Expression and characterization of a low molecular weight recombinant human gelatin: development of a substitute for animal-derived gelatin with superior features.
Gelatin is used as a stabilizer in several vaccines. Allergic reactions to gelatins have been reported, including anaphylaxis. These gelatins are derived from animal tissues and thus represent a potential source of contaminants that cause transmissible spongiform encephalopathies. We have developed a low molecular weight human sequence gelatin that can substitute for the animal sourced materials. A cDNA fragment encoding 101 amino acids of the human proalpha1 (I) chain was amplified, cloned into plasmid pPICZalpha, integrated into Pichia pastoris strain X-33, and isolates expressing high levels of recombinant gelatin FG-5001 were identified. Purified FG-5001 was able to stabilize a live attenuated viral vaccine as effectively as porcine gelatin. This prototype recombinant gelatin was homogeneous with respect to molecular weight but consisted of several charge isoforms. These isoforms were separated by cation exchange chromatography and found to result from a combination of truncation of the C-terminal arginine and post-translational phosphorylation. Site-directed mutagenesis was used to identify the primary site of phosphorylation as serine residue 546; serine 543 was phosphorylated at a low level. A new construct was designed encoding an engineered gelatin, FG-5009, with point mutations that eliminated the charge heterogeneity. FG-5009 was not recognized by antigelatin IgE antibodies from children with confirmed gelatin allergies, establishing the low allergenic potential of this gelatin. The homogeneity of FG-5009, the ability to produce large quantities in a reproducible manner, and its low allergenic potential make this a superior substitute for the animal gelatin hydrolysates currently used to stabilize many pharmaceuticals.
Olsen
David
D
FibroGen, Inc. 225 Gateway Boulevard, South San Francisco, CA 94080, USA. dolsen@fibrogen.com
Jiang
Jenny
J
Chang
Robert
R
Duffy
Robert
R
Sakaguchi
Masahiro
M
Leigh
Scott
S
Lundgard
Robert
R
Ju
Julia
J
Buschman
Frank
F
Truong-Le
Vu
V
Pham
Binh
B
Polarek
James W
JW
D000906:Antibodies; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D004342:Drug Hypersensitivity / Q000276:immunology; D005079:Excipients; D005780:Gelatin / Q000009:adverse effects* / Q000235:genetics* / Q000627:therapeutic use; D006801:Humans; D007073:Immunoglobulin E; D008970:Molecular Weight; D010766:Phosphorylation; D010843:Pichia / Q000235:genetics; D017354:Point Mutation; D015202:Protein Engineering; D020033:Protein Isoforms; D014613:Vaccines, Attenuated / Q000009:adverse effects / Q000737:chemistry*
D000906:Antibodies; D018076:DNA, Complementary; D005079:Excipients; D020033:Protein Isoforms; D014613:Vaccines, Attenuated; D007073:Immunoglobulin E; D005780:Gelatin