PreDictor RoboColumn Capto S ImpAct, 200 μL
Minicolumns packed with Capto S ImpAct, an ion exchange chromatography resin for the polishing step in downstream processes.
FAQ
Properties of Capto media, such as matrix rigidity, allow a wide working range of flow velocities, bed heights, and sample viscosities. High flow velocities increase volume throughput and decrease process time. Higher bed heights mean smaller diameters and a reduced column footprint.
Figure 6 shows the relationship between column bed height and operating flow velocity for Capto S ImpAct. The size of the area below the pressure-limit curve represents the window of operation, that is, the available operating range for the medium.
The pressure limits for Capto S ImpAct shown in Figure 6 are based on a production-scale column and are calculated for 20 cm bed height and maximum operating flow velocities of 220 cm/h.
At this flow velocity, the pressure is equal to or less than 3 bar (0.3 MPa) measured using process buffers with the same viscosity as water, 20°C, which is the highest recommended operating pressure for this medium at this scale. Capto S ImpAct chromatography medium can normally be run at maximum pressure ratings of low-pressure columns (3 bar [0.3 MPa]).
Note that the maximum operating velocity of Capto S ImpAct is dependent of the column used as well as the viscosity of the liquid. For recommended columns, see data file, 29-0670-18.
Fig 6. The pressure/flow window of operation (area under the curve) of Capto S ImpAct. Data corresponds to a production-scale column at 20°C and a viscosity equivalent to water. Green lines show the residence time in the column (4, 8, and 16 min).
Table 5 shows some application examples of purification of different MAbs using Capto S ImpAct post-Protein A purification. See also data file, 29-0670-18.
Table 5. Results from the purification of four MAbs using Capto S ImpAct.
| MAb A | MAb C | MAb D | MAb E | |
|---|---|---|---|---|
| Dynamic binding capacity (DBC) (mg/mL medium) | 108 | 92 | 118 | 122 |
| Load (mg/mL medium) | 80 | 64 | 80 | 85 |
| Start aggregate content (%) | 7 | 2 | 4 | 2 |
| Aggregates at 90% monomer recovery (%) | 0.9 | 0.6 | 1.2 | 0.6 |
| Start HCP* content (ppm) | 34 | 1800 | 300 | 454 |
| HCP at 90% monomer recovery (ppm) | 8 | 42 | 25 | 43 |
| Start protein A content (ppm) | 4 | 1 | 1 | <1 |
| Protein A at 90% monomer recovery (ppm) | <1 | <1 | <1 | <1 |
| Elution pool volume (CV) | 5.1 | 5.4 | 4.0 | 6.3 |
| Elution pool concentration at 90% monomer recovery (mg/mL) | 13.6 | 10.6 | 17.8 | 12.3 |
* HCP = host cell proteins
See Figure 7.
| Running conditions | |
|---|---|
| Medium: | Capto S ImpAct |
| Column: | Tricorn 5/50, CV 1 mL |
| Sample: | ~ 10 mg/mL MAb purified on MabSelect SuRe medium, buffer exchanged into starting buffer |
| Load: | Until 20% breakthrough |
| Start buffer: | MAb A: 50 mM sodium acetate, pH 5.5 50 mM NaCl MAb B: 50 mM sodium acetate, pH 5.5 MAb C: 50 mM sodium acetate, pH 5.25 MAb D: 50 mM sodium acetate, pH 5.5 MAb E: 50 mM sodium acetate, pH 5.25 pIgG: 50 mM sodium acetate, pH 5.5 |
| Gradient: | Step elution including 500 mM NaCl in respective start buffer |
| Flow rate: | 0.185 mL/min, 5.4 min residence time |
| Cleaning in place (CIP) | 1 M NaOH |
Fig 7. Dynamic binding capacity for five different MAbs and one polyclonal IgG (pIgG).
See Figure 8. See also data file 29-0670-18, and application note “Polishing of monoclonal antibodies using Capto S ImpAct”, 29-1083-27.
Figure 8 shows the resolution for Capto S ImpAct at 70% of DBC at 10% breakthrough (64 mg/mL for Capto S ImpAct). The chromatogram shows the analysis of the aggregate (in red) and host cell protein (HCP) contents (in green).
| Running conditions | |
|---|---|
| Medium: | Capto S ImpAct |
| Column: | Tricorn 5/100, bed height 10 cm |
| Sample: | 9.6 mg/mL MAb C, purified on MabSelect SuRe medium. pH adjusted to pH 5.3 |
| Sample volume: | 12 mL |
| Start buffer: | 50 mM sodium acetate, pH 5.3 |
| Elution buffer: | 50 mM sodium acetate, 500 mM NaCl, pH 5.3 |
| Gradient: | Linear, 0 to 350 mM NaCl (0% to 70% elution buffer) in 20 CV |
| Flow rate: | 0.35 mL/min, 5.4 min residence time |
| System: | ÄKTA system |
Fig 8. Chromatogram from purification of a MAb on Capto S ImpAct, demonstrating the high resolution even at high load. Blue: absorbance at 280 nm, red: MAb aggregates, green: HCP, orange: conductivity. Table 6 shows a summary of the results.
Table 6. Summary of results for chromatogram shown in Figure 8.
| Summary of results | |
|---|---|
| DBC (mg/mL medium) | 92 |
| Load selectivity run (mg/mL) | 64 |
| Aggregates at 90% monomer recovery (%) | ~ 0.6 |
| HCP at 90% monomer recovery (ppm) (start 1800 ppm) | 42 |
| Protein A at 90% monomer recovery (ppm) (start ~ 1 ppm) | < 1 |
| Elution pool volume (CV) | 5.4 |
| Elution pool concentration at 90% monomer recovery (mg/mL) | 10.6 |
For screening of a wide range of buffer conditions we recommend prepacked 96-well filter PreDictor plates or PreDictor RoboColumn, prepacked miniaturized columns. Further optimization is preferably done in laboratory-scale columns before scale-up.
Typical running conditions:
| Medium: | Capto S ImpAct |
| Sample: | MAb, partially purified by MabSelect SuRe or Mab Select SuRe LX. pH adjusted to start condition pH |
| Start buffer: | 50 mM sodium acetate, pH 5.3 (has to be optimized for every individual MAb) |
| Elution buffer: | 50 mM sodium acetate, 500 mM NaCl, pH 5.3 (has to be optimized for every individual MAb) |
| Gradient: | Linear, 0 to 350 mM NaCl (0% to 70% elution buffer) in 20 CV |
| Residence time: | 5.4 min |
| System: | ÄKTA™ chromatography system |
Not to the same extent, but in some cases yes. We recommend screening of pH and salt conditions to evaluate each protein that is going to be purified.
All are prepacked, convenient, ready-to-use formats for fast and reproducible process development and small-scale purifications. The small format volumes give fast results and low sample/buffer consumption.
PreDictor plates
• Format: Prefilled 96-well filter plate, several different media volumes/well depending on the specific media
• Excellent for high-throughput condition/parameter screening
• Excellent for high-throughput chromatography media screening
• Minimal sample/buffer consumption
• To be used with a robot or manually (centrifugation)
• Sold as four plates per package
PreDictor RoboColumn miniaturized columns
• Column format: Prepacked as 200 μL or 600 μL
• Excellent for high-throughput condition/parameter screening
• Excellent for high-throughput chromatography media screening
• Minimal sample/buffer consumption
• To be used with a robot
• Sold as eight columns in row per package
HiTrap columns
• Column formats: 0.7 × 2.5 cm (1 mL) and 1.6 × 2.5 cm (5 mL)
• Suitable for condition screening and small-scale purifications
• Broad range of prepacked chromatography media
• To be used manually (syringe), with a peristaltic pump, or a system such as ÄKTA
• Sold as 5 × 1 mL, 2 × 1 mL, 1 × 5 mL and 5 × 5 mL depending on the medium
HiScreen columns
• Column format: 0.77 × 10 cm (4.7 mL)
• Excellent for method optimization due to the 10 cm bed height
• Easy connection of two columns in series for 20 cm bed height
• Scalable results to BioProcess columns using the same linear flow velocity
• To be used with a system such as ÄKTA
• Sold as one column per package
Yes, the same buffer systems can be used for Capto S ImpAct as for Capto S and Capto SP ImpRes. Capto S ImpAct is a CIEX medium and can be used as any other CIEX media.
Testing and optimization should always be done before replacement. In general Capto S ImpAct will give similar resolution as Capto SP ImpRes, but at higher loads.
