Detection and characterization of altered conformations of protein pharmaceuticals using complementary mass spectrometry-based approaches

Abstract

Unlike small-molecule drugs, the conformational properties of protein biopharmaceuticals in solution are influenced by a variety of factors that are not solely defined by their covalent chemical structure. Since the conformation (or higher order structure) of a protein is a major modulator of its biological activity, the ability to detect changes in both the higher order structure and conformational dynamics of a protein, induced by an array of extrinsic factors, is of central importance in producing, purifying, and formulating a commercial biopharmaceutical with consistent therapeutic properties. In this study we demonstrate that two complementary mass spectrometry-based approaches (analysis of ionic charge-state distribution and hydrogen/deuterium exchange) can be a potent tool in monitoring conformational changes in protein biopharmaceuticals. The utility of these approaches is demonstrated by detecting and characterizing conformational changes in the biopharmaceutical product interferon beta-1a (IFN-beta-1a). The protein degradation process was modeled by inducing a single chemical modification of IFN-beta1a (alkylation of its only free cysteine residue with N-ethylmaleimide), which causes significant reduction in its antiviral activity. Analysis of IFN-beta1a ionic charge-state distributions unequivocally reveals a significant decrease of conformational stability in the degraded protein, while hydrogen/deuterium exchange measurements provide a clear indication that the higher order structure is affected well beyond the covalent modification site. Importantly, neither technique required that the location or indeed the nature of the chemical modification be known prior to or elucidated in the process of the analysis. In contrast, application of the standard armamentarium of biophysical tools, which are commonly employed for quality control of protein pharmaceuticals, met with very limited success in detection and characterization of conformational changes in the modified IFN-beta1a. This work highlights the role mass spectrometry can and should play in the biopharmaceutical industry beyond the presently assigned task of primary structure analysis.

Figure 1

Comparison of IFN-β1a (blue traces) and NEM-IFN-β1a (red traces) using classical biophysical techniques: SEC profiles (A), fluorescence emission spectra (B), far-UV CD spectra (C) and near-UV CD spectra (D). Gray trace in panel A corresponds to incompletely alkylated protein. Inset in panel C s show the difference between the two spectra (normalized to protein concentration).

Figure 2

Charge state distributions of NEM-IFN-β1a (top) and intact IFN-β1a (bottom) ions in ESI MS acquired under near-native conditions following buffer exchange of protein solutions to 100 mM ammonium acetate.

Figure 3

Global HDX kinetics of intact IFN-β1a (blue) and NEM-IFN-β1a (red). The top panel shows raw HDX MS data (charge state +9), where the m/z scale for IFN-NEM ions was offset such that the observed difference in the ion peak position is due to the different rates of deuterium incorporation. Using the same color coding, the bottom panel shows the incorporation of deuterium by each protein versus time, calculated as described in the materials and methods.

Figure 4

Evolution of isotopic distributions of a peptide fragment (88-102) derived from unmodified IFN-β1a (blue) and NEM-IFN-β1a (red) at different time points of the exchange reaction. The gray trace shows the isotopic distribution of the fully exchanged peptide (HDX end-point).

Figure 5

Levels of deuterium incorporation following 5 min of exchange within segments representing all observed proteolytic fragments derived from unmodified IFN-β1a (blue) and NEM-IFN-β1a (red). Gray bars plot the difference between IFN-β1a and NEM-IFN-β1a. Peptides that exhibited bimodal exchange in the NEM-IFN-β1a sample are indicated with an asterisk.

Figure 6

Segments representing all observed proteolytic fragments of unmodified NEM-IFN-β1a (A) and IFN-β1a (B) colored according to their deuteration level following 5 min of exchange mapped on the 1° and 3° structures of the protein. Color coding reflects flexibility of various segments ranging from high protection levels (blue) to very low protection (red). The bottom diagram (C) shows protein segments where flexibility is changed as a result of alkylation. These are colored by difference in % exchange as indicated by the scale, except for the residues in pink, which exhibited a marked difference in exchange though the fast exchange kinetics in this region prevented an accurate difference calculation. In all 3° structures residues not observed were colored in black. Helical segments are indicated by wavy lines above the amino acid sequence and labeled alphabetically by convention. Cysteine residues involved in a disulfide bond are colored in orange circles, the free (alkylated) cysteine residue is marked with a red pentagon, and the first residue of the CHO chain is marked with a blue hexagon.