Localized Hydration in Lyophilized Myoglobin by Hydrogen–Deuterium Exchange Mass Spectrometry. 1. Exchange Mapping

Sophocleous, Andreas M.; Zhang, Jun; Topp, Elizabeth M.

doi:10.1021/mp3000088

Cited by 36 publications

(63 citation statements)

References 51 publications

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“…20,21 For intact Mb, peak width steadily increased with time for formulations MbB, MbC, and MbD but showed smaller increases in MbA and MbE (Figure 6A). To establish a level of deuterium uptake for comparing different formulations, peak width was plotted against percentage deuterium uptake (Figure 6B), and 15% deuterium incorporation was selected for comparison.…”

Section: Resultsmentioning

confidence: 96%

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

et al. 2014

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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 The remaining samples were then placed on stability at 25 °C and 60% RH or 40 °C and 75% RH for up to 1 year, withdrawn at intervals, and analyzed for aggregate content by SEC and DLS.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.

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Section: Resultsmentioning

confidence: 96%

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

et al. 2014

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

2015

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Protein structure and local environment in lyophilized formulations were probed using high-resolution solid-state photolytic crosslinking with mass spectrometric analysis (ssPC-MS). In order to characterize structure and microenvironment, protein-protein, protein-excipient and protein-water interactions in lyophilized powders were identified. Myoglobin (Mb) was derivatized in solution with the heterobifunctional probe succinimidyl 4,4’-azipentanoate (SDA) and the structural integrity of the labeled protein (Mb-SDA) confirmed using CD spectroscopy and liquid chromatography / mass spectrometry (LC-MS). Mb-SDA was then formulated with and without excipients (raffinose, guanidine hydrochloride (Gdn HCl)) and lyophilized. The freeze-dried powder was irradiated with ultraviolet light at 365 nm for 30 min to produce crosslinked adducts that were analyzed at the intact protein level and after trypsin digestion. SDA-labeling produced Mb carrying up to 5 labels, as detected by LC-MS. Following lyophilization and irradiation, crosslinked peptide-peptide, peptide-water and peptide-raffinose adducts were detected. The exposure of Mb side chains to the matrix was quantified based on the number of different peptide-peptide, peptide-water and peptide-excipient adducts detected. In the absence of excipients, peptide-peptide adducts involving the CD, DE and EF loops and helix H were common. In the raffinose formulation, peptide-peptide adducts were more distributed throughout the molecule. The Gdn HCl formulation showed more protein-protein and protein-water adducts than the other formulations, consistent with protein unfolding and increased matrix interactions. The results demonstrate that ssPC-MS can be used to distinguish excipient effects and characterize the local protein environment in lyophilized formulations with high resolution.

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“…5). Since sorption and diffusion of D 2 O from the vapor phase into the solid must precede the hydrogen-deuterium exchange reaction in the solid state, the observed ssHDX rate can be affected by the rate and extent of sorption 30 . To determine the effect of D 2 O sorption on ssHDX kinetics, moisture uptake was measured using TGA to simulate D 2 O uptake at 43 % humidity.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Methodsmentioning

confidence: 99%

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

Iyer

Sacha

Moorthy

et al. 2016

Journal of Pharmaceutical Sciences

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Myoglobin (Mb) was lyophilized in the absence (Mb-A) and presence (Mb-B) of sucrose in a pilot-scale lyophilizer with or without controlled ice nucleation. Cake morphology was characterized using scanning electron microscopy (SEM) and changes in protein structure were monitored using solid-state Fourier-transform infrared spectroscopy (ssFTIR), solid-state hydrogen-deuterium exchange-mass spectrometry (ssHDX-MS) and solid-state photolytic labeling-mass spectrometry (ssPL-MS). The results showed greater variability in nucleation temperature and irregular cake structure for formulations lyophilized without controlled nucleation. Controlled nucleation resulted in nucleation at ~ −5 °C and uniform cake structure. Formulations containing sucrose showed better retention of protein structure by all measures than formulations without sucrose. Samples lyophilized with and without controlled nucleation were similar by most measures of protein structure. However, ssPL-MS showed the greatest pLeu incorporation and more labeled regions for Mb-B lyophilized with controlled nucleation. The data support the use of ssHDX-MS and ssPL-MS to study formulation and process-induced conformational changes in lyophilized proteins.

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Localized Hydration in Lyophilized Myoglobin by Hydrogen–Deuterium Exchange Mass Spectrometry. 1. Exchange Mapping

Cited by 36 publications

References 51 publications

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

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

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

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

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