University of Limerick
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Effect of lyophilizate collapse on the stability of protein biopharmaceuticals

Version 2 2022-05-24, 10:24
Version 1 2022-05-24, 07:02
posted on 2011-12-09, 15:51 authored by Timothy McCoy
In recent years, freeze drying of protein preparations above the collapse temperature of amorphous or partially amorphous formulations has been the subject of great interest (Leukel et al. 1998. Effects of formulation and process variables on the aggregation of interleukin-6 (IL- 6) after lyophilization and on storage, Pharm. Dev. Technol., 3(3): 337-346; Pikal, M.J., Shah,S. 1990. The collapse temperature in freeze-drying: dependence on measurement methodology and rate of water removal from the glassy phase. Int. J. Pharm., 62: 165-168; Wang, D.Q. et al.,2003. Effect of collapse on the sability of freeze-dried recombinant factor VIII and α- amylase. J. Pharm. Sci., 93(5): 1253-1263; Passot, S. et al. 2007. Effect of product temperature during primary drying on the long-term satability of lyophilized proteins. pharm. dev. Technol., 12(6): 543-533; Schersch K. et. al. 2010. Systematic Investigation of the effect of lyophilizate collapse on pharmaceutically relevant proteins I: stability after freeze drying. J. Pharm. Sci., 99(5): 2256-2277). It is understood that freeze drying in this way would generally result in shorter freeze drying cycles and therefore significant savings in operating costs. Freeze drying protein preparations above and below the critical temperature was assessed using two model proteins made up of either a mannitol or glycine based formulation in the presence of a variety of other excipients, such as sucrose and NaCl. All bulking agents were crystallized using an annealing step during freezing, as crystalline bulking agents are known to act as structural supports in lyophilized cake during the primary drying step (Shalaev, E. Y.; Franks, F. 1996. Changes in the physical state of model mixtures during freezing and drying: Impact on product quality. Cryobiology. 33: 14-26 ). Protein X (150kDa proprietary biopharmaceutical) was lyophilized using only a mannitol based formulation while Protein B (Fraction V BSA, 66kDa) was lyophilized in conjunction with one of four formulations which were either mannitol or glycine based. It was found that freeze drying above the critical temperature resulted in a maximum 7 fold improvement in the degradation (aggregation) rate of BSA when freeze dried in a glycine: sucrose (4%w/v :1%w/v) based buffer and stored for 6 months at 25°C and 60% relative humidity or at 40°C and 75% relative humidity. Other formulations provided varied results and the improvement in protein degradation seemed to be a function of excipient choice when dried below or above the critical temperature of the formulation. Formulations with mannitol: sucrose (4%w/v: 1%w/v) and NaCl seemed to experience improved degradation rates when dried above collapse than when using mannitol alone. Proprietary protein X with a mannitol: sucrose (4%w/v: 1%w/v) based formulation showed comparable degradation (aggregation) rates when dried below and above collapse. It was found that choice of excipients was a key factor in the ability of a protein formulation to dry above the collapse temperature without negatively affecting stability. Overall, the use of glycine: sucrose (4%w/v: 1%w/v) combination as excipients resulted in improved protein stability when dried above the collapse temperature and the presence of sodium chloride also was found to be a factor.



  • Master (Research)

First supervisor

Pembroke, Tony J.

Second supervisor

McMonagle, Seamus





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