Predicting Protein Aggregation during Storage in Lyophilized Solids Using Solid State Amide Hydrogen/Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS)

Abstract

Solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS) was used to assess the conformation of myoglobin (Mb) in lyophilized formulations, and the results correlated with the extent of aggregation during storage. Mb was colyophilized with sucrose (1:1 or 1:8 w/w), mannitol (1:1 w/w), or NaCl (1:1 w/w) or in the absence of excipients. Immediately after lyophilization, samples of each formulation were analyzed by ssHDX-MS and Fourier transform infrared spectroscopy (FTIR) to assess Mb conformation, and by dynamic light scattering (DLS) and size exclusion chromatography (SEC) to determine the extent of aggregation. The remaining samples were then placed on stability at 25 °C and 60% RH or 40 °C and 75% RH for up to 1 year, withdrawn at intervals, and analyzed for aggregate content by SEC and DLS. In ssHDX-MS of samples immediately after lyophilization (t = 0), Mb was less deuterated in solids containing sucrose (1:1 and 1:8 w/w) than in those containing mannitol (1:1 w/w), NaCl (1:1 w/w), or Mb alone. Deuterium uptake kinetics and peptide mass envelopes also indicated greater Mb structural perturbation in mannitol, NaCl, or Mb-alone samples at t = 0. The extent of deuterium incorporation and kinetic parameters related to rapidly and slowly exchanging amide pools (Nfast, Nslow), measured at t = 0, were highly correlated with the extent of aggregation on storage as measured by SEC. In contrast, the extent of aggregation was weakly correlated with FTIR band intensity and peak position measured at t = 0. The results support the use of ssHDX-MS as a formulation screening tool in developing lyophilized protein drug products.

Photolytic Cross-Linking to Probe Protein–Protein and Protein–Matrix Interactions in Lyophilized Powders

“…ssHDX-MS is able to distinguish the effects of different formulation excipients on structure in lyophilized solids 15,16 , and, in a recent study of lyophilized myoglobin formulations, provided significantly higher correlation with aggregation during storage than FTIR 17 . ssHDX-MS is not without its limitations, however.…”

Process and Formulation Effects on Protein Structure in Lyophilized Solids Using Mass Spectrometric Methods

“…Previous ssHDX-MS studies in our lab have shown a correlation between deuterium incorporation in freshly lyophilized samples and aggregation during storage over a year, with greater stability for formulations showing lower deuterium incorporation 29 . It is reasonable to expect a similar correlation for process-induced differences in ssHDX-MS, though extended storage stability studies were not conducted here.…”

“…FTIR spectra were acquired for all lyophilized samples using a Tensor 37 spectrometer (Bruker Optics, Billerica, MA), as described previously 29 . 128 scans were obtained at 4 cm −1 resolution and spectra were processed using OPUS software (v. 6.5, Bruker Optics), by cutting around 1600–1700 cm −1 , smoothing and baseline correcting before obtaining second derivative spectra.…”

“…HPLC-MS (1200 series HPLC, ESI-qTOF 6520, Agilent Technologies, Santa Clara, CA) was used to measure deuterium uptake at the intact level, as described previously 29, 30 . Deuterated samples were reconstituted with 2 mL of ice-cold quench buffer (5 % methanol, 0.2 % formic acid in LC-MS-grade water, pH 2.5) and injected into a refrigerated box housing the HPLC valves, tubing and protein microtrap at ~ 0 °C to reduce back-exchange.…”

Characterization of Aggregation Propensity of a Human Fc-Fusion Protein Therapeutic by Hydrogen/Deuterium Exchange Mass Spectrometry

“…By monitoring protein amide backbone HDX, which depends on the protein local solvent accessibility and hydrogen bonds, one can characterize the solution conformation and dynamics of proteins, including changes due to the external environment [18] (e.g., chemical modifications [19][20][21][22], buffer components [23,24], and the presence of a binding protein/ligand [25,26]). Regarding the characterization of protein aggregation, HDX-MS has shown its potential for our understanding of protein self-association interfaces and aggregation-induced protein conformational changes at the molecular level in either the lyophilized state [27] or the solution state [28][29][30].…”