Steven J. Prestrelski

Application of infrared spectroscopy to development of stable lyophilized protein formulations

The preparation of protein therapeutics as lyophilized (freeze-dried) products is often essential to obtain the requisite stability during shipping and long-term storage. When prepared properly, lyophilized proteins can retain stability for months or even years at ambient temperature [1]. A crucial aspect of proper, rational development of a lyophilized formulation is the recognition that the most sensitive element in the system is the protein itself. Unfortunately, in the early days of preparing lyophilized protein formulations, it appears that frequently the focus was primarily on obtaining an acceptable dried cake structure, with the apparent assumption that a protein would be resistant to freezing and dehydration stresses. For example, when only mannitol is employed as an excipient and crystalline bulking agent, the resulting cake structure is usually excellent, but proteins derive essentially no protection from crystalline mannitol [1]. Such approaches, which can be sufficient for more stable low molecular weight drugs, led to dried products in which the protein was so susceptible to physical and/ or chemical degradation that shipping and storage at subzero temperatures was required. Obviously, the potential advantages of a lyophilized product are mostly lost when there are such stringent requirements for control of product temperature. It is now well known that without adequate stabilization by the appropriate amorphous excipients (e.g. sucrose), the protein can be irreversibly denatured after lyophilization and reconstitution and/or after long-term storage in the dried solid and rehydration [1–8]. Since most protein drugs are delivered parenterally, even if only a small fraction of the total protein molecular population (e.g. a few percent) is irreversibly denatured and aggregated, then the product will not be acceptable. Until recently the changes in protein structure arising during the processing steps of lyophilization, which were usually manifested as protein aggregation after rehydration, were unknown. However, with infrared spectroscopy it is now possible to examine directly the secondary structure of a protein in the initial aqueous solution, and in both the frozen state and the final dried solid. This analysis, combined with exploitation of stabilizers that are specific for either freezing or drying stresses, has documented that both freezing and dehydration can induce protein unfolding [1–8]. Unfolding not only can lead to irreversible protein denaturation, if the sample is rehydrated immediately, but, perhaps more importantly for industrial development of lyophilized protein drugs, can also reduce storage stability in the dried solid [1,6,7]. Moreover, simply obtaining a native protein in samples rehydrated immediately after lyophilization is not necessaEuropean Journal of Pharmaceutics and Biopharmaceutics 45 (1998) 231–238

A New Strategy for Enhancing the Stability of Lyophilized Protein: The Effect of the Reconstitution Medium on Keratinocyte Growth Factor

AbstractPurpose. Protein stabilization during lyophilization has previously focused on optimization of the formulation as well as the freezing and dehydration process parameters. However, the effect of the reconstitution medium has been largely neglected. We have investigated its effect on aggregate formation using recombinant keratinocyte growth factor (KGF).nMethods. The protein was lyophilized under suboptimal conditions to induce aggregation and precipitation upon reconstitution with water. A series of additives were examined by UV spectrophotometry and size exclusion chromatography (SEC-HPLC) for their effects on decreasing the degree of KGF aggregation and precipitation by the increase in recovery of soluble monomer.nResults. Several additives resulted in a significant reduction of aggregation, including sulfated polysaccharides, surfactants, polyphosphates, and amino acids. A similar effect was achieved by adjusting the ionic strength of the reconstitution medium. SEC-HPLC indicated that the amount of soluble monomer was also increased by these additives suggesting that the recovery of the soluble protein correlates with the native, monomeric protein.nConclusions. These results suggest that optimization of reconstitution conditions will be a useful methodology for increasing the recovery of soluble, active proteins and that for KGF, the recovery of the soluble protein correlates with the native, monomeric form.