(240b) Design of Experiments Reveals Critical Parameters for Bulk Freeze-Thaw of Lactate Dehydrogenase

Purpose: Nearly half of biotherapeutics bulk drug substance are stored frozen. Storage in a frozen state provides operational flexibility for fill-finish of drug products and extends the shelf life of protein solutions. However, the freezing and thawing (F/T) processes themselves can destabilize the complex structure of proteins. Formulations and processes, therefore, must be designed to be protective against potential F/T-induced stresses to proteins. The overall goal of this research project was to investigate the impact of F/T process parameters on the stability of a model protein under pharmaceutically relevant conditions at a large scale.

Methods: A response surface model was designed and the results were evaluated using JMP® 13 (SAS Institute, Cary, NC) to investigate how F/T process parameters affect the lactate dehydrogenase (LDH) protein stability in 1-L polycarbonate containers. The LDH protein solutions in histidine buffer were frozen at -20°C, -50°C, or -80°C and thawed at room temperature after 24 hours. The F/T cycles applied either single or full loading configuration, i.e., a total of nine bottles arranged in a 3x3 array. The following process parameters were applied: solution fill volume (50%, 70%, and 90%), F/T loading distance (1 cm, 5 cm, and 10 cm), and the thawing method (with or without forced air flow). The F/T rate was monitored using type-T thermocouples inserted at different axial and radial locations inside the solutions. After each F/T cycle, a sample withdrawn from the homogenized bulk was analyzed for protein concentration, LDH tetramer purity, and LDH enzymatic activity.

Results: Analysis from 40 experimental runs indicate that increasing the loading distance and lowering the solution fill volume to 50% significantly increased the freezing rate with 95% confidence interval. Furthermore, decreasing the solution fill volume and applying a forced air flow at 102 fpm during thawing significantly increased the thawing rate. The F/T runs performed at faster F/T rates also correlated with improved protein stability. F/T cycles performed at faster rates resulted in enhanced retention of the protein enzymatic activity and native structures percentage. For the faster F/T processes completed in less than 6.0 hours of freezing and 8.7 hours of thawing, the percentage of LDH initial activity was greater or equal to 97.4%, correlating with approximately 99.9% of LDH native structures. However, longer F/T processes requiring more than 9.0 hours of freezing and 25.5 hours of thawing resulted in a poor protein stability: less than 70.7% of the LDH initial activity and approximately 98.2% of LDH native structures.

Conclusions: This study confirms the feasibility of a faster freezing rate and forced-air thawing procedure to enhance the stability of proteins exposed to F/T unit operations. Moreover, the data obtained provided a mechanistic understanding of possible stresses arising during the process and their ultimate effect on the protein stability. Overall, the data obtained shed light on how F/T process parameters impact on bulk protein stability and can be applied to establish F/T process guidelines.