Critical process and formulation factors in the freezing of lactate dehydrogenase

Date of Completion

January 2006


Health Sciences, Pharmacology|Chemistry, Pharmaceutical




Therapeutic proteins are subjected to routine freeze-thaw studies as a part of preformulation and formulation development of lyophilized proteins. The overall objective of this research was to determine the individual contributions of solute concentration, temperature, and time, on the process of irreversible protein denaturation during freezing. ^ A temperature-step approach method was developed and validated to study isothermal degradation of lactate dehydrogenase (LDH) during freezing in the absence and presence of stabilizers, hydroxyethyl starch (HES) or sucrose. In addition to a rapid cooling followed by seeding with ice to control ice crystallization and thawing at the end of an experiment, a post-thaw aging step was employed to control the variability of the enzyme activity assay. A thawing rate of 2°C/min followed by 15 minutes aging was established as a part of the temperature-step protocol for isothermal stability studies of frozen LDH. ^ A combined experimental and theoretical approach was developed to reliably determine the extent of freeze concentration in frozen sucrose solutions using differential scanning calorimetry (DSC). When compared to the values for the offset temperature, Toffset, and the peak temperature, Tpeak , at a finite scan rate, values for Toffset extrapolated to a zero scan rate showed a far better agreement with the literature data on binary melting points from the International Critical Tables (ICT). Good agreement was observed between calculations of the freeze concentrate composition using the liquidus curve generated from the extrapolated Toffset data and those obtained from the ICT unlike those obtained using the T offset and Tpeak data at a finite heating rate.^ A faster degradation of LDH was observed at lower subzero temperatures in the absence and presence of HES and sucrose using the temperature-step methodology. The data further implicate ice as the destabilizing stress. Stability studies on LDH using the temperature-step methodology provide concrete experimental evidence that ice formation is critical to LDH destabilization during freezing, and that freeze concentration is not responsible for activity losses in frozen solutions of LDH:sucrose:citrate buffer. (Abstract shortened by UMI.)^