Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation

Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation

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 pro...

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Main Authors: Faber, Bart W., Hellwig, Stephan, Houard, Sophie, Havelange, Nicolas, Drossard, Jürgen, Mertens, Hubert, Croon, Alexander, Kastilan, Robin, Byrne, Richard, van der Werff, Nicole, van der Eijk, Marjolein, Thomas, Alan W., Kocken, Clemens H. M., Remarque, Edmond J.
Format: Online
Language:English
Published: Public Library of Science 2016
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5047445/
id pubmed-5047445
recordtype oai_dc
spelling pubmed-50474452016-10-27 Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation Faber, Bart W. Hellwig, Stephan Houard, Sophie Havelange, Nicolas Drossard, Jürgen Mertens, Hubert Croon, Alexander Kastilan, Robin Byrne, Richard van der Werff, Nicole van der Eijk, Marjolein Thomas, Alan W. Kocken, Clemens H. M. Remarque, Edmond J. Research Article 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. Public Library of Science 2016-10-03 /pmc/articles/PMC5047445/ /pubmed/27695087 http://dx.doi.org/10.1371/journal.pone.0164053 Text en © 2016 Faber et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
repository_type Open Access Journal
institution_category Foreign Institution
institution US National Center for Biotechnology Information
building NCBI PubMed
collection Online Access
language English
format Online
author Faber, Bart W.
Hellwig, Stephan
Houard, Sophie
Havelange, Nicolas
Drossard, Jürgen
Mertens, Hubert
Croon, Alexander
Kastilan, Robin
Byrne, Richard
van der Werff, Nicole
van der Eijk, Marjolein
Thomas, Alan W.
Kocken, Clemens H. M.
Remarque, Edmond J.
spellingShingle Faber, Bart W.
Hellwig, Stephan
Houard, Sophie
Havelange, Nicolas
Drossard, Jürgen
Mertens, Hubert
Croon, Alexander
Kastilan, Robin
Byrne, Richard
van der Werff, Nicole
van der Eijk, Marjolein
Thomas, Alan W.
Kocken, Clemens H. M.
Remarque, Edmond J.
Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation
author_facet Faber, Bart W.
Hellwig, Stephan
Houard, Sophie
Havelange, Nicolas
Drossard, Jürgen
Mertens, Hubert
Croon, Alexander
Kastilan, Robin
Byrne, Richard
van der Werff, Nicole
van der Eijk, Marjolein
Thomas, Alan W.
Kocken, Clemens H. M.
Remarque, Edmond J.
author_sort Faber, Bart W.
title Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation
title_short Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation
title_full Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation
title_fullStr Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation
title_full_unstemmed Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation
title_sort production, quality control, stability and pharmacotoxicity of a malaria vaccine comprising three highly similar pfama1 protein molecules to overcome antigenic variation
description 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.
publisher Public Library of Science
publishDate 2016
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5047445/
_version_ 1613670205346545664

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