A scalable and reproducible manufacturing process for Phlebotomus papatasi salivary protein PpSP15, a vaccine candidate for leishmaniasis

Protein Expr Purif. 2021 Jan;177:105750. doi: 10.1016/j.pep.2020.105750. Epub 2020 Sep 10.


Cutaneous leishmaniasis is a parasitic and neglected tropical disease transmitted by the bites of sandflies. The emergence of cutaneous leishmaniasis in areas of war, conflict, political instability, and climate change has prompted efforts to develop a preventive vaccine. One vaccine candidate antigen is PpSP15, a 15 kDa salivary antigen from the sandfly Phlebotomus papatasi that facilitates the infection of the Leishmania parasite and has been shown to induce parasite-specific cell-mediated immunity. Previously, we developed a fermentation process for producing recombinant PpSP15 in Pichia pastoris and a two-chromatographic-step purification process at 100 mL scale. Here we expand the process design to the 10 L scale and examine its reproducibility by performing three identical process runs, an essential transition step towards technology transfer for pilot manufacture. The process was able to reproducibly recover 81% of PpSP15 recombinant protein with a yield of 0.75 g/L of fermentation supernatant, a purity level of 97% and with low variance among runs. Additionally, a freeze-thaw stability study indicated that the PpSP15 recombinant protein remains stable after undergoing three freeze-thaw cycles, and an accelerated stability study confirmed its stability at 37 °C for at least one month. A research cell bank for the expression of PpSP15 was generated and fully characterized. Collectively, the cell bank and the production process are ready for technology transfer for future cGMP pilot manufacturing.

Keywords: 15 kDa sandfly (Phlebotomus papatasi) salivary protein; Cutaneous leishmaniasis; Fermentation; Pichia pastoris; PpSP15; Process development; Purification; Vaccine.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cloning, Molecular
  • Female
  • Fermentation
  • Gene Expression
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Humans
  • Insect Proteins / genetics
  • Insect Proteins / immunology*
  • Insect Proteins / metabolism
  • Leishmania / chemistry
  • Leishmania / immunology*
  • Leishmaniasis Vaccines / genetics
  • Leishmaniasis Vaccines / immunology*
  • Leishmaniasis Vaccines / metabolism
  • Leishmaniasis, Cutaneous / prevention & control
  • Molecular Weight
  • Phlebotomus / chemistry*
  • Phlebotomus / physiology
  • Protein Stability
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Recombinant Proteins / metabolism
  • Saccharomycetales / genetics
  • Saccharomycetales / metabolism
  • Salivary Proteins and Peptides / genetics
  • Salivary Proteins and Peptides / immunology*
  • Salivary Proteins and Peptides / metabolism


  • Insect Proteins
  • Leishmaniasis Vaccines
  • Recombinant Proteins
  • Salivary Proteins and Peptides

Supplementary concepts

  • Komagataella pastoris