An Improved Manufacturing Process for a Recombinant Polyprotein Vaccine - Combating the devastating global disease of leishmaniasis with a new therapeutic and prophylactic vaccine - BioPharm

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An Improved Manufacturing Process for a Recombinant Polyprotein Vaccine
Combating the devastating global disease of leishmaniasis with a new therapeutic and prophylactic vaccine


BioPharm International


Characterization of the Protein


Figure 6
The final product was tested for endotoxins, residual moisture, pH, concentration, physical appearance, identity, and quality as measured by SDS-PAGE. These data are shown in Table 1.

Immunogenicity of the cGMP Material in a Murine Model


Table 1. Summary of in-process contaminant clearance using CEX for capture and HCI for polish
We compared the Leish-111f protein with the improved version Leish-110f in a murine potency assay. Mice were immunized with a single dose of 10 g of Leish-111f or Leish-110f adjuvanted with MPL-SE. Seven days post-injection, mice were bled and antigen-specific IgG1 and IgG2a antibody titers were measured by enzyme-linked immunosorbent assay (ELISA) (Figure 7). The two forms of the protein induced comparable titers of both IgG1 and IgG2a antigen cross-reactive antibody. These results suggest that Leish-110f made using our new process and construct induced antibody responses comparable to that obtained with Leish-111f in mice.

Discussion


Figure 7
Researchers at IDRI have chosen to develop a Leishmania vaccine that is a fusion of three distinct, conserved Leishmania antigens. These three antigens—TSA, LmSTI1, and LeIF—were selected for the development of a subunit vaccine based on their demonstrated abilities to induce at least partial protection in the BALB/c mouse model of L. major in either prophylactic (TSA and LmSTI1) or therapeutic (LeIF) applications.13 All three antigens are present in the amastigote and the promastigote forms of the parasite and are highly conserved among Leishmania species, a requisite for ensuring cross-species protection. Immunization with a single recombinant antigen, in addition to having a simplified manufacturing process, may insure equivalent uptake of the components by individual antigen-presenting cells; in turn, it may generate an immune response that is broadly specific for all the immunogenic epitopes spanning the polyprotein.

Creating a vaccine composed of three or more independently produced recombinant proteins would be prohibitively expensive to manufacture and formulate, compared to the single polypeptide vaccine described here. The manufacturing cost of a vaccine is particularly important in developing countries, and it was one of the considerations in producing Leish-110f. The original Leish-111f fermentation was different from that used in the new process. Conditions were developed for Leish-110f to attain a higher cell density before induction and thus, a higher product yield. This outcome was accomplished by using a richer, semi-defined media, and a controlled feeding strategy to manage the growth of the culture. These two changes allowed for a substantial increase in recombinant protein production per liter of culture.


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