Helios 30S

Helios 30S is an IEX resin based on highly cross-linked porous polystyrene-divinylbenzene (PS/DVB) monodisperse microspheres, with chemical compounds bonded to the microsphere surface to give a highly stable hydrophilic layer. It combines a high flow rate, high resolution, high capacity, high rigidity, good biocompatibility and physical and chemical stability. Compared with traditional chromatography media it significantly improves the production efficiency and sample recovery of the downstream purification process, and is widely used in the capture, intermediate purification and fine purification of biomolecules such as antibodies, proteins, peptides and nucleic acids.

Method of use

Chromatographic conditions

Buffer selection. Select buffer salts whose buffer groups do not interact with the resin. Use a low-salt equilibration buffer (less than 5 mS/cm) at a pH about 1 unit below the isoelectric point of the target molecule to promote binding, and consider the stability of the sample in that buffer. Elution buffers are usually prepared by adding a high concentration of salt, such as 1M NaCl, or a high-pH buffer to the equilibration buffer.

Flow rate. Select a linear flow rate of 300–600 cm/h according to the column bed height; the greater the bed height, the lower the flow rate.

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 match the equilibration buffer, using dilution, ultrafiltration or desalting with Persedex G-25.

Chromatography steps

1. Equilibration. Wash the column with equilibration buffer until the pH and conductivity at the column outlet are essentially the same as the equilibration buffer. This usually requires 3–5 column bed volumes (CV).
2. Sample loading. Determine the loading volume from the mass of the sample and the binding capacity of Helios 30S.
3. Rinsing. Wash the column with equilibration buffer until the UV absorbance falls to an appropriate value.
4. Elution. Use a linear or step gradient to increase the elution strength, typically a salt gradient of 0.5–1.0M NaCl, so that substances of different binding strength are eluted in turn. Collect the fractions and identify those containing the target molecule. As a rule of thumb, use a shallow gradient across the elution zone of the target molecule and a steep gradient in the region used to wash out contaminants and impurities.
5. Regeneration. Flush the column with a high concentration of salt, such as 2M NaCl.
6. Re-equilibration. After rinsing with equilibration buffer, the next sample can be loaded and the cycle repeated.

In large-scale production, step gradients are often preferred: they are technically simpler and more reproducible than linear gradients, reduce buffer consumption, shorten process time and elute the target molecule at a high concentration.

Cleaning and sterilisation

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. The resin can also be autoclaved at 121°C (pH 7) for 20 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 containing 0.2M NaAc and store at 2–8°C.

Destruction and recycling

Helios 30S is difficult to degrade in nature, so incineration of waste 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

• 114-00025 — 25 ml
• 114-00100 — 100 ml
• 114-00500 — 500 ml
• 114-01000 — 1 L
• 114-05000 — 5 L
• 114-10000 — 10 L
• 114-20000 — 20 L