Peer Reviewed: Facility Sanitation

www.ivtnetwork.com

ABSTRACTMaintaining environmental control

including microbiological contamina-tion in a pharmaceutical manufacturing environment is primarily dependent on the facility sanitization program. Saniti-zation considerations are specific for fa-cility rooms, equipment, and personnel. Sanitization comprises cleaning and dis-infection. Cleaning is necessary prior to the application of disinfectant to enable sufficient contact time of the disinfecting agent with the surface. Disinfectants vary in their spectrum of activity, modes of ac-tion, sites of action in microorganisms, and efficacy. Disinfectants kill vegetative mi-cro-organisms but do not necessarily kill bacterial spores. There are many different types and categorizations of disinfectants such as non-oxidizing disinfectants and oxydizing agents. Many pharmaceutical manufacturers will have two in-use disin-fectants and a third disinfectant for major contamination incidents. Rotation of dis-infectants is often implemented to satisfy the requirements of regulators. Cleaning and disinfection must be detailed in a Stan-dard Operating Procedure (SOP) to ensure consistency of practice. The effectiveness of cleanroom sanitization is assessed through the site environmental monitoring pro-gram. Viable monitoring is undertaken us-ing microbiological growth medium. Reg-ulatory agencies expect the pharmaceutical manufacturer to have evaluated the efficacy of disinfectants. While suspension testing is useful for initial screening, comparative surface (or carrier) testing is more relevant.

USP lists common materials used in clean rooms that should be considered when developing disinfectant surface test-ing. To demonstrate the effectiveness of a disinfectant, it must be challenged using a panel of organisms that is reflective of the natural microflora of the facility. The bio-cidal activity of the disinfectant should be taken into account when selecting the panel of organisms. The use of microbial isolates from the manufacturing facility is increas-ingly becoming a regulatory expectation. Surface tests cannot demonstrate the effect of a range of environmental factors in ac-tual environmental conditions. Field tri-als are an important part of the qualifica-tion of a sanitizer to determine if cleaning techniques are suitable and if the cleaning frequencies of cleanrooms require modifi-cation.

INTRODUCTIONThis article provides an introduction to

the sanitization and bio-decontamination of pharmaceutical manufacturing facilities. This topic is especially relevant for manu-facturing of sterile products.

Pharmaceutical facilities used for manu-facturing of sterile products are comprised of a series of rooms called cleanrooms. Cleanrooms and zones are typically classi-fied according to their use or main activity within each room or zone. Cleanrooms are confirmed by the cleanliness of the air by the measurement of particles. Pharmaceu-tical cleanrooms are classified by standards in either EU and WHO GMP guidance for aseptically filled products (alphabet-

ic notation) or by International Standard ISO14644 (numeric classification). The cleanliness of the air is controlled by the HVAC system (Heating, Ventilation and Air-Conditioning) in the facility.

Cleanrooms are designed to minimize and to control contamination. There are many sources of contamination. The at-mosphere contains dust, microorganisms, condensates, and gases. Manufacturing processes will also produce a range of contaminants. Wherever there is a pro-cess which grinds, corrodes, fumes, heats, sprays, turns, etc., particles and fumes are emitted and will contaminate the sur-roundings (1). The foremost concern in pharmaceutical manufacturing of sterile products is microbial contamination.

Maintaining environmental control in a pharmaceutical manufacturing environ-ment is primarily dependent on the facil-itys cleaning and disinfection program. The program requires the selection of the appropriate disinfectants, their proper ap-plication, and validation of their capability to inactivate vegetative cells.

TYPES OF SANITIZATIONSanitization differs depending on the

specific area of concern. Three areas are discussed: Rooms, equipment, and person-nel.

Sanitation of Pharmaceutical FacilitiesTim Sandle

Tim Sandle

Journal of GXP Compliance Volume 18 Number 3

Room SanitizationCleanrooms and clean areas must be

regularly cleaned and disinfected. This is normally undertaken by cleaning with de-tergent followed by the application of a dis-infectant. Sometimes more than one dis-infectant application is necessary such as following a production shutdown. It may be necessary to remove the residue of the disinfectant using water.

Arguably the most effective cleaning and disinfection process is undertaken manual-ly by use of wiping techniques. Some facil-ities utilize fumigation or fogging methods. These methods are effective when surfaces are clean and the sanitizing agent can reach all of the cleanroom surfaces.

When cleaning rooms manually, the equipment used (mops and buckets) should be of an appropriate design for the grade of cleanroom. Cleaning procedures require a strict cleaning regime. Clean-ing and disinfection using cloths and mop heads is ideally performed by saturating the cleaning item and wiping the area using a series of parallel overlapping strokes with an approximate one quarter overlap. Cir-cular motion should never be used. The direction of the cleaning should be towards the operator (from top to bottom, from back to front). Only one application of the disinfectant or detergent should be applied to avoid over-concentration. Cleaning and disinfection should begin with the visually cleanest area first and towards the dirtiest area last.

Cleaning is normally undertaken in each process area before use. In general, the frequency of cleaning should be estab-lished through risk assessment based on a review of environmental monitoring data before and after the disinfection process. The review should consider the numbers of microorganisms recovered and the types of species. With species, a check should be made on the frequency of recovery of spore-forming microorganisms. A high recovery of microbial contamination could signal a concern with the disinfectants used or with their frequency of rotation.

Equipment SanitizationEffective cleaning and sanitization of

equipment is important because equipment may not be amenable to visual inspection. Also, equipment may be prone to biofilm formation.

The main method for cleaning industri-al equipment is by making the mechanism for cleaning integral to the equipment itself. This can be achieved by use of pressure, heat, steam sterilization, mechanical re-moval, or chemical cleaning agents. Auto-mated methods are termed Clean-in-Place (CIP) or Steam-in-Place (SIP). Prior to chemical or heat treatment, attempts must be made to remove process residues and particles using steam or high pressure wa-ter cleaning. Alkali-based disinfectants and detergents are commonly used for CIP systems; sodium hydroxide is among the most widely used. Such caustic alkalis can readily remove organic deposits without af-fecting the equipment. It is important that the equipment cleaning process is validat-ed.

Glove SanitizationThe gloved hands of staff undertaking

critical activities should be sanitized on a frequent basis using an effective hand san-itizer. Aseptic techniques performed by trained personnel are most important. The sanitizing agent itself is not a replacement for poor aseptic techniques.

There are many commercially avail-able hand sanitizers. Most commonly used types are alcohol-based gels contain-ing either ethanol or isopropanol. An EU standard (EN 1499 (2) and EN 150025A (3) provides test methodology to validate the efficacy of the hand sanitizer. Testing determines if a hand sanitizer can reduce the number of transient microflora under simulated practical conditions. There are three key variables which affect the use of hand sanitizers. These are the agitation and rubbing the hand sanitizer into the glove, the frequency of application, and the quan-tity applied (5). Of these, consistency of the rubbing technique in ensuring that all surfaces of the hand come into contact with the sanitizer is most important.

TYPES OF DISINFECTION AGENTS

A disinfectant is one of a diverse group of chemicals which reduces the number of micro-organisms present on an inanimate object. Disinfectants kill vegetative mi-cro-organisms but do not necessarily kill bacterial spores. To be effective, disinfec-tants must meet either European standards (the CEN series) or US standards (the AOAC standards) for disinfectant efficacy. These standards involve challenging disin-fectants with high populations of a range of different microorganisms and noting the log reduction after a period of time. Such studies are undertaken for the disinfectant solution (the suspension test), on surfaces, and in the field to develop appropriate cleaning frequencies.

Disinfectants vary in their spectrum of activity, modes of action, and efficacy. Some are bacteriostatic in which the ability of the bacterial population to grow is halt-ed. Here the disinfectant can cause selective and reversible changes to cells by interact-ing with nucleic acids, inhibiting enzymes, or permeating into the cell wall. Once the disinfectant is removed from contact with bacteria cells, the surviving bacterial pop-ulation could potentially resume growth. Other disinfectants are bactericidal in that they destroy bacterial cells through differ-ent mechanisms including causing struc-tural damage to the cell, lysis, and leakage or coagulation of cytoplasm (6). The mech-anisms of action are not always completely known and continue to be investigated.

Surface disinfectants have varying modes of action against microbial cells due to their chemical diversity. Different dis-infectants target different sites within the microbial cell. These include the cell wall, the cytoplasmic membrane (where the ma-trix of phospholipids and enzymes provide various targets) and the cytoplasm. Some disinfectants, on entering the cell either by disruption of the membrane or through diffusion, proceed to act on intracellular components.

Peer Reviewed: Facility Sanitation

www.ivtnetwork.com

There are different approaches to the categorization and sub-division of disin-fectants, including grouping by chemical nature, mode of activity, or by bacteristatic and bactericidal effects on micro-organ-isms (7). These and other factors such as efficacy, compatibility, cost, and current health and safety standards (8) must be considered when selecting disinfectants for use in the facility. The following describes the primary types of disinfectants currently in use categorized as non-oxidizing and ox-idizing agents.

Non-Oxidizing Disinfectants

The non-oxidizing disinfectants include alcohols, aldehydes, amphoterics, phe-nolics, and quaternary ammonium com-pounds (QACs or quats).

Alcohols. The effectiveness of alcohols against vegetative bacteria and fungi in-creases with their molecular weight (eth-anol is more effective than methanol and isopropyl alcohols are more effective than ethanol). Alcohols act on the bacterial cell membrane by making it permeable. Effica-cy is increased with the presence of water leading to cytoplasm leakage, denaturation of protein, and eventual cell lysis. The ad-vantages of employing alcohols include a relatively low cost, little odor, and rapid evaporation (9).

Aldehydes. Aldehydes include form-aldehyde and glutaraldehyde. Aldehydes have a non-specific effect in the denatur-ing of bacterial cell proteins and can cause coagulation of cellular protein. There are safety concerns about the use of some alde-hydes (10).

Amphoterics. Amphoterics have both anionic and cationic character and pos-sess a relative wide spectrum of activity. Amphoterics are limited by their inability to damage endospores. Amphoterics are frequently used as surface disinfectants. Examples include the alkyl di(aminoethyl) glycine group of compounds.

Phenolics. Synthetic phenols are widely available such as the bis-phenols (triclosan) and halophenols (chloroxylenol). Phenols are bactericidal and antifungal, but are not

effective against spores. Some phenols cause bacterial cell damage through disrup-tion of proton motive force, while others at-tack the cell wall and cause leakage of cellu-lar components and protein denaturation.

Quaternary ammonium compounds. QACs or quats are cationic salts of organ-ically substituted ammonium compounds that have a fairly broad range of microbial activity. They are ineffective against bac-terial spores. QACs are possibly the most widely used of the non-oxidizing disinfec-tants. Example QACs include cetrimide and benzalkonium chloride. Their mode of action is on the cell membrane leading to cytoplasm leakage and cytoplasm coag-ulation through interaction with phospho-lipids (11).

Oxidizing disinfectantsThis group includes oxygen-releasing

compounds (peroxygens) like peracetic acid and hydrogen peroxide. They func-tion by disrupting the cell wall, causing cytoplasm leakage, and denature bacterial cell enzymes through oxidation. Oxidiz-ing agents have advantages in that they are clear and colorless, thereby avoiding sur-face staining.

SANITIZATION REGIMEThere are several important process

considerations involved with sanitization. These include cleaning, disinfection, selec-tion and rotation disinfecting agents, and the specific procedures utilized.

CleaningCleaning, in the context of pharmaceuti-

cal manufacturing, is the process of remov-ing residues and soil (dirt, grease, protein etc.) from surfaces to the extent that they are visually clean. This involves clearly de-fined procedures that often require use of a detergent or other cleaning agent. Deter-gents generally work by penetrating the soil and reducing the surface tension (which fixes the soil to the surface) to allow its removal. Hence many of the products are surfactants (surface active agents).

Cleaning as described above is necessary for cleanrooms prior to the application of disinfectant. It is essential that a surface

or item of equipment has been properly cleaned before disinfectant application in order for the disinfectant to work efficient-ly. This is particularly important for spori-cidal disinfectants, many of which have limited ability to effectively penetrate soil.

DisinfectionThe critical aspect for disinfectant effi-

cacy is the contact time. The disinfectant is only effective when left in contact with the surface for the validated time. This can be achieved more easily when the dis-infectant is applied in overlapping strokes. When rotation of disinfectants is required, a water rinse normally employing Water for Injection (WFI) is employed between the change-over of disinfectants. This rinse re-moves traces of disinfectant and detergent residue such as anions which may reduce the efficacy of the subsequent disinfectant.

A disinfectant will achieve the desired effectiveness if it remains on the targeted surface for an appropriate length of time. Determining the optimal contact time of-ten means striking a balance between what is necessary to achieve the desired micro-bial reduction and what is practical for re-al-time use in the facility. At minimum, the manufacturers recommended contact time should be tested. Additional contact times may also be evaluated if the manufacturers recommended time is demonstrated to be ineffective or if a shorter contact time is de-sired (12).

Rotation of DisinfectantsMany pharmaceutical manufacturers

will routinely use two in-use disinfec-tants in a specified rotational sequence for the site disinfection program. A third disinfectant will be available in reserve in case a major contamination incident arises. For example, a bioburden contamination increase which appears resistant or diffi-cult to eliminate using the routinely used disinfectants may necessitate use of an ad-ditional disinfectant. The reserve disinfec-tant such as an oxidizing agent will often be more powerful and sporicidal, the routine use of which is restricted because of like-ly damage to the equipment and premises. The rotation of two primary disinfectants is a requirement of regulatory bodies. The

Tim Sandle

Journal of GXP Compliance Volume 18 Number 3

EU GMP Guide states that where disinfec-tants are used, more than one type should be employed (Annex 1). Nevertheless, the case for rotation has not been scientifical-ly proven in that there are very few studies providing empirical evidence. However, it remains that rotation is often implemented to satisfy the requirements of regulators.

Cleaning and disinfection proceduresCleaning and disinfection must be de-

tailed in a Standard Operating Procedure (SOP) to ensure consistency of practice. Furthermore, sufficient detail in SOPs is important because detergents and disin-fectants are only partially effective if they are not applied correctly. An SOP should describe: The type of detergents and disinfec-

tants to be used. These agents must be compatible

The frequency of rotation of disinfec-tants

A list of suitable cleaning materials Cleaning techniques Contact times Rinsing Frequency of cleaning and disinfection Procedure for the transfer of cleaning

agents and disinfectants into and out of clean areas

Procedure for sterilization of disinfec-tants

Holding times for detergents and dis-infectants.

ASSESSING SANITIZATION EFFECTIVENESS

The effectiveness of cleanroom saniti-zation is assessed through environmental monitoring. Environmental monitoring is a program which examines the numbers and occurrences of viable micro-organ-isms and non-viable particles such as dust or skin cells. Environmental monitoring is ideally targeted to those areas of the pro-duction process where the risk cannot be adequately controlled. Trend analysis of environmental monitoring data provides an indication if the cleanroom disinfection program is moving out-of-control (13). Viable monitoring of surfaces is the most relevant approach for assessing the effec-tiveness of surface sanitization.

Viable monitoring is designed to detect levels of bacteria and fungi present in de-fined locations during a particular stage in the processing and filling of product. Vi-able monitoring is designed to detect mi-cro-organisms and answer questions such as how many, how frequent, when do they occur and why do they occur? (14)

Viable monitoring is undertaken using microbiological growth medium (agar or other substances) in different presenta-tions. It is important that the culture media used for environmental monitoring con-tains a neutralizer to eliminate any residues of the disinfectant.

The environmental monitoring program is normally controlled by the site microbi-ology department who establish the appro-priate frequencies and durations for moni-toring based on a risk assessment approach. The sampling plan takes into account the cleanliness level required at each site to be sampled.

QUALIFICATION OF DISINFECTANTS

Regulatory agencies expect the phar-maceutical manufacturer to have evaluated the efficacy of disinfectants. While suspen-sion testing is useful for initial screening, it is surface (or carrier) testing that is more relevant. Qualification of a disinfectant is demonstrated through performance testing to show that the disinfectant is capable of reducing the microbial bioburden found on manufacturing area surfaces. Representa-tive manufacturing surface samples are in-oculated with a selection of microbial chal-lenge organisms. A disinfectant is applied to the inoculated surfaces and exposed for a predetermined contact time after which surviving organisms are recovered using a qualified disinfectant-neutralizing broth and test method (surface rinse, contact plate, or swab). The number of challenge organisms recovered from the test samples (exposed to a disinfectant) is compared to the number of challenge organisms re-covered from the corresponding control sample (not exposed to a disinfectant) to determine the ability of the disinfectant to reduce the microbial bioburden. Success-ful completion of the validation qualifies the disinfectant evaluated for use. The dis-infectant efficacy validation should provide

documented evidence that the disinfectant demonstrates bactericidal, fungicidal, and/or sporicidal activity necessary to control microbial contamination in the facility (15).

The selection of surfaces to be assessed for disinfectant efficacy is an important consideration. Given the multitude of available surfaces in a facility, a pragmatic view should be taken. Where the surface is considered critical in terms of cleaning and disinfection, i.e., contact with product and personnel, it should be considered for disinfectant surface testing. USP chap-ter lists common materials used in clean rooms that should be considered when developing disinfectant surface test-ing. Stainless steel and other surfaces within the manufacturing facility should be tested such as different grades of vinyl and stainless steel, different types of plastic, glass from windows and vessels, and other materials as appropriate.

The test involves examining prepa-rations of micro-organisms dried onto surfaces. A prepared sample of the disin-fectant is added to the dried surface con-taining and microbial suspension. The surface is then transferred to a previously validated neutralization medium and test-ing performed to measure the reduction in viable counts. One variation of the test involves drying 0.05 ml suspensions of the micro-organisms with interfering sub-stances such as bovine serum albumin onto different surfaces. The micro-organisms should have a population range of 1.5 - 5.0 x 108 for bacteria and 1.5 - 5.0 x 107 for fungi and are equilibrated to 25oC before use. Once applied to the surface, the drying of the micro-organisms maybe accelerated using an incubator operating at 36-38oC. Disinfectant solutions (where disinfectants are made with Water of Standard Hard-ness) are added to the surfaces. After the specified contact time (five minutes is the target), the surfaces are transferred to the validated neutralization medium and then pour plates are prepared for incubation and counting. An alternative method is avail-able using a soaked swab step (16).

To demonstrate the effectiveness of a disinfectant, it must be challenged using a panel of organisms that is reflective of the natural microflora of the facility. The bio-

Peer Reviewed: Facility Sanitation

www.ivtnetwork.com

cidal activity of the disinfectant should be taken into account when selecting the panel of organisms. Some organizations use type cultures, some use environmental isolates, and others a combination of the two. The use of isolates from the manufacturing fa-cility is increasingly becoming a regulatory expectation.

The surface test described above cannot demonstrate the effect of a range of envi-ronmental factors like temperature, pH, de-tergent residues, mechanical stress, and at-tachment in the facility. For these reasons, a disinfectant which appears effective for the surface test can show marked variability when applied to practical conditions. This is due to problems in drying and differenc-es between surfaces. In terms of drying microbial suspensions, there is a marked loss in the viability of a population when dried onto a surface. Attempts to speed the drying process do not significantly reduce the variability of the actual number of mi-cro-organisms challenged. Surfaces intro-duce another variation because surfaces. Surfaces of the same grade of material are not truly identical. There have been marked problems in achieving reproducibility and repeatability for the surface test between laboratories particular in estimating the concentration of disinfectant required to be effective. Some of these limitations can be addressed through field trials.

Field trials (or in situ studies) are an im-portant part of the qualification of a sani-tizer. These trials determine if cleaning techniques are suitable and if the cleaning frequencies of cleanrooms require modifi-cation. Filed trials involve a considerable amount of environmental monitoring for the counts before and after disinfection and the types of microorganisms recovered must each be evaluated (17).

SUMMARYThis paper has presented an overview

of the application of sanitization of phar-maceutical facilities. Sanitization is a key part of contamination control within cleanrooms. It has examined some of the techniques and control required, and has compared different types of disinfectants available for use. It has also emphasized the importance of qualifying disinfectant in order to demonstrate their effectiveness,

and for undertaking a vigorous monitoring regime in order to show that the cleanroom environment remains in control. The em-phasis has been upon the assessment of surfaces and the evaluation of disinfectants in the field. While the article has focused on microbiological assessments, there are other variables at play including wiping technique, expiry times, and chemical sta-bility that must also be considered. This acknowledged, it is hoped that this article has provided information and advice useful to compliance practitioners that can be ap-plicable within the manufacturing facility.

References1. Halls, N. (2004): Effects and causes of contami-

nation in sterile manufacturing in Hall, N. (ed.): Microbiological Contamination Control in Pharmaceutical Clean Rooms, CRC Press, Boca Raton, pp1-22

2. EN 1499. 1997; Chemical disinfectants and an-tiseptics. Hygienic handwash. Test method and requirements (phase 2/step 2)

3. EN 1500. 1997; Chemical Disinfectants Quan-titative Carrier Test to Evaluate the Bactericidial Activity of a Hygenic Handrub Solution (Phase 2/2). Chemical disinfectants and antiseptics. Hy-gienic handrub. Test method and requirements (phase 2/step 2)

4. Best M and Kennedy ME. Effectiveness of hand-washing agents in eliminating Staphylococcus aureus from gloved hands, Journal of Applied Bacteriology, 1992; 73: 6366.

5. Larson E, Mayur K and Laughon B. Influence of two handwashing frequencies on reduction in colonizing flora with three handwashing prod-ucts used by health care personnel, American Journal of Infection Control, 1989; 17(2): 8388.

6. Sandle, T.: Selection and use of cleaning and disinfection agents in pharmaceutical manu-facturing in Hodges, N and Hanlon, G. (2003): Industrial Pharmaceutical Microbiology Stan-dards and Controls, Euromed Communications, England

7. Block S. 1977; Disinfection, Sterilisation and Preservation, Third Edition, Lea and Febiger, Philadelphia

8. Denyer SP and Stewart GSAB. Mechanisms of action of disinfectants, International Biodeteri-oration and Biodegradation, 1998; 41: 261-268

9. McDonnell G and Russell A. Antiseptics and Disinfectants: Activity, Action and Resistance, Clinical Microbiology Reviews, Jan. 1999; 147179

10. Angelillo IF, Bianco A, Nobile CGA and Pavia M. Evaluation of the efficacy of glutaraldehyde and peroxygen for disinfection of dental instru-ments, Letters in Applied Microbiology, 1998;

27: 29229611. Bergan T and Lystad A. Evaluation of Disin-

fectant Inactivators, Acta Path Microbiol Scand Section B, 1972; 80: 507510.

12. Bessems, E.: The effect of practical conditions on the efficacy of disinfectants, International Biodeterioration and Biodegradation, 41, 1998, pp177-183

13. Johnson, S. M. (2004): Microbiological Environ-mental Monitoring in Hodges, N. and Hanlon, G. Microbiological Standards and Controls, Eu-romed, London

14. Vincent, D.(2002): Validating, Establishing and Maintaining A Routine Environmental Moni-toring Program for Cleanroom Environments: Part 1, Journal of Validation Technology, August 2002, Vol. 8, No.4

15. Vina, P., Rubio, S. and Sandle, T. (2011): Selec-tion and Validation of Disinfectants, in Saghee, M.R., Sandle, T. and Tidswell, E.C. (Eds.) (2011): Microbiology and Sterility Assurance in Phar-maceuticals and Medical Devices, New Delhi: Business Horizons, pp219-236

16. Bloomfield, S.F., Arthur, M., Van Klingeren, B., Pullen, W., Holah, J.T. and Elton, R.: An evalua-tion of the repeatability and reproducibility of a surface test for the activity of disinfectants, Jour-nal of Applied Bacteriology, 1994, 76, pp86-94

17. Sandle, T. (2012). Application of Disinfectants and Detergents in the Pharmaceutical Sector. In Sandle, T. (2012). The CDC Handbook: A Guide to Cleaning and Disinfecting Clean-rooms, Grosvenor House Publishing: Surrey, UK, pp168-197

About the AuthorTim Sandle, Ph.D., is the head of the microbiology department at Bio Products Laboratory Limited, a pharmaceutical organization owned by the UK Department of Health. Dr. Sandle is, additionally, a visiting tutor at the School of Pharmacy, Manchester University. E-mail: Tim.Sandle@bpl.co.uk