PHARMACEUTICAL TECHNOLOGY ASIA. Cleaning, Validating and Monitoring Aseptic Fill Areas

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1 PHARMACEUTICAL TECHNOLOGY ASIA September/October 1997 Cleaning, Validating and Monitoring Aseptic Fill Areas Douglas W. Cooper, Texwipe Pharmaceuticals that cannot be given terminal sterilization are packaged using aseptic fill, where clean, sterile materials are placed into sterile containers in a clean room. Contamination control prevents the introduction of contaminants and involves the cleaning of surfaces such as floors, walls, tables and equipment. Cleaning must be performed systematically, validated for effectiveness and monitored regularly. Aseptic filling strategy seems simple: sterilize the containers and everything that enters them and fill them in a virtually sterile environment. The aseptic fill area, typically controlled to Federal Standard 209E Class 100 or better for air cleanliness, should contain fewer than 100 particles larger than 0.5 µm per cubic foot (28.3 L) of air. The planning and building of such clean rooms has been detailed by Hansz and Linamen, 1 and useful details and standards concerning sterilization and cleaning aseptic fill areas are available elsewhere. 2 6 Contaminants enter the clean room and are also generated within the room by processes and personnel. Cleaning the aseptic fill area is made more difficult by the variety of surfaces that require attention: barrier curtains, walls, windows, floors, ceilings, table-tops and machinery with complicated inner and outer surfaces. General principles of cleaning and making surfaces sterile can help guide the cleaning of aseptic fill rooms. 5, 6 Successful aseptic filling requires the following elements: design, materials, separation of areas, product flow, progression in cleanliness and exclusion of pests. Design includes obtaining a smooth (laminar) flow of filtered air to keep particles from the product and arrangement of the equipment to facilitate cleaning. Clean room materials should be non-porous, easily cleaned and resistant to abrasive release of particles and to outgassing. The essence of good product flow is to keep contaminated and uncontaminated regions clearly delimited and to prevent interchange. As the fill area is approached, the environment should become progressively more clean. 7 Cleaning efforts often centre on viable and non-viable particles. Particles can be hard to remove because of capillary forces, electrostatic forces, hardened chemical bridges, thermal solidification, tackiness, Van der Waals forces, deformation and imbedding, surface roughness and gravity. Viable particles are often on, or under, non-viable particles on surfaces and cannot be killed until the covering particles are removed. Cleaning materials The general strategy is to make those areas closest to the product the cleanest. Because cleaning materials themselves become less clean as they are used, one should start near the product and move to the less clean areas, such as toward where personnel work. The clean room floors should be cleaned toward the entrance and from there to the changing/gowning room. Cleaning of equipment may involve dismantling, cleaning and then re-assembly. Vacuum cleaners, squeegees and brushes can be used for gross cleaning.

2 Whether or not there has been gross cleaning, precision cleaning needs to be performed, almost always requiring the use of fabric wipers or swabs that are among the cleanest available. Wipers for cleaning aseptic fill areas are typically knitted from continuous filament polyester and may have sealed edges to further reduce particle release. Wipers designed for aseptic fill areas are often purchased pre-sterilized, typically by gamma radiation at a dose level adequate to assure a less than one in a million chance of a viable organism being present on any particular sterilized wiper. Sometimes the users sterilize unsterilized wipers by autoclaving. Sterilization by steam or radiation may reduce, but will not destroy, pyrogens (fever-causing outer coats of Gramnegative bacteria), so pyrogens must be minimized by clean manufacturing. Double and triple bagging of the wipers is common to allow the wipers to pass through contaminated areas before reaching the site of use. Wet wiping is used not only to disinfect but also to dissolve contaminating materials and to weaken bonds between the particles and the surface. Damp (rather than saturated) wiping is often best. De-ionized water can remove ionic contaminants, but usually it must be supplemented by other chemicals. Successful liquid cleaners should provide: reduced surface tension to wet surfaces; quick evaporation and minimal residue. They should possess an aqueous component; a hydrocarbon solvent; an alkaline component to help dissolve oils and greases; and should be non-toxic, non-flammable and not ozone-depleting. The liquid must remain sterile, which is not easy, as organisms can live even in de-ionized water. Water and isopropyl alcohol, commonly used for cleaning clean room surfaces, allow spores to survive. Less than 50% (v/v) alcohol concentration is not a particularly effective disinfectant. The use of pre-saturated wipers may avoid the mixing of chemicals, ensure the correct chemical concentrations in the cleaning liquid, prevent wiper-liquid incompatibility, reduce the consumption of cleaning solutions, reinforce good technique and simplify sterilization. The containers should allow easy removal of single wipers, avoid contaminating those remaining and limit evaporation. Disinfectants and liquids put into a dispenser should be filtered through a hydrophilic 0.2 µm filter, as should any air that replaces displaced liquid, and the opening of the container should not be allowed to touch other surfaces. A recommended practice of the Institute of Environmental Sciences 5 outlined the following options for clean room disinfection: wiping and mopping, flooding and vacuuming, fogging and the submerging of small objects. Cleaning methods Cleaning personnel should have clean room garments, presumably boots, cover-alls (typically woven polyester), masks, hoods and gloves, for cleanliness and safety. The recommended steps for wiping are: Figure 1: Folding of a sealed-edge wiper into quarters. - 2

3 1. Fold the wiper in quarters, obtaining several clean wiper surfaces. Folding helps to make the pressure of the hand and fingers more uniform (Figure 1). Figure 2: Absorbing a spill. Figure 3: Pre-wetting, then wiping with overlapping strokes. 2. Wipe in a pattern of parallel strokes with some overlap, changing the surface of the wiper or the wiper itself at the beginning of each stroke (Figures 2 and 3). 3. Start each stroke at the cleaner end of the planned path. 4. Change the wiper surface approximately every 10 wiper lengths of wiping (more often if the surface is visibly dirty). 5. To disinfect, use a nearly saturated wiper and leave a visible disinfectant film. Wiping a surface transfers contaminants to and from the wiper, roughly in proportion to the difference in the concentration of contaminants on the wiper versus the concentration on the surface being wiped. The wiper should be kept as clean as possible while cleaning, so the pattern for wiping should be from the cleaner regions to the dirtier regions. To conserve cleaning liquid and prevent wiper-to-wiper contamination, put the liquid onto the wiper rather than onto the surface being cleaned or submerging the wiper in the liquid. For floors, use two buckets, one with the cleaning solution, the second to rinse the dirty mop before putting it into the first bucket. Keep people off wet floors, for safety and cleanliness. Perform periodic cleaning audits to find any areas missed and to highlight problems of materials, - 3

4 techniques or personnel. It is best if the measuring technique is sensitive to what is of practical interest. Validation Validation is a formalized demonstration of the capabilities of the processing. 8 The US Food and Drug Administration (FDA) has stated: In the end, the test of any validation process is whether scientific data shows that the system consistently does as expected and produces a result that consistently meets pre-determined specifications. 9 This Guide to Inspections, intended to cover equipment cleaning for chemical residues only, suggests: 1. Written procedures are required that detail the cleaning processes and how these cleaning processes will be validated, addressing who is responsible for performing and approving the validation study, the acceptance criteria and when revalidation will be required. 2. The sampling and analysis combination should be challenged to determine what fraction of the target material is actually sampled and then detected. 3. Direct sampling (for example, with swabs) is most desirable, although rinse sampling may be satisfactory. Sterilization validation is a specialty with its own extensive literature and is not covered in detail here. The broader topic of the validation of aseptic pharmaceutical processes has been explored in detail by the book of that title. 8 Cleaning validation Cleaning validation means obtaining proof that the cleaning procedures are adequate. J.P. Agalloco s points to consider include: What is being removed? What sampling methods are being used? What analysis is being performed? When are evaluations performed? Which cleaning parameters should be evaluated? What final level of residuals is acceptable, and on what basis? How difficult is the cleaning task? A book on cleaning and cleaning validation 6 covers process equipment design, cleaning mechanisms, automatic cleaning equipment, validation studies, sampling methods, analytical methods, validation acceptance criteria and special considerations for multi-product facilities. Sampling methods listed there, to provide various levels of assurance concerning cleaning, include visual inspection, rinse water sampling and analysis, surface sampling and analysis and surface sampling from coupons. Sampling The three major sampling methods for cleaning validation are swabbing, rinse sampling and placebo sampling. 10 Smith 11 noted that analysis of the rinse water, to determine the - 4

5 thoroughness of rinsing, often involves assuming that the compound being measured is proportional to the compounds of interest. Often, what is required to be monitored is the compound that is last to be rinsed. Smith showed that for a particular cleaning compound used with either stainless steel or glass, the last compound to be released was the surfactant in the cleaner. An evaluation of the effectiveness of cleaning has been done successfully by adding dye to product surrogates, then swabbing the surfaces and analysing the swab for traces of the dye or by testing for unmodified product residues or detergent residues. 12 The closeness of the match between product and surrogate is crucial. The direct method, using swabs, has the advantage of physical removal of contaminants, but the swab and solvent must be chosen so as to be effective and not to interfere with the analysis. Rinsing can deal with a larger area and reach otherwise inaccessible areas, but it may not have the required removal efficiency. Validation techniques depend in part on the surfaces to be cleaned. Flat surfaces are relatively easy to sample, and it is easy to determine the size of the area that has been sampled. There is a tendency to avoid corners and depressions, but these are often critical areas, requiring small or pointed swabs. Areas that are hard to sample well are often hard to clean to a sufficient standard and cannot be ignored. Even if swabbing is chosen, some rinsing may be required as a back-up, particularly if there are areas that swabs cannot reach. Sampling is similar to cleaning. Factors that aid in the removal of contaminants aid in sampling them, as long as interferences are avoided. To minimize contamination, sampling should be performed wearing gloves. The area to be sampled should be clearly delineated and its dimensions recorded. If a template is used, it must not contaminate the sample. Some facilities make repeated passes over the same area to increase the efficiency of removal, being less concerned with any contamination deposited by the sampling procedure. The quantity and areas for sampling must be decided. Guidelines published by a joint committee, established by the International Pharmaceutical Federation, 13 concerning the validation and environmental monitoring of aseptic processing, included as sampling options either contact plates (25 50 cm 2 ), swabbing or rinsing, with limits (see below) generally placed on the basis of 25 cm 2 areas. The recommended practice of the Institute of Environmental Sciences (RP-23) also uses 25 cm 2. Sample recovery Recovery efficiency is the fraction of material originally present on the test surface that is subsequently quantified by the analysis. It involves removing the contaminant from the original surface, extracting and measuring it. Usually, swabs are used to remove the materials and some form of immersion in agitated liquid is used to extract it from the swab (Figure 4). Zeller 14 noted that swabs are hard to use to sample inside equipment and that it is unclear how to combine those swab results from almost inaccessible locations with those from convenient, flat locations. He recommended that rinse-fluid sampling be used if it can be shown to be adequate; otherwise, a combination of rinsing and swabbing was favoured. He advised that a guiding substance should be chosen on which to base quantification. Recovery from swabs spiked with the guiding substance was found to be 92% to 99%, and the limit of quantitation was approximately 1 ng/cm 2 of surface. Ideally, to determine the recovery and analysis efficiency of a method, a known amount of contaminant of interest would be deposited on the surface, then the surface would be swabbed, the swab extracted, the extract quantified and the quantity thus found compared with the original, known amount. Otherwise, lacking a reference, the method itself can be used to estimate its own - 5

6 efficiency, through the technique of exhaustive recovery, so that repetitive samples and extractions eventually recover all the material that was originally present. Monitoring Monitoring is related to the word warning. Various locations are sampled and evaluated at various times with techniques of adequate sensitivity to warn that the process is not performing as designed. Many of the techniques used in validation would be candidates for use in monitoring. The difference is analogous to the difference between a statistical quality control chart that follows a process over time and the initial tests that qualify a process for use. One approach to monitoring would be to use the same sampling and analysis methods as used in validation, but to do them less intensively that is, fewer, or more widely spaced or less frequently. Another approach to monitoring would be to use somewhat less sensitive, precise or accurate methods that are still adequately correlated with the variables of concern. Analytical options There are many suitable physical, chemical and biological techniques for validating and monitoring cleaning 6 that include assays specific to particular molecules, as well as many nonspecific assays. A typical cleaning validation study might employ ph, conductivity, total organic carbon (TOC), detergent assays and, if a multi-product facility, a product-specific assay. 6 Analyses should be compared for: precision, accuracy, detection limits, quantitation limits, selectivity, linearity, range and sensitivity. Many of the most sensitive and convenient methods of detecting and measuring contaminants rely on light or, more specifically, electromagnetic radiation. Atoms and molecules may absorb light, and this absorption can be measured for materials on surfaces or in fluids (liquids and gases). The material s absorption coefficient will depend on its structure (electronic and molecular) and on the wave-length of the light. Similar measurements can be performed using light transmitted through, or reflected from, solids. Molecules have specific fingerprints, but the presence of several species can cause overlapping that makes interpretation difficult. An extensive presentation with many tables is available. 15 Exceptional detection sensitivity can be obtained with fluorescent materials, often used as tracers. Fluorescence is excited with relatively short wavelength radiation and measured at relatively long wavelengths, using optical filters to remove the excitation wavelengths, greatly reducing the background signal. Jenkins and Vanderwielen 10 matched common methods (high performance liquid chromatography [HPLC], thin-layer chromatography [TLC], UV spectrophotometry, TOC, enzymatic analysis [ELISA], gel electrophoresis, ph, conductivity and gravimetric analysis) against common targets: drug residue, excipient, cleaning agent residue and biopharmaceutical, and stated the advantages and disadvantages of the methods. Most cleaning validation work can be performed using chemical or physical assays, but there are instances where biological assays are useful. Large, flat surfaces can be sampled and the bioburden enumerated by contacting them with a sterile contact plate (RODAC plate) that contains solidified agar gel-growth media. (Unfortunately, the unknown sampling efficiency and the agar residues left on the sampled surface are problems with this technique). The plate is removed, incubated, and the number of colony-forming units (CFUs) is determined. Laboratories often have their own standard procedures, and there is one offered in the monograph, Microorganisms in Clean Rooms. 5 Flat surfaces and irregular surfaces can be swabbed with a sterile swab, the material captured on the swab extracted into biological growth medium, incubated and the CFUs counted. - 6

7 Figure 4: Swabs having material extracted in a bath. Pyrogens will not be detected as CFUs and are now usually determined by the limulus amebocyte lysate (LAL) assay. A reagent obtained from the horseshoe crab is mixed with various dilutions of the samples, and the formation of a gel clot or an increase in the liquid s turbidity is used to detect and quantify the pyrogen level, as endotoxin units. 16 A trend toward TOC? Much pharmaceutical concern regarding cleaning validation centres on non-biological contamination, such as residues of previously made products or their intermediates or carriers or cleaning agents. Jenkins et al. 17 made a strong case for the use of TOC analysis for cleaning validation: TOC has low level detection, rapid analysis time, is low cost as compared to other methods, and can detect all carbon-based residuals. They compared TOC, HPLC, TLC, spectrophotometric (UV), ELISA, electrophoresis, ph, conductivity and visual analysis. TOC analysis following swab sampling was the method selected. Quartz wool had high recovery but high residue, and a hydroentangled polyester gave low sample recovery. The foam tested had high TOC. Glass fibre filters had high particulate contamination and moderate recovery. Products with cellulose had overly high TOC values. Knitted polyester was selected on the basis of cleanliness and cost, cut into 2 2 cm squares to use as swabs to recover drugs placed on 225 cm 2 stainless steel surfaces and allowed to dry. Swab recoveries were greater than 50% in all cases, which was considered acceptable. TOC did as well or better than HPLC and spectrophotometric methods tested. Strege et al. 18 selected TOC analysis in preference to total protein analysis formerly used for cleaning validation. They reported having applied TOC successfully to cleaning validation for equipment used with water-soluble drugs, various excipients and cleaning agents. Five large biotechnology companies in the US were surveyed to determine the assays in use: ph, conductivity and visual inspection were used throughout. Late-stage processing steps were also assayed with endotoxin (LAL), gel electrophoresis (PAGE) and immunoassays (ELISA). Although swab samples have the disadvantage of obtaining only small amounts of contaminants, - 7

8 there is a trend toward coupling this with TOC analysis: the TOC swab assay may eventually replace the need for more specific protein swab assays such as fluorescamine and ninhydrin. Summary Thorough cleaning is crucial to aseptic fill operations, but it must be validated as well as monitored. This article has presented some sampling, extraction and analysis options that have proved successful in their application. References 1. T.E. Hansz and D.R. Linamen, Planning, Programming, Designing, and Constructing a Clean Room, Med. Dev. Diag. Ind. 17(2), (1995). 2. AAMI Standards and Recommended Practices: Vol. 1: Sterilization, Association for the Advancement of Medical Instrumentation, Arlington, Virginia, USA, D.W. Cooper, Cleaning Aseptic Fill Areas, Pharm. Tech. 20(2), (1996). 4. D.W. Cooper, Using Swabs for Cleaning Validation: A Review, Cleaning Validation (Institute of Validation Technology, Royal Palm Beach, Florida, USA, 1997). 5. Institute of Environmental Sciences, Micro-organisms in Clean Rooms, IES-RP-CC023.1 (Mount Prospect, Illinois, USA, 1993). 6. Cleaning and Cleaning Validation: A Biotechnology Perspective, Parenteral Drug Association (Bethesda, Maryland, USA, 1996). 7. E.R.L. Gaughran, R.L. Morrisey and Y. Wang, Eds., Sterilization of Medical Products, Volume IV (Polyscience, Montreal, Canada, 1986). 8. F.J. Carleton and J.P. Agalloco, Eds., Validation of Aseptic Pharmaceutical Processes (Marcel Dekker, New York, USA, 1986). 9. Guide to Inspections of Validation of Cleaning Processes (FDA, Washington DC, USA, July 1993). 10. K.M. Jenkins and A.J. Vanderwielen, Cleaning Validation: An Overall Perspective, Pharm. Tech. 18(4), (1994). 11. J.A. Smith, Selecting Analytical Methods to Detect Residue from Cleaning Compounds in Validated Process Systems, Pharm. Tech. 17(6), (1993). 12. T. Myers et al., Approaches to Cycle Development for Clean-In-Place Processes, J. Parenteral Sci. Tech., 41(1), 9 15 (1987). 13. FIP Committee on Microbial Purity, Validation and Environmental Monitoring of Aseptic Processing, J. Parenteral Sci. Tech. 44(5), (1990). 14. A.O. Zeller, Cleaning Validation and Residue Limits: A Contribution to Current Discussions, Pharm. Tech. 17(10), (1993). 15. J.A. Dean, Ed., Lange s Handbook of Chemistry, 12th Edn. (McGraw Hill, New York, USA, 1985). - 8

9 16. Guideline on Validation of the Limulus Amebocyte Test as an End-Product Endotoxin Test for Humans and Animal Parenteral Drugs, Biological Products, and Medical Devices (FDA, Washington DC, USA, December 1987). 17. K.M. Jenkins et al., Application of Total Organic Carbon Analysis to Cleaning Validation, PDA J. Pharm. Sci. & Tech. 50(1), 6 15 (1996). 18. M.A. Strege et al., Total Organic Carbon Analysis of Swab Samples for the Cleaning Validation of Bioprocess Fermentation Equipment, BioPharm 9(4), (1996). Douglas W. Cooper, PhD, is the Director of Contamination Control at The Texwipe Company, 650 East Crescent Avenue, Upper Saddle River, New Jersey 07458, USA. Tel , ext. 397 Fax