Effects of excipients on protein conformation in lyophilized solids by hydrogen/deuterium exchange mass spectrometry.
OBJECTIVE
Excipients are added to lyophilized protein drug formulations to protect the protein during processing and storage, but the mechanisms are poorly understood. Here, hydrogen/deuterium (H/D) exchange with mass spectrometry was used to assess protein conformation and excipient interactions in lyophilized solids.


METHODS
Calmodulin (CaM, 17 kD) was co-lyophilized with carbohydrate excipients (sucrose, mannitol, trehalose, raffinose, dextran 5,000, dextran 12,000) or guanidine hydrochloride (negative control) and exposed to D2O vapor at 33% RH and RT. Samples were then dissolved and analyzed by mass spectrometry (+ESI/MS). Peptic digestion provided additional, site-specific information on H/D exchange. Solids were further characterized by powder x-ray diffraction (PXRD), differential scanning calorimetry (DSC), infrared spectroscopy (FTIR) and water vapor sorption.


RESULTS
Excipients protected CaM from H/D exchange, increasing in the order guanidine hydrochloride < no excipient, mannitol < dextran 5,000, dextran 12,000 < sucrose < raffinose < trehalose. Effects were exerted primarily in the protein's alpha-helical segments.


CONCLUSIONS
The effects of carbohydrate excipients on protein conformation in lyophilized solids are not exhibited uniformly along the protein sequence, but instead are exerted in a site-specific manner. The results also demonstrate the utility of H/D exchange with ESI/MS for protein structure characterization in lyophilized samples.
Li
Yunsong
Y
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, 66047, USA.
Williams
Todd D
TD
Topp
Elizabeth M
EM
D000595:Amino Acid Sequence; D002626:Chemistry, Pharmaceutical; D003903:Deuterium; D005079:Excipients / Q000737:chemistry*; D005612:Freeze Drying; D006859:Hydrogen; D008969:Molecular Sequence Data; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared
D005079:Excipients; D006859:Hydrogen; D003903:Deuterium