A Systematic Approach to Selecting Disinfecting Agents for Aseptic Pharmaceutical Manufacturing Operations

Picking the right disinfecting agent means examining environmental test data in your aseptic area. This helps establish standard operating procedures which are aligned with the U.S. Pharmacopoeia.

By Art Vellutato, Jr.

The successful operation and maintenance of an aseptic pharmaceutical manufacturing area, in accordance with mandated guidelines, is dependent on the proper selection of disinfecting agents based on historical environmental data as well as the precautionary measures implemented to reduce contamination from being introduced into the area by personnel or components.

Proper selection of disinfecting agents, based on environmental test data combined with incoming inspection testing of these agents, provides the necessary framework for establishing required standard operating procedures. These procedures should follow the protocol set forth in the United States Pharmacopoeia (USP, Vol. 23 NF18). Testing should also include the use of efficacy performance data presented by the manufacturer of the disinfecting agent following the Association of Official Analytical Chemists (AOAC) protocol specifications approved by the Environmental Protection Agency (EPA). Efficacy performance together with assured sterility of the solution, is essential.

For many years, pharmaceutical operations worldwide have used this approach concept to assure that manufacturing facilities meet the stringent requirements set forth by current GMPs. Incorporating a relationship between testing and disinfecting agents focuses cleaning efforts on what needs to be addressed in the facility. Developing the “Personality of an Aseptic Area” provides a systematic framework for selecting disinfecting agents, assuring sterility levels are maintained, and it provides a concrete methodology for addressing possible future contamination.

The relationship between testing and selection of disinfecting agents

The relationship between testing and selecting disinfecting agents requires tracing contaminates. And, there are many possible sources and types of contamination. Microorganisms are transferred by air, people, and liquids into the aseptic area, and in fact, contamination levels of an aseptic area are minimal, prior to the introduction of personnel. To combat contamination from personnel, pharmaceutical operations have begun using isolation barrier technology in various operations. In a majority of process manufacturing operations, however, this separation of personnel and product is not feasible, thus, systems consequently must be designed to accommodate them.

At the same time personnel are introduced, the aseptic area may also be invaded by contamination from components, liquids or production suppliers. To assure contamination is combated, viable surface testing should be conducted routinely throughout the aseptic area using rodac plates. Testing of personnel, introduced parts, walls, equipment and filling line surfaces, etc. should be completed. Viable air testing must also be conducted simultaneously with surface testing to assure airborne contamination is at a suitable level prior to and during manufacturing operations. The critical area to be tested is the point where product is exposed to the environment before being filled or processed. Once testing is completed, incubation of the exposed plates is recommended for a period of 72 hours.

Selection based on recorded environmental testing results

Viable air and surface testing of the aseptic area is required under current GMPs. This information tells what organisms were present in various areas of the aseptic manufacturing environment and will allow you to discern whether the present disinfecting agents are working sufficiently or whether using another disinfecting agent is required. Periodic rotation of disinfecting agents assures a broad spectrum of efficacy performance–disinfecting agents are implemented to reduce a broad spectrum of contaminants.

Validated sterilization processes such as gamma radiation, ethylene oxide (ETO), steam, and heat are all methods which qualify components as sterile prior to entering the aseptic area, however, existing organisms in the aseptic area cannot be exposed to these processes. Thus, appropriate disinfecting agents are the designed cure. The key to a disinfecting agent`s effectiveness is dependent upon its saturation and penetration of the organism`s cell wall over time. Assuring efficacy requires penetrating the organism`s cell wall to denature the cellular fluid with the disinfectant agent. Efficacy of the disinfecting agent is dependent upon the saturation and penetration over a specified time period.

Cleaning agent manufacturers present efficacy performance data based on AOAC protocol testing which is required by the EPA. Various AOAC protocol tests are conducted for varying levels of efficacy performance ratings. The most commonly used test, the AOAC Use Dilution Test, requires a challenge organism to be placed in a 5 percent blood serum, used to simulate organic soil load. The organism is then subjected to a use dilution strength of the disinfecting agent for a contact time of 10 minutes at 20&#176C. Three lots of testing are performed. Confirmed efficacy data is then presented to the EPA for its evaluation. Chemicals deemed suitable for the efficacy performance against specific organisms versus the appropriate EPA approved test method are then classified, as presented in the table labeled, “EPA-approved Disinfectant Test Methods.”

Disinfecting agent selection to meet the regulated requirements

The selection of a disinfecting agent is not only dependent upon the efficacy of the agent but also the requirements set forth in USP. The USP specifications require preacceptance testing against “indicator organisms” prior to introduction of a disinfecting agent into an aseptic area. Indicator organisms are listed in USP as Candidida albicans, Apergillis niger, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. These organisms should be tested against the disinfecting agent following AOAC protocol testing specifications.

Filtration of disinfecting agents

Cleaning agents will be used on walls, ceilings, floors, and equipment in the aseptic area and, unless assurances are in place, may themselves lead to contamination. Cleaning agents should, therefore, be filtered through a hydrophobic filter at 0.2 micron. These agents should be filtered directly into pre-sterilized containers which have been pre-validated and tested. Filtration directly to the aseptic area is acceptable provided that the reception vessel has been sterilized. Upon completion of filtration, sample testing of the solution must be recorded on a lot-by-lot basis to assure use concentrations (analytical) and sterility of the solution. Testing protocol is presented in the USP for the solution in question. Use of an unfiltered and untested product in the aseptic area warrants future problems both with respect to established guidelines and possible introduction of contaminates from the solution itself.

Handling of various disinfecting agents

Cleaning agents require different handling and packaging prior to their introduction to and during use in the aseptic area. Due to differing use patterns, handling and packaging for each must also be done differently.

One of the most commonly used disinfecting agents in aseptic operations is sterile alcohol. Characteristically, alcohol does not leave a residue and is an excellent choice for use on gloves, surfaces, component parts, carts and close to point-of-fill areas. Sterile Isopropyl Alcohol or Sterile Denatured Ethanol are two commonly used disinfecting agents. For optimal efficacy performance, dilution of these two disinfecting agents is recommended at 70 percent v/v Isopropyl or Ethyl and 30 percent water (USP purified water or water for injection, if available). These products must be formulated and analytically tested according to USP specifications.

The use of alcohol in the aseptic area requires the utmost concern for container style, handling, shelf life and filtration at 0.2 microns (per USP specifications) through a sterile, validated hydrophobic filter. Containers are the most important concern. The aerosol container provides a pressure vessel which eliminates the aspiration of room air (the return from the exterior) to the master reservoir. Alternate methods such as squeeze bottles or trigger sprayers aspirate room air into the reservoir of alcohol and can cause contamination to the bulk of the material. Due to their non-aspirating design, aerosol containers have longer sterility shelf life data than that of an open or aspirating container. An important advantage for the use of the aerosol container is hands-free dispensing. This eliminates the possible cross-contamination from user to user.

Internal studies of various containers using USP protocols for sterile alcohol have validated sterility in the aerosol container for a period of at least three years. During internal studies of trigger sprayers and squeeze bottles containing sterile alcohol, contamination to the container and the alcohol inside was recorded in a majority of the samples tested throughout the normal work operation (6 hours) within the aseptic area. Regulated operations must be able to assure sterility throughout the use of the sterile alcohol container. Validation for sterility of an open container is variable and may be unachievable and reproducible from process to process. Thus, if alternate containers are to be used, it is suggested that they be removed and reprocessed on a frequent basis.

While validated studies have confirmed the sterility of alcohol contained in the container after filtration and the sterility of the container prior to filling operations, it is advisable that the entire contents be double bagged, and subjected to gamma radiation through a validated cycle. This will assure the exterior of the container, air inside the first bag, the first bag and air inside the second bag are sterile. Particulate analysis of the final form should be rendered to determine contamination levels of the product prior to use.

Disinfecting agents are also required to be filtered through a sterile 0.2-micron filter into presterilized containers. This aseptic filling operation must also be completely validated including sterility testing per lot/batch as described in USP. Removal of contaminates at 0.2microns will ensure a pyrogen-reduced solution. Once processed, they should be double-bag packaged, transferred to the sterile manufacturing area and mixed directly with water for injection, or USP purified water.

Sporicidal disinfecting agents are one of the most commonly misunderstood chemicals in the pharmaceutical industry. As shown in the table, to be recognized by the EPA as having sporicidal capabilities chemicals must undergo soaking of specific spores over an extended time period which sometimes takes from 5 1/2 to 12 hours. Soaking is the key to sporicidal properties, since spore forming organisms change their morphology from a vegetative state to a protective state. In the protective state their cell walls require soaking in an approved sporicidal chemical for an extended time period to assure its demise. Due to the laminar air flow in cleanrooms, however, most operations cannot maintain a wet (soaking) condition on walls, ceilings, floors and equipment for this extended time period. Thus, sporicidal efficacy claims on surfaces that involve short drying times (less than EPA approved efficacy performance data for the agent) may be flawed. Filtration of sporicidal chemicals should also be done through a sterile 0.2-micron filter into presterilized containers to ensure removal of particulate and microbial contaminates.

Residue Removal

One of the most important functions within the aseptic manufacturing area is the removal of residues. Most disinfecting agents leave a residue on the surface which it is applied. Residue build-up presents a critical problem to future disinfection operations since it becomes the surface being decontaminated instead of the original surface. Cleaning operations designed to remove residues are critical.

While time consuming, assuring product effectiveness and systematically approaching the operation and maintenance of an aseptic area will have its rewards in attained maximum final sterile production quotas.n

References

1. U.S. Pharmacopeia, Vol. 23, NF18, U.S. Pharmacopoeia Convention, Washington, DC, Mar. 8-10, 1990, Published by the Board of Trustees, Jan. 1, 1995.

2. EPA, “General Information on Applying for Registration of Pesticides,” 2nd Ed., EPA/737/b-92-001, Aug. 1992, Pesticide Labeling Requirements, Appendix 2-1.

3. Block, Seymour, S., “Disinfectant, Sterilization, and Preservation,” 1991, Fourth Ed., Philadelphia, Lea & Zebiger.

4. Prescott, Harley & Klien, Microbiology, 2nd Ed., 1993, Wm. C. Brown Publishers, Dubuque, IA.

5. Vellutato, A., Sr., Project 010056 Alcohol Containers, VAI Internal Testing Report, 1989, File 010056-AC, P. 2-4.

6. Vellutato, A. Sr., Project 010097 Sterility Shelf Life, DECON-AHOL, VAI Internal Testing Report, 1990-1996, File 010097-SSL-A, P. 9-12.

Art Vellutato Jr. is the vice president of sales and marketing for Veltek Associates, Inc. (Exton, PA), which designs, manufactures and markets contamination control products for cleanrooms.