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

Abstract

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.

Spatially Resolved Effects of Protein Freeze-Thawing in a Small-Scale Model Using Monoclonal Antibodies

“…These changes are a result of the temperature dependence of the pH value and phosphate crystallization [24], which may have also effects on the titer/conductivity relation. Furthermore, applications of surface microelectrodes in the solid ice state may be reasonable to get high local information about protein concentrations and especially enrichment hot spots as shown in literature [21,31]. Bradford results are very similar to the HPLC titer.…”

“…Especially in phosphate-based buffers, this crystallization effect results in high deviations from the set pH upon freezing. All these effects are considered to affect protein stability and promote aggregation of proteins during freezing [8,[21][22][23][24][25].…”

Interfacial Stress and Container Failure During Freezing of Bulk Protein Solutions Can Be Prevented by Local Heating

“…Freezing is typically slower and less controlled, and hydrostatic pressure can build up causing the bottles to deform or rupture (6). Some of these adversities are associated with the cooling of the liquid phase at the air-liquid interface, which causes the top of the liquid to freeze first, forming an ice crust, enclosing the remaining liquid phase (6,7). As the enclosed liquid freezes, the liquid phase is pushed through the ice crust towards the air interface, creating an ice mound (7,8).…”

“…Some of these adversities are associated with the cooling of the liquid phase at the air-liquid interface, which causes the top of the liquid to freeze first, forming an ice crust, enclosing the remaining liquid phase (6,7). As the enclosed liquid freezes, the liquid phase is pushed through the ice crust towards the air interface, creating an ice mound (7,8). It has been shown that this ice mound is almost pure ice and rich in entrapped air bubbles, and therefore, it is a region of high interfacial stress for proteins, which has been correlated with higher aggregation of monoclonal antibodies (7).…”

“…As the enclosed liquid freezes, the liquid phase is pushed through the ice crust towards the air interface, creating an ice mound (7,8). It has been shown that this ice mound is almost pure ice and rich in entrapped air bubbles, and therefore, it is a region of high interfacial stress for proteins, which has been correlated with higher aggregation of monoclonal antibodies (7).…”