Lepta SuperA

Lepta SuperA is an affinity chromatography resin for antibody purification, separating through the specific interaction between antigen and antibody. It is designed to handle high concentrations of monoclonal antibodies and combines high capacity, high flow rate and alkali resistance. It is suitable for capturing monoclonal antibodies or Fc-fusion proteins from large volumes of cell culture medium, as well as for capturing polyclonal antibodies from ascites or plasma. Main features:

• High dynamic binding capacity.
• The modified alkali-resistant rProtein A ligand tolerates 0.5–1.0M NaOH for cleaning-in-place (CIP).

Method of use

Chromatographic conditions

Buffer selection. Use a neutral buffer as the binding buffer, such as 50 mM PB with 0.15M NaCl, pH 7.0–7.6. Use a low-pH buffer as the eluent, such as 0.1M citric acid, pH 3.0–4.0. Since the ability of Lepta SuperA to bind IgG depends on the source and subtype of the antibody, high salt and high pH promote binding of the antibody to the resin: increasing the salt concentration reduces electrostatic repulsion, which would otherwise hinder the affinity reaction, and enhances the hydrophobic effect. For different antibodies, optimise the binding and washing conditions by changing the salt type, salt concentration and pH of the buffer. When optimising the elution conditions, explore the highest pH that still gives effective desorption.

Flow rate. Select a linear flow rate of 60–300 cm/h according to the column bed height. The higher the column bed, the slower 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 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. Rinse. After loading, wash with equilibration buffer until the UV absorption falls to an appropriate value. If necessary, add high salt or slightly lower the pH to remove non-specifically adsorbed impurities as far as possible.
4. Elution. Apply a linear gradient of 10 CV from equilibration buffer to elution buffer, such as 1M sodium citrate, pH 3.0, and determine the optimal elution pH from the peak position of the antibody. If the antibody is unstable under acidic conditions, neutralise the eluate with a neutralising solution such as 1.0M Tris-HCl, pH 9.0.
5. Re-equilibration. Re-equilibrate the column with equilibration buffer.

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:

• Pre-wash: wash the column with 2–3 CV of binding buffer.
• Alkali wash: wash the column with 0.1–0.5M NaOH, with a contact time of 10–15 minutes.
• Final rinse: immediately flush with at least 5 CV of binding buffer.

Lepta SuperA tolerates 0.5–1.0M NaOH. NaOH concentration, contact time and contact frequency all affect the cleaning result: a high concentration and a long contact time increase the CIP effect, but also accelerate the loss of IgG binding capacity. Choose 0.1–0.5M NaOH as a rule, and reserve 0.5–1.0M NaOH for severe contamination.

If antibody bound to the resin has not been completely eluted, regenerate the column before CIP. Before performing CIP with NaOH, equilibrate the column with a neutral-pH solution to avoid direct contact between the low-pH buffer and the high-pH NaOH solution, which can raise the temperature inside the column.

Since 20% ethanol preservation solution neither sterilises nor depyrogenates, treat the resin with 0.5M NaOH for 15–30 minutes before and during use to reduce the risk of microbial contamination and pyrogens.

Storage

Keep unopened resin in the original container at 2–8°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

• 151-00025 — 25 ml
• 151-00100 — 100 ml
• 151-00500 — 500 ml
• 151-01000 — 1 L
• 151-05000 — 5 L
• 151-10000 — 10 L