A microbial immersion research approach
By Richard Prince
The Parenteral Drug Association is conducting research in the area of standardizing a microbial immersion test that may one day be used by firms to establish (direct) conclusive evidence that container/closure systems provide a suitable barrier against the ingress of adventitious microbial contamination. PDA`s interest extends to examining both microbiological and physical test models. This column offers a research approach for validating a microbial immersion model.
1. The minimum container/closure system breach or “defect” necessary to induce ingress from a target challenge microorganism should be identified. It would be nice to prove a form of linearity, so that the percentage of positive units increases with increasing size of the container/closure defect. From a test systems perspective, the Minimum Defect Size (MDS) could represent X, with 1/2 X and 2 X defects also artificially introduced so as to represent potential negative controls and positive controls, respectively.
2. A target microorganism should be selected on the basis of relative size, motility, substrate adsorption properties, and hardiness. We should understand the microbial penetration process. Scanning Electron Microscopy would seem to be a useful tool in this regard.
3. A Standardized Microbial Challenge (SMC) should be established. The starting inoculum should represent a “substantial” challenge relative to typical bioburden levels associated with practitioner and user settings. A scale-up of three logs is suggested. For starters, medicines are typically stored in bathroom cabinets. If a drug product fell into a user`s toilet bowl, a highly contaminated area, a microbial test challenge increase of three logs over the average toilet water bioburden might represent a good example of the standardized challenge being based on realistic epidemiological data.
4. The minimum time necessary for the challenge microorganism to achieve container/closure ingress (possessing a standardized “defect”) should be identified. From a test systems perspective, the Minimum Ingress Time (MIT) could represent Y, with 1/2 Y and 2 Y representing additional potential negative controls and positive controls, respectively.
5. The “delivery” of the SMC to the container/closure system should be understood. Factors such as water reservoir type, ideal water temperature, apparatus holding the completely submerged container/closure system, and recirculation (mixing) time should be examined and hopefully standardized. Static vs. dynamic mixing systems using MIT, MDS and SMC is suggested.
6. Modeling would be conducted to study the interrelationships between variables such as MDS, MIT, and SMC. It would be determined whether time and defect size can synergistically effect microbial ingress within container/closure systems. This presumes that a challenge organism and an SMC have been utilized. Can a container/closure system, with a 1/2 X MDS, be defeated with the passage of time? Can a container/closure system, with a 1/2 Y MIT, be defeated with a 2 X MDS?
7. Container/closure test systems should be filled with sterile media and/or representative sterile drugs.
8. Container/closure systems should be treated with a suitable chemical germicide possessing non-corrosive, non- staining, broad-spectrum, fast-acting antimicrobial properties.
9. Container/closure test systems should be incubated for up to 28 days at 20 to 25 degrees Celsius and 30 to 35 degrees Celsius. This will evaluate the effects of time and temperature on microbial outgrowth rates.
10. Microbial recovery testing could include comparing polymerase chain reaction (PCR) and flourescein-labeled scanning technology versus conventional broth (turbidity) systems. Standard USP <1225> assay validation parameters such as linearity, accuracy, precision, and limit of detection should be studied.
11. Post Bacteriostasis and Fungistasis testing and chemical germicidal identity testing should be performed to assure no chemical leakage into the container/closure test system thus preventing the possibility of false negative sterility results.
12. A rapid-immersion screening test may be a part of or a prelude to the ideas presented in points 1 through 11.
It seems appropriate to extensively study the molecular relationships of microbes in aqueous systems (they are purported to shrink up to 40 percent according to data from a leading filter manufacturer) and then relate to the physical mass used as a challenge in any physical model. Beyond sizing considerations, the influence of electrostatic charges, pH and other physical forces upon microbe/substrate interfaces needs to be investigated. Should microbial model standardization efforts fail, it could signal an impressive confirmatory argument in favor of the (presumed validatable) physical testing model. CR
Richard Prince, Ph.D., is president of Richard Prince Associates Inc. (Short Hills, NJ), a compliance and technical based consultancy, and an officer of Microgen, a provider of qual ity disinfectant-cleaner products. He can be reached at (973) 564-8565, fax at (973) 564-8731 or by e-mail: [email protected].