Distribution of Protein Content and Number of Aggregates in Monoclonal Antibody Formulation After Large-Scale Freezing

Cryoconcentration of an in-house IgG(1) and number of aggregates in a formulation containing trehalose were determined in dependence on freezing protocol and volume. Morphology changes of ice crystals depending on cooling rates were captured by optical cryomicroscopy (OCM) images. UV-Vis and affinity chromatography (ALC) was used to determine protein content and size-exclusion chromatography (SEC) for detection of aggregates. Cooling to − 80°C rather than − 20°C is beneficial in avoiding hot spots of high protein concentration. An upscaling of 250 ml to 2 L bottles results in an up to fourfold increase of macroscopic cryoconcentration. There is no direct correlation between number of aggregates and macroscopic cryoconcentration. Aggregate formation of that specific mAb is not caused by macroscopic cryoconcentration but can be directly linked to microscopic cryoconcentration in between the ice dendrites. Slower cooling with set-point and storage temperatures below T(g)’ has proven to be advantageous for the prevention of aggregate formation. We reveal that the subcooling prior to freezing plays a key role in avoiding aggregates. The lower the solution is supercooled the more likely aggregates form. As a consequence, we suggest controlled initiation of the freezing process to avoid large supercooling. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1208/s12249-018-1281-z) contains supplementary material, which is available to authorized users.