(200ag) Effect of Process Parameters on Stability of Lactate Dehydrogenase during Bulk Freeze-Thaw

Nearly half of the commercial biotherapeutics are stored in frozen state. Storage in the frozen state provides operational flexibility during the drug product fill-finish, and extends the protein solution shelf life. However, the freezing and thawing (F/T) processes themselves can destabilize the complex structure of proteins. Cold-denaturation, osmotic stress, and ice-interfacial stress are inevitable consequences of freezing. Formulations and processes, therefore, must be designed to be protective against all these freezing-induced stresses. Hence, 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 (1 L polycarbonate bottle), through a DoE approach. To properly uncover how process parameters affect the response, i.e., the protein stability after F/T, a response surface model was designed and results evaluated using JMP® software version 12.1.0 (SAS Institute, Cary, NC). 1 L square polycarbonate bottles containing the protein solution of 10 µg/mL lactate dehydrogenase (LDH) in 20 mM histidine buffer at pH 7.0 were frozen at -50°C. All F/T runs applied a full loading configuration, i.e., total of nine bottles in a 3x3 array configuration. The following parameters were varied during the study: solution fill volume (50 %, 70 %, and 90 %), thawing rates (thawing was performed at room temperature with or without forced air flow, inducing either a fast or slow thawing rate, respectively), and F/T loading distance (containers were placed at 1, 4, or 10 cm apart during both F/T). Moreover, the F/T rate was monitored using type T thermocouples inserted at different axial and radial locations inside the solutions. The stability of the model protein formulated solutions was evaluated after each F/T cycle. Samples were analyzed by size-exclusion chromatography (SE-UPLC), and FlowCam® to study the effect of F/T process variables on the tetramer purity and sub visible particle formation, respectively. Moreover, protein concentration by UV-spectroscopy and LDH enzymatic activity bioassay analysis was also performed. Analysis from 22 experimental runs indicated the correlation between the required time to complete the F/T process, the distance among the containers, and the fill volume. The array with the smallest separation distance and higher fill volume required the longest time to both F/T processes to complete. In addition, a direct correlation was observed between the F/T rate and protein stability: process parameters yielding faster F/T rates showed the highest LDH stability; increased stability was observed as an increased enzyme activity and percentage of protein native structure. This study confirms the feasibility of a faster freezing rate and forced-air procedure during thawing to enhance the stability of proteins exposed to F/T unit operations. Data obtained may lead to a more thorough understanding of how F/T critical process parameters impact on protein stability. Also, the obtained results can be applied to establish optimal manufacturing conditions, to generate F/T process guidelines, and to guide the evaluation of novel bulk storage technologies.