In pharmaceutical manufacturing, disinfection and sanitization refer to the killing, inactivation, removal or reduction of contaminating microorganisms to levels considered as safe, as set by industry standards and regulations.  However, in any laboratory or facility, different surfaces and materials will be found, as will different microorganisms and different disinfecting agents and procedures. 

Disinfection qualification (sometimes, incorrectly, referred to as disinfection validation or cleaning validation) is required to certify that disinfecting agents and procedures are effective enough to meet regulatory requirements.  These tests are an essential part of current good manufacturing practices (cGMP’s), and the FDA in its 2004 Guidance for Sterile Drug Products Produced by Aseptic Processing requires that all manufacturers of sterile drug products perform these studies to determine how effective their disinfecting agents and procedures are.

Disinfection qualifications formally evaluate the efficacy and suitability of antimicrobial products and procedures used to eliminate contaminant microorganisms on various surface types and components within an aseptic, sterile or otherwise controlled manufacturing environment. Disinfection qualifications are critical in assuring the microbial control of a manufacturing environment by qualifying the appropriate use and effectiveness of disinfection products and procedures.

However, disinfection qualifications are not disinfection validations.  A disinfection validation assures that the sterile, aseptic and even non-sterile manufacturing environments are under microbial control, as measured by a comprehensive and continuous environmental monitoring program. Cleaning validations, meanwhile, are designed to measure a procedure’s effectiveness at removing by-products or residual chemicals which may result during the manufacturing process.

Disinfection qualifications are critical because they ensure patient safety by preventing the microbial contamination that may occur during the manufacture of a product. Control of microbial contamination is required by the FDA’s Current Good Manufacturing Practice For Finished Pharmaceuticals as defined in 21 CFR §211.113 which states:

Control of Microbiological Contamination
a. Appropriate written procedures, designed to prevent objectionable microorganisms in drug products not required to be sterile, shall be established and followed.
b. Appropriate written procedures, designed to prevent microbiological contamination of drug products purporting to be sterile, shall be established and followed. Such procedures shall include validation of all aseptic and sterilization processes.

Enforcement of these regulations can be seen through GMP inspections and some of the FDA Form 483 warning letters that have resulted. The extent of the deficiencies listed in a 483 warning letter can mean the difference between a plant being able to operate, or being shut down.

Testing is also required because any disinfectant’s standard label claim for efficacy reflects testing on stainless steel or glass under set conditions that often don’t reflect real world materials or conditions in pharmaceutial manufacturing labs and facilities.
Therefore, disinfection assessment techniques must be modified to evaluate the true uses of the product in these environments.
This article will review key requirements of disinfection qualification.

Table 1 displays the differences found in log10 reductions using the same test organism and disinfectant on multiple surface types. The table illustrates that with the particular disinfectant studied, microbial reduction from plastic-based surfaces was relatively less efficient than the obtained reduction from stainless steel. Anomalies are highlighted in red.

Unfortunately, there is no standard method outlining step-by-step instructions of how disinfection qualifications studies should be conducted. However, general guidance can be found in the United States Pharmacopeia (USP) document and in the ASTM International E2614 guidance document.

Before performing a comprehensive disinfectant qualification, a simple test tube study is recommended, to screen various disinfectants to determine which will be most effective against the specific organisms present in the company’s facility.

Companies should then select surfaces that are present in the facility’s cleanrooms.  While some similar surfaces, such as 304- and 310-grade stainless steel can be viewed as a single surface (using one as a worst-case scenario), most surfaces will need to be challenged separately.

All disinfectants currently used, or being evaluated for use at the facility, must be reviewed. If multiple forms of the same disinfectant are used, wall must be qualified.

The biggest and most important part of qualification is selecting the organism panel for the study, which should evaluate the entire spectrum of organisms most commonly found in the facility’s controlled environment.  At least one gram positive cocci, gram positive rods with spores, gram negative rods, yeast and mold should all be included.

Suspension-based Testing vs. Coupon-based Testing
In general, evaluations are made using either suspension-based methods or coupon/surface-based methods. Suspension methods evaluate the reduction of a known organism population inoculated directly into a sample of the liquid disinfectant.
Following inoculation and the observation of a pre-determined contact  time, samples of the inoculated substance are removed, neutralized and evaluated for survivors as compared to an untreated control suspension. Since the simulation of organism films on the specific surface types used in the space is not accounted for in this method, it is recommended that suspension-based tests be used only for initial disinfectant screening purposes.

In contrast, coupon/surface-based testing is more rigorous and is recommended. In this method, known populations of each organism are applied to coupons made of surface materials present in the facility. 

The inoculated coupons are then exposed to the disinfectant being qualified. After the desired contact time, the coupons are extracted to determine the log reduction of the challenge organism.  The log reduction value is then used to determine whether the disinfectant is effective against that specific organism on that surface material.

Running through the procedures in greater detail, the coupons are placed into an incubator to allow the test organism to dry as a film. Once dried, each coupon is treated and exposed to the disinfectant. Following careful monitoring of the exposure, each coupon is transferred to a pre-selected solution designed to neutralize the disinfectant and elute or rinse off any surviving test organisms. This solution is quantitatively evaluated to enumerate the number of survivors onto an appropriate agar plate medium.
Untreated, inoculated, coupons are similarly enumerated to determine the starting level of test organism on each surface type prior to treatment. Appropriate controls should be included with the study to assess the sterility of the materials used in testing and to confirm the adequacy of the neutralization techniques used.

After incubation, the recovery plates are enumerated and the study controls are evaluated to assure study validity. Survivors found on the treated coupons are compared to survivors recovered on the untreated control coupons to determine the log10 reductions. The level of reduction observed can then be used to assess the success of the disinfection procedure.

Disinfectant qualification can be extremely time consuming and resource intensive. Timelines are typically based on the quantities of each organism, and turnaround times for the studies can range from one to two months, for simpler studies, to one full year or longer for more extensive swab recovery studies.

Studies should be conducted:

1. Before full-scale manufacturing
2. When changing disinfectants
3. When changing procedures
4. Before FDA audits

The ideal time to conduct a disinfection qualification study is at the construction of the manufacturing facility, prior  to operation, when disinfection processes and products are being considered. At a minimum, a qualification should be performed prior to starting full scale GMP manufacturing operations and prior to an FDA GMP audit.

Unfortunately, disinfection qualifications are often performed reactively instead of proactively, either in response to a product contamination, an environmental monitoring excursion, or to the observations listed in the feared FDA Form 483 warning letter. Waiting to perform a qualification study in these scenarios can lead to plant shutdown and the commissioning of a disinfection qualification study much larger than necessary.

Once the procedures have been qualified, the manufacturing environment should be continuously monitored to identify newly trending environmental isolates. This allows manufacturing facilities to successfully determine when additional disinfection qualification testing is necessary. In addition, supplemental qualifications should be performed following a change in a disinfectant product, a modification in cleaning or disinfection procedures and  the incorporation of any new surfaces into the cleanroom or aseptic manufacturing area.

Testing Scope
Each manufacturing facility is different and therefore each qualification study will be different. Considering the various combinations of surfaces, organisms, disinfectant products and disinfection procedures, these studies can become rather complex. Tables 2, 3 and 4 list common considerations that contribute to the complexity of these tests and  help illustrate why careful scientific rationale should be used in the design of the studies.

Again, a proactive approach and constant monitoring will help any facility prevent noncompliance, and help ensure smooth and safe operations. 

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Dave Rottjakob, MT (ASCP), is Director of Business Development at Accuratus Lab Services, where Shawn Sherry is Account Development Manager, Deborah Ensign is Principal R&D Scientist, and Kate Bloomrose is Supervisor of Specialized Microbiology.

References

1. United States Pharmacopeia (USP) 34, Chapter Disinfection and Antiseptics – General Information pp.579-580, May 1, 2011.
2. American Society for Testing and Materials (ASTM). Test Method, Standard Guide for Evaluation of Cleanroom Disinfectants, E2614-08.
3. U.S. Food and Drug Administration (FDA), Guidance for Industry Sterile Drug Products Produced by Aseptic Processing- Current Good Manufacturing Practice, 2004.
4. Block, SS, Disinfection, Sterilization, and Preservation. Lippincott, Williams and Wilkins, Philadelphia, PA., 2001.
5. Madsen, R.E and Moldenhauer, J. Contamination Control in Healthcare Product Manufacturing,  Volume 1., DHI Publishing, River Grove, IL. (2013).
6. Inspections, Compliance, Enforcement, and Criminal Investigations, Retrieved http://www.fda.gov/iceci/enforcementactions/WarningLetters/default.htm, 2013.
7. Current Good Manufacturing Practice for Finished Pharmaceuticals, 21 CFR Part 211.
8. Madsen, R.E and Moldenhauer, J. Contamination Control in Healthcare Product Manufacturing, Volume 3., DHI Publishing, River Grove, IL. (2014).