Unambiguous Identification of Pyroglutamate in Full-Length Biopharmaceutical Monoclonal Antibodies by NMR Spectroscopy.

Abstract

Biotherapeutic proteins are an indispensable class of pharmaceuticals that present a high degree of structural complexity and are prone to chemical modifications during production, processing, and storage, which have to be tightly controlled. Pyroglutamate (pGlu), a cyclization product of N-terminal Gln or Glu residues, is a widespread post-translational modification in proteins, including monoclonal antibodies (mAbs). The unambiguous identification and quantification of this modification in proteins is challenging, since the mass difference of -17 Da or -18 Da, when formed from Gln or Glu, respectively, is not unique. Moreover, deamidation and dehydration occur not only during cyclization to pGlu, but also during other reactions leading to different types of modifications, like succinimide or isopeptide bond moieties due to cross-linking between Asn or Gln and Lys side chains. Here we report the unambiguous identification and quantification of pGlu in intact mAbs with natural isotope distribution by NMR spectroscopy. The assignment of all 1H, 13C and 15N random coil chemical shifts of pGlu in short reference peptides led to the identification of unique chemical shift pairs that are distinct from the random coil chemical shifts of the natural amino-acid residues. These characteristic correlations are suited for the detection of pGlu in denatured proteins. We achieved complete denaturation of mAbs using a straightforward protocol, and could detect and quantify pGlu, in agreement with available mass spectrometric data. The application to the mAbs rituximab and adalimumab illustrates the potential of our approach for the characterization of biotherapeutics containing isotopes at natural abundance.

• Authors: Arthur Hinterholzer, Vesna Stanojlovic, Chiara Cabrele, Mario Schubert
• Journal: Analytical chemistry (Anal Chem) Vol. 91 Issue 22 Pg. 14299-14305 (11 19 2019) ISSN: 1520-6882 [Electronic] United States
• PMID: 31589410 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Anti-Inflammatory Agents
• Antibodies, Monoclonal
• Antineoplastic Agents, Immunological
• Rituximab
• Adalimumab
• Pyrrolidonecarboxylic Acid

Topics

• Adalimumab (chemistry)
• Anti-Inflammatory Agents (chemistry)
• Antibodies, Monoclonal (chemistry)
• Antineoplastic Agents, Immunological (chemistry)
• Magnetic Resonance Spectroscopy (methods)
• Pyrrolidonecarboxylic Acid (analysis)
• Rituximab (chemistry)