Accelerating the clinical development of protein-based vaccines for malaria by efficient purification using a four amino acid C-terminal 'C-tag'.
Development of bespoke biomanufacturing processes remains a critical bottleneck for translational studies, in particular when modest quantities of a novel product are required for proof-of-concept Phase I/II clinical trials. In these instances the ability to develop a biomanufacturing process quickly and relatively cheaply, without risk to product quality or safety, provides a great advantage by allowing new antigens or concepts in immunogen design to more rapidly enter human testing. These challenges with production and purification are particularly apparent when developing recombinant protein-based vaccines for difficult parasitic diseases, with Plasmodium falciparum malaria being a prime example. To that end, we have previously reported the expression of a novel protein vaccine for malaria using the ExpreS2Drosophila melanogaster Schneider 2 stable cell line system, however, a very low overall process yield (typically <5% recovery of hexa-histidine-tagged protein) meant the initial purification strategy was not suitable for scale-up and clinical biomanufacture of such a vaccine. Here we describe a newly available affinity purification method that was ideally suited to purification of the same protein which encodes the P. falciparum reticulocyte-binding protein homolog 5 - currently the leading antigen for assessment in next generation vaccines aiming to prevent red blood cell invasion by the blood-stage parasite. This purification system makes use of a C-terminal tag known as 'C-tag', composed of the four amino acids, glutamic acid - proline - glutamic acid - alanine (E-P-E-A), which is selectively purified on a CaptureSelect™ affinity resin coupled to a camelid single chain antibody, called NbSyn2. The C-terminal fusion of this short C-tag to P. falciparum reticulocyte-binding protein homolog 5 achieved >85% recovery and >70% purity in a single step purification directly from clarified, concentrated Schneider 2 cell supernatant under mild conditions. Biochemical and immunological analysis showed that the C-tagged and hexa-histidine-tagged P. falciparum reticulocyte-binding protein homolog 5 proteins are comparable. The C-tag technology has the potential to form the basis of a current good manufacturing practice-compliant platform, which could greatly improve the speed and ease with which novel protein-based products progress to clinical testing.
The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK. Electronic address: jing.jin@ndm.ox.ac.uk.
Jing
J
Jin
The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK.
Kathryn A
KA
Hjerrild
The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK.
Sarah E
SE
Silk
The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK.
Rebecca E
RE
Brown
The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK.
Geneviève M
GM
Labbé
The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK.
Jennifer M
JM
Marshall
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Katherine E
KE
Wright
Thermo Fisher Scientific, J.H. Oortweg 21, 2333 CH Leiden, Netherlands.
Sandra
S
Bezemer
ExpreS(2)ion Biotechnologies, SCION-DTU Science Park, Agern Allé 1, Hørsholm DK-2970, Denmark.
Stine B
SB
Clemmensen
The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK.
Sumi
S
Biswas
The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK.
Yuanyuan
Y
Li
The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK.
Aadil
A
El-Turabi
The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK.
Alexander D
AD
Douglas
Thermo Fisher Scientific, J.H. Oortweg 21, 2333 CH Leiden, Netherlands.
Pim
P
Hermans
Thermo Fisher Scientific, J.H. Oortweg 21, 2333 CH Leiden, Netherlands.
Frank J
FJ
Detmers
ExpreS(2)ion Biotechnologies, SCION-DTU Science Park, Agern Allé 1, Hørsholm DK-2970, Denmark.
Willem A
WA
de Jongh
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Matthew K
MK
Higgins
The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK.
Rebecca
R
Ashfield
The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK. Electronic address: simon.draper@ndm.ox.ac.uk.
Simon J
SJ
Draper
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D017780:Malaria Vaccines / Q000276:immunology*; D010963:Plasmodium falciparum / Q000378:metabolism*; D011817:Rabbits
D002352:Carrier Proteins; D017780:Malaria Vaccines; C531020:RH5 protein, Plasmodium falciparum
Biomanufacture; Blood-stage; Malaria; Plasmodium falciparum; Protein purification; RH5; Vaccine