Production, quality control, stability, and potency of cGMP-produced Plasmodium falciparum RH5.1 protein vaccine expressed in Drosophila S2 cells

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 AS01_B 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 AS01_B following addition of RH5.1. Subsequent immunization of mice confirmed the RH5.1/AS01_B vaccine was immunogenic and could induce functional growth inhibitory antibodies against blood-stage P. falciparum in vitro. The RH5.1/AS01_B 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.

Fig. 1

RH5.1 protein vaccine monoclonal stable S2 cell line generation. a Schematic of RH5.1 encoding from the N-terminus: a BiP insect signal peptide (green) followed by PfRH5 (aa E26-Q526) (blue), followed by a C-terminal four amino acid C-tag (EPEA). The protein was based on the P. falciparum 3D7 clone sequence, which has cysteine (C) at polymorphic position 203 (yellow circle). The other cysteine residues in PfRH5 are indicated by small black boxes (C224, C317, C329, C345, and C351). Threonine (T) to alanine (A) substitutions to remove N-linked glycan sequons are indicated by red asterisks. The predicted molecular weight (Mw) is 60.2 kDa. b A total of 124 single clones were selected and expanded in shake flasks. RH5.1 expression levels in the medium were assessed by quantitative ELISA. The distribution of expression levels is shown from highest through to lowest. c A total of 37 clonal cell lines were assessed for stability with respect to growth, viability, and productivity for up to 73 days. Example productivity data are shown for two clones (38 and 77) over a 39-day period. RH5.1 expression levels were assessed at indicated time points by ELISA, and are normalized to the concentration of RH5.1 measured on day 1 (set as 1.0)

Fig. 2

Analysis of the purified final RH5.1 drug product. a Overview of RH5.1 protein vaccine cGMP production process. b SDS-PAGE and c western blot (under reducing conditions) of the final RH5.1 drug product produced to cGMP (G) run alongside the comparator engineering batch (E). The western blot used the anti-PfRH5 4BA7 mouse mAb. Within each panel, the gels derive from the same experiment and were processed in parallel. d HPLC-SEC analysis of the final RH5.1 drug product to assess aggregation. M molecular weight markers

Fig. 3

Characterization of RH5.1 clinical vaccine. a SPR analysis of the interaction of RH5.1 protein with basigin. b Anti-RH5.1 ELISA using a panel of eight PfRH5-specific mouse mAbs. Each sample was tested in triplicate. Bars show the mean plus range. c Potency ELISA using RH5.1 test sample versus RH5.1 protein standards (100, 50, and 20% concentration). Each point is the mean of triplicate readings. pAb mouse anti-PfRH5 polyclonal antibody serum control

Fig. 4

Stability testing of RH5.1 protein. RH5.1 protein vaccine was assessed for stability over time. The engineering batch was tested for a protein degradation by SDS-PAGE and b aggregation by analytical SEC following storage at −80 °C. Results are shown at the 0, 1, 2, 3, 6, 9, 12, and 19-month time points. In b, each colored line shows a different time point. The engineering batch was also tested for c protein degradation by SDS-PAGE and d aggregation by analytical SEC following storage at 4 °C as part of an accelerated stability study. Results are shown at the 1, 2, 4, 8, 10, and 14-day (D) time points. Within a and c, the gels for each time point derive from different experiments, but are shown aligned here for ease of comparison. m molecular weight markers

Fig. 5

Characterization of the RH5.1/AS01_B vaccine formulation. Stability of the RH5.1 protein was assessed by SDS-PAGE at various time points after formulation in AS01_B. Lanes 1–3 = immediate testing or after 1 and 4 h post mixing, respectively. Samples were also run on the same gel following testing of RH5.1 dilution using a mixing vial as required for clinical vaccine administration. Lane 4 = RH5.1 diluted 1:5 with 0.9% saline in a clinical mixing vial. Lane 5 = RH5.1 undiluted standard, and lane 6 = RH5.1 1:5 diluted standard. The gel in this figure derives from a single experiment with all samples processed in parallel

Fig. 6

Immunological analysis of RH5.1/AS01_B in mice. a BALB/c mice (n = 6 per group) were immunized with 2 µg RH5.1/AS01_B using the cGMP-produced clinical vaccine batch (GMP) or the engineering batch of RH5.1 (Eng), or AS01_B alone. Two weeks after the last immunization, spleens were collected and T-cell responses were measured from spleen samples by ex vivo IFN-γ ELISpot following re-stimulation with RH5 peptides or RH5.1 protein. Median and individual data points are shown. b Serum IgG responses were measured by ELISA against RH5.1 using pooled serum samples taken 4 weeks after the first or second immunization using the cGMP-produced or engineering batches of RH5.1 (1-E, 1-G, 2-E, 2-G, respectively). Responses were measured in all mice 2 weeks after the third and final immunization (3-G, 3-E). There was no detectable IgG (N.D.) in any mouse following three immunizations with AS01_B alone (3-A). Individual and median responses are shown. c Functional GIA of purified IgG was assessed against 3D7 clone P. falciparum parasites. GIA is plotted against RH5.1 responses measured by ELISA in the purified IgG samples used for the assay, in order to assess quality of the vaccine-induced antibody response. The dashed line indicates 50% GIA. Non-linear least squares regression line is shown; r² = 0.99, n = 16