Abstract
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.
Highlights
The production of recombinant antigen remains central to the development of many types of subunit vaccines, and especially⇑ Corresponding author.for those seeking to induce antibody (Draper et al, 2015)
Jin et al / International Journal for Parasitology 47 (2017) 435–446 notable success in humans including hepatitis B virus surface antigen (HBsAg) and bacterial toxoids. These approaches are further exemplified by ongoing efforts to develop a highly effective vaccine against infection, disease or transmission caused by the Plasmodium falciparum human malaria parasite (Halbroth and Draper, 2015)
We have previously reported the production of a full-length PfRH5 protein vaccine with C-terminal His6 tag using a stable Drosophila Schneider 2 (S2) stable cell line (Hjerrild et al, 2016)
Summary
The production of recombinant antigen remains central to the development of many types of subunit vaccines, and especially⇑ Corresponding author.for those seeking to induce antibody (Draper et al, 2015). J. Jin et al / International Journal for Parasitology 47 (2017) 435–446 notable success in humans including hepatitis B virus surface antigen (HBsAg) and bacterial toxoids (tetanus and diphtheria). Jin et al / International Journal for Parasitology 47 (2017) 435–446 notable success in humans including hepatitis B virus surface antigen (HBsAg) and bacterial toxoids (tetanus and diphtheria) These approaches are further exemplified by ongoing efforts to develop a highly effective vaccine against infection, disease or transmission caused by the Plasmodium falciparum human malaria parasite (Halbroth and Draper, 2015). In this case multiple stages of the parasite’s complex lifecycle are susceptible to functional antibodies – including sporozoites, merozoites, infected red blood cells, gametocytes and sexual stages within the mosquito
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