Compared with manual purifications and user-packed columns, the time required for purification of a protein can be greatly reduced using automation and prepacked columns. In this post, Abhijit describes the purification of a mAb in one step from culture supernatant to high purity using the ÄKTA start chromatography system and a HiTrap Protein G HP column.
Purify your mAb in a simple workflow
A popular method for purifying antibodies and their fragments is affinity chromatography, using a resin that contains an immobilized ligand such as protein A, protein G, or protein L. Protein G is a good general choice, as it binds to more subclasses of IgG than protein A and thus will capture a broad range of IgG molecules from various eukaryotic species.Using a manual workflow, achieving consistent purity and yield can be a challenge. When column packing is performed manually by different operators, for example, there is a risk of obtaining variation between packings, which will be reflected in the outcome. Also, manually loading of large sample volumes can be both time- and resource-consuming. Often, a syringe is used and full sample load requires multiple loadings. As a result, the throughput is typically low, with only one single batch of mAb purified per day.
ÄKTA start chromatography system, quipped with a HiTrap Protein G HP column, allows you to purify your mAb in an automated manner following the simple workflow described in Fig 1.
Fig 1. Workflow illustrating the steps involved in purifying mAbs.
You define your method in the affinity template of UNICORN start system control software, as described in Table 1, and use the System Control module to run the method. The ÄKTA start pump can load 20 mL of diluted culture supernatant onto a 1 mL HiTrap Protein G HP column. Peak fractions are automatically collected using the Frac30 fraction collector. The whole run is completed in only a few hours.
Table 1. UNICORN start method overview
| Method flow | Run parameters |
|---|---|
| Method settings | CV
Pressure limit 0.3 MPa Flow rate 1 ml/min |
| Prime and equilibration | 5 CV |
| Sample application | Apply sample using pump
Sample volume 20 ml |
| Wash out unbound | 10 CV |
| Elution and fractionation | Gradient elution:
Step 1: 70% B 5 CV Step 2: 100% B 10 CV Fractionation: Peak fractionation level based (start/end: 5 mAU) |
| Prime and equilibration | 5 CV |
CV = column volume, 0.3 MPa = 3 bar (43.5 psi)
Additional application-specific HiTrap prepacked columns for use with ÄKTA start are available, including the protein A resin column for purification of human IgG as well as columns for histidine- or GST-tagged proteins and untagged proteins.
Learn more about how ÄKTA start can simplify your mAb purification and reduce the time required from here.