Persefose 6FF

Persefose 6FF is a gel filtration SEC resin built on a highly cross-linked agarose matrix, with a cross-linking ratio of 6% and a median particle size of 45–165 µm. It separates molecules by differences in molecular weight and conformation. The resin is used for the separation and purification of recombinant proteins, antibodies, nucleic acids, viruses, virus-like particles and polysaccharides.

Persefose 6FF scales up on two counts:

• The highly cross-linked agarose matrix is rigid. This reduces the restriction on column bed height, and allows a high process flow rate under low back pressure.
• An improved bead-making process reduces the diffusion effect and gives better resolution.

Method of use

Chromatographic conditions

Buffer selection. Consider the stability of the sample in the buffer. Use a salt-containing buffer rather than ultrapure or pure water, to avoid non-specific adsorption.

Flow rate. Select a linear flow rate of 90–200 cm/h according to the column bed height. Where the column bed height is 60 cm or more, reduce the flow rate to 45–60 cm/h.

Sample pretreatment. Filter the sample through a 0.45 µm microporous membrane before loading to prevent the column from clogging.

Chromatography steps

1. Equilibration. Use the buffer to fully equilibrate the column until the pH and conductivity are stable and consistent with the equilibration buffer. This step usually requires 1–2 column bed volumes (CV).
2. Sample loading. The usual loading volume is 1–5% of the column volume. Do not use too high a sample concentration, to avoid overpressure and loss of resolution.
3. Elution. Elute with buffer and collect the peaks at different positions, usually over 1–1.5 CV.
4. Regeneration. Rinse the column with a high-salt buffer, such as 1M NaCl.
5. Re-equilibration. Re-equilibrate the column with buffer.

Cleaning and regeneration

Contaminants such as lipids, endotoxins and proteins accumulate on the column as the number of uses increases. Regular cleaning-in-place (CIP) keeps the column in a stable working condition. Determine the frequency of 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 at pH 2 or above, such as 1M NaAc 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. Keep the flow rate at 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

• 702-00025 — 25 ml
• 702-00100 — 100 ml
• 702-00500 — 500 ml
• 702-01000 — 1 L
• 702-05000 — 5 L
• 702-10000 — 10 L
• 702-20000 — 20 L

‍