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

Minatovicz, B., University of Connecticut
Chaudhuri, B., University of Connecticut
Bogner, R., University of Connecticut
Sun, L., Regeneron Pharmaceuticals
Fan, T. H., University of Connecticut
Li, J. Q., University of Connecticut
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.