Conformation and dynamics of biopharmaceuticals: transition of mass spectrometry-based tools from academe to industry

Abstract

Mass spectrometry plays a very visible role in biopharmaceutical industry, although its use in development, characterization, and quality control of protein drugs is mostly limited to the analysis of covalent structure (amino acid sequence and post-translational modifications). Despite the centrality of protein conformation to biological activity, stability, and safety of biopharmaceutical products, the expanding arsenal of mass spectrometry-based methods that are currently available to probe higher order structure and conformational dynamics of biopolymers did not, until recently, enjoy much attention in the industry. This is beginning to change as a result of recent work demonstrating the utility of these experimental tools for various aspects of biopharmaceutical product development and manufacturing. In this work, we use a paradigmatic protein drug interferon beta-1a as an example to illustrate the utility of mass spectrometry as a powerful tool not only to assess the integrity of higher order structure of a protein drug, but also to predict consequences of its degradation at a variety of levels.

Figure 1

Higher order structure (top) and amino acid sequence (bottom) of IFN with elements of secondary structure labeled according to a commonly accepted nomenclature (44). Colored residues are common targets of non-enzymatic or designer PTMs: orange, Cys-17 (alkylation, oxidation, formation of external disulfides, internal disulfide scrambling, replacement with Ser in interferon β1b, PEGylation via thiol-reactive group); yellow, Cys-31 and Cys-141 (disulfide scrambling); cyan, Lys-19 (glycation (86)); magenta, methionine residues (oxidation); and green, Asn-90 (variable glycosylation and deglycosylation).

Figure 2

ESI mass spectra of intact (blue) and NEM-alkylated IFN (red) buffer-exchanged to aqueous 100 mM ammonium acetate prior to MS analysis. The inset shows limited heterogeneity of IFN due to the presence of several glycoforms. Black trace shows an ESI mass spectrum of intact IFN acquired under strongly denaturing conditions (50% methanol, 6% acetic acid).

Figure 3

Evolution of isotopic distributions of peptic fragments [88-102] derived from intact (blue) and NEM-alkylated (red) IFN throughout the course of HDX. The endpoint of the exchange reaction is indicated with a gray trace (isotopic distribution of a fully exchanged peptide). Location of this peptide within the amino acid sequence of IFN is shown on the right. Adapted with permission from (42).

Figure 4

Location of the [88-102] segment (highlighted in red) within the crystal structure of IFN (1AU1, panel A) with respect to Cys-17 (highlighted in orange). Alkylation of Cys-17 inevitably leads to steric clashes within the native structure, which can be removed by unfolding of the helix D containing the [88-102] segment (panel B). Side chains of hydrophobic residues within helix D are sequestered in the protein interior (highlighted in green, panel C), but become exposed to solvent upon unfolding of this structural element (panel D).

Figure 5

A schematic representation of the JAK/STAT pathway activation by IFN initiated by its binding with the receptors IFNAR1 and IFNAR2 (A), adapted from (45); and the proposed sequence of events leading to the assembly of the ternary complex IFNAR2/IFN/IFNAR1 (B).

Figure 6

A: flexibility map of intact IFN generated by mapping the extent of deuterium incorporation in various peptic fragments (following 120 sec. HDX) onto the tertiary structure of the protein. B: regions of the protein exhibiting the difference in backbone protection between the intact and NEM-alkylated forms of IFN. C: receptor binding interfaces of IFN from earlier mutagenesis work (56).

Figure 7

ESI MS of IFN/IFNAR2 interaction in the presence of excess IFN. Note that free IFNAR2 is absent from the spectrum (a reference mass spectrum of free IFNAR2 is shown in gray).

Figure 8

A: ESI MS of IFN/IFNAR1 interaction in the presence of excess IFN. Note that free IFNAR1 is absent from the spectrum (a reference mass spectrum of free IFNAR1 is shown in gray). B: ESI MS of NEM-IFN/IFNAR1 interaction in the presence of excess NEM-IFN. Note that both free NEM-IFN and IFNAR1 are present in the spectrum alongside the NEM-IFN·IFNAR1 complex.

Figure 9

ESI MS of NEM-IFN interaction with IFNAR1 and IFNAR2 in the presence of excess NEM-IFN and IFNAR2. Note that both free IFNAR1 and a binary complex NEM-IFN·IFNAR2 are present in the spectrum alongside the ternary complex. Mixing intact IFN with the two receptors results in complete elimination of either free IFNAR1 or the binary complex IFN·IFNAR2 depending on the relative abundance of each of the three proteins (blue trace).

Figure 10

HDX MS comparability studies of two different preparations of IFN grown in different culture media (A) and NEM-alkylated vs. intact IFN (B). The fractional exchange for each peptide at each HDX time point (plotted in different colors) is represented as a single point in a graph where the x-axis shows the sequential order of each peptic fragment in IFN sequence and the y-axis represents the fraction of exchanged amides for each peptide. To allow for better visualization, the data for the two samples are plotted in opposite directions (the so-called butterfly plot). Differences in total HDX levels (summations of all time points) for each peptide are plotted as vertical bars across the x-axis (the average standard deviation of these values is below 2% of the total exchange level). The vertical lines at the bottom of the graph A are scaled to represent the relative size of each peptic fragment.

Figure 11

Heterogeneity of protein-polymer conjugates exemplified by ESI MS of mono-PEGylated ubiquitin (A) and mono-PEGylated IFN (B). The average molecular weights of the PEG chains are 5 kDa (ubiquitin) and 20 kDa (IFN). Insets show mass spectra of unconjugated proteins.