For best performance of multimodal chromatography media over a long working lifetime, follow the procedures described below.
After packing, and before a chromatographic run, equilibrate with loading buffer by washing with at least 5 bed volumes or until the column effluent shows stable conductivity and pH values.
After each step, elute any reversibly bound material with
Regenerate the medium by washing until the column effluent shows stable conductivity and pH values.
CIP is a procedure for removal of contaminants such as lipids, endotoxins, nucleic acids, and precipitated or denatured proteins that remain in the packed column after regeneration. Regular CIP prevents the build-up of contaminants in the medium and helps to maintain capacity, flow properties, and general performance. The frequency of CIP depends on the nature and the condition of the feedstock.
However, for capture steps CIP is normally recommended after each cycle. A specific CIP protocol should be designed for each process according to the type of contaminants present. CIP protocols should always be applied in reverse flow because contaminants are usually found in the first part of the column.
Typically, it is recommended to perform a CIP:
The nature of the sample will ultimately determine the final CIP protocol so the CIP procedure below may require optimization. NaOH concentration, contact time, and frequency are typically the main parameters to vary during the optimization of the CIP.
The CIP procedure that follows removes common contaminants.
For increased contact time and due to the viscosity of the CIP solutions it is recommended to use a lower flow rate than during the purification.
Regular CIP is necessary to remove captured contaminants and allow re-use of Capto Core 700 with maintained capacity. Use of 1 M NaOH in 27% 1-propanol is recommended for effective CIP and sanitization of the medium after every cycle. Due to the strong binding of a wide range of contaminants to the ligand, an organic solvent will be needed for CIP with most samples. However, this will be sample dependent, and it may be possible to use CIP solutions without organic solvents. CIP protocols are dependent on the feed material and running conditions, and optimization is therefore recommended for the chosen application.
To reduce microbial contamination in the packed column, sanitization using 0.5 to 1 M NaOH with a contact time of at least 1 h is recommended (Table A2.1). For spore-forming bacteria (e.g., Bacillus spp.), including 20% ethanol will improve the efficiency of the sanitization significantly (Table A2.2). Including propanol instead of ethanol will also improve the sanitization efficiency (Table A2.3).
1 for reduction to below detection limit of < 3 organisms/mL
2 for reduction to below detection limit of 10 organisms/mL
3 for reduction to below detection limit of 100 organisms/mL
Store used medium in the container at a temperature of 4 °C to 30 °C. Recommended storage solutions is: