Investigation of histidine stabilizing effects on LDH during freeze-drying

The objective of this study was to investigate the effect of histidine on the stability of the model protein lactate dehydrogenase (LDH) during freeze-drying. Several parameters were varied including pH of the bulk solution, histidine concentration, and performance of an annealing step during freezing. First, histidine was used as a buffer in the protein formulations and compared with "conventional" potassium phosphate and citrate buffer systems. For this purpose, sucrose or mannitol was used as stabilizers. Second, the possibility of using histidine as both buffer and stabilizer (cryoprotectant and lyoprotectant) in the protein formulations was evaluated with focus on protein stability and the physical state of histidine in the final product, in addition to cake elegance. Protein stability was evaluated both functionally by measuring the activity recovery of the model protein LDH after freeze-drying and structurally by analyzing the protein secondary structure. LDH showed improved stability in histidine buffer in comparison with other buffers. Protein stability and the tendency of histidine to crystallize during freeze-drying were pH dependent. Annealing destabilized LDH and resulted in a decrease of the activity recovery. However, the extent of protein destabilization caused by annealing appears to be also pH dependent.