Intact Protein Mass Spectrometry for Therapeutic Protein Quantitation, Pharmacokinetics, and Biotransformation in Preclinical and Clinical Studies: An Industry Perspective

Abstract

Recent advancements in immunocapture methods and mass spectrometer technology have enabled intact protein mass spectrometry to be applied for the characterization of antibodies and other large biotherapeutics from in-life studies. Protein molecules have not been traditionally studied by intact mass or screened for catabolites in the same manner as small molecules, but the landscape has changed. Researchers have presented methods that can be applied to the drug discovery and development stages, and others are exploring the possibilities of the new approaches. However, a wide variety of options for assay development exists without clear recommendation on best practice, and data processing workflows may have limitations depending on the vendor. In this perspective, we share experiences and recommendations for current and future application of mass spectrometry for biotherapeutic molecule monitoring from preclinical and clinical studies.

Top-Down Characterization and Intact Mass Quantitation of a Monoclonal Antibody Drug from Serum by Use of a Quadrupole TOF MS System Equipped with Electron-Activated Dissociation

“…For intact protein LC-MS applied to biopharmaceutical in-life studies (and perhaps mAb analysis in general), the LC-MS/MS platform presented here adds extra flexibility and benefit to analytical LC-MS workflows already in use. 25 The ZenoTOF 7600 system was able to achieve good sequence coverage at the reduced mAb level, especially for the light chain. The GSKmAb light chain (25 kDa) was within size range for MS/MS ions to cover 100% of the protein sequence; however, thorough top-down sequencing coverage for the intact mAb (150 kDa) or heavy chain (50 kDa) is not achieved, partly due to higher mass.…”

The Human Proteoform Project: Defining the human proteome

“…Prior initiatives such as those describing first drafts of the human proteome in 2014 ( 33 , 34 ) and ongoing work under the aegis of the Human Protein Atlas and the Human Proteome Project ( 35 ) have accomplished a great deal over the past several years, and the Human Proteome Organization has called for the community to “systematically map all human proteoforms” ( 36 ). There has also been an industry-led call from several pharmaceutical companies underscoring the need for major improvements in proteoform measurement ( 37 ).…”

Approaches to Heterogeneity in Native Mass Spectrometry

“…Peak-sharpening algorithms are subsequently used on the deconvolved data to resolve remaining overlapped features in the zero-charge spectrum. PMI includes a “comb filter” to identify peak series equally spaced in m / z , such as those arising from polydisperse adduction of a subunit, which can greatly improve analysis of mass spectra representing samples of this type, including those of great interest in the biopharmaceutical industry , such as highly disperse antibody-drug conjugates. ,,,− PMI is coded and compiled in C++ for increased speed, has batch processing capabilities, allows the user to easily select different liquid chromatography-MS (LC-MS) retention data to analyze, and can be used to automatically assign peaks based on protein sequence data or other user-supplied information. It is also vendor-neutral and can produce user-friendly, customizable reports for non-MS users, features important for its use in industry .…”

“…Although many online solution-phase separation approaches (such as online chromatography methods − and capillary zone electrophoresis − ) have been introduced to help solve this problem, our discussion is confined to instrumentation and techniques commonly available within mass spectrometers themselves or with small modifications. As this review is written from an academic viewpoint, we refer readers not only to recent native MS work on biotherapeutics ,− but also to many recent efforts and helpful perspectives on this topic from industry scientists representing a variety of biopharmaceutical companies, drawing upon these insights where possible. ,,,− Importantly, though implementation of the methods we describe below faces unique challenges in industry (namely, rigorous standardization and commercialization), we hold that educating potential users on the theoretical aspects of these approaches is important and beneficial to all who utilize native MS, regardless of background. We conclude by reflecting upon the progress of native MS with respect to the problem of heterogeneity, discussing remaining challenges and future strategies for the field, and providing our view of optimal approaches to facilitate analysis and interpretation of the complicated mass spectra of heterogeneous biomolecules, which is paramount for continued growth of this technique as a tool in structural biology.…”