Evidence of partial unfolding of proteins at the ice/freeze-concentrate interface by infrared microscopy.
The goal of this research was to use infrared spectroscopy in combination with a freeze drying stage to gain a better understanding of the mechanism of loss of protein integrity due to the stresses associated with freezing. Infrared spectra were collected in triplicate for the interstitial space between ice crystals and through ice crystals in a partially frozen system. Spectra were collected for lactate dehydrogenase (LDH) and human immune globulin (IgG) both in the presence and absence of an added surfactant (polysorbate 80). Spectra collected in the interstitial space, distant from the surface of ice crystals, were very similar to spectra collected from the initial solution regardless of the presence of a surfactant. Spectra collected through ice crystals, without added surfactant, were significantly different than spectra collected from the initial solution. An increase in bands characteristic of intermolecular beta-sheet structures (main component of aggregates) were present in these spectra. The presence of surfactant in both protein formulations resulted in a decrease in intermolecular beta-sheet signals in spectra of the proteins on the ice crystal surface. Additionally, much of the native state structure of LDH initially lost on the surface of ice crystals returned when surfactant was added to the formulation prior to freezing.
Schwegman
J Jeff
JJ
Department of Industrial and Physical Pharmacy, School of Pharmacy, Purdue University, 575 W. Stadium Mall Dr., West Lafayette, Indiana 47907, USA.
Carpenter
John F
JF
Nail
Steven L
SL
D000818:Animals; D005079:Excipients / Q000737:chemistry; D005612:Freeze Drying; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis* / Q000737:chemistry; D007770:L-Lactate Dehydrogenase / Q000032:analysis* / Q000737:chemistry; D011136:Polysorbates / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D011817:Rabbits; D017550:Spectroscopy, Fourier Transform Infrared / Q000295:instrumentation / Q000379:methods*
D005079:Excipients; D007074:Immunoglobulin G; D011136:Polysorbates; D007770:L-Lactate Dehydrogenase