Maintenance of Multimodal Chromatography Media and Storage Conditions

For best performance of multimodal chromatography media over a long working lifetime, follow the procedures described below.

Equilibration

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.

Regeneration/strip

After each step, elute any reversibly bound material with

• Capto adhere/Capto adhere ImpRes: low pH (e.g., 0.1 to 0.5 M acetic acid)
• Capto MMC/Capto MMC ImpRes: high ionic strength solution (e.g., 2 M NaCl in buffer) and at the same time increase pH to > 9 (e.g., Tris buffer with 2 M NaCl)

Regenerate the medium by washing until the column effluent shows stable conductivity and pH values.

Cleaning-in-place (CIP)

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:

• when an increase in back pressure is seen
• if reduced column performance is observed
• before first-time use or after long-term storage
• between runs when the same column is used for purification of different batches of protein to prevent possible cross-contamination
• after every run if media is used for capture

CIP protocol—Capto adhere/Capto adhere ImpRes and Capto MMC/Capto MMC ImpRes

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.

1. Wash with at least 2 CV of 2 M NaCl at a pH > 9 (Capto MMC/Capto MMC ImpRes) or 0.5 M acetic acid (Capto adhere/Capto adhere ImpRes).
2. Wash with at least 4 CV of 1 M NaOH.
3. Wash with 5 CV of start buffer or until eluent pH and conductivity have reached the required values.

CIP protocol—Capto Core 700

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.

Sanitization

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).

Table A2.1. Inactivation of microorganisms by NaOH

Organism	NaOH (M)	Time1	Temp. (°C)
E. coli	0.01	2 h	4 or 22
S. aureus	0.1	1 h	4 or 22
C. albicans	0.5	1 h	4 or 22
A. niger	0.5	1 h	4 or 22
B. subtilis spores	1.0	48 h2	22
B. subtilis spores	1.0	8d3	4
P. aeruginosa	0.5	1 h	22

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

Table A2.2. Antimicrobial effect (log10 reduction) of NaOH with the addition of 20% ethanol on Bacillus subtilis spores

Time	0.5 M NaOH	0.5 M NaOH with 20% ethanol	0.1 M NaOH	0.1 M NaOH with 20% ethanol
24 h	3 log	7 log	-	-
300 h	-	-	2 log	4 log

Table A2.3. Sanitization effect of solutions containing 1-propanol or 2-propanol on Bacillus subtilis spores

Solutions	Remaining B. subtilis (ATCC 6633) spores after treatment with propanol, Log10 values
	Initial	0 h	1 h	2 h	5 h	10 h
0.5 M NaOH, 20% 1-Propanol	5.8	5.1	4.4	3.6	1	0
0.5 M NaOH, 40% 1-Propanol	5.8	4.9	3.2	1.2	0	0
0.5 M NaOH, 20% 2-Propanol	5.8	5.1	4.2	3.4	1	0
0.6 M NaOH, 20% 2-Propanol	5.8	5.2	4	2.4	0.6	0
0.7 M NaOH, 20% 2-Propanol	5.8	5.2	3.7	2.6	0	0
0.8 M NaOH, 20% 2-Propanol	5.8	5.1	3.1	1.3	0	0

Storage

Store used medium in the container at a temperature of 4 °C to 30 °C. Recommended storage solutions is:

• 20% ethanol in 0.2 M sodium acetate for Capto MMC ImpRes
• 20% ethanol in water for Capto MMC, Capto adhere, Capto adhere ImpRes, and Capto Core 700
• Do not freeze.