Native mass spectrometry combined with enzymatic dissection unravels glycoform heterogeneity of biopharmaceuticals

Abstract

Robust manufacturing processes resulting in consistent glycosylation are critical for the efficacy and safety of biopharmaceuticals. Information on glycosylation can be obtained by conventional bottom–up methods but is often limited to the glycan or glycopeptide level. Here, we apply high-resolution native mass spectrometry (MS) for the characterization of the therapeutic fusion protein Etanercept to unravel glycoform heterogeneity in conditions of hitherto unmatched mass spectral complexity. Higher spatial resolution at lower charge states, an inherent characteristic of native MS, represents a key component for the successful revelation of glycan heterogeneity. Combined with enzymatic dissection using a set of proteases and glycosidases, assignment of specific glycoforms is achieved by transferring information from subunit to whole protein level. The application of native mass spectrometric analysis of intact Etanercept as a fingerprinting tool for the assessment of batch-to-batch variability is exemplified and may be extended to demonstrate comparability after changes in the biologic manufacturing process.

Editorial: antigenic response to CT‐P13 and infliximab originator in IBD shows similar epitope recognition—evidence from basic science supports safe switching to biosimilars

“…There is still insufficient evidence for multiple switching, and this relates to the complex manufacturing of biological medicines, which are very large, highly complex molecules that are derived from living systems as bacteria and mammalian cells. Conformational changes, side‐chain additions or subtractions, oligomerization and glycosylation all can differ between the biosimilar and its originator, and might theoretically alter their immune response . Further postmarketing pharmacovigilance, together with prospective and retrospective observational studies, will therefore remain important to detect rare potential immunogenic events.…”

Towards middle-up analysis of polyclonal antibodies: subclass-specificN-glycosylation profiling of murine immunoglobulin G (IgG) by means of HPLC-MS

“…In this context, intact protein analysis reveals protein heterogeneity arising from glycosylation and other post-translational modifications (PTMs) as has been demonstrated for several therapeutic mAbs and an Fc-fusion protein. [2][3][4][5][6][7][8] Alternative strategies for mAb characterization are based on protein subunits obtained by reduction of disulphide bonds or limited proteolysis. [9][10][11] Subsequent mass determination of protein subunits is referred to as "middle-up", additional gas phase fragmentation and fragment mass determination as "middle-down" analysis.…”

Site-specific N- and O-glycosylation analysis of atacicept

“…17,18 The analysis of intact glycoproteins provides complementary information on overall glycoform distributions and relative abundances of the corresponding proteoforms for comprehensive characterization. 19,20 A comparison of glycan-, glycopeptide-and glycoprotein-based MS approaches has been recently shown for the analysis of IgG Fc-glycosylation. 21 Here, we present a detailed N-and O-glycosylation site characterization of two individual production batches of atacicept using a set of MS-based workflows, which may be also applied to a variety of other glycosylated biopharmaceutical products.…”