title: Production, quality control, stability and pharmacotoxicity of cGMP-produced Plasmodium falciparum AMA1 FVO strain ectodomain expressed in Pichia pastoris.
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 35L 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 5.8g 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 70kDa 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.
authors.affiliation: Department of Parasitology, Biomedical Primate Research Center, Lange Kleiweg 139, 2288 GJ Rijswijk, The Netherlands.
authors.forename: Bart W
authors.initials: BW
authors.lastname: Faber
authors.forename: Edmond J
authors.initials: EJ
authors.lastname: Remarque
authors.forename: Clemens H M
authors.initials: CH
authors.lastname: Kocken
authors.forename: Pascale
authors.initials: P
authors.lastname: Cheront
authors.forename: Doriano
authors.initials: D
authors.lastname: Cingolani
authors.forename: Florence
authors.initials: F
authors.lastname: Xhonneux
authors.forename: Margarita
authors.initials: M
authors.lastname: Jurado
authors.forename: Michele
authors.initials: M
authors.lastname: Haumont
authors.forename: Soren
authors.initials: S
authors.lastname: Jepsen
authors.forename: Odile
authors.initials: O
authors.lastname: Leroy
authors.forename: Alan W
authors.initials: AW
authors.lastname: Thomas
mesh_terms: D000276:Adjuvants, Immunologic; D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000096:biosynthesis* / Q000633:toxicity; D015153:Blotting, Western; D003001:Cloning, Molecular; D004345:Drug Industry / Q000592:standards*; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005285:Fermentation; D005612:Freeze Drying; D006168:Guinea Pigs; D017780:Malaria Vaccines / Q000096:biosynthesis* / Q000592:standards* / Q000633:toxicity; D008297:Male; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000592:standards* / Q000633:toxicity; D051379:Mice; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010963:Plasmodium falciparum / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000592:standards* / Q000633:toxicity; D011786:Quality Control; D011817:Rabbits; D014614:Vaccines, Synthetic / Q000096:biosynthesis / Q000592:standards / Q000633:toxicity
chemical_list: D000276:Adjuvants, Immunologic; D000953:Antigens, Protozoan; D017780:Malaria Vaccines; D008565:Membrane Proteins; D015800:Protozoan Proteins; D014614:Vaccines, Synthetic; C090077:apical membrane antigen I, Plasmodium