title: Photolytic Cross-Linking to Probe Protein-Protein and Protein-Matrix Interactions in Lyophilized Powders.
abstract: Protein structure and local environment in lyophilized formulations were probed using high-resolution solid-state photolytic cross-linking 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 cross-linked adducts that were analyzed at the intact protein level and after trypsin digestion. SDA-labeling produced Mb carrying up to five labels, as detected by LC-MS. Following lyophilization and irradiation, cross-linked 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.
authors.affiliation: Department of Industrial and Physical Pharmacy, Purdue University , West Lafayette, Indiana 47907-2091, United States.
authors.forename: Lavanya K
authors.initials: LK
authors.lastname: Iyer
authors.affiliation: Department of Industrial and Physical Pharmacy, Purdue University , West Lafayette, Indiana 47907-2091, United States.
authors.forename: Balakrishnan S
authors.initials: BS
authors.lastname: Moorthy
authors.affiliation: Department of Industrial and Physical Pharmacy, Purdue University , West Lafayette, Indiana 47907-2091, United States.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Topp
mesh_terms: D000818:Animals; D002626:Chemistry, Pharmaceutical; D002853:Chromatography, Liquid / Q000379:methods; D003432:Cross-Linking Reagents / Q000378:metabolism*; D005079:Excipients / Q000737:chemistry*; D005612:Freeze Drying / Q000379:methods*; D019791:Guanidine / Q000737:chemistry; D006736:Horses; D013058:Mass Spectrometry / Q000379:methods; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010782:Photolysis; D011208:Powders / Q000737:chemistry*; D014867:Water / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D005079:Excipients; D009211:Myoglobin; D010446:Peptide Fragments; D011208:Powders; D014867:Water; D019791:Guanidine
keywords: lyophilized formulations; mass spectrometry; myoglobin; photolytic cross-linking; protein−excipient interactions; protein−protein interactions; protein−water interactions; raffinose; succinimidyl 4,4′-azipentanoate