It is a regulatory requirement that the disinfection procedures used in cleanrooms have to be shown to be effective. Qualification of disinfectants by the vendor is not sufficient for full validation, and it is also essential to establish that disinfectants perform adequately in the cleanroom.

Airborne Gram-positive bacteria cause major problems in pharmaceutical cleanrooms, as they contaminate all surfaces within the manufacturing area and also tend to form spores, which can be extremely difficult to destroy with disinfectants. Gram-negative bacteria, usually introduced to the environment by human operators, can also cause problems and are difficult to eradicate.

Which disinfectant to use depends on the type of microbial contamination that is present or is possible in the cleanroom, but in general, quaternary ammonium disinfectants (quats) give good results. These bind to the outer negatively-charged surface of Gram-positive bacteria, disturbing the cell membrane and killing the bacteria. They are not as effective against Gram-negative bacteria, which have a less negatively-charged surface. This can be overcome by adding guanidine or glucoprotamin components to the quats, which also helps in removing yeasts and molds.

Severe contamination, including the presence of spores, requires much stronger disinfectants, but there are disadvantages with this. For example, peracetic acid oxidizes bacterial membrane proteins and phospholipids and disturbs components inside the cell, but it is not suitable for routine use because it is corrosive and damages cleanroom surfaces. It is therefore not suitable for routine disinfection.

Alcohol-based disinfectants are available but are not suitable for large surface areas because of their tendency to form aerosols and their ignitability. However, they work quickly and do not damage surfaces. Chlorinated bleach-type disinfectants are also commonly used.

The Road to Validation

Validation of a disinfection process typically takes place in three stages:

1. The disinfectant used must be qualified with vendor certification appropriate for the intended use
2. Its action needs to be verified within the manufacturing environment
3. The disinfection process has to be monitored on an on-going basis through the collection of data and statistical analysis to create stringent alert and action limits that ensure good control of the cleaning and disinfection process

In general, vendors test disinfectants on standard surfaces such as stainless steel, from which it is relatively easy to remove bacteria and other microorganisms. But this is not the case for many other surfaces, such as floors, sidings, and curtaining. Disinfectant activity has to be verified on all materials used in a cleanroom.

Testing Disinfectants

Disinfectant tests are usually performed using 5×5 cm tiles of the surface material, referred to as test carriers, contaminated with the relevant microorganism. The disinfectant is applied at a strength used in practice, left on the test carrier for an appropriate time, dropped into a neutralizing solution, and rinsed. The microorganisms in the rinsing solution are then investigated, either by incubating the solution on agar plates, or the filter obtained after membrane filtration is incubated on agar. This is followed by an enumeration procedure.

The tests are designed to mimic manufacturing area conditions in a laboratory setting, which means that a larger number of parameters than specified in standard methods need to be tested. Regulatory agencies also require testing of isolates from the actual environment. Because of this, each disinfectant has to be tested against eight to 12 different microorganisms.

The contact time to be used is particularly important, as many manufacturers have different recommended times for the same disinfectant. Some may suggest 10 minutes, extended to 60 minutes if necessary, so both contact times should be tested.

Three controls are required:

1. A positive control with no disinfectant
2. A control to confirm that the neutralizing solution does not affect the bacteria
3. A control for recovery validation

The difference in bacteria numbers between the treated sample and the positive control indicates the effectiveness of the disinfection, allowing the reduction log factor to be calculated.

Tests need to be run on all the materials present in the cleanroom, which means sample tiles of surfaces such as glass, Plexiglass, aluminum, PVC fabrics, and flooring, for example, are required. With eight or more materials needing to be tested with different contact times, and the need for controls, a single disinfectant needs to undergo at least 100 tests. In fact, USP and some other guidelines require tests to be run in triplicate, bringing the number up to more than 300. When all of the disinfectant products in use are tested, more than 1,000 individual tests are required for qualification.

This is a lot of manual work, representing a massive time and cost investment, and the large number of steps involved means the process is susceptible to errors, hence the need to run tests in triplicate to evaluate reproducibility. Such work can only be achieved by employing experienced analytical scientists.

Difficult Process

There are other problems that make the process difficult and challenging—for example, some of the carriers can only be used once. PVC is almost impossible to decontaminate for a second test without damaging the carrier, whereas stainless steel and glass carriers are often used more than once. Surface wettability may also be an issue. It is very difficult to spread aqueous solutions containing microorganisms evenly on silicone materials—the surface tension causing the liquid to pool rather than spread.

Microbial isolates from a cleanroom can also be difficult to work with. They may have been damaged and therefore do not grow as well as reference strains. This may be a problem with the positive control, and if results for this are not reliable, then neither are any of the other test results.

Pseudomonas species are particularly problematic, as they are resistant to being spread on surfaces, preferring to be in a water-based environment. Thus, isolates can die within the normal contact time even in the absence of disinfection, producing inaccurate results for the test sample and the positive control. This can be countered if the test environment is highly humid because the kill rate resulting from drying will be reduced. These problems demonstrate that there is no substitute for experience when running these tests.

Detailed Protocol

When employing a contractor for the validation of disinfection protocols, it is important that everything discussed regarding the studies is recorded. The large amount of work and the possible variability of results mean that the requirements for any repeated tests necessary should be specified. It pays to invest some time in developing the protocol to address the GMP-compliant handling of possible incidents.

While there is a very wide range of standardized tests prescribed by different bodies, each of their requirements differ, and there is no single standard covering all possibilities. Often, the best way to proceed when designing a validation procedure is to combine the best and most relevant elements from several standards into one protocol. This should then be discussed with the client, and if it meets all these requirements, the authorities should be satisfied and accept it.

The final step in a disinfection validation program is to monitor ongoing success. This is usually done using procedures and techniques such as contact plates, swabs, and the rinsing of material. Statistical comparisons are needed to evaluate the data created and determine the resulting extent of disinfection within the facility. Alert and action levels are also set, specifying when remedial action is required. The regulatory guidelines do not give precise specifications for these, as they are assessed and determined specifically for each individual facility using the data generated during the validation and ongoing test work.

Only one guideline, USP Chapter 1072, lays down specifications for the effectiveness of disinfection. USP says that a 3 log reduction is the norm, with a 2 log reduction for bacterial spores. This reduction is particularly difficult to achieve for spores since only a 1 log or even 1.5 log is usually attainable, which is likely to suffice for a pharmaceutical cleanroom, except in the case of unusually high contamination with spores.

All regulatory guidelines emphasize the importance of validation studies for proper disinfection, but the variability of materials, conditions, and layout of different sites means that each facility needs to be treated as a unique environment. Collecting the data that demonstrate the effectiveness of a disinfection protocol requires a lot of experience, and reproducible results can only be expected when the work is performed by an experienced team. Lack of experience, or inattention to detail, almost certainly leads to failure, and if disinfection cannot be proved, risks to patients will be significant. This view has been reflected by several warning letters being issued by the FDA in recent years.

This feature story can also be found in the November/December 2016 issue of Pharmaceutical Processing. 

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