Formulation protein stability

Because of the multiple degradation pathways that may take place at elevated temperature, protein stability monitoring data may not conform to the Arrhenius relationship, and the maximum temperature selected for accelerated stability studies must be carefully selected. Gu et al. [32] described the different mechanisms of inactivation of interleukin-1 (3 (IL-1 (3) in solution above and below 39°C. In this example, the multiple mechanisms precluded the prediction of formulation shelf life from accelerated temperature data. In contrast, by working at 40° C and lower, Perlman and Nguyen [33] were able to successfully extrapolate data from stability studies of tissue plasminogen activator down to 5°C. 

M. A. Hanson and S. K. E. Rouan, Introduction to formulation of protein pharmaceuticals, in Stability of Protein Pharmaceuticals, Part B In Vivo Pathways of Degradation and Strategies for Protein Stabilization (T. J. Ahem and M. C. Manning, eds.), Plenum Press, New York, 1992, pp. 209-233. 

M. Pikal, Freeze drying of proteins Process, formulation, and stability, in Formulation and Delivery of Proteins and Peptides (J. Cleland and R. Langer, eds.), ACS Symposium Series 567, 1993, pp. 120-133. 

Hora et al. [3.19] described the complexity of protein stabilization by the example of recombinant, human Interleukin-2 (rhIL-2). Formulations with amino acids and mannitol/ sucrose are sensitive to mechanical stress e. g. by pumping. Hydroxypropyl-beta-cyclodextrin (HPcD) provides stability, but increases the sensitivity to oxygen. Polysor-bate 80 forms a mechanically stable product, but results in oxidation. In both cases contamination in the HPcD or traces of H202 in the Polysorbate may have been the starter for the oxidation. Brewster [3.20] reports, that HPcD stabilizes interleukin without forming aggregations and this results in 100 % biopotency. 

Pikal, M. J., Dellermann, K., Roy, M. L Formulation and stability of freeze-dried proteins Effects of moisture and oxygen on the freeze-dried formulation of human growth hormones. Developments in Biological Standardization, Vol. 74, p. 21-38. Acting Editors Joan C. May - F. Brown. S. Karger AG, CH-4009 Basel (Switzerland), 1992... 

In addition to the direct absorbance methods, colorimetric methods are suited for relatively pure proteins as purification progresses. They are accurate if calibrated from a standard curve of the test protein reference sample and fast if automated. However, they are not as simple to perform as direct absorbance methods. Hence they are not as suitable for production as direct absorbance methods. The relative simplicity of colorimetric methods makes them more suited to automated formulation and stability studies and total-protein assays of complex mixtures. Microtiter plate versions of colorimetric assays allow for automation and consumption of relatively small sample sizes while requiring little specialized equipment or training. 

Inclusion of a cryoprotectant in the formulation can stabilize the protein during the freezing and drying stages of lyophilization.17 Excipients frequently... 

The choice of protein stabilizer stemmed from the necessity of burying the hybrid formulate in a water pool structured by a strongly hydrophilic coating shell. This appears to be an extremely efficient option allowing for... 

TABLE 5.8. Examples of excipients used to enhance protein stability in solution and lyophilized formulations... 

Specific formulation strategies need to be employed for macromolecule compounds. An excellent review of protein stability in aqueous solutions has been published by Chi et al. (92). In addition to solution stability of proteins and peptides, aerosolization may result in significant surface interfacial destabilization of these compounds if no additional stabilization excipients are added. This is due to the fact that protein molecules are also surface active and adsorb at interfaces. The surface tension forces at interfaces perturb protein structure and often result in aggregation (92). Surfactants inhibit interface-induced aggregation by limiting the extent of protein adsorption (92). 

Experimental studies of protein stabilities are numerous and there are still points of serious disagreement concerning the conclusions to be drawn from these studies. Areas of discord include the extent to which thermal denaturation corresponds to denaturation by chemical agents and the extent to which hydrophobic, van der Waals, and/or hydrogen bonds stabilize the native state. In this discussion, we will focus on the work of Peter L. Privalov.k Privalov has developed much of the microcalorimetric instrumentation that has made the calorimetric studies of proteins feasible. He has also published numerous review articles that summarize experimental data and formulate general observations concerning protein denaturation. His 1995 paper in Advances in Protein Chemistry9 presents a recent, comprehensive, review of the experimental results... 

Herman AC, Boone TC, Lu HS. Characterization, formulation, and stability of Neupogen (filgrastim), a recombinant human granulocyte-colony stimulating factor. In Pearlman R.WangYJ (Eds.), Formulation, Characterization, and Stability of Protein Drugs. New York, NY Plenum Press 1996 303-328. 

Determan, A. S., Wilson, J. H., Kipper, M. J., Wannemuehler, M. J., and Narasimhan, B. (2006), Protein stability in the presence of polymer degradation products Consequences for controlled release formulations, Biomaterials, 27, 3312-3320. 

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