Investigating the conformation and conformational dynamics of protein biopharmaceuticals with hydrogen/deuterium exchange mass spectrometry

The structure of a protein will ultimately dictate its function. A consequence of this structure-function relationship is: any structural change has the potential to cause a change in function. Therefore, it is important to investigate and understand protein structure. It is well known that the structure of a protein is predominately controlled by the amino acid sequence, a large array of weak secondary interactions, and, at times, by some covalent interactions. For biopharmaceutical proteins, it is particularly important to investigate the protein structure-function relationship; since during production, these proteins can be exposed to a wide range of chemical and physical environments that may perturb some of these interactions and potentially alter protein structure, function, and efficacy. Consequently, good analytical tools capable of characterizing protein conformation are highly desirable. Hydrogen/deuterium exchange mass spectrometry (H/DX-MS) can be used to probe and investigate the conformational and dynamic properties of proteins in solution with small sample quantities (picomoles), with peptide level resolution, in essentially any buffer system, in a few days or less. These attributes make H/DX-MS an attractive tool for characterizing the conformation and dynamics of protein biopharmaceuticals. Experiments were designed to probe and characterize protein posttranslational modifications, physical stress, and protein-protein interactions. As well, the feasibility of H/DX-MS in protein biopharmaceutical comparability studies was demonstrated.;Two representative biopharmaceuticals were investigated using H/DX-MS: a recombinant monoclonal antibody (rmAB) immunoglobulin gamma 1 (IgG1), and interferon-beta1a (IFN). These experiments demonstrated for the first time that H/DX-MS could be applied to a protein as large and complicated as an antibody (IgG1). The data showed that IgG1 glycans strongly influence the conformation and dynamics of the IgG1 Fc region and these changes indirectly impact antibody effector function.;An important stability attribute of many protein biopharmaceuticals is oxidation. Using H/DX-MS orthogonally with other techniques, the conformational impact of oxidation on both the IgG1 and IFN was examined. These data showed that while both proteins experienced a conformational change, the IgG1 suffered only a mild effect, with destabilization localized to a small segment of the CH2 domain. IFN however experienced a large structural change, with nearly fifty percent of the proteins amino acid sequence destabilized. Even though methionine oxidation affected the stability of both proteins, there was no detectable effect on the IgG1s ability to bind specific effector proteins in a binding assay or IFN to produce antiviral activity in a screening assay.;The ability of an antibody to engage specific Fc receptors (i.e., effector function) is a critical component of many therapeutic antibodies. Binding experiments were preformed using H/DX-MS to investigate the interaction between two IgG1 variants and the Fc receptor, FcgammaRIIIa. In one experiment, the binding between a native IgG1 with weak affinity (∼1 muM) for FcgammaRIIIa was investigated. In the other experiment, the binding between a higher affinity (∼20 nM) IgG1 glycovariant (afucosylated and hypergalactosylated) and FcaRIIIa was studied. These experiments revealed that the mechanisms of IgG1 and FcgammaRIIIa binding are different, and these results indicated that the glycans are involved in indirectly controlling the interaction between the two proteins.;Finally, the ability to consistently manufacture a biopharmaceutical is both an industrial and a regulatory requirement. Using IFN as a model biopharmaceutical, H/DX-MS was used to compare the conformation and dynamics of four different forms and preparations of IFN, relative to an IFN control. These data showed that the conformation of different preparations of IFN, IFN produced in different media, and N-terminally pegylated IFN were comparable to the control IFN, whereas the oxidized IFN was not comparable. A unique data display format was also developed, which displays all of the data acquired in a typical H/DX-MS experiment (where two proteins are being compared) on a single graph. This data display format significantly reduces data evaluation time and will improve biopharmaceutical comparability studies, making H/DX-MS data more accessible for regulatory groups and other departments across the biopharmaceutical industry.