The pharmaceutical industry is slow to change, even when new technologies are superior to the methods that are currently being used. One of the areas that shows this is the practices used for the cleaning and disinfection of cleanrooms. The specific cleaning agents used have many known weaknesses, but they are the same agents that have been used for decades. In some cases, they have been updated to be provided as sterile formulations, or perhaps in containers and packaging easier to use in isolators. However, there have not been significant improvements to the actual agents used for these processes. Today, there are many agents available that can be used that are superior to the existing methods, as well as agents that significantly reduce the weaknesses associated with the traditional methods.

Background

“Cleanrooms are vital when it comes to ensuring that medicines are contamination free — and yet the technology behind them is well over 50 years old. Is it time for a makeover?”¹ Cleanrooms are relatively new to the pharmaceutical industry. One of the fathers of the modern cleanroom is Willis Whitfield, who was given the nickname “Mr. Clean” by Time Magazine. Whitfield originated the laminar-flow cleanroom. This design was a thousand times better than the technology in use prior to this time. His designs were actually originated to help with the creation of nuclear weapons during the Cold War.¹

Many of the improvements in contamination control have come from the methods used to fabricate the cleanrooms; e.g., flush-glazing systems and architectural changes to enhance cleanroom performance.¹ In the last couple of decades there have been many improvements in the methods used for detection of both particles and viable microorganisms present in cleanrooms. Many new technologies, whose origin came from products developed for detection of biological warfare contaminant, have been adapted for use in pharmaceuticals; e.g., systems that detect viable contaminants using Mie Scattering; systems that detect, enumerate, and identify microorganisms using RAMAN spectroscopy; and growth-based systems that utilize computer-aided imaging to obtain results in significantly shorter time periods than conventional methods.

The pharmaceutical industry has been slow to implement these methods in spite of the obvious advancements associated with using these technologies. For routine non-viable particulate counting, the systems have advanced to allow for the use of remote probes to detect the counts, computerized systems for documenting the results, and the like. Newer systems are even available to count particulates in the nanoparticle size range.

However, the actual cleaning and disinfection agents used have been the same for many years in spite of new and superior agents that have been developed.

Typical cleaning and disinfection agents

There are a variety of disinfectants that are utilized in the biopharma industry.

Alcohols

Both ethyl and isopropyl alcohol are used as disinfectants. They are typically diluted to 70 percent.² These agents are not sporicidal. In fact, biological indicators are frequently shipped in alcohol solutions. They also do not have activity against prions. These products are not corrosive and do not leave residues.

Aldehydes

There are two popular aldehydes used, primarily in facilities outside the U.S. They are formaldehyde and glutaraldehyde.

• Formaldehyde (methanal)
Formaldehyde (methanol) can be used as a sterilant (37 percent) or a disinfectant (3 to 8 percent) It is not typically used in the U.S. Formaldehyde vapor is often used under very controlled conditions for disinfecting enclosed spaces such as rooms and laminar flow hoods while they are out of service and with appropriate safety precautions.²

• Glutaraldehyde (pentanedial)
Glutaraldehyde can be used as a disinfectant or sterilizing agent. Glutaraldehyde cannot be used as a general surface disinfectant because of its irritating vapor and required long contact time. Glutaraldehyde is generally obtainable as a 2 percent, 25 percent, or 50 percent solution at an acidic pH that must be activated by making the solution alkaline (pH 7.4 to 8). It is more active at alkaline pH than acidic pH. For disinfection purposes glutaraldehyde is used at a 2 percent concentration. Alkaline 2 percent solutions have bactericidal, sporicidal, fungicidal, and virucidal activity. The literature indicates that it requires up to 10 hours’ exposure to kill dried spores of Bacillus subtilis. Vegetative bacteria are generally killed in 10 to 20 minutes. This cleaning agent can leave residues, so proper rinsing is required.² Gluteraldehyde is not widely used in the U.S. pharmaceutical industry.

Ortho-phthalaldehyde (OPA)
Ortho-phthalaldehyde, a newer aldehyde disinfectant, is not meant for general surface decontamination but can be used to disinfect items that can be immersed. The advantages of OPA over glutaraldehyde are that it requires no activation, it has minimal odor, it is stable over a wide range of pH (3 to 9), it is not irritating to the eyes or nose, and it has excellent material compatibility. A disadvantage is that it stains material containing proteins gray (including exposed skin).²

Halogens
The halogens consist of bromine, chlorine, fluorine, and iodine, of which chlorine and iodine are the more commonly used for general disinfection. Chlorine and iodine as pure substances are unstable, corrosive, and toxic. To partially negate, these characteristics halogens have been combined with other chemicals.²

Chlorine and chlorine-containing compounds
Chlorine is commonly available as a solution of sodium hypochlorite (NaOCl) in a concentration range of 1 percent (equivalent to 10,000 ppm chlorine) to 5 percent (equivalent to 50,000 ppm chlorine), although some are as much as 35 percent sodium hypochlorite. The 35 percent and higher concentrations of chlorine are extremely corrosive and can be explosive it not handled correctly. Sodium hypochlorite solutions are corrosive even to the best grades of stainless steel. An aqueous solution of sodium hypochlorite does not leave a residue upon drying. Concentrations of 1 percent to 5 percent are bactericidal, fungicical, sporicidal, mycobactericidal, and virucidal against lipid and non-lipid containing viruses when the wet contact time is appropriate.²

Iodine
Commercial preparations of iodine coupled with carriers are known as iodophors. Povidone-iodine is probably the most recognized formulation. These carrier iodine combinations allow for stability of the iodine and the sustained release of the iodine. Iodophors must be properly diluted in order for free iodine to be present in solution in the appropriate concentration. The advantage of an iodophor over tinctures of iodine (iodine diluted with alcohol) is that they’re non-staining and relatively free of toxicity and irritancy. Iodophors are used at a concentration of 500 mg active iodine/L, which is bactericidal and fungicidal. Contact time for iodine containing preparations is at least 10 minutes. Iodine is virucidal for certain viruses.²

Peroxygen compounds
Peroxygen compounds include hydrogen peroxide and peracetic (peroxyacetic) acid. Both compounds are considered thermodynamically unstable. Peracetic acid is less stable than hydrogen peroxide. A one-percent solution of peracetic acid loses half of its strength in six days. Both hydrogen peroxide and peracetic acid are environmentally friendly in that they decompose into oxygen and water. Neither compound leaves a residue.² However, they need additional safety precautions due to their irritating properties. 

Hydrogen peroxide (H₂O₂)
Hydrogen peroxide is available as a liquid. The solutions are generally designated as 20 or 10 volumes of oxygen that are evolved from one volume of the peroxide solution. Hydrogen peroxide has been shown to be bactericidal, virucidal, tuberculocidal, sporicidal, and fungicidal. Hydrogen peroxide kills vegetative cells much more quickly than it kills spores, thus requiring an increased contact time for spores. The activity of hydrogen peroxide is increased with temperature and concentration.²

Peracetic acid (CH₃COOOH)
Peracetic acid is usually provided as a 35 percent aqueous solution. Peracetic acid has activity against a variety of vegetative microorganisms, viruses, fungi, mycobacteria, and spores, for concentrations of 50 to 500 ppm (0.05 percent to 0.5 percent) when the microorganisms have been exposed for at least five minutes.²

Phenolics
Phenol is toxic, carcinogenic, and corrosive. It is not used as a disinfectant. Phenolic derivatives are commonly used as disinfectants. The derivative results in products that are much less corrosive, toxic, and carcinogenic. However, skin irritation and absorption through the skin still occurs so proper safety precautions need to be taken when working with these materials.²

Since detergents do not inactivate phenolic derivatives, they can be added to the base formulation producing products that clean and disinfect in one step. Phenolic compounds do leave residues so rinsing of items that have come in contact with phenolic compounds is important. Phenol compounds when used at a concentration of 2 percent to 5 percent for the appropriate amount of time are considered bactericidal, tuberculocidal, virucidal, and fungicidal. They are not sporicidal.²

Quaternary ammonium compounds (quats)
Quats are bacteriostatic against a variety of bacteria; certain Gram-negative bacteria are known to be intrinsically resistant to quats. Pseudomonas aeruginosa, Burkholderia (Pseudomonas) cepacia, and Providencia stuartii are resistant to the quats. Quats have some activity against lipophilic viruses and poor activity against hydrophobic viruses. Quats can be bactericidal at medium to high concentrations but they are not sporicidal or tuberculocidal at any concentration. Quats are fungistatic and mycobacteriostatic.²

Alternative cleaning and disinfectant products

As in many other areas of pharmaceuticals, companies have been slow to implement “newer” methods of cleaning and disinfection. Some of these products come from other regulated industries while others have been newly developed.

Ozonated water
Ozone was first used in the Netherlands for treating drinking water. Ozone is a used for disinfection and chemical contamination oxidation in water systems. It is also reported to be more effective than chlorine at inactivating bacteria and viruses with a shorter contact time.³

It has also been reported that ozonated water is effective in depyrogenation, able to show a 3-log reduction in endotoxin in less than 30 minutes.⁴

Ozonated water has been widely used in the food industry, for both contamination control and elimination of odors. In fact, the FDA website for foods identifies ozonated water as acceptable for use on food products.

Hubka⁵ presented various contamination control case studies showing the effectiveness of ozonated water in resolving contamination issues with both sterile and non-sterile manufacturing processes. Ozonated water systems are available in handwashing sinks, mobile portable ozonated water generators, and in-line skid systems. One of the common problems is the difficulty of eradicating an out-of-limits contamination event. Many of our cleaning and disinfection procedures are validated to show a 2- to 3- log reduction of microorganisms. Unfortunately, the level of organisms present in many contamination events is much higher, after detection in product. As such, use of a routine disinfection program a single time may not be sufficient to eradicate a contamination event.

Some of the portable ozonated water units have been used to clean walls, tanks, filling lines, floors, and the actual solution or powder processing line. Some companies have found cost savings by using this technology in place of other sporicidal cleaners due to the reduced cost, no residues, and no corrosiveness. Additionally, the only ongoing cost for the ozonated water system is replacement of a few items as part of the yearly maintenance, and the use of purified water to operate the system.

USP 1229.6⁶ identifies ozonated water as a type of chemical sterilant that may be qualified to show a 12-log reduction of microorganisms. The ability to qualify this technology with bacterial spores may be easier to implement than disinfectant efficacy testing. Routine testing of disinfectants is difficult with ozonated water due to the short half-life of about 20 minutes and the time required for testing. As such, the USP method can be utilized to kill to the desired spore log reduction. In most cases a 12-log reduction can be achieved in a short time period, typically less than 30 minutes. A 3-log reduction of endotoxin can typically be achieved in the same time period at ambient conditions.

Hand sinks
Ozonated water hand sinks can be used anywhere people need to wash their hands. Studies conducted in the U.K. properly taught healthcare professional how to wash their hands. The professionals were evaluated to show that they could do it properly. In spite of knowing they were being watched, 75 percent continued to wash their hands improperly. One of the problems is the amount of time it takes to wash your hands with soap and water. Rinsing your hands in ozonated water can decontaminate quickly at ambient temperatures and keep down the risk of human contamination from their hands in subsequent steps. The reduction in bacterial content and endotoxin concentration can be demonstrated in validation studies.

Hydrogen-activated water
Isopropyl alcohol and ethyl alcohol are routinely used for sanitizers throughout the pharmaceutical facility. These products are used for hand sanitizers, surfaces, and many other things. It is a well-known fact that neither of these products are sporicidal. Today there are other hand sanitizers available that are relatively inexpensive and leave no residues, but are superior in microbial killing power.

U.S. sanitizers data has shown that hydrogen-activated water is effective against both bacteria and spores. In hospital and nursing home facilities, the risk of infection from Clostridium difficile can be significant. It is a major concern. This hydrogen-activated water product was tested with Clostridium difficile ATCC 43598 to evaluate the effectiveness of the disinfectant. In this testing, spore cultures (shown to be 97 percent spore purity) were evaluated and showed reduction of these spores.7 Since bacterial and mold spores occur as typical contamination events, going to a sporicidal sanitizer can offer many benefits.

Moving to prevention technologies
The real answer to contamination control is to move to prevention technologies. There are paints available that include nanoparticles (usually silver) that prevent a painted surface from becoming contaminated by bacteria. There are similar nanoparticles that are available for materials that could be used with our cleanroom gowning. Depending upon the nanoparticles selected, a variety of conditions can be addressed; e.g., preventing moisture from getting out or in, aiding in keeping personnel cool, antimicrobial properties, antifungal properties, and the like.

Another type of preventive measure is the use of fungal barriers; some building materials are made with these properties. If not, there are products like MoldGuardian that can be applied to a surface. It can be wiped, sprayed, or fogged onto the surface. It attracts the mold and prevents mold growth for at least a year, providing strong caustic cleaners are not used on it. If they are, the product needs to be re-applied.

Conclusion

While many wonderful cleaning and disinfection agents exist and have been used for years, that does not preclude the use of new methods and technologies. The ideal approach is to never have contamination, so prevention methods are critical. If you can’t use prevention methods, go to the methods that provide the greatest assurance of contamination control. Sterilizing grade disinfectants that can be used inexpensively and be sporicidal — this significantly improves your risk of contamination. This is especially true in situations that have issues with those hot topic organisms — e.g., Burkholderia cepacia, Clostridium difficile, and mold contaminants. 

References

1. Sutton, S. (2016) Keep It Clean. The Medicine Maker. July/August, #21. 20-27.
2. Denny, V. (2005) Disinfectants. In. Environmental Monitoring Volume 2: A Comprehensive Guide, Moldenhauer, J., Editor. PDA/DHI Publishers. Bethesda, Md. 151-163
3. Saha, R., Martin, R., and Donofrio, R. Efficacy of Ozone Treatment Systems Against Microorganisms: Current Methodology and Future Approaches for Evaluating Novel Disinfection Technologies. Water Conditioning & Purification. 2013.
4. Cooper, J. (2015) Personal Communication.
5. Hubka, B.G. (2016) Contamination Control Case Studies. Contamination Prevention Technologies. Boston, Mass. Presented at the NE PDA Chapter Meeting on May 18, 2016.
6. USP (2016) Monograph <1229.6>
7. Weaver, D. (2016) Personal Communication showing testing data.

 

Jeanne Moldenhauer is Vice President of Excellent Pharma Consulting Inc. in Mundelein, Ill. www.excellpharma.com

Brian G. Hubka is CEO of Contamination Prevention Technologies Inc. in Las Vegas. www.contaminationprevention.com