Table 1.
Columns and corresponding conditions used for the development of the new chromatographic purification process for QS-21. Listed gradients are the main separation gradients of each method. For full gradient profiles, refer to figure captions. FA: formic acid, AA: ammonium acetate, AF: ammonium formate.
| Column Name | Column Specifications | Column Chemistry | Flowrate (mL/min) | Injection Volume | Sample Concentration (mg/mL) | Condition(s) | Figure(s) |
|---|---|---|---|---|---|---|---|
| First Chromatographic Step | |||||||
| Gemini C18 | 250 × 4.6 mm, 5 μm, 110 Å | C18 | 1 | 50 μL | 1 | Gradient: 20–80% MeCN with 0.1% FA | Fig. 1A |
| Synergi Hydro RP | 250 × 4.6 mm, 4 μm, 80 Å | Polar C18 | 1 | 50 μL | 1 | Gradient: 20–80% MeCN with 0.1% FA | Fig. 1A |
| Luna Omega Polar C18 | 250 × 4.6 mm, 5 μm, 100 Å | Polar C18 | 1 | 50 μL | 1 | Gradients: 20–80%, 30–70%, 40–50% MeCN with 0.1% FA | Fig. 1 |
| Luna Omega Polar C18 | 250 × 50 mm, 5 μm, 100 Å | Polar C18 | 85 | 1, 3, 5, 10 mL | 100 | Gradients: 35–55%, 37–50%, 38–49%, 40–47%, 40–45% MeCN with 0.1% FA |
Fig. 2, SI Fig. 1B |
| Second Chromatographic Step | |||||||
| Kinetex HILIC | 250 × 4.6 mm, 5 μm, 100 Å | Unbonded silica | 1 | 20 μL | 1 | Isocratic: 80:20 MeCN:H 2 O with 5 mM AA (pH 5.8) or AF (pH3.2) |
SI Fig. 3 |
| TSKgel-Amide 80 | 250 × 4.6 mm, 5 μm, 100 Å | Amido | 1 | 20 μL | 1 | Isocratic: 80:20 MeCN:H 2 O with 5 mM AA (pH 5.8) |
Fig. 3 |
| Zorbax NH 2 | 250 × 4.6 mm, 5 μm, 70 Å | Amino | 1 | 20 μL | 1 | Isocratic: 50:50, 60:40, 65:35 MeCN:H 2 O with 5 mM AA (pH 5.8) |
Fig. 4 |
| Luna NH 2 | 250 × 4.6 mm, 5 μm, 100 Å | Amino | 1 | 20 μL | 1 | Isocratic: 50:50 MeCN:H 2 O with 5 mM AA (pH 5.8) |
SI Fig. 4 |
| Luna Omega SUGAR | 250 × 4.6 mm, 3 μm, 100 Å | Amido/amino | 1 | 20 μL | 1 | Isocratic: 70:30, 75:25 MeCN:H 2 O with 5 mM AA (pH 5.8) |
SI Fig. 5 |
| Syncronis HILIC* | 250 × 4.6 mm, 5 μm, 100 Å | Sulfobetaine | 1 | 20| 2, 5, 30, 60 μL | 1| 40 | Isocratic: 80:20 MeCN:H 2 O with 5 mM AA (pH 5.8) or AF (pH3.2) |
Fig. 5, SI Fig. 6 |
| Syncronis HILIC | 250 × 21.2 mm, 5 μm, 100 Å | Sulfobetaine | 20 | 250, 600 μL | 40 | Isocratic: 80:20 MeCN:H 2 O with 5 mM AA (pH 5.8) |
Fig. 6, SI Fig. 8A |
| Sequant ZIC-HILIC | 250 × 4.6 mm, 5 μm, 200 Å | Sulfobetaine | 1 | 5, 30 μL | 40 | Isocratic: 80:20 MeCN:H 2 O with 5 mM AA (pH 5.8) |
SI Fig. 7 |
*
On the analytical Syncronis HILIC column, testing of run conditions was performed with 20 μL sample injection at 1 mg/mL, while sample loading study was performed with 2, 5, 30 and 60 μL injection volumes at 40 mg/mL.