Protein Manufacturing and Formulation

PROTEIN MANUFACTURING AND FORMULATION

rhDNase I is expressed in mammalian cell culture and purified to homogeneity using a variety of chromato-graphic steps. The development of the formulation of rhDNase I is especially important in that a suitable formulation is required to take into account protein stability, aerosolization properties, tonicity and the sealed container for storage (Shire, 1996). rhDNase I (Pulmozyme dornase alpha) is manufactured by Genentech, Inc. and formulated as a sterile, clear, colorless aqueous solution containing 1.0 mg/mL dor-nase alpha, 0.15 mg/mL calcium chloride dihydrate and 8.77 mg/mL sodium chloride. The solution contains no preservative and has a nominal pH of 6.3. Pulmozyme is administered by the inhalation of an aerosol mist produced by a compressed air-driven nebulizer system. Pulmozyme is supplied as single-use ampoules, which deliver 2.5 mL of solution to the nebulizer.

The choice of formulation components was determined by a need to provide one to two years of stability and to meet additional requirements unique to aerosol delivery (Shire, 1996). A simple colorimetric assay for rhDNase I activity was used to evaluate the stability of rhDNase I in various formulations (Sinicropi et al., 1994b). In order to avoid adverse pulmonary reactions, such as cough or bronchoconstriction, aero-sols for local pulmonary delivery should be formulated as isotonic solutions with minimal or no buffer components and should maintain pH > 5.0. rhDNase I has an additional requirement for calcium to be present for optimal enzymatic activity. Limiting formulation components raised concerns about pH control, since protein stability and solubility can be highly pH-dependent. Fortunately, the protein itself provided sufficient buffering capacity at 1 mg/mL to maintain pH stability over the storage life of the product.