Mass spectrometry platforms for higher order structural measurements of protein biotherapeutics Reference procedures for establishing the reproducibility and sensitivity of measurements

Monitoring changes in protein higher order structure (HOS) is an important regulatory requirement for reasons of safety, efficacy and potency of the resultant therapeutic. In particular, as monoclonal antibodies (mAbs) emerge rapidly as a dominant class of therapeutics, so does the need for suitable analytical technologies to monitor for changes in HOS of these complex large biomolecules. Reference materials (RM) serve a key analytical purpose of benchmarking the suitability and robustness of analytical procedures for both drug producers and regulators. Here two different model systems, of increasing complexity based on commercially available RMs, have been developed and used to illustrate the assessment of repeatability and reproducibility of a range of mass spectrometry based analytical platforms for protein HOS measurements. The simplest model protein RM, recombinant human growth hormone, rhGH, was used to illustrate the development and systematic evaluation of more established platforms hydrogen deuterium exchange-mass spectrometry (HDX-MS) and ion mobility spectrometry-mass spectrometry (IMS-MS) and a more novel platform, fast photochemical oxidation of proteins (FPOP-MS) for protein HOS measurements. Both global and localised changes in rhGH HOS, induced due to the presence of zinc ligands were identified using the methods developed and cross-platform measurements compared. Structural conclusions were underpinned using a statistical approach developed for HDX-MS measurements in which measurement variability was differentiated from small but significant changes in HOS. These HDX-MS and IMS-MS methods approaches were validated using a mAb-based RM from the National Institute of Science and Technology (NISTmAb) and two Fc-glycan variants generated using a simple enzymatic protocol and structural changes characterised. Measurements reproducibly demonstrated decreases in structural stability as a result of loss of Fc-glycan structure. These data promote the use of these, rhGH and NISTmAb RM based model systems, for both validating and establishing the sensitivity of analytical methods for the detection of HOS changes of mAbs and other protein therapeutics. These data also demonstrate the suitability of HDX-MS, IMS-MS and FPOP-MS as valuable analytical methods within the biopharma toolbox.