Post-thaw aging affects activity of lactate dehydrogenase.
Freeze-thawing is routinely used to study freezing-induced irreversible protein denaturation in the formulation characterization and development of lyophilized proteins. In most cases, the temperature profiles of the samples are not fully monitored during freeze-thawing and therefore, the sample thermal histories are largely unknown. The objective of this study was to develop experimental protocols for the study of isothermal protein degradation using a temperature-step apparatus. Freeze-thaw experiments were performed at a freezing rate of 10 degrees C/min and various thawing rates (0.5-3.3 degrees C/min) using a temperature-step apparatus. In our efforts to design validation studies, we encountered anomalies in the recovered enzyme activity data of an enzyme, lactate dehydrogenase at the end of freeze-thawing. The effect of thawing rate was studied to explain the variability in the data. In addition, post-thaw "aging" of freshly frozen and thawed samples was performed at 5 degrees C to reduce the variability in the recovered enzyme activity. Results from these experiments implicate the use of aging of dilute multimeric enzymes at the end of freeze-thawing to control the variability in enzyme assays.
Bhatnagar
Bakul S
BS
School of Pharmacy, U2092, University of Connecticut, Storrs, CN 06269, USA.
Nehm
Sarah J
SJ
Pikal
Michael J
MJ
Bogner
Robin H
RH
D005615:Freezing; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
D007770:L-Lactate Dehydrogenase