Effect of process conditions on recovery of protein activity after freezing and freeze-drying.
The objective of this research was to gain a better understanding of the degree to which recovery of activity of model proteins after freeze-drying can be maximized by manipulation of freeze-dry process conditions in the absence of protective solutes. Catalase, beta-galactosidase and lactate dehydrogenase (LDH) were used as model proteins. All of the three proteins exhibited a concentration-dependent loss of activity after freezing, with significantly higher recovery at higher concentration. The freezing method and the type of buffer were also important, with sodium phosphate buffer and freezing by immersion of vials in liquid nitrogen associated with the lowest recovery of activity. Differential scanning calorimetry was predictive of the onset of collapse during freeze-drying only for beta-galactosidase. For the other proteins, either no Tg' transition was observed, or the apparent glass transition did not correlate with the microscopically-observed collapse temperature. The time course of activity loss for beta-galactosidase and LDH was compared during freeze-drying under conditions which produced collapse of the dried matrix and conditions which produced retention of microstructure in the dried solid. Recovery of activity decreased continuously during primary drying, with no sharp drop in recovery of activity associated with the onset of collapse. The most important drying process variable affecting recovery of activity was residual moisture level, with a dramatic drop in activity recovery associated with residual moisture levels less than about 10%.
Jiang
S
S
Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, IN 47907, USA.
Nail
S L
SL
D000818:Animals; D001236:Aspergillus oryzae / Q000201:enzymology; D002374:Catalase / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D005612:Freeze Drying / Q000379:methods*; D005615:Freezing; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008855:Microscopy, Electron, Scanning; D018482:Muscle, Skeletal / Q000201:enzymology; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D013696:Temperature; D001616:beta-Galactosidase / Q000737:chemistry / Q000378:metabolism
D011506:Proteins; D007770:L-Lactate Dehydrogenase; D002374:Catalase; D001616:beta-Galactosidase