Lyophilized formulation

In addition to the additives used in a formulation to help stabilize the protein to freezing, the residual moisture content of the lyophilized powder needs to be considered. Not only is moisture capable of affecting the physicochemical stability of the protein itself, equally important is the ability of moisture to affect the Tg of the formulation. Water acts as a plasticizer and depresses the Tg of amorphous solids [124,137,138]. During primary drying, as water is gradually removed from the product, the Tg increases accordingly. The duration and temperature of the secondary drying step of the lyophilization process determines how much moisture remains bound to the powder. Usually lower residual moisture in the finished biopharmaceutical product leads to enhanced stability. Typically, moisture content in lyophilized formulations should not exceed 2% [139]. The optimal moisture level for maximum stability of a particular product must be demonstrated on a case-by-case basis. 

S. P. Duddu, and P. R. Dal Monte, Effect of glass transition temperature on the stability of lyophilized formulations containing a chimeric therapeutic antibody, Pharm. Res., 14(5), 591 (1997). 

De Luca, P. P. Development of lyophilization formulations. International Colloquium on Ind.-Pharm. Lyophilization, Gent, 1992... 

Page, C., P. Dawson, D. Woollacott, R. Thorpe, and A. Mire-Sluis (2000). Development of a lyophilization formulation that preserves the biological activity of the platelet-inducing cytokine interleukin-11 at low concentrations. J Pharm Pharmacol 52(1) 19-26. 

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

Buffers for lyophilized formulations need additional consideration. Some buffers like sodium phosphate can crystallize out of the protein amorphous phase during freezing resulting in rather large shifts in pH. Other common buffers such as acetate and imidazole should be avoided since they may sublime or evaporate during the lyophilization process, thereby shifting the pH of formulation during lyophilization or after reconstitution. 

The PEG could stabilize proteins by two different temperature-dependent mechanisms. At lower temperatures, it is preferentially excluded from the protein surface but has been shown to interact with the unfolded form of the protein at higher temperatures, given its amphipathic nature (57). Thus, at lower temperatures, it may protect proteins via the mechanism of preferential exclusion, but at higher temperatures possibly by reducing the number of productive collisions between unfolded molecules. PEG is also a cryoprotectant and has been employed in Recombinate, a lyophilized formulation of recombinant Antihemophilic Factor, which utilizes PEG 3350 at a concentration of 1.5mg/mL. The low-molecular weight liquid PEGs (PEG 300-600) can be contaminated with peroxides and cause protein oxidation. If used, the peroxide content in the raw material must be minimized and controlled throughout its shelf life. The same holds true for polysorbates (discussed below). 

Roy S, Jung R, Kerwin BA, Randolph TW, Carpenter JF. Effects of benzyl alcohol on aggregation of recombinant human interleukin-1-receptor antagonist in reconstituted lyophilized formulations. J Pharm Sci 2005 94(2) 382-396. 

Assay methods for monitoring any degradation products may be used to justify the time limits for bulk storage. This time would include the period from when the product is formulated to the end of the filling operation. Because most lyophilized formulations do not contain a biological preservative, microbiological quality before sterilization by filtration must also be monitored. The unfiltered solution bioburden would include microorganisms and endotoxin levels. 

Dubost, D., Kaufman, M., Zimmerman, J., Bogusky, M. J., Coddington, A. B., Pitzenberger, S. M. Characterization of a solid state reaction product from a lyophilized formulation of a cyclic heptapeptide. A novel example of an excipient induced oxidation. Pharm Res 13 1811-1814 (1996). 

Kirsch L, Zhang S, Muangsiri W, Redmon M, Wurster D. Development of a lyophilized formulation for (R,R)-formoterol (L)-tartrate. Drug Dev Ind Pharm 2001 27 89-96. 

Henrar, R.E.C., Kettenes-van den Bosch, J.J., Bult, A., Beijnen, J.H. Pharmaceutical development of a parenteral lyophilized formulation of the novel in-doloquinone antitumor agent E 09. Cancer Chemother. Pharmacol. 34, 416 122,... 

T. A. Jennings, The effect if resistivity probe design on the measurement of the freezing temperature of lyophilized formulations. 7. Parent. Drug Assoc., 109-126(1980). 

S. J. Prestrelski, T. Arakawa, and J. F. Carpenter, The structure of proteins in lyophilized formulations using Fourier transform infrared spectroscopy, in Formulation and Delivery of Proteins and Peptides, (J. L. Cleland and R. Danger, eds.) American Chemical Society Symposium Series No. 567, 1994, pp. 148-169. 

Oliyai et al. [23] studied an Asn-hexapeptide (Val-Tyr-Pro-Asn-Gly-Ala) in lyophilized formulation as a function of pH of the bulk solution, temperature, and residual moisture content in a factorial study. A statistically significant two-factor interaction indicated that the pH of formulation solution determined the extent to which this peptide stability depends on moisture level and temperature. 

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