Abstract
Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is a leading asexual blood-stage vaccine candidate for malaria. In preparation for clinical trials, a full-length PfRH5 protein vaccine called “RH5.1” was produced as a soluble product under cGMP using the ExpreS2 platform (based on a Drosophila melanogaster S2 stable cell line system). Following development of a high-producing monoclonal S2 cell line, a master cell bank was produced prior to the cGMP campaign. Culture supernatants were processed using C-tag affinity chromatography followed by size exclusion chromatography and virus-reduction filtration. The overall process yielded >400 mg highly pure RH5.1 protein. QC testing showed the MCB and the RH5.1 product met all specified acceptance criteria including those for sterility, purity, and identity. The RH5.1 vaccine product was stored at −80 °C and is stable for over 18 months. Characterization of the protein following formulation in the adjuvant system AS01B showed that RH5.1 is stable in the timeframe needed for clinical vaccine administration, and that there was no discernible impact on the liposomal formulation of AS01B following addition of RH5.1. Subsequent immunization of mice confirmed the RH5.1/AS01B vaccine was immunogenic and could induce functional growth inhibitory antibodies against blood-stage P. falciparum in vitro. The RH5.1/AS01B was judged suitable for use in humans and has since progressed to phase I/IIa clinical trial. Our data support the future use of the Drosophila S2 cell and C-tag platform technologies to enable cGMP-compliant biomanufacture of other novel and “difficult-to-express” recombinant protein-based vaccines.
Highlights
Malaria caused by the parasite Plasmodium falciparum continues to exert a huge burden on global public health, with over 200 million clinical cases annually and approximately half a million deaths.[1]
We reported the production of soluble full-length PfRH5 protein using a current good manufacturing practice (cGMP)-compliant platform called ExpreS2, based on a Drosophila melanogaster Schneider 2 (S2) stable cell line system.[33,34]
We have previously reported the production of preclinical-grade full-length PfRH5 protein vaccines using polyclonal Drosophila S2 stable cell lines.[33,35]
Summary
Malaria caused by the parasite Plasmodium falciparum continues to exert a huge burden on global public health, with over 200 million clinical cases annually and approximately half a million deaths.[1]. Immunization of animals with full-length PfRH5 protein antigen leads to the induction of neutralizing antibodies,[15,16,17,18] in contrast to the earliest vaccination studies that used PfRH5 fragments made in Escherichia coli, which failed to induce functional antibodies.[19,32] In order to progress clinically, it has been critical to develop a protein expression and purification process that (i) allowed for production of full-length PfRH5 protein, and (ii) was scalable and compliant with current good manufacturing practice (cGMP) In this regard, we reported the production of soluble full-length PfRH5 protein using a cGMP-compliant platform called ExpreS2, based on a Drosophila melanogaster Schneider 2 (S2) stable cell line system.[33,34] Full-length PfRH5 protein was expressed from stable S2 cell lines and secreted into the supernatant from where it was purified using a newly available affinity purification system that makes use of a C-terminal tag known as “C-tag,” composed of the four amino acids (aa) glutamic acid–proline–glutamic acid–alanine (E-P-E-A).[35] This C-tag is selectively captured on a resin coupled to a camelid single-chain antibody specific for this short sequence[36] that has been developed into a CaptureSelectTM affinity resin by Thermo Fisher Scientific. This work led to the successful production of >400 mg RH5.1 protein vaccine, which has subsequently progressed to phase I/IIa clinical testing in healthy adults in Oxford, UK formulated with GSK’s adjuvant AS01B (Clinicaltrials.gov NCT02927145)
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