It is evident from the discussion in this chapter that although several excipients can be used, each has its advantages and disadvantages. If an excipient can provide the desired buffering capacity while enhancing protein solubility and stabilizing the formulation, that would be close to an ideal excipient. However, usually more than one excipient may be required in the formulation to provide all these desirable attributes. The best excipients must be found for each individual protein, and it is hard to make generalizations. This is not to say that guiding principles cannot be used, but only that these principles provide a starting point. Even a simple formulation component such as sodium chloride has been reported to cause extensive aggregation of hGH on lyophilization.97
Also, the choice of stabilizer depends on the goals, such as whether the primary goal is to prevent aggregation or to stabilize against hydrolysis. Different stabilizers working by different mechanisms may be required. Sugars are good stabilizers in both solution and dry state. Glycerol and other polyols are very good stabilizers but may induce self-association. Amino acids and salts may be unpredictable because of their ionic nature and consequent interactions with charged proteins. In general, a strongly excluded excipient leaves the protein in an aqueous environment, which is conducive to the stability of the native state.
The quantity of the excipient used is also important. As an example, although carbohydrates can stabilize proteins in the dried state, excess carbohydrates may eliminate the interaction responsible for such stabilization.27
Also, it should be recognized that each excipient in the formulation has the potential to contribute to impurities. Rigorous quality control is required to ensure that the excipient is 100% pure or that any impurity present would not compromise the stability or efficacy of the formulation. For example, the contamination of clinical albumin solutions with adventitious metal ions can damage the metal-binding sites of albumin, which in turn will affect its ability to mediate drug metabolism. Hemodialysis patients receiving vanadium-contaminated albumin will not be able to excrete vanadium, resulting in serious body accumulation of the metal.98
Differential scanning calorimetry (DSC) (see Chapter 2 for a discussion of the DSC technique) is gaining increasing use as a tool to screen potential excipients that can stabilize the desired protein.99 The thermal transition in this case represents the unfolding of the native structure, which results in an endothermic peak. Stabilizing excipients, sugars such as glucose and sucrose and polyols such as sorbitol and glycerol, were found to increase the denaturation temperature Tm of the proteins, ovalbumin, lysozyme, and a-chymotrypsinogen.37 For tPA, the melting temperature Tm in phosphate buffer was about 66°C. In the presence of arginine, a stabilizer used in the formulation, the Tm shifted to 71°C.100 The thermogram for this observation is shown in Figure 4.3. Similarly, the transition temperature of aFGF was found to rise from 63 to 67 to 74°C as the concentration of trehalose increased from 0 to 0.5 to 1.5 M, respectively.43 In studies with a humanized monoclonal antibody, preservatives such as benzyl alcohol or chorobutanol were found to destabilize the protein, with denaturation temperatures decreasing by 2
---t - PAin phosphate buffer
-t - PAin arginine phosphate buffer
Figure 4.3 Plot of heat capacity (Cp) versus temperature for a solution of tissue plasminogen activator (tPA) in either phosphate buffer or arginine phosphate buffer. (Replotted from Pearlman, R. and Nguyen, T., J. Pharm. Pharmacol., 44: 178-185, 1992, with permission of Pharmaceutical Press, the Publications Division of the Royal Pharmaceutical Society of Great Britain.)
to 8°C in a concentration-dependent manner. However, the addition of methyl or propylparaben did not affect protein stability.31 In contrast, sorbitol has been reported to be a stabilizer for antibodies, with Tm shifting to higher temperatures.101 It should also be realized that Tm is pH dependent.
A combination of excipients will often be required to stabilize a peptide or protein formulation. A combination of mannitol and amorphous glycine provided the greatest protection against degradation and aggregation of hGH.97 The utility of DSC to predict protein stability can be confirmed by verifying the predictions by a more direct measure. In a study with IL-1 receptor type I, direct measurement by size exclusion chromatography was used to confirm that DSC data correctly predicted the rank and order of stability in the presence of preservatives.102
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