Helios 50-HS

Helios 50-HS is a strong cation-exchange IEX resin that separates molecules by differences in the nature and magnitude of their charge under defined conditions. Its base frame is a porous polymer microsphere prepared by suspension and emulsion polymerisation of styrene and divinylbenzene. The unique through-hole structure makes it especially suitable for the separation and purification of large-scale samples such as recombinant proteins, antibodies, nucleic acids, viruses and virus-like particles, and polysaccharides.

Helios 50-HS scales up on three counts:

• The polystyrene-divinylbenzene frame gives high stability and resistance to aggressive CIP cleaning.
• The through-hole design achieves a high flow rate.
• It combines high capacity, high resolution and high flow rate to improve process efficiency.

Method of use

Chromatographic conditions

Buffer selection. Select buffer salts whose buffer groups do not interact with the resin. In bind and elute mode, the equilibration buffer should be a low-salt (less than 5 mS/cm) and low-pH buffer, usually about 1 pH unit below the isoelectric point of the target molecule, to promote binding. Consider the stability of the sample in the buffer. The elution buffer is usually the equilibration buffer with a high concentration of salt, such as 1M NaCl, added. 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 high-concentration salt.

Flow rate. Select a linear flow rate of 200–600 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, and desalt it to adjust its pH and conductivity.

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 of Helios 50-HS 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 eluent, applied as a linear or step gradient, to separate molecules of different binding strength, and collect fractions from the eluate. pH gradient elution or mixed elution can also be used.
5. Regeneration. Rinse the column with a buffer containing high salt, 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 impurity 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 at not lower than pH 2, 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. 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

Helios 50-HS chromatography resin is difficult to degrade in nature, so incineration of discarded resin is recommended to protect the environment. 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

• 801-00025 — 25 ml
• 801-00100 — 100 ml
• 801-00500 — 500 ml
• 801-01000 — 1 L
• 801-05000 — 5 L
• 801-10000 — 10 L
• 801-20000 — 20 L