SEC columns for analysis and purification of antibodies

Superdex 200 Increase is suitable for both preparative and analytical purposes.

Superdex 200 Increase’s fractionation range allows separation of a large variety of proteins (M_r ~ 10 000 to ~ 600 000) with an optimized resolution for antibodies (M_r ~ 100 000 to ~ 300 000). It is well suited for antibody dimer, monomer, and aggregate detection and separation in mAb preparations.

These columns also enable protein characterization, including aggregate analysis of antibodies, size homogeneity analysis of membrane proteins, and protein-protein interactions, where high-quality data can be achieved.

Cleaning-in-place (CIP) capabilities

This agarose-based resin is alkali resistant and supports cleaning-in-place (CIP) procedures. This capability allows the same column to be used for different proteins, with minimal risk of carry-over between samples.

Regular cleaning can also increase column lifetime.

Three SEC column sizes

Three size exclusion chromatography (SEC) column sizes are designed to serve different purposes:

• 10/300 GL column, 10 mm × 300 mm, for high resolution analysis as well as for small-scale (25–500 µL) preparative purification, such as the polishing step in a purification protocol
• 5/150 column, 5 mm × 150 mm, for rapid purity check and screening of small volumes (4–50 µL)
• 3.2/300 column, 3.2 mm × 300 mm, for high resolution analysis and purification of sample volumes of 4–50 µl, when sample amount is limited and low buffer consumption is important

Feedback on Superdex 200 Increase column performance

“We use SEC MALS (Size Exclusion Chromatography Multi Angle Light Scattering) to determine absolute molecular weight for proteins and protein complexes. It is a very powerful technique and for the SEC part we need a column that will give us good separation and good resolution. We have tried a column that we bought with the system, this gives reasonable results but fractionation range was limiting.

"We also tried Superose 6 3.2/300 but this required such a slow flow rate the signal to noise was too high. We also tried a silica based column for a better fractionation range but were a bit limited with pH range and buffer choice due to it being silica based. Now we use the Superdex 200 Increase 10/300 and the Superose 6 Increase regularly for this technique. The Increase column gives us better pressure limits, increased flow rate, buffer compatibility and good resolution.

The one area we do run into problems is if we have very large complexes, up to 1 MDa and our sample comes out in the void volume. In this case the sample can be mixed with aggregates and contaminants in the void and give an impure sample for downstream analysis.” Ciaran McKeown

Successful use in published research

These prepacked columns are also used in a variety of applications, including:

• Analysis of protein oligomerization states
• Study of replicative helicase machinery
• Examination of the structure, assembly, and function of cytoplasmic pore filaments
• Initiation of pulsed gene expression by LytTR transcription regulators