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

Protein freeze-thawing is frequently used to stabilize and store recombinantly produced proteins after different unit operations in upstream and downstream processing. However, freeze-thawing is often accompanied by product damage and, hence, loss of product. Different effects are responsible, including cold denaturation, aggregation effects, which are caused by inhomogeneities in protein concentration, as well as pH and buffer ingredients, especially during the freeze cycle. In this study, we tested a commercially available small-scale protein freezing unit using immunoglobin G (IgG) as monoclonal antibody in a typical formulation buffer containing sodium phosphate, sodium chloride, and Tween 80. Different freezing rates were used respectively, and the product quality was tested in the frozen sample. Spatially resolved tests for protein concentration, pH, conductivity, and aggregation revealed high spatial differences in the frozen sample. Usage of slow freezing rates revealed high inhomogeneities in terms of buffer salt and protein distribution, while fast rates led to far lower spatial differences. These protein and buffer salt inhomogeneities can be reliably monitored using straight forward analytics, like conductivity and photometric total protein concentration measurements, reducing the need for HPLC analytics in screening experiments. Summarizing, fast freezing using steep rates shows promising results concerning homogeneity of the final frozen product and inhibits increased product aggregation.