September 22-24, 2003
Formulation Strategies for Biopharmaceuticals, Philadelphia, PA.

Development of Recombinant Human Gelatin for Use as a Stabilizer in Biopharmaceuticals. D. Olsen, R. Chang, M. Sakaguchi, S. Leigh, R. Lundgard, F. Buschman, M. Lonergan, H. McMullin, C. Luehrs, A. Beardsley, T. Revak, and J. Polarek, FibroGen Inc., South San Francisco, CA 94080.

Gelatins are widely used in the pharmaceutical industry as stabilizers in vaccines and other biopharmaceuticals. These gelatins are a heterogeneous mixture of different sized polypeptides derived from bovine or porcine bones or hides. The heterogeneous nature of these protein mixtures creates a significant challenge with respect to their analytical characterization. These gelatins are also a potential source of contaminants that cause transmissible spongiform encephalopathies. Furthermore, several cases of allergic reactions to bovine and porcine gelatins have been reported, including anaphylaxis. To eliminate these challenges and concerns associated with the use of animal-derived gelatin, we have developed a low molecular weight recombinant human gelatin fragment that can substitute for the animal sourced materials as a stabilizer.

A cDNA fragment encoding 98 amino acids of the human pro alpha1 (I) chain was amplified by PCR and cloned in-frame to the mating factor alpha prepro sequence of S. cerevisiae in plasmid pPICZalphaA (Invitrogen). The construct was integrated into the AOX1 loci of Pichia pastoris strain X-33 and isolates expressing and secreting high levels of the 8.5 kd gelatin fragment were identified. A fermentation process, which does not use any animal derived components, was established that resulted in gram/liter expression levels. The recombinant gelatin was efficiently secreted into the extracellular media, allowing the development of a simple, scalable purification process. The process has been run at the 400 liter scale.

A battery of analytical tests was established to characterize product purity, charge heterogeneity, endotoxin levels, sterility, and the levels of various host cell and non-host contaminants. The reactivity of our recombinant human gelatin with anti-gelatin IgE antibodies from children with confirmed gelatin allergies was tested in an in vitro binding assay. These antibodies recognized bovine and human collagens but not the 8.5 kd gelatin, establishing the low allergenic potential of our recombinant gelatin. We have tested the ability of our recombinant gelatin to stabilize several live attenuated viral vaccines and have found its efficacy to be equivalent to the porcine gelatin used in several marketed vaccines. We are currently in the process of testing this gelatin as a stabilizer for other biopharmaceuticals. Finally, we have performed a clinical study in 66 normal healthy volunteers to test the safety and tolerability of this recombinant gelatin in humans. No adverse events or reactions were seen in any of the three dose groups tested. We will be submitting an Excipient Master File to the FDA in 2004 and preparing a monograph for the USP describing this 8.5 kd human gelatin fragment.