available today are in the form of freeze-dried powders since freeze drying results in a stable formulation with good shelf-life (Tang and Pikal 2004; Tsinontides et al. 2004). Selection of the dosage form also helps in selecting the vehicle and process parameters. Chang and Hershenson have summarized a list of strategies useful in designing formulation studies. Information such as clinical indications, dose requirement, and drug interactions can help in narrowing down the formulation and dosage form (Chang and Hershenson 2002).

The second step of preformulation studies comprises identification of different mechanisms that lead to degradation of the protein. Mechanisms of degradation are determined by subjecting the protein to conditions that protein might encounter during processing and final formulation (Table 17.5). Preformulation studies help in making rational decisions about excipients and conditions to be used in formulation studies and also give relevant information about handling and storage of the protein. A reproducible stability indicating assay is also developed during preformulation studies to assess the loss of protein integrity and activity during testing. Reversed phase HPLC and mass spectrometry are good techniques for development of stability indicating assays to identify amino acid modifications, sequence variations, and degradation products (Hoffman and Pisch-Heberle 2000; Srebalus Barnes and Lim 2007).

17.6.3  Selection of Excipients

The third step in developing a formulation involves selection of various excipients that can potentially be used for preserving and stabilizing the formulation. In addition, a set of formulations are identified at this stage for stability and process compatibility studies. The different excipients that are to be optimized are given below. Excipients listed below are mostly of pharmacopoeial grade (United States Pharmacopoeia) and generally regarded as safe (GRAS). Use of new excipients requires additional studies, time, and cost to meet regulatory standards.

Buffer ingredients:  pH is the most significant parameter to be stabilized as it is a major cause of degradation of proteins and therefore, buffers should be selected judiciously (Khossravi and Borchardt 2000; Liu et al. 2008). Akers and DeFelippis have listed the buffering agents that are useful for protein formulations (Table 17.6). Based on the optimum pH selected during preformulation, the pH range is defined and the buffer ingredients are selected accordingly.

Stabilizing agents:  As most of the protein formulations are in the form of a lyophilized cake, the protein has to be protected from degradation due to the stresses encountered during freezing and dehydration steps of the lyophilization cycle. The main cause of protein aggregation during lyophilization is the removal of bound water which results in breakage of hydrogen bonds between the protein molecule and the water molecule (Kim et al. 2003). Stabilizing agents such as sucrose and trehalose are added to the formulation to minimize protein aggregation. It has been observed that sugars prevent unfolding during dehydration because they form hydrogen bonds with protein in the place of lost water molecules (Carpenter et al. 1997).

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Table 17.5  Preformulation studies to monitor degradation products in protein formulation

Based on information provided in Chang et al. (Chang and Hershenson 2002)

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Temperature: Incubating protein formulations at abnormal temperatures can cause irreversible denaturation due to aggregation (Saishin et al. 2003). A range of temperatures need to be assessed during formulation development to determine an optimum temperature range that does not affect the protein. As protein can denature at both high and low temperatures, temperatures that are both higher and lower than the protein’s optimum temperature need to be assessed. The temperature study has to be done with the formulation that was finalized by selecting suitable excipients from the above list.

Agitation during manufacturing (shaking and stirring):  Studying protein stability at different agitation rates is necessary to predict the behavior of the formulation during shipping and transport. Aggregation can occur during the mechanical stresses encountered, thereby causing protein unfolding (Nie et al. 2006). Agitation studies need to be done at temperature extremes to determine the effect of temperature on agitation induced aggregation.

Freeze thawing:  During lyophilization and also during shipping of the formulation, the product is subjected to intermittent freezing and melting. During freezing, the fall in temperature can perturb protein’s secondary and tertiary structures. This can lead to aggregation (Chang et al. 1996). The formulation has to be subjected to freezing and thawing cycles to study the loss of protein during temperature fluctuations.

Photodegradation:  During formulation preparation, proteins are subjected to light exposure either during purification via UV-based column chromatography or during fill finish operations wherein inspection of filled vials is performed under light. Light can cause damage to the protein if an amino acid such as methionine that is prone to oxidative degradation in presence of light is present in the protein structure (Hovorka and Schoneich 2001). Typically, light exposure of 1.2 million lux hours and 200 W h/square meter of ultra violet (UV) light is needed to induce degradation of proteins

Container closure system:  Container closure system entails the entire packaging that protects and contains the product. The final immediate pack of the formulation is of critical importance because of the direct interaction that occurs between the protein and container material. The material of the container may leach into the protein formulation and contaminate the formulation, causing degradation of the protein. Decisions regarding the choice of material for the container are taken at an early stage of the development cycle. Some inputs are expected from the marketing department (based on the market appeal of a pack) and some inputs are put forth by the formulation scientist (based on the interaction study of the container material with the protein and also based on the physical strength of the pack to withstand the stress applied during filling, packing, and transport). Most lyophilized formulations are supplied in glass vials made of borosilicate glass type I. Glass is able to withstand stress and is also physically appealing. Prefilled syringes can also be used to improve patient compliance if self-administration is an option.