Abstract
Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, three Diversity Covering (DiCo) PfAMA1 ectodomain proteins, designed to overcome the intrinsic polymorphism that is present in PfAMA1, were produced under Good Manufacturing Practice (GMP) in Pichia pastoris. Using identical methodology, the 3 strains were cultivated in 70-L scale fed-batch fermentations and PfAMA1-DiCos were purified by two chromatography steps, an ultrafiltration/diafiltration procedure and size exclusion chromatography, resulting in highly pure (>95%) PfAMA1-DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3, with final yields of 1.8, 1.9 and 1.3 gram, respectively. N-terminal determinations showed that approximately 50% of each of the proteins lost 12 residues from their N-terminus, in accordance with SDS-PAGE (2 main bands) and MS-data. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident. The three proteins quantitatively bound to the mAb 4G2 that recognizes a conformational epitope, suggesting proper folding of the proteins. The lyophilized Drug Product (1:1:1 mixture of PfAMA1-DiCo1, DiCo2, DiCo3) fulfilled all pre-set release criteria (appearance, dissolution rate, identity, purity, protein content, moisture content, sub-visible particles, immuno-potency (after reconstitution with adjuvant), abnormal toxicity, sterility and endotoxin), was stable in accelerated and real-time stability studies at -20°C for over 24 months. When formulated with adjuvants selected for clinical phase I evaluation, the Drug Product did not show adverse effect in a repeated-dose toxicity study in rabbits. The Drug Product has entered a phase Ia/Ib clinical trial.
Highlights
The battle against malaria has been going on for centuries and since half way through the last century the parasite has been eradicated from large parts of the world, there are indications that this progress has come to a halt, despite huge efforts of many companies, governmental and non-governmental organizations and individual people, that have, at the same time, led to a strong decrease in malaria mortality and morbidity [1]
The upstream process development preceding the Good Manufacturing Practice (GMP) production of the Plasmodium falciparum apical membrane antigen 1 (PfAMA1)-Diversity Covering (DiCo) described below aimed at improving the stability, while not substantially lowering the yield
The production process for Pichia-produced PfAMA1 DiCo proteins as well as the fundamental downstream process unit operations were successfully transferred from the first generation PfAMA1 GMP processes, with minor modifications
Summary
The battle against malaria has been going on for centuries and since half way through the last century the parasite has been eradicated from large parts of the world, there are indications that this progress has come to a halt, despite huge efforts of many companies, governmental and non-governmental organizations and individual people, that have, at the same time, led to a strong decrease in malaria mortality and morbidity [1]. With the phase III clinical evaluation of the RTS,S vaccine [3, 4] and the promising attenuated sporozoite vaccines [5, 6] considerable progress has been made in this area which will hopefully lead to the powerful tools that are needed in the combat against malaria. With an efficacy of approximately 30% and “protection against clinical and severe disease”, instead of the anticipated sterile protection, there is room for improvement for the RTS,S vaccine [3]. The efficacy in infants wanes quickly, with time and with increasing exposure [7]
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