Production, quality control, stability and pharmacotoxicity of cGMP-produced Plasmodium falciparum AMA1 FVO strain ectodomain expressed in Pichia pastoris

Vaccine. 2008 Nov 11;26(48):6143-50. doi: 10.1016/j.vaccine.2008.08.055. Epub 2008 Sep 17.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adjuvants, Immunologic
  • Amino Acid Sequence
  • Animals
  • Antigens, Protozoan / biosynthesis*
  • Antigens, Protozoan / toxicity
  • Blotting, Western
  • Cloning, Molecular
  • Drug Industry / standards*
  • Drug Stability
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme-Linked Immunosorbent Assay
  • Female
  • Fermentation
  • Freeze Drying
  • Guinea Pigs
  • Malaria Vaccines / biosynthesis*
  • Malaria Vaccines / standards*
  • Malaria Vaccines / toxicity
  • Male
  • Mass Spectrometry
  • Membrane Proteins / biosynthesis*
  • Membrane Proteins / standards*
  • Membrane Proteins / toxicity
  • Mice
  • Molecular Sequence Data
  • Pichia / genetics*
  • Pichia / metabolism
  • Plasmodium falciparum / genetics*
  • Plasmodium falciparum / immunology
  • Plasmodium falciparum / metabolism
  • Protozoan Proteins / biosynthesis*
  • Protozoan Proteins / standards*
  • Protozoan Proteins / toxicity
  • Quality Control
  • Rabbits
  • Vaccines, Synthetic / biosynthesis
  • Vaccines, Synthetic / standards
  • Vaccines, Synthetic / toxicity

Substances

  • Adjuvants, Immunologic
  • Antigens, Protozoan
  • Malaria Vaccines
  • Membrane Proteins
  • Protozoan Proteins
  • Vaccines, Synthetic
  • apical membrane antigen I, Plasmodium