A specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody

The selection of the appropriate excipient and the amount of excipient requ ired to achieve a 2-year shelf-life is often done by using iso-osmotic conc entrations of excipients such as sugars (e.g., 275 mM sucrose or trehalose) and salts. Excipients used for freeze-dried protein formulations are selec ted for their ability to prevent protein denaturation during the freeze-dry ing process as well as during storage. Using a model recombinant humanized monoclonal antibody (rhuMAb HERS), we assessed the impact of lyoprotectants , sucrose, and trehalose, alone or in combination with mannitol, on the sto rage stability at 40 degreesC. Molar ratios of sugar to protein were used, and the stability of the resulting lyophilized formulations was determined by measuring aggregation, deamidation, and oxidation of the reconstituted p rotein and by infrared (IR) spectroscopy (secondary structure) of the dried protein. A 360:1 molar ratio of lyoprotectant to protein was required for storage stability of the protein, and the sugar concentration was 3-4-fold below the iso-osmotic concentration typically used in formulations. Formula tions with combinations of sucrose (20 mM) or trehalose (20 mM) and mannito l (40 mM) had comparable stability to those with sucrose or trehalose alone at 60 mM concentration. A formulation with 60 mM mannitol alone provided s lightly less protection during storage than 60 mM sucrose or trehalose. The disaccharide/mannitol formulations also inhibited deamidation during stora ge to a greater extent than the lyoprotectant formulations alone. The reduc tion in aggregation and deamidation during storage correlated directly with inhibition of unfolding during lyophilization, as assessed by IR spectrosc opy. Thus, it appears that the protein must be retained in its native-like state during freeze-drying to assure storage stability in the dried solid. Long-term studies (23-54 months) performed at 40 degreesC revealed that the appropriate molar ratio of sugar to protein stabilized against aggregation and deamidation for up to 33 months. Therefore, long-term storage at room temperature or above may be achieved by proper selection of the molar ratio and sugar mixture. Overall, a specific sugar/protein molar ratio was suffi cient to provide storage stability of rhuMAb HER2. (C) 2001 Wiley-Liss, Inc .