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Vaccine ; 26(48): 6143-50, 2008 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-18804135


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.

Antígenos de Protozoários/biossíntese , Indústria Farmacêutica/normas , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/normas , Proteínas de Membrana/biossíntese , Proteínas de Membrana/normas , Pichia/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/normas , Adjuvantes Imunológicos , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/toxicidade , Western Blotting , Clonagem Molecular , Estabilidade de Medicamentos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Fermentação , Liofilização , Cobaias , Vacinas Antimaláricas/toxicidade , Masculino , Espectrometria de Massas , Proteínas de Membrana/toxicidade , Camundongos , Dados de Sequência Molecular , Pichia/metabolismo , Plasmodium falciparum/imunologia , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/toxicidade , Controle de Qualidade , Coelhos , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/normas , Vacinas Sintéticas/toxicidade