DEAE Persefose BB

DEAE Persefose BB is a highly cross-linked agarose IEX resin formed by bonding diethylaminoethyl groups to a high-flow agarose base matrix. This weak anion exchanger has a large 200 µm bead, combining a very high flow rate and low back pressure with high dynamic capacity, good chemical and mechanical stability, low non-specific adsorption and a high recovery rate. It scales up conveniently, shortening production time and improving production efficiency, and is widely used in the ion-exchange separation of proteins, nucleic acids and polypeptides downstream in biopharmaceutical and bioengineering processes.

Method of use

Chromatographic conditions

Buffer selection. Select a buffer salt whose buffer group does not interact with the resin. In bind and elute mode, the equilibration buffer should be low in salt (less than 5 mS/cm) and high in pH — usually 1 pH unit above the isoelectric point of the target molecule — to support binding of the target molecule. Consider the stability of the sample in the buffer. The elution buffer is usually the equilibration buffer with a high concentration of salt added, such as 1M NaCl. In flow-through mode, the equilibration buffer should use conditions that favour the binding of impurities; once the target molecule has completely flowed through, wash directly with a high-concentration salt buffer.

Flow rate. Select a linear flow rate according to the column bed height. The recommended operating range for this resin is 1200–1800 cm/h.

Sample pretreatment. Filter the sample through a 0.45 µm microporous membrane before loading to prevent the column from clogging. Adjust the pH and conductivity of the sample to be consistent with the equilibration buffer, using dilution, ultrafiltration or desalting.

Chromatography steps

1. Equilibration. Use equilibration buffer to fully equilibrate the column until the pH and conductivity are stable and consistent with the equilibration buffer. This step usually requires 3–5 column bed volumes (CV).
2. Sample loading. Determine the loading volume and loading amount from the binding capacity measured in a small-scale test.
3. Impurity washing. Use equilibration buffer or another suitable buffer to wash the column until the UV signal stabilises and returns to baseline.
4. Elution. Elute by increasing the concentration of salt ions in the elution buffer, applied as a linear or step gradient, to separate molecules of different binding strength. pH gradient elution or mixed elution can also be used.
5. Regeneration. Rinse the column with a high-salt buffer such as 2M NaCl.
6. Re-equilibration. Re-equilibrate the column with equilibration buffer.

In flow-through mode, collect during the sample loading step. Continue collecting through the washing step until all target molecules have passed through, then stop. Use high-salt buffer directly at the elution step to wash impurities away.

Cleaning and regeneration

Contaminants such as lipids, endotoxins and proteins accumulate on the column as the number of uses increases. Determine the frequency of cleaning-in-place (CIP) according to the degree of contamination. Where contamination is considerable, CIP after each use is recommended to ensure repeatability of results and to prolong the working life of the resin.

Recommended cleaning conditions by contaminant type:

• Strongly binding proteins: wash with 5 CV of 2M NaCl solution, or use a high-salt buffer no lower than pH 2, such as 1M sodium acetate solution.
• Strongly hydrophobic and precipitated proteins: wash with 5 CV of 1M NaOH solution, then wash out the alkali with 5–10 CV of ultrapure or pure water.
• Lipoproteins and lipids: wash with 5 CV of 70% ethanol or 30% isopropanol, then rinse with 5–10 CV of ultrapure or pure water.

Degas 70% ethanol or 30% isopropanol before use. Use a flow rate of 30–60 cm/h during CIP. Use reverse cleaning where clogging is severe.

To reduce microbial load, treat the resin with 0.5–1M NaOH solution for 15–30 minutes.

Storage

Keep unopened resin in the original container at 4–30°C in a well-ventilated, dry and clean place. Do not freeze. Wash the used column with 2–3 CV of 20% ethanol solution and store at 2–8°C.

Destruction and recycling

Chromatography resin is difficult to degrade in nature, so incineration of waste resin is recommended. For resin that has been in contact with biologically active samples such as viruses and blood, follow local biosafety requirements before destroying or disposing of it.

Packing method

Detailed information on resin packaging is available on request. Please contact your local distributor.

Ordering information

• 296-00025 — 25 ml
• 296-00100 — 100 ml
• 296-00500 — 500 ml
• 296-01000 — 1 L
• 296-05000 — 5 L
• 296-10000 — 10 L