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 DoEbased 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 largescale F/T.

Reversible Self-Association in Lactate Dehydrogenase during Freeze–Thaw in Buffered Solutions Using Neutron Scattering

“…Due to the limitations, both in analytical techniques and in molecular modeling, the stresses experienced by the proteins during cryo-concentration have not been thoroughly studied. However, in certain cases, molecular dynamic simulation and design of experimental approaches can aid in estimating the degree of protein saturation in the freeze concentrate, gain insights into the mechanism of unfolding and aggregation at the ice–air or ice–freeze concentrate interface, and determine the optimum freezing rates which will result in maximum protein stability during the freeze-thawing processes. , …”

“…However, in certain cases, molecular dynamic simulation and design of experimental approaches can aid in estimating the degree of protein saturation in the freeze concentrate, gain insights into the mechanism of unfolding and aggregation at the ice−air or ice− freeze concentrate interface, and determine the optimum freezing rates which will result in maximum protein stability during the freeze-thawing processes. 10,12 Among the numerous potential degradation pathways, aggregation is one of the most well-known and highly studied phenomena which can occur in diverse protein modalities differing with respect to their conformations and molecular size. Non-native aggregation, conventionally referred to as aggregation, is the formation of oligomeric units as a result of the interactions either due to conformational changes or chemical modifications in the monomeric unit.…”