Use of a Design of Experiments (DoE) Approach to Optimize Large-Scale Freeze-Thaw Process of Biologics.

Abstract

Large volumes of protein solutions are commonly stored in a frozen state before further drug product fill and finish. This study aimed to establish a design space to perform large-scale freeze-thaw (F/T) processes of biotherapeutics without inducing protein destabilization. A response surface model was designed to evaluate the following main factors and interactions: fill volume of the protein solution in 1-L containers, distance among nine containers during both F/T, freezer set temperature, and a novel forced air flow methodology during thawing. The analysis from 46 experimental runs indicated over 4-fold increase in the freezing rate by lowering the freezing temperature from -20 to -80°C, and the forced air flow at 98 fpm doubled the thawing rate. Furthermore, multivariate linear regression modeling revealed the significant impact of all main factors investigated on lactate dehydrogenase (LDH) quality attributes. The factor that most strongly affected the retention of LDH activity was the loading distance: ≥ 5 cm among containers positively affected the LDH activity response in 50.6%. The factor that most strongly retained the LDH tetramers was the set freezer temperature towards the lower range of -80°C (2.2% higher tetramer retention compared to -20°C freezing, due to faster freezing rate). In summary, this DoE-based systematic analysis increased F/T process understanding at large scale, identified critical F/T process parameters, and confirmed the feasibility of applying faster freezing and forced air thawing procedures to maintain the stability of LDH solutions subject to large-scale F/T.