Effects of Excipients on Protein Conformation in Lyophilized Solids by Hydrogen/Deuterium Exchange Mass Spectrometry

Li, Yunsong; Williams, Todd D.; Topp, Elizabeth M.

doi:10.1007/s11095-007-9365-6

Cited by 43 publications

(36 citation statements)

References 22 publications

(30 reference statements)

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“…Effects of excipients on protein conformation in lyophilized solids have been studied using hydrogen/deuterium exchange with mass spectrometry analysis [74]. The combination of hydrogen/deuterium exchange with mass spectrometry can provide useful information about protein structure in amorphous solids with the aid of enzymatic digestion which is capable of providing site-specific information about protein interactions with excipients [75].…”

Section: Mass Spectrometrymentioning

confidence: 99%

Development of Stable Lyophilized Protein Drug Products

Remmele¹,

Krishnan²,

Callahan³

2012

CPB

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Freeze drying, or lyophilization is widely used for biopharmaceuticals to improve the long term storage stability of labile molecules. This review examines general theory and practice of rational lyophilization of biopharmaceuticals. Formulation development involving the selection of appropriate excipients, their associated physical properties, and mechanism of action in achieving a stable drug product are primary considerations for a successful lyophilization program. There are several parameters considered critical on the basis of their relationship to lyophilization cycle development and protein product stability. This along with the importance of analytical methods to provide insight toward understanding properties of drug product stability and cake structure are discussed. Also, aspects of instability found in lyophilized biopharmaceutical products, their degradation pathways and control are elucidated. Finally, container-closure requirements and drug product handling are described in context of the caveats to avoid compromising drug product quality.

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Section: Mass Spectrometrymentioning

confidence: 99%

Development of Stable Lyophilized Protein Drug Products

Remmele¹,

Krishnan²,

Callahan³

2012

CPB

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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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“…HX-MS has been widely used in solution phase for analysis of protein conformation and dynamics (23)(24)(25), as well as structural changes upon a variety of state transitions, such as folding/unfolding (26), ligand binding (27), and self-association (28,29). Further, Li et al carried out hydrogen/deuterium exchange in protein powders to measure the effects of lyophilization and excipients on protein structure (30).…”

Section: Introductionmentioning

confidence: 99%