A Reproducible and Scalable Process for Manufacturing a Pfs48/45 Based Plasmodium falciparum Transmission-Blocking Vaccine
The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has been advancing as a candidate antigen for a transmission-blocking vaccine (TBV) for malaria. However, Pfs48/45 contains multiple disulfide bonds, that are critical for proper folding and induction of transm...
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doaj-abac308fce534339b9649af5cb94636e2021-01-11T14:18:58ZengFrontiers Media S.A.Frontiers in Immunology1664-32242021-01-011110.3389/fimmu.2020.606266606266A Reproducible and Scalable Process for Manufacturing a Pfs48/45 Based Plasmodium falciparum Transmission-Blocking VaccineSusheel K. Singh0Susheel K. Singh1Jordan Plieskatt2Bishwanath K. Chourasia3Bishwanath K. Chourasia4Amanda Fabra-García5Asier Garcia-Senosiain6Asier Garcia-Senosiain7Vandana Singh8Vandana Singh9Karin Lövgren Bengtsson10Jenny M. Reimer11Robert Sauerwein12Matthijs M. Jore13Michael Theisen14Michael Theisen15Department for Congenital Disorders, Statens Serum Institut, Copenhagen, DenmarkCentre for Medical Parasitology at Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, DenmarkPATH’s Malaria Vaccine Initiative, Washington, DC, United StatesDepartment for Congenital Disorders, Statens Serum Institut, Copenhagen, DenmarkCentre for Medical Parasitology at Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, DenmarkDepartment of Medical Microbiology, Radboud University Medical Center, Nijmegen, NetherlandsDepartment for Congenital Disorders, Statens Serum Institut, Copenhagen, DenmarkCentre for Medical Parasitology at Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, DenmarkDepartment for Congenital Disorders, Statens Serum Institut, Copenhagen, DenmarkCentre for Medical Parasitology at Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, DenmarkNovavax AB, Uppsala, SwedenNovavax AB, Uppsala, SwedenDepartment of Medical Microbiology, Radboud University Medical Center, Nijmegen, NetherlandsDepartment of Medical Microbiology, Radboud University Medical Center, Nijmegen, NetherlandsDepartment for Congenital Disorders, Statens Serum Institut, Copenhagen, DenmarkCentre for Medical Parasitology at Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, DenmarkThe cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has been advancing as a candidate antigen for a transmission-blocking vaccine (TBV) for malaria. However, Pfs48/45 contains multiple disulfide bonds, that are critical for proper folding and induction of transmission-blocking (TB) antibodies. We have previously shown that R0.6C, a fusion of the 6C domain of Pfs48/45 and a fragment of PfGLURP (R0), expressed in Lactococcus lactis, was properly folded and induced transmission-blocking antibodies. Here we describe the process development and technology transfer of a scalable and reproducible process suitable for R0.6C manufacturing under current Good Manufacturing Practices (cGMP). This process resulted in a final purified yield of 25 mg/L, sufficient for clinical evaluation. A panel of analytical assays for release and stability assessment of R0.6C were developed including HPLC, SDS-PAGE, and immunoblotting with the conformation-dependent TB mAb45.1. Intact mass analysis of R0.6C confirmed the identity of the product including the three disulfide bonds and the absence of post-translational modifications. Multi-Angle Light Scattering (MALS) coupled to size exclusion chromatography (SEC-MALS), further confirmed that R0.6C was monomeric (~70 kDa) in solution. Lastly, preclinical studies demonstrated that the R0.6C Drug Product (adsorbed to Alhydrogel®) elicited functional antibodies in small rodents and that adding Matrix-M™ adjuvant further increased the functional response. Here, building upon our past work, we filled the gap between laboratory and manufacturing to ready R0.6C for production under cGMP and eventual clinical evaluation as a malaria TB vaccine.https://www.frontiersin.org/articles/10.3389/fimmu.2020.606266/fullmalariavaccinePfs48/45R0.6Ctransmission-blockingLactococcus lactis |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Susheel K. Singh Susheel K. Singh Jordan Plieskatt Bishwanath K. Chourasia Bishwanath K. Chourasia Amanda Fabra-García Asier Garcia-Senosiain Asier Garcia-Senosiain Vandana Singh Vandana Singh Karin Lövgren Bengtsson Jenny M. Reimer Robert Sauerwein Matthijs M. Jore Michael Theisen Michael Theisen |
spellingShingle |
Susheel K. Singh Susheel K. Singh Jordan Plieskatt Bishwanath K. Chourasia Bishwanath K. Chourasia Amanda Fabra-García Asier Garcia-Senosiain Asier Garcia-Senosiain Vandana Singh Vandana Singh Karin Lövgren Bengtsson Jenny M. Reimer Robert Sauerwein Matthijs M. Jore Michael Theisen Michael Theisen A Reproducible and Scalable Process for Manufacturing a Pfs48/45 Based Plasmodium falciparum Transmission-Blocking Vaccine Frontiers in Immunology malaria vaccine Pfs48/45 R0.6C transmission-blocking Lactococcus lactis |
author_facet |
Susheel K. Singh Susheel K. Singh Jordan Plieskatt Bishwanath K. Chourasia Bishwanath K. Chourasia Amanda Fabra-García Asier Garcia-Senosiain Asier Garcia-Senosiain Vandana Singh Vandana Singh Karin Lövgren Bengtsson Jenny M. Reimer Robert Sauerwein Matthijs M. Jore Michael Theisen Michael Theisen |
author_sort |
Susheel K. Singh |
title |
A Reproducible and Scalable Process for Manufacturing a Pfs48/45 Based Plasmodium falciparum Transmission-Blocking Vaccine |
title_short |
A Reproducible and Scalable Process for Manufacturing a Pfs48/45 Based Plasmodium falciparum Transmission-Blocking Vaccine |
title_full |
A Reproducible and Scalable Process for Manufacturing a Pfs48/45 Based Plasmodium falciparum Transmission-Blocking Vaccine |
title_fullStr |
A Reproducible and Scalable Process for Manufacturing a Pfs48/45 Based Plasmodium falciparum Transmission-Blocking Vaccine |
title_full_unstemmed |
A Reproducible and Scalable Process for Manufacturing a Pfs48/45 Based Plasmodium falciparum Transmission-Blocking Vaccine |
title_sort |
reproducible and scalable process for manufacturing a pfs48/45 based plasmodium falciparum transmission-blocking vaccine |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Immunology |
issn |
1664-3224 |
publishDate |
2021-01-01 |
description |
The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has been advancing as a candidate antigen for a transmission-blocking vaccine (TBV) for malaria. However, Pfs48/45 contains multiple disulfide bonds, that are critical for proper folding and induction of transmission-blocking (TB) antibodies. We have previously shown that R0.6C, a fusion of the 6C domain of Pfs48/45 and a fragment of PfGLURP (R0), expressed in Lactococcus lactis, was properly folded and induced transmission-blocking antibodies. Here we describe the process development and technology transfer of a scalable and reproducible process suitable for R0.6C manufacturing under current Good Manufacturing Practices (cGMP). This process resulted in a final purified yield of 25 mg/L, sufficient for clinical evaluation. A panel of analytical assays for release and stability assessment of R0.6C were developed including HPLC, SDS-PAGE, and immunoblotting with the conformation-dependent TB mAb45.1. Intact mass analysis of R0.6C confirmed the identity of the product including the three disulfide bonds and the absence of post-translational modifications. Multi-Angle Light Scattering (MALS) coupled to size exclusion chromatography (SEC-MALS), further confirmed that R0.6C was monomeric (~70 kDa) in solution. Lastly, preclinical studies demonstrated that the R0.6C Drug Product (adsorbed to Alhydrogel®) elicited functional antibodies in small rodents and that adding Matrix-M™ adjuvant further increased the functional response. Here, building upon our past work, we filled the gap between laboratory and manufacturing to ready R0.6C for production under cGMP and eventual clinical evaluation as a malaria TB vaccine. |
topic |
malaria vaccine Pfs48/45 R0.6C transmission-blocking Lactococcus lactis |
url |
https://www.frontiersin.org/articles/10.3389/fimmu.2020.606266/full |
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