4.7 Article

Development of a process for large scale production of PfRH5 in E. coli expression system

Journal

INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
Volume 188, Issue -, Pages 169-179

Publisher

ELSEVIER
DOI: 10.1016/j.ijbiomac.2021.08.014

Keywords

PfRH5; SEC-HPLC; RP-HPLC; SDS-PAGE; GLA-SE; E; coli

Funding

  1. Gennova Biopharmaceuticals Ltd, Pune, Maharashtra, India

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This study successfully expressed full-length PfRH5 in E. coli, optimized the purification and refolding process to obtain high purity antigen. Furthermore, PfRH5 formulated with adjuvant GLA-SE in mice showed high antibody titers, indicating its potential as a malaria vaccine.
The Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5) has recently shown great promise to be developed as a vaccine candidate to prevent blood-stage malaria. However, because of its molecular complexity, most previous efforts were focused on expressing PfRH5 in its native and soluble form. Here, we describe the E. coli expression of full-length PfRH5 as inclusion bodies (IBs), followed by its high cell density fermentation at 1, 5 and 30 L scale. Denatured full-length PfRH5 was purified using a two-step chromatography process before being refolded using design of experiments (DoE). Refolded PfRH5 was further purified using size exclusion chromatography (SEC), recovering high purity antigen with an overall yield of 102 mg/L from fermentation cell harvest. Purified PfRH5 was further characterized using orthogonal analytical methods, and a short-term stability study revealed -80 degrees C as an optimum storage temperature. Moreover, refolded, and purified PfRH5, when formulated with adjuvant Glucopyranosyl A lipid stable emulsion (GLA-SE), elicited high antibody titers in BALB/c mice, proving its potential to neutralize the blood-stage malarial parasite. Here, we establish an E. coli-based process platform for the large-scale cGMP production of full-length PfRH5, enabling global malaria vaccine development efforts.

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