Inclusion Body Isolation
Following fermentation harvest, the wet cell paste was resuspended in lysis buffer (150 mM NaCl, 20 mM Tris pH 8.0, 5 mM EDTA)
at a defined volume-to-mass ratio of 5 mL of lysis buffer to 1 g of cell paste. The resuspended E. coli were disrupted with an M-110S microfluidizer (Microflluidics, Newton, MA). The resulting lysate was centrifuged at 18,000
grams for 30 min at 4° C, which pelleted the insoluble inclusion bodies (IBs). The IBs were washed with a 2% CHAPS (3[(3 cholamidopropyl)
di-methylammonio]-propanesulfonic acid) wash, followed by an isopropanol wash. The washed IB pellets were stored at –80° C
The inclusion body pellets were solubilized with 8 M urea, 50 mM Tris pH8, and 25 mM dithiothreitol at 150 mL/g of inclusion
body pellet. The solubilized inclusion bodies were clarified using centrifugation. The first column, Amersham Biosciences'
Q Sepharose Fast Flow resin (QFF), was equilibrated using buffer A (8 M urea, 50 mM Tris pH8). The clarified inclusion body
preparation was loaded onto the anion exchange column at a linear velocity of approximately 120 cm/h. The full-length Leish-110f
protein was eluted in a single peak from the resin, using buffer B (8 M urea, 50 mM Tris pH8, 150 mM NaCl). The second column
was a BioRad Macro-Prep ceramic hydroxyapatite (CHT), type I, 40 mM. The CHT column was equilibrated using buffer B at a linear
velocity of 120 cm/h. The reduced QFF elution peak was loaded onto the CHT column and washed with buffer B for 10 column volumes.
The Leish-110f protein was eluted using buffer C (8 M urea, 100 mM potassium phosphate, pH 7.5, 150 mM NaCl).
Diafiltration was performed on the CHT elution peak to remove the urea and replace the buffer with 20 mM Tris pH 8.0. The
diafiltration was performed using a tangential flow filtration device with a 30 kDalton molecular weight cut off (MWCO) polyethersulfone
ultra filtration membrane, and with a final protein concentration of 0.5 mg/mL as determined by A280. The purified Leish-110f
bulk protein was then filter-sterilized using a 0.2 µm filter. The final purified bulk protein was stored at –80° C until
Fill and Finish
The final configuration was a lyophilized cake, containing sucrose mannitol and polysorbate 80. The formulated product was
then loaded into a Virtis lyophilizer and lyophilized.
Testing for Process-Related Contaminants
General safety and residual amounts of process-related contaminants, such as kanamycin, urea, and endotoxins, were measured
using standard procedures. In the case of general safety and residual kanamycin, United States Pharmacopeia (USP) methods
were applied. For urea, a modification of the method of Knorst, Neubert, and Wohlrab was used, adapting the test to a 96-well
format.19 For endotoxins, the limulus amebocyte assay was used.
SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was performed according to the Laemmli method.20 Samples were diluted 1:1 in reducing 2x Laemmli sample buffer (Sigma) and boiled for 5 min. Proteins were then loaded on
4–20% acrylamide gradient gels (Bio-Rad Laboratories, Hercules, CA) and were run at 30 milliamperes (mA) constant for 60 min.
The gels were stained using Coommassie blue, with standard techniques.