken together, the reported approaches and data form a solid framework for the comparability of antibodies and their biosimilar candidates that could be further applied to routine structural assessments of these and other antibody-based products.