Protein quality during freezing or thawing is governed by several well known chemical and physical phenomena linked to the
way that the protein is frozen or thawed.3–5 It is mandatory to characterize as much as possible the impact that these different process parameters may have on the protein
quality. Traditional freeze–thaw approaches are based on stability studies, carried out during process development, which
use simple and uncontrolled procedures to freeze and thaw the sample protein. These small-scale freeze–thaw systems are a
simple reduction of the container used for freezing the sample at process scale (usually the scale down is based on the surface
or volume ratio, which is kept constant between scales).4,5 Therefore, the freezing and thawing velocities (probably the two main parameters affecting protein quality) are clearly faster
compared with a larger scale that involves larger volumes and therefore larger freezing path lengths.4–9
In this study, we have shown the possibility of characterizing the stability of the protein over a range of process parameters
typical of a manufacturing scale. Scale-down studies were designed to capture a fully controlled freeze–thaw scenario when
short freezing and thawing times, as well as very long freezing and thawing times (typical of uncontrolled freeze–thaw large-scale
systems) can be generated. After the design space was created, it was easier to define the most appropriate freeze–thaw solution
to be used at manufacturing scale.
Finally, the same freeze–thaw QbD approach can be applied to formulation studies where small scale-down models, representative
of the manufacturing scale, are needed.2,4–5 In this case, factorial screening of different excipients stabilizing the molecule can be carried out using the freezing
and thawing times as block parameters, including the direct effect of the freezing or thawing procedure in the formulation
study. Formulation development studies also are critical for protein therapeutics development and can clearly benefit from
this freeze–thaw QbD approach. Indeed, in process development, intermediates and final drug substance formulation are defined
using buffer and additives screening approaches. To select the best candidates, storage and freeze–thaw has to be taken into
account. In this context, the Celsius S3 concept can be used to test several formulations in parallel, allowing good throughput,
good knowledge of the freeze–thaw procedure, and good scalability for the transfer to the production site.
Xavier Le Saout is an assistant scientist DSP & Preformulation, biotech process sciences, Hervé Broly is a vice president of the Center of Expertise Manufacturing and head of biotech process sciences, and Matteo D. Costioli is a bioprocess and innovation DSP specialist, Centre of Expertise Manufacturing, all at Merck Serono SA, CH, Switzerland.
Eric Youssef is an application specialist, fluid management Technologies, Sartorius Stedim Biotech SA.
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