Lepta 400

Lepta 400 is an MMC resin. Different from traditional chromatographic media, it consists of two different structures: the outer shell is a porous passivation layer, which ensures that macromolecules above 400 kDa will not enter the pores and directly flow through the outer layer; the core is coupled and has hydrophobic and positive charge adsorption.

The spherical core with the adsorption group can bind host proteins, nucleic acids and other impurities to the greatest possible extent. The chromatographic medium can be used effectively for the separation and purification of various macromolecular biological samples, such as viruses and virus-like particles. Compared with traditional single-mode chromatography media, Lepta 400 shows better performance:

(1) The unique double-layer structure design can process macromolecules above 400 kDa in flow-through mode, making process optimisation easier and more convenient for linear amplification.

(2) The improved Lepta base frame is more rigid, so it can achieve higher process flow rate at lower back pressure and improve process efficiency.

(3) Compared with traditional gel filtration chromatography, it has a larger loading volume and reduces costs.

Method of use

Chromatographic conditions

• Buffer selection: a buffer salt whose buffer group does not interact with the chromatographic resin should be selected. If the binding and elution mode is used, the equilibration buffer should be a low pH (usually 1 pH unit lower than the isoelectric point of the target molecule) to facilitate the binding of the target molecule. Consider the stability of the sample in the buffer; the elution buffer is usually a buffer with a high concentration of salt (such as 1M NaCl) added to the equilibration buffer. If the flow-through mode is used, the equilibration buffer should adopt conditions that are conducive to the binding of impurities. After the target molecule has completely flowed through, it should be washed directly with high-concentration salt.
• Flow rate: generally, choose a linear flow rate of 90–500 cm/h according to the bed height of the column.
• Sample pretreatment: to prevent the sample from clogging the column, the sample needs to be filtered with a 0.45 μm microporous membrane before loading. It is recommended that the pH and conductivity of the sample is adjusted to be consistent with the equilibrium buffer (dilution, ultrafiltration can be used and desalting to adjust the pH and conductivity of the sample).

Chromatography steps

• Equilibration: use equilibration buffer to fully equilibrate the chromatography column until the pH and conductivity are stable and basically consistent with the equilibration buffer. This step usually requires 3–5 column bed volumes (CV).
• Sample loading*: according to the binding capacity measured in the small test, determine the sample loading volume and loading amount of the sample on Lepta 400.
• Wash: use equilibration buffer or another suitable buffer to wash the column until the UV becomes stable and returns to the baseline.
• Regeneration: rinse the column with a high-salt buffer (such as 2M NaCl), or 1M NaOH, 30% isopropanol solution.
• Re-equilibration: re-equilibrate the column with equilibration buffer.

Cleaning and regeneration

Contaminants (e.g. lipids, endotoxins and proteins) accumulate on the column as the number of uses of the chromatography resin increases. Determine the frequency of CIP according to the degree of contamination of the chromatography resin (if the contamination is considerable, CIP is recommended after each use to ensure repeatability of results and to prolong the working life of the chromatography resin). For different types of impurities and contaminants, the recommended cleaning conditions are as follows:

• Removal of strongly binding proteins: wash with 5 CV of 2M NaCl solution or use a high salt buffer not lower than pH 2, such as 1M NaAc solution.
• Removal of strongly hydrophobic proteins and precipitated proteins: first wash with 5 CV of 1M NaOH solution, then wash the lye with 5–10 CV of ultra pure or pure water.
• Removal of lipoproteins and lipids: first wash with 5 CV of 70% ethanol or 30% isopropanol, then rinse with 5–10 CV of ultra pure or pure water.

Note: 70% ethanol or 30% isopropanol should be degassed before use; the flow rate should be 30–60 cm/h during CIP; reverse cleaning should be used when the clogging is severe. To reduce the microbial load, it is recommended that 0.5~1M NaOH solution is used to treat the chromatography resin for 15~30 minutes.

Storage

Keep the unopened chromatography resin in the original container and store 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

Since chromatography resin is difficult to degrade in nature, it is recommended that the waste chromatography resin is incinerated to protect the environment. For chromatography resin that has been in contact with biologically active samples such as viruses and blood, follow the 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.