Case study : in-process sterility testing
. Because of the ubiquity of AK in living organisms, AK-amplified assays can detect bacteria, molds, and yeasts, and this
microbial testing is used in sterility testing to determine product release (4, 5). A company was testing its sterile, in-process
materials using a 14-day sterility test. A comparability test was performed to see if equivalent results could be obtained
using a rapid method. Spiking studies were conducted by inoculating samples at a level approximating 1 colony-forming unit
(CFU). Following inoculation, the samples were enriched for 24 h, and contamination was detected using a rapid microbial system
(Celsis AKuScreen). Microbial hold times and the corresponding time for quarantined inventory was 48 h, a savings of 12 days
over traditional microbial testing.
Case study: mold detection.
Pharmaceutical companies use microbial specifications that are based on United States Pharmacopeia, European Pharmacopoeia, and Japanese Pharmacopoeia guidelines, product history, and risk profile. With respect to mold, this typically means the ability to detect Aspergillus brasiliensis. Slow-growing, molds are often the rate-determining step for product release. AK-amplified assays (e.g., Celsis AKuScreen)
can detect yeast and mold in 24 h compared with 48 h if standard bioluminescence is used or 4–7 days if using traditional
One company used standard ATP screening placed microbial specifications on certain products to include the detection of Penicillum expansum. The company modified its bioluminescence protocol using a rapid microbial system. A study was performed on a water-based
lotion, examining how broth type, incubation temperature, and conditions affect A. brasiliensis and P. expansum detection using standard bioluminescence and AK-amplified reageants (e.g., Celsis AKuScreen). Preliminary studies indicated
that static incubation at room temperature optimized the growth of P. expansum. Table 1 (rapid microbial testing) summarizes the study data in which the samples were inoculated with < 50 spores of P. expansum and incubated statically at room temperature (24–27 °C). The modifications of room temperature incubation and broth changes
to letheen or malt extract broth (MEB) enabled detection with the AKuScreen reagents within 24 h. Detection with standard
bioluminescence required 48 h using MEB and 72 h in letheen under the same incubation conditions.
Table I (rapid microbial testing): Detection times for slow-growing mold.* (TABLE 1 (RAPID MICROBIAL TESTING) IS COURTESY
Rapid microbial testing section references
1. B.S. Riley, Am. Pharm. Rev,
7 (2), 28–31 (2004).
2. D.J. Squirrel et al., Anal. Chim. Acta
457 (1), 109–114 (2002).
3. D.J. Squirrel and M.J. Murphy, "A Practical Guide to Industrial Uses of ATP-Luminescence" in Rapid Microbiology (Cara Technology, Lingfield, Surrey, UK, 1997).
4. L. Noda, Adenylate Kinase: The Enzymes (Vol. 8) ( Academic Press, New York, 1973), p. 279–305.
5. G.E. Shultz, Cold Spring Harbor Symposia on Quantitative Biology, 52 , 429–439 (1987).
6. L. Dane, "Study Results: Modifications to Detect P. expansum in 24 Hours Using AKuScreen Reagents" (Celsis, Chicago, 2006).