Phenyl Persefose HP

Phenyl Persefose HP is a HIC resin with a median particle size of 34 µm. It separates molecules by differences in the nature and degree of their hydrophobicity under defined conditions. The resin is used for the separation and purification of recombinant proteins, antibodies, viruses and vaccines.

Phenyl Persefose HP scales up on four counts:

• The highly cross-linked agarose matrix is rigid, so it achieves a high process flow rate under low back pressure.
• The fine particle size improves resolution.
• The hydrophilic base frame minimises the influence of the base frame on the hydrophobicity of the ligand.
• Chemical modification gives it chemical compatibility and resistance to cleaning-in-place (CIP) agents such as sodium hydroxide.

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 a high-salt buffer, such as one containing 1.5–2M ammonium sulfate, to support binding of the target molecule. Consider the stability of the sample in the buffer. The elution buffer is usually a buffer with a low salt content.

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 low-concentration salt.

Flow rate. Select a linear flow rate of 90–150 cm/h according to the column bed height.

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 decreasing 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 buffer containing low salt.
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 low-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 ultrapure or pure water.
• 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

• 730-00025 — 25 ml
• 730-00100 — 100 ml
• 730-00500 — 500 ml
• 730-01000 — 1 L
• 730-05000 — 5 L
• 730-10000 — 10 L
• 730-20000 — 20 L

‍