Columns
Ion exchange chromatography (IEX) is widely used for analysis and purification of biomolecules. BioPro ion exchange columns are specifically designed for separation of proteins, peptides and nucleic acids.
BioPro IEX columns are based on porous and non-porous hydrophilic polymer beads with low nonspecific adsorption.
They also show higher binding capacity and higher recovery of biomolecules compared to conventional ion exchange columns. The completely spherical, monodisperse beads, together with optimal packing technology, provide high theoretical plate numbers and symmetrical peak shapes!
| Accura BioPro IEX QF BioPro IEX QF |
Accura BioPro IEX SF BioPro IEX SF |
BioPro IEX QA | BioPro IEX SP | |
|---|---|---|---|---|
| Matrix | hydrophilic non-porous polymer beads | hydrophilic porous polymer beads | ||
| Particle size (µm) | 3, 5 | 5 | ||
| Charged group | -CH2N+(CH3)3 | -CH2CH2CH2SO3- | -CH2N+(CH3)3 | -CH2CH2CH2SO3- |
| Counter ion | Cl- | Na+ | Cl- | Na+ |
| Ion-exchange capacity [reference value] (meq/mL-resin) |
0.09 | 0.24 | 0.09 | 0.09 |
| Dynamic binding capacity [reference value] (mg/mL-resin) |
>12 (BSA) | >10 (human-IgG) | >110 (BSA) | >70 (human-IgG) |
| Usable temperature | 4-80°C (Accura BioPro IEX) 4-60°C (BioPro IEX) |
4-60°C | ||
| Usable pH range | 2-12 | |||
| Column material | bioinert coated stainless steel (Accura BioPro IEX) PEEK (BioPro IEX) |
PEEK | ||
The separation of MAb is compared on Accura BioPro IEX SF and competitor’s column under same conditions.
Accura BioPro IEX SF provides sharp peak shape and good resolution of charge variants.
| Eluent | A) 10 mM MES-NaOH (pH 6.6) B) 10 mM MES-NaOH (pH 6.6) containing 1.0 M NaCl 0-20%B (0-15 min) |
|---|---|
| Flow rate | 0.5 mL/min for 4.6 mmI.D., 0.378 mL/min for 4.0 mmI.D. |
| Temperature | 25°C |
| Detection | UV at 280 nm |
| Sample | Bevacizumab |
[All PS], [13PS, 1PO] and [12PS, 2PO] of DNA 15mer are clearly separated by BioPro IEX column.
1. DNA 15mer, 12PS, 2PO (5’-T^T^T^T^T^T^T^T^T^T^T^T^TTT-3’)
2. DNA 15mer, 13PS, 1PO (5’-T^T^T^T^T^T^T^T^T^T^T^T^T^TT-3’ )
3. DNA 15mer, All PS
(5’-T^T^T^T^T^T^T^T^T^T^T^T^T^T^T-3’ )
^=Phosphorothioated
| Column | BioPro IEX QF (5 µm), 100 X 4.6 mmI.D. |
|---|---|
| Eluent | A) 10 mM NaOH B) 10 mM NaOH containing 1.0 M NaClO4 40-70%B (0-15 min), 100%B (15-20 min) |
| Flow rate | 1.0 mL/min |
| Temperature | 25°C |
| Detection | UV at 260 nm |
| Injection | 6 µL (each 3.3 nmol/mL) |
Accura BioPro IEX columns are available with different lengths for specific purposes. Short columns provide short run times and high throughput, whilst retaining good resolution. Longer columns offer a higher resolution that is required for challenging samples, such as the separation of single- and double-stranded RNA.
1. antisense strand
2. siRNA duplex
| Column | Accura BioPro IEX QF (5 µm) |
|---|---|
| Eluent | A) 20 mM Tris-HCl (pH 8.1) B) 20 mM Tris-HCl (pH 8.1) containing 1.0 M NaClO4 |
| Flow rate | 0.5 mL/min |
| Temperature | 60°C |
| Detection | UV at 260 nm |
| Injection | 8 µL (each 5 nmol/mL) |
Oligonucleotides generally exhibit poor peak shape and therefore low recovery in AEX analysis, mainly due to adsorption onto the column hardware. Accura BioPro IEX columns provide high recovery and very good peak shapes from the first injection. This makes Accura BioPro IEX QF columns ideal for the analysis of oligonucleotides with reproducible results. The columns show stable peak areas from the 1st injection, so that no preconditioning is required.
| Column | 5 µm, 100 X 4.6 mmI.D |
|---|---|
| Eluent | A) 20 mM Tris-HCl (pH 8.1) B) 20 mM Tris-HCl (pH 8.1) containing 1.0 M NaClO4 25-40%B (0-15 min), 40%B (15-20min) |
| Flow rate | 1.0 mL/min |
| Temperature | 60°C |
| Detection | UV at 260 nm |
| Injection | 4 µL (5 nmol/mL) |
| Sample | 21mer RNA |
BioPro IEX SF column exhibits excellent batch-to-batch reproducibility for MAb analysis, including the resolution of peaks for small charge variants. BioPro IEX Columns are the best choice for the quality control of MAb and other biopharmaceuticals.
| Column | BioPro IEX SF (5 µm), 100 X 4.6 mmI.D. |
|---|---|
| Eluent | A) 20 mM NaH2PO4-Na2HPO4 (pH 6.5) B) 20 mM NaH2PO4-Na2HPO4 (pH 6.5) containing 0.2 M NaCl 0-50%B (0.5-30 min) |
| Flow rate | 0.5 mL/min (180 cm/hr) |
| Temperature | 25°C |
| Detection | UV at 215 nm |
| Injection | 10 µL |
| Sample | Humanized monoclonal IgG1 |
BioPro IEX QA gives the superior DBC and recovery compared with conventional porous polymer anion exchange columns. The surface structure of porous type BioPro IEX, which is designed for maximum interaction with proteins, provides high binding capacity, and the hydrophilic property of polymer beads significantly reduces nonspecific adsorption of proteins.
Comparisons of dynamic binding capacity (DBC) and recovery for BSA
| Dynamic binding capacity (mg/mL-resin, 10% breakthrough) |
Eluted amount (mg/mL-resin) |
Recovery* (%) |
|
|---|---|---|---|
| BioPro IEX QA | 126 | 120 | 95 |
| Brand T (porous Q type) | 73 | 58 | 79 |
| Brand G (porous Q type) | 100 | 35 | 35 |
*Recovery: (Eluted amount/DBC) X 100
Breakthrough curves
| Column | BioPro IEX QA, 50 X 4.6 mmI.D. Brand T (porous Q Type) 50 X 4.6 mmI.D. Brand G (porous Q Type) 50 X 5.0 mmI.D. |
|---|---|
| Linear velocity | 180 cm/hr |
| Equilibration buffer | 20 mM Tris-HCl (pH 8.6) |
| Elution buffer | 20 mM Tris-HCl (pH 8.6) containing 1.0 M NaCl |
| Detection | UV at 280 nm |
| Sample | 1 mg/mL Bovine serum albumin (BSA) in equilibration buffer |
