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Production, quality control, stability and pharmacotoxicity of cGMP-produced Plasmodium falciparum AMA1 FVO strain ectodomain expressed in Pichia pastoris
- Source :
- Vaccine. 26:6143-6150
- Publication Year :
- 2008
- Publisher :
- Elsevier BV, 2008.
-
Abstract
- Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, PfAMA1 ectodomain (amino acid 25–545, FVO strain) was produced in Pichia pastoris by 35 L scale fed batch fermentation under current Good Manufacturing Practice (cGMP). Fermentation was followed by a three-step chromatographic purification procedure resulting in a yield of 5.8 g of purified protein. As judged by size exclusion chromatography, the cGMP-product comprised >95% PfAMA1 monomer, the remainder being predominantly PfAMA1 dimer. In SDS-PAGE two main bands of 68 and 70 kDa and some minor bands were evident. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident; less than 15% of the protein had this internal cleavage. By mass-spectrometric analysis, all bands analyzed in overloaded SDS-PAGE gels comprised PfAMA1 derived products. The protein was quantitatively bound by immobilized 4G2, a monoclonal antibody reactive with a reduction sensitive conformational determinant. The lyophilized product was stable for over 1 year. Immunopotency did not diminish, and storage did not lead to alterations in the behaviour of the protein upon formulation with adjuvants selected for Phase I clinical evaluation. These formulations also showed no pharmacotoxicity in rabbits. The final product conformed to preset criteria and was judged suitable for use in human clinical trials.
- Subjects :
- Male
Quality Control
Drug Industry
Blotting, Western
Guinea Pigs
Molecular Sequence Data
Plasmodium falciparum
Protozoan Proteins
Antigens, Protozoan
Enzyme-Linked Immunosorbent Assay
Cleavage (embryo)
Mass Spectrometry
Pichia
Pichia pastoris
Mice
Adjuvants, Immunologic
Drug Stability
Malaria Vaccines
Animals
Amino Acid Sequence
Cloning, Molecular
Apical membrane antigen 1
chemistry.chemical_classification
Vaccines, Synthetic
General Veterinary
General Immunology and Microbiology
biology
Malaria vaccine
Public Health, Environmental and Occupational Health
Membrane Proteins
biology.organism_classification
Amino acid
Freeze Drying
Infectious Diseases
Ectodomain
chemistry
Biochemistry
Fermentation
Molecular Medicine
Electrophoresis, Polyacrylamide Gel
Female
Rabbits
Subjects
Details
- ISSN :
- 0264410X
- Volume :
- 26
- Database :
- OpenAIRE
- Journal :
- Vaccine
- Accession number :
- edsair.doi.dedup.....ce3c699d360cc6225be349bc7a9f1d84