Post-thaw aging affects activity of lactate dehydrogenase

✍ Scribed by Bakul S. Bhatnagar; Sarah J. Nehm; Michael J. Pikal; Robin H. Bogner

• Book ID: 102402929
• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 128 KB
• Volume: 94
• Category: Article
• ISSN: 0022-3549
• DOI: 10.1002/jps.20345

✦ Synopsis

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 108C/min and various thawing rates (0.5-3.38C/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 58C 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.