TABLE 1.
Comparison of biochemical attributes of Shigella variable LPS InvaplexAR products
| Shigella serotype | LPS multiplier a | Endotoxin (×106 EU/mL) b |
IpaC:IpaB ratio (gel densitometry) c |
SEC-HPLC retention time (min) d | DLS (DH, nm) e | % Yield efficiency f |
|---|---|---|---|---|---|---|
| S. flexneri 2a | 1× | 15 | 3.8:1 | 16.2 | 21.4 ± 5.5 | 40.3 |
| 2× | 29 | 6.9:1 | 16.0 | 17.8 ± 4.1 | 57.1 | |
| 4× | 63 | 7.8:1 | 15.6 | 19.5 ± 7.7 | 67.2 | |
| 8× | 61 | 9.4:1 | 15.6 | 21.4 ± 26.4 | 42.5 g | |
| S. flexneri 3a | 1× | 7.3 | 2.5:1 | 16.4 | 20.7 ± 3.4 | 20.6 |
| 2× | 16 | 3.8:1 | 16.2 | 19.5 ± 4.8 | 30.8 | |
| 4× | 55 | 5.8:1 | 15.8 | 22.4 ± 3.5 | 51.8 | |
| 8× | 78 | 5.8:1 | 15.6 | 21.4 ± 2.4 | 50.3 | |
| S. flexneri 6 | 1× | 16 | 2.1:1 | Not done | 20.3 ± 1.3 | 40.0 |
| 2× | 35 | 4.1:1 | 16.3 | 21.4 ± 0.7 | 64.2 | |
| 4× | 62 | 4.0:1 | 16.2 | 25.4 ± 1.4 | 66.9 | |
| 8× | 220 | 3.9:1 | 16.0 | 28.2 ± 0.4 | 72.2 | |
| S. sonnei | 1× | 5.7 | 2.6:1 | 16.1 | 19.5 ± 4.0 | 32.4 |
| 2× | 11 | 5.6:1 | 16.0 | 19.5 ± 8.2 | 43.6 | |
| 4× | 32 | 9.4:1 | 15.8 | 19.5 ± 8.0 | 51.4 | |
| 8× | 89 | 7.2:1 | 15.7 | 22.4 ± 4.8 | 52.0 |
Lipopolysaccharide (LPS) multiplier = the fold-increase in the amount of LPS used in the initial reaction mixture of the four formulations for each serotype. The 1× amount is 0.56 mg total LPS/mg total protein.
Endotoxin was measured using the Charles River Laboratories EndoSafe-PTS endotoxin reader and FDA-licensed cartridges with a sensitivity of 0.001 EU/mL.
The IpaC:IpaB ratio was determined by scanning Coomassie-stained SDS-PAGE gels using a Bio-Rad GS900 calibrated flatbed scanner and the pixel densitometric ratios calculated using the Bio-Rad Image Lab software.
Size exclusion chromatography (SEC)-HPLC.
Dynamic light scattering (DLS) measurements of hydrodynamic diameter (DH) in nanometers.
Percent yield efficiency is defined as the total protein, in milligram, of the resulting InvaplexAR product divided by the total amount, in milligram, of the quantities of protein (IpaB and IpaC) used in the initial reaction mixture, multiplied by 100.
It was noted that the dialysis cassette containing the 8× product had swollen during dialysis and indeed almost twice the volume of product was recovered from the cassette as compared to the 1×, 2×, and 4× products. Total protein concentration of the 8× product was lower understandably due to this volume dilution, but the product yield efficiency was comparable to that of the 1× product. It is unclear as to the reason for this as the phenomenon was not exhibited by other 8× products of other serotypes.