Chromatography columns frequently are located at the end of the process train, where purity is essential. Columns have certain
intrinsic cleaning challenges, making them an exceptional test case for cleaning effectiveness. Packed beds influence hydraulic
dynamics within a column, making it difficult to achieve the high linear velocities necessary for clean-in-place (CIP) procedures.
Intraparticulate accessibility varies, creating sampling difficulties because the effectiveness of CIP is based on exposure.
Ligands may be sensitive to cleaning agents and resin affinities may bind biomolecules that should be removed. O-rings, seals,
and bed supports can be difficult to clean thoroughly and ancillary equipment such as pumps, valves, hoses, and safety devices
must be sanitized in isolation. T-intersections and dead-legs in connecting tubing also may require special attention.
Despite the difficulties, CIP procedures are desirable to minimize cleaning-associated downtime, reduce material expense,
reduce operator exposure, and avoid repacking. Also, standardized CIP procedures prevent repeated tests and judgment errors.
Cleaning out of place (manually) requires additional equipment and time, resulting in efficacy variance. Disassembly is labor
intensive and excludes resin from concurrent sanitization.
The column tested in this study was built to minimize inherent cleanability problems. It includes a new adjuster arrangement
instead of a conventional O-ring and a wedge-shaped seat seal designed to maximize flow in the seal area. Other design enhancements
result in improved flow rate and eliminated settling.
Test plan. Following the design and prototype manufacture, the column's cleanability was tested at a small scale using a variety of
tools and challenges. The tests are described in detail. This plan can be applied to maximize equipment performance and to
Figure 1. Washout of phenol red from a chromatography column.
- Dye testing was administered to visually inspect flow coverage.
- Computational flow design helped identify problem areas and model potential design solutions.
- Riboflavin clearance testing and visual inspection with UV excitation helped evaluate the cleaning strength needed and to
assess the ability to remove strongly adhesive contaminants.
- Endotoxin and bacteria assays were conducted to test inactivation of pyrogens and removal of microbiological organisms.
Dye removal test. To determine the overall flow coverage in the system and how well-swept the wetted parts were, packed resin was slurried
in a dye–salt solution, and then washed out with water. The washout was monitored with conductivity and the bed dissected
for traces of dye. The derived peak is indicative of pack symmetry. The test is inexpensive, simple to perform, and non-hazardous.
It should be noted that its qualitative nature might irreversibly bind media and identify only gross flow problems.
Dye testing was performed with phenol red. Figure 1 shows that no visible dye remained in the column; the column passed the
Figure 2. The riboflavin residue detected on a screw showed a design weakness, which was corrected
Riboflavin Clearance Testing. Rib-oflavin clearance testing was conducted to test relative cleanability and identify problem areas in the system. Accessible
wetted surfaces were spray-painted with riboflavin ethanol (EtOH) solution; 100 ppm riboflavin was recirculated throughout
the system. The system was then flushed with a cleaning solution. Removal was monitored with inline UV measurements. All wetted
surfaces were examined with UV and the system was disassembled to inspect seal interfaces. The industry's standard acceptance
criteria require that no riboflavin be present under visual or UV excitation; UV traces return to baseline. Level of detection
(LOD) is 500 ppb for absorbance, –5 ppb for fluorescence. This test is very sensitive, relatively inexpensive, and identifies
specific problem areas. It is also non-hazardous and an industry standard. However, it is a comparative, indirect test because
riboflavin is not a natural contaminant.