Freeze concentration during freezing: How does the maximally freeze concentrated solution influence protein stability?

During freeze drying of biologics, a highly viscous freeze concentrate (FC) is formed upon the initial freezing due to the crystallisation of ice. Protein stability in this freeze concentrated phase is not yet well understood, but can decide upon the success of the lyophilisation itself. Protein stability may be high below the Tg' as it is typically the case during primary drying but decreases above Tg', e.g. during annealing or during aggressive freeze drying above Tg' in presence of a crystalline bulking agent or, beyond freeze drying, during storage of frozen bulk. Different FCs containing monoclonal antibody, sucrose, histidine or phosphate buffer and sodium chloride were prepared via partial freeze drying and analysed for protein aggregation. No solute crystallisation is visible and the systems are vitrifying during cooling. Increasing sugar or buffer concentration showed positive effects on either melting and aggregation temperature or on protein self-interaction as indicated by A2 values. Protein integrity in the FC was not affected by 1 month storage at temperatures above Tg'. Thus, upconcentration of solutes during freezing does not negatively impact protein stability. Exceeding Tg' during freeze drying e.g. upon annealing or, intentionally or unintentionally, during primary drying does not lead to protein aggregation.