October 29 - November 2, 2000
American Association of Pharmaceutical Scientists (AAPS) Annual Meeting and Exposition, Indianapolis, Indiana.

Expression and Characterization of Recombinant Human Gelatin Fragments. David Olsen¹, Robert Chang¹, Asta Pirskanen³, Johanna Myllyharju³, Chunlin Yang¹, Juliana Balan¹, Marko Jarvinen², Tuija Nevalainen², Eija-Riitta Hamalainen², Maritta Perala-Heape², Minna Nokelainen³, Kari I. Kivirikko³, and Jim Polarek^1,2.
¹FibroGen Inc. South San Francisco, CA 94080. ²FibroGen Europe Oy, Oulu, Finland, ³Collagen Research Unit, Biocenter and the Department of Medical Biochemistry, University of Oulu, Oulu, Finland.

Purpose. Gelatin is a common excipient in numerous pharmaceutical preparations including vaccines, capsules, and plasma substitutes and is used in surgical sponges and other medical devices. Gelatin is typically derived from animal tissue and therefore poses potential problems due to immunologic reactions and contaminants which cause transmissible spongiform encephalopathy (TSE). The purpose of our study was to develop a recombinant expression system that could produce homogenous, well-defined human gelatins of different sizes to test as vaccine stabilizers.

Methods. The host used to express recombinant gelatin was a Pichia pastoris strain which contains a single copy of the alpha and beta subunits of prolyl hydroxylase (P4H), the enzyme that catalyzes the post-translational conversion of peptide bound proline to hydroxyproline. Co-expression of P4H and gelatin fragments results in the expression of gelatin with the native human sequence. The expression plasmids used in our studies contain alpha1 (I) cDNA sequences of different sizes fused to the mating factor alpha prepro sequence and protein expression is regulated by the methanol-inducible alcohol oxidase promoter (P_AOX1). Transformants were selected using zeocin or by complementation of a his4 auxotrophy. Gelatin producing strains were identified by SDS-PAGE analysis of conditioned media and P4H activity in extracts from shake flask cultures. Strains were then grown in 10 liter fermentors to generate media for large scale purification. Gelatin was purified from the media by ion-exchange chromatography and characterized by amino acid sequence and composition and tested in bioassays.

Results. Gelatin fragments of discrete sizes, ranging from 5-50 kd, were expressed and secreted into the media. Expression level in the 10 liter fermentor exceeded 3 grams/liter. High purity gelatin was obtained by a combination of anion and cation exchange chromatography. N-terminal sequence analysis demonstrated correct signal sequence processing. Some of the gelatin fragments demonstrated in vitro cell attachment activity.

Conclusions.These gelatin fragments represent a novel biomaterial which can be used in a variety of applications, such as vaccine stabilization, were animal derived gelatins are currently used. These new materials provide a safe, well-defined, non-immunogenic replacement for currently used gelatins.