Table 1. Organisms recommended by the USP for challenge testing
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Figure 2 shows that the riboflavin test revealed a weakness in the original design of the column, a dead space where the bed
supports were attached to the column. The edge supports were redesigned and the problem resolved.
Endotoxin spiking. To determine the capacity to inactivate or remove endotoxins from a column in situ, the column was spiked with endotoxins
and subsequently cleaned. Columns are acrylic 450 mm, glass 100 mm; and the medium used was Matrex Cellufine CG700. To simulate
a worst-case scenario, the test used a higher concentration of endotoxins than would ever be encountered in production and
more volume than the column could hold. The system was spiked with >1.0 x 104 EU/mL (670 ng/mL) solution. A typical cleaning procedure followed, after which elluent samples were analyzed for endotoxins
with a kinetic assay (limulus amebocyte lysate, LAL). Industry criteria are 5 log reduction in concentration (endotoxin units
per millimeter). The United States Pharmacopoeia (USP) recommends a <0.25 EU/mL for WFI.2 This test is a quantitative, realistic challenge, but relatively expensive. It also exposes a column to material that could
contaminate the entire system; thus, endotoxin spiking is prohibited.
The static inactivation trials, performed with no solution replenishment, and gentle shaking at room temperature with this
buffer, showed that 1.0 M offered significant enhancement over 0.5 M NaOH. Positive product controls (PPCs) demonstrated negligible
enzymatic inhibition. The study of concentration-dependent inactivation kinetics indicate that only 1.0 M NaOH achieves inactivation
< 0.005 EU/mL.
Figure 3. Endotoxin removal from chromatography columns.
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Lipopolysaccharide (LPS) spiking results. All final rinse values were >0.24 EU/mL. A trial with a 450-mm column filled with reverse osmosis (RO) H2O to a bed height of 5 cm resulted in a log removal value (LRV) of 7.2, with final traces below LAL LOD. LRVs of 5.8 and 5.9
were obtained with the 100 mm and 450 mm columns when packed with Matrex Cellufine GC700 media (nominal 17 cm packed bed).
The tests demonstrated scalable cleanability with these columns and this methodology. Media have a negative impact on inactivation
kinetics due to mass transfer and the shielding effect. However, this test proves that not only the column itself, but also
the Matrex Cellufine CG700 resin can be cleaned on different scales. Dynamic, realistic cleaning is much more effective at
inactivation than merely static exposure alone. For all trials, feed water LPS content was below the LOD for the LAL assay
(0.100 EU/mL.) This is further evidence that negligible, if any, levels of contaminants were held up in the column and would
be released through diffusion (Figure 3).
Microbial challenge. Microbial challenges were developed to characterize the susceptibility of various microorganisms to treatment conditions,
to develop a cleaning procedure that can effectively sanitize the column, and to determine the ability of the column to be
sanitized after severe bacterial and fungal contamination. Typical acceptance criteria include a 5 log reduction in microorganism
concentration in the effluent (CLU/mL), insignificant levels of growth in swab, media, and component samples, and 10 CFU per
100 mL WFI.2 Specific biologics may contain customized information on the appropriate USP monograph. Microbial challenge tests are quantitative,
realistic challenge scenarios, but they are very expensive and preliminary assay development is required.
The USP compendium recommends specific microorganisms for use in microbial challenge testing.
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