Biopharmaceuticals are one of the fastest growing areas within the pharmaceutical industry. Protein drugs are widely used to treat several diseases, such as chronic viral hepatitis, rheumatoid arthritis. For protein molecules that are not stable in aqueous media, freeze-drying represents the method of choice in the manufacture of stable biopharmaceutical products. Nowadays, the most commonly used crystalline bulking agents are mannitol and glycine, but they require high bulking agent to stabilizer ratios to ensure their crystallization during the freeze-drying process. A few amino acids (AA) are known for their crystallization thendency upon freeze-drying and could be a potential alternative bulking agents in low AA to sucrose ratios.
Within the master thesis, highly concentrated protein formulations (monoclonal antibodies) for subcutaneous application with different AA as bulking agents were freeze-dried. Firstly the formulations of different mAbs concentration without bulking agents were freeze-dried under aggressive freeze-drying cycle. Hypothesis that collapse in mAbs concentration correlated, which means that high concentrated formulations could be freeze-dried under more aggressive conditions that low concentrated formulations was accepted. Formulations were analyzed before and after different aggressive cycles and after 1 and 3 months stability on 40 °C and 75 % relative humidity by appropriate analythical methods where cake appearance, reconstitution time, thermal propertis of formulations to be freeze-dried were evaluated. Stability of a monoclonal antibody was investigated with size exclusion chromatography. The potential AA as bulking acents in freeze-dried highly concentrated monoclonal antibody sformulations for subcutaneous application were selected based on literature and results of conventional drying, where formulations with monoclonal antibody in concentration of 90 mg/ml and seven different AA as bulking agents in different AA to sucrose ratios (i.e. 1 : 15, 1 : 7 and 1 : 3) were freeze-dried. Different aggressive cycles by varying primary drying conditions were used for low concentrated monoclonal antibody formulations with the most suitable AA (isoleucine and phenylalanine) in different AA to sucrose ratios. Based on cake appearance and reconstitution time the optimal AA to sucrose ratio was selected as bulking agents in highly concentrated protein formulations that were freeze-dried. Those AA to sucrose ratios were: Phenylalanine : sucrose = 1 : 4 and Isoleucine : sucrose = 1 : 4. Hypothesis that mannitol as bulking agents enables freeze-drying under aggressive freeze-dried cycles at mannitol to sucrose ratio 2 : 1, and that AA, such as phenylalanine and isoleusine can be used as alternative bulking agents at lower bulking agent to sucrose ratios, was accepted.
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