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Zippelius, Ralf (2002): Untersuchungen zum Einfrier- und Auftauverhalten pharmazeutischer Humanproteinlösungen im Großmaßstab. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

The purpose of the study was to determin the major stress factors during large-scale freezing of pharmaceutical protein solutions. The stability of four protein bulk solutions during several freeze-thaw cycles was studied, in order to characterise two different large scale freezing systems and identify the specific stress factors. In the third part of the study, a stable formulation for a bulk protein solution during large scale freezing could be developed. The stability of a monoclonal antibody (Daclizumab) could be obtained by the addition of disaccharides or the replacement of the existing buffer media. Results and Conclusions The KVS-System is a completely new system for freezing and thawing of therapeutic protein solution in industrial scale. The Systems provides the possibility to freeze and thaw bulk solutions very quickly. The results showed, that freezing processes in the KVS-System may be associated with an increased risk of protein loss. With an increasing volume of bulk solution cumulative stress factors were observed: A volume of 15 to 20 % of the bulk solution is freezing at a significantly higher concentration of protein, because of the cryoconcentration phenomena. Additionally, considerable interactions of the solution with the headspace takes place, leading in some cases to interface induced aggregation. Protein loss (precipitation) can be eliminated by optimization of the bulk formulation. The results suggest, that precipitation is a pH-dependent phenomenon. After optimization of the bulk formulation, a high stability of the protein could be demonstrated for all tested bulk substances. The stability of four proteins during freezing in the Integrated Biosystems® CryoWedge was studied. The amount of precipitated protein during freezing in the CryoWedge was significantly lower than in the KVS-System. The dimension of cryoconentration during freezing processes in the CryoWedge was reduced in comparison to the KVS-System. But within a small worste case area (0.5 %), considerable cryoconcentration could be detected as well. The results from the preformulation study of a monoclonal antibody point out, that detremental effects of the buffer media, e. g. pH-shift, cannot be settled by the use of the CryoWedge. Studies on the stability of the monoclonal antibody Daclizumab showed a significant increase of aggregates after freeze-thaw-cycles in a PBS-buffer media. It could be demonstrated that the reason for the instability was the pH-shift of the freezing buffer. Replacing sodium phosphate with potassium phosphate stabilized the protein. The same effect could be detected by adding sucrose (120 mM) or trehalose (40 mM) to the PBS-buffered antibody solution. The formation of insoluble protein particles could be reduced significantly by the addition of polysorbate 80 (0.01% m/V), whereas the amount of soluble aggregates detected by SE-HPLC could not be affected. The results demonstrate, that a prerequisite for high protein stability is an optimized bulk formulation. Modern freezing systems can not stabilize the protein against unfavorable conditions caused by the formulation. Buffer media showing pH-shift can lead to dimerization and precepitation. Disaccharides such as trehalose and sucrose can prevent dimerization in spite of the detected pH-shift, whereas polysorbate reduced the amount of precipitated protein significantly.