Toxineraser endotoxin removal resin

Three P-types of S₆₀-VP8* nanoparticles (P[8], P[6], and P[4]) were produced and purified as previously described [31 (link),33 (link)] and mixed as the trivalent vaccine in the liquid formulation in phosphate buffer saline (PBS). The individual nanoparticles and the trivalent vaccine were assessed by (1) SDS-PAGE for protein quality; (2) gel filtration chromatography and EM inspection for nanoparticle formation and structural integrity [31 (link),33 (link)]. Bacterial endotoxin was removed by using ToxinEraser™ Endotoxin Removal Resin (GenScript, Cat. No L00402. Piscataway, NJ, USA). The vaccines were formulated by mixing each S₆₀-VP8* P[8], P[6], and P[4] or S₆₀ alone with 600 µg/dose of aluminum hydroxide adjuvant (Alhydrogel, InvivoGen, San Diego, CA, USA). The vaccine dose was originally designed to contain 200 µg of each P-type. However, due to the need for filtration to eliminate contaminating bacteria, the actual amount of antigen in the vaccine was reduced. The actual amount of antigens in the vaccine measured with Nanodrop was 94 µg of P[4], 52 µg of P[6], and 143 µg of P[8]. Sterility of antigens was confirmed using blood agar and thioglycolate broth prior to inoculation of Gn pigs.

A single clone was reinoculated in LB medium containing 100 mg/ml of ampicillin, induced by 1 mmol/L isopropyl β-D-thiogalactopyranoside (IPTG) at 37°C and harvested 24 hours later. Both bacteria lysate and sonicated supernatant were detected by SDS-PAGE with Coomassie blue staining. The soluble hTLR4-Fab01 was purified from the periplasm by immobilized metal affinity chromatography (IMAC) using His-trap Lambda Fab Select column (GE healthcare, Madison, WI, USA) according to the manufacturer’s instructions. The purity of the hTLR4-Fab01 was analyzed by SDS-PAGE (12%) or native-page (Bio-Rad, CA, USA) with Coomassie Blue staining.
The endotoxin concentration during the Fab preparation was examined with ToxinSensor^™ Chromogenic LAL Endotoxin Assay Kit (Genscript, Nanjing, China). The hTLR4-Fab01 solution was purified with ToxinEraser^™ endotoxin removal resin (Genscript, Nanjing, China) The final endotoxin level of Fab solution was decreased to less than 0.1 EU/ml.