Basics of Isolator Cleaning

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Basics of Isolator Cleaning

By Dr. Thomas H. Treutler





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.Isolators are increasingly installed in pharmaceutical production laboratories due to the increased handling of hazardous drug ingredients as well as the need for smaller batches and more flexible production environments. Isolators can potentially lower the installation and maintenance costs compared to large scale cleanroom environments. While manufacturing facilities have established SOPs for isolators, this article focuses on the importance of proper cleaning and wiping procedures.



ISOLATORS AND DECONTAMINATION

Decontamination is the reduction or removal of biological or chemical agents, including non-active particles to non-hazardous levels to products, processes, or the environment by means of physical or chemical procedures.



Specifically in pharmaceutical manufacturing environments, research laboratories, and hospital pharmacies, the effective decontamination of biological agents like bacteria, viruses, fungi, protozoa, prions, and spores is essential.



Isolators like fume hoods, biosafety cabinets, and gloveboxes are used to create environments with low levels of environmental pollutants such as biological agents, aerosol particles, and dust. These separative devices have a controlled level of contamination, specified by the number of particles with a defined size per cubic meter, providing controlled environments that are specifically tailored to the needs of its operator. This classification of cleanrooms and isolators, however, is not taking into account specific requirements regarding biological contamination. In order to maintain the low levels of environmental pollutants, isolators have to be decontaminated on a regular basis.



ISOLATOR CLEANLINESS

Isolator cleanliness levels are defined by different classifications, shown in Table 1 and Table 2. These classifications are evaluating the environmental pollution by particles, however, not taking into account specific requirements regarding biological contamination. In order to maintain the low levels of environmental pollutants, isolators have to be decontaminated on a regular basis.



Quality supervisors in facilities using isolators have to determine the acceptable level of biological agents in their respective environment and decide on the method to achieve these levels. Several factors influence the choice of method and materials.











POTENTIAL CONTAMINANTS

Isolators are used in a variety of industries working with different material and under different requirements. Potential contaminants in isolators can therefore range from biological contaminants (e.g. pharmaceutical industry, hospital pharmacies), radionuclides (e.g. pharmaceutical industry, research laboratories) to general particulate contaminants (e.g. semiconductor industry).



CHEMICAL AGENTS: INACTIVATION

Spills of hazardous chemical agents in isolators or potential reaction products immobilized on isolator surfaces have to be inactivated or diluted to non-hazardous levels. The chemicals and chemical processes used for inactivation depend on the contaminant.



BIOLOGICAL AGENTS: DISINFECTION AND STERILIZATION

To reduce the level of biological agents in an environment, disinfectants/sanitizers and sterilants can be used. Sanitizers and disinfectants are terms used in different industries for the same kind of product. Whereas the food and foodprocessing industry uses the term sanitizers, the pharmaceutical industry, laboratories, and hospitals are predominantly using the term disinfectant.



Disinfection describes a process that eliminates many or all pathogenic microorganisms on inanimate objects, except bacterial spores.1 On the other hand, sterilization describes a process that destroys or eliminates all forms of microbial life and is carried out by physical or chemical methods.1 Depending on the biological agent and the material or media holding it, sterilization can be achieved through the application of heat, chemicals, irradiation, high pressure, or filtration. It is essential to understand the difference between both processes to assure that contamination level requirements of work environments are met. Whereas some commercial and technical literature is confusing readers by using both terms interchangeably, it should be noted that disinfection and sterilization describe two processes with very different requirements in outcome. It is not appropriate to talk about partial sterilization or even replace the word disinfection with sterilization.



The efficacy of sterilization depends on a number of factors like:



•prior physical cleaning (effective surface and biofilm reduction)

•presence of organic and inorganic load-level and type of microbial contaminants

•concentration of sterilant

•exposure time of sterilant

•pH, temperature, and humidity of environment

•geometry of objects and spaces

•physical properties of objects

Frequent application of sterilization processes is facing two major challenges; the potential build-up of resistance against the used sterilization agent as well as disadvantageous interactions with humans and surfaces that get in direct contact with these agents. The applied processes have to be well understood in order to avoid these detrimental effects.



The efficacy of different sterilization methods has been evaluated and reported by a number of publications. Tested microbial agents include bacteria, spores, and viruses.3,4,5 As discussed in these articles, microbiological agents may show a significant difference in resistance to the discussed sterilization methods. Therefore previously mentioned factors (the efficacy of sterilization depends on a number of factors such as in list one as well as the specific resistance of microbiological agents) play a vital role in the selection of the appropriate sterilization method.





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CLEANING OF ISOLATORS

Decontamination or cleaning, the reduction or removal of biological or chemical agents, including non-active particles is a multi-step process that depends on the contaminant and the required cleanliness level.



In isolators with processes using chemical agents the successful inactivation of these agents precedes any removal attempt in order to avoid further contamination of the environment or reaction with the isolator surfaces and cleaning materials. After successfully inactivating hazardous chemicals, high absorbency wipes are used to physically remove the reaction products.



When choosing isolator cleaning tools and materials, it is recommended that operators introduce the least amount of particle and fiber generating materials into the isolator. Typically a cleanroom laundered 100% continuous filament polyester knit material with sealed edges is recommended for use to clean surfaces inside the isolator. Additionally isolator cleaning tools with replacement covers that have been tested for particle and fiber release are appropriate to extend the reach of the cleaning area as well as providing ergonomic benefits to the operator.



One can also use cleanroom wipes with specific surface treatments to allow the wiper to capture and retain particulate contamination, resulting in more efficient cleaning and reduced likelihood of recontamination of critical surfaces.



The recommended steps to be performed when cleaning a contaminated surface do not change and are the same for all kinds of contaminants.



1.Always clean from the cleanest to the dirtiest surface.

2.Clean with overlapping strokes and change wiper surface with each stroke.

3.If using an isolator cleaning tool or mop, change out cover material with each surface side of the isolator.

In the case of isolators with biological contaminants, like bacteria, spores, and viruses, regular sterilization might seem to be sufficient in killing the microbial agents. However, it is extremely important that prior to sterilization, a physical removal of these contaminants is done in order to avoid a subsequent buildup of biofilms that would increase the resistance to sterilization attempts in the future. Biofilm is composed of polysaccharides that consist of carbon, hydrogen, and oxygen. Hydrogen and oxygen are most likely to be found in most isolators with natural atmosphere, leaving killed microbial agents behind would provide the required carbon for bacteria to reproduce and form new biofilms.



CLEANING PROCESS SOP

Developing a Standard Operating Procedure (SOP) for your isolators is a difficult task and depends on the very specific requirements of a facility’s processes and regulation in its industry. As a rule of thumb, Table 3 can serve as a general guideline to develop your own SOP.6 Questions you should ask yourself are:



•What contaminants am I concerned about?

•Would they contaminate my processes (inside) or the environment (outside)?

•Are these contaminants inert, chemically-, biologically-, or radio-active?

•What contamination limits have to be considered?

The use of an isolator cleaning tool should also be considered to allow efficient cleaning6 of hard-toreach areas and guarantee an equal pressure distribution of your cleaning material (wipes/pads) on the isolator surface. The applied pressure is a determining factor in the physical removal of contaminants from a surface.











SUMMARY

Proper decontamination and cleaning of isolators is critical to the long term success of materials produced in these environments. Reducing the risk of cross contamination starts with a full understanding of the type of potential contaminants introduced before, during, and after the production process. Sterilization and spraying with disinfectants alone are not enough to remove residual particles that could result in the buildup of biofilms. Proper wiping and rinsing protocols are needed to ensure the total removal of contaminants and the cleanliness of the isolator.



References



1.Healthcare Infection Control Practices Advisory Committee (HICPAC), “Guideline for Disinfection and Sterilization in Healthcare Facilities,” 2008

2.McDonnell, G.; Russell, A.D.; “Antiseptics and Disinfectants: Activity, Action, and Resistance” Clinical Microbiological Reviews, Jan. 1999, p. 147-179

3.Mehmi, M.; Marshall, L.J.; Lambert, P.A.; Smith J.C.; “Evaluation of Disinfecting Procedures for Aseptic Transfer in Hospital Pharmacy Departments” PDA Journal of Pharmaceutical Science and Technology, Vol. 63, No. 2, p. 123-138

4.Block, S.S. Disinfection, Sterilization, and Preservation. Philadelphia: Lea & Febiger 1991

5.Siegerman, H. “Wiping Surfaces Clean” A2C2 Magazine, April 2003

6.“Isolator Cleaning Guide” 01 Aug 2010 Berkshire Corporation     Dr. Thomas H. Treutler is CTO for Berkshire Corporation. He has years of experience in nanotechnology and applications in contained environments. Berkshire Corporation, 21 River Street, Great Barrington, MA 01230; (413) 931-3468 begin_of_the_skype_highlighting (413) 931-3468 end_of_the_skype_highlighting; ttreutler@berkshire.com; www.berkshire.com



REF: http://www.cemag.us/print/5090 Acessado em 05/09/12

Verticillium sp.

Verticillium sp.


Colonies are fast growing, suede-like to downy, white to pale yellow in colour, becoming pinkish brown, red, green or yellow with a colourless, yellow or reddish brown reverse. Conidiophores are usually well differentiated and erect, verticillately branched over most of their length, bearing whorls of slender awl-shaped divergent phialides. Conidia are hyaline or brightly coloured, mostly one-celled, and are usually borne in slimy heads (glioconidia).

Conidiophores, phialides and conidia of Verticillium sp.
Clinical significance:

Members of this genus are often isolated from the environment. It has been reported as a rare agent of mycotic keratitis.

Mycosis: Hyalohyphomycosis
Further reading:

Domsch, K.H., W. Gams, and T.H. Anderson. 1980. Compendium of soil fungi. Volume 1. Academic Press, London, UK.

Rippon, J.W. 1988. Medical Mycology. 3rd Edition. W.B. Saunders Co., Philadelphia, USA.

REF: http://www.mycology.adelaide.edu.au/Fungal_Descriptions/Hyphomycetes_(hyaline)/Verticillium/ Acessado em 12/06/12

Staphylococcus sciuri

We previously characterized over 100 Staphylococcus sciuri isolates, mainly of animal origin, and found that they all carried a genetic element (S. sciuri mecA) closely related to the mecA gene of methicillin-resistantStaphylococcus aureus (MRSA) strains. We also found a few isolates that carried a second copy of the gene, identical to MRSAmecA. In this work, we analyzed a collection of 28 S. sciuri strains isolated from both healthy and hospitalized individuals.

REF: http://jcm.asm.org/content/38/3/1136.full Acessado em 12/06/12

Staphylococcus hominis

Staphylococcus hominis is a coagulase-negative member of the bacterial genus Staphylococcus, consisting of Gram-positive, spherical cells in clusters. It occurs very commonly as a harmless commensal on human and animal skin. However, like many other coagulase-negative staphylococci, S. hominis may occasionally cause infection in patients whose immune systems are compromised, for example by chemotherapy or predisposing illness.




DescriptionColonies of S. hominis are small, usually 1–2 mm in diameter after 24 hours' incubation at 35 °C, and white or tan in colour. Occasional strains are resistant to novobiocin and may be confused with other resistant species (e.g. S. saprophyticus.)




It is one of only two species of Staphylococcus that display sensitivity to desferrioxamine, the other being S. epidermidis. Unlike S. epidermidis, S. hominis produces acid from trehalose, so the two tests together serve to identify the species.



[edit] BiologyNumerous coagulase-negative staphylococci appear commonly on the skin of human. Of these species, Staphylococcus epidermidis and S. hominis are the most abundant. While S. epidermidis tends to colonize the upper part of the body, S. hominis tends to colonize in areas with numerous apocrine glands, such as axillae and the pubic region. In a certain study, S. hominis was calculated to account for 22% of the total staphylococci species recovered from individuals, second to S. epidermidis at 46%. S. hominis is the predominant species on the head, axillae, arms, and legs. S. hominis, as well as most other staphylococci species common on the human skin, is able to produce acid aerobically from glucose, fructose, sucrose, trehalose, and glycerol. Some strains were also able to produce acid from turanose, lactose, and galactose, melezitose, mannitol, and mannose. Most strains colonize on the skin for relatively short periods of time compared to other Staphylococcus species. They, on average, stay on the skin for only several weeks or months. The cell wall contains low amounts of teichoic acid and glutamic acid. The cell wall teichoic acid contains glycerol and glucosamine. S. hominis cells are Gram-positive cocci, usually 1.2 to 1.4 micrometers in diameter. They appear normally in tetrads and sometimes in pairs.[1]



[edit] ResistanceBased on a total of 240 strains, all were resistant to lysozyme, some were slightly resistant to lysostaphin, 77% were susceptible to penicillin G, 97% to streptomycin, 93% to erythromycin, 64% to tetracycline, and 99% to novobiocin.[2]



[edit] CulturingWhen grown in agar cultures, colonies are usually circular, 4.0 to 4.5 micrometers in diameter. Agar colonies usually have wide edges and an elevated center. They are commonly smooth with dull surfaces, and are yellow-orange pigmented in the center of the opaque colonies. They grow both in aerobic and anaerobic conditions, but tend to grow significantly less in the latter. Optimal NaCl concentrations of the agar culture for the growth of S. hominis seems to be around 7.5%, and a salt concentration of 15% yielded poor growth to no growth at all. The optimal growth temperature range was around 28 to 40 °C, but good growth is still observed at 45 °C, while no growth is observed at 15 °C. S. hominis can be differentiated from staphylococci by its colony morphology and pigmentation patterns, predominant tetrad cell arrangement, poor growth in thioglycolate, low tolerance of NaCl, and carbohydrate reaction pattern. Each species is also significantly different in cell wall composition, lactic acid configuration, temperature extremes of growth, coagulase activity, hemolysis acetylmethylcarbinol production, nitrate reduction, and phosphatase, DNase, and bacteriolytic activities. Similarities in these properties between S. hominis and several other species suggest there is a close relationship between S. hominis and S. epidermidis, S. haemolyticus, and S. warneri.[3]



[edit] Antibiotic-resistant subspeciesS. hominis is normally found on human skin and is usually harmless, but can sometimes cause infections in people with abnormally weak immune systems. Most, if not all, strains are susceptible to penicillin, erythromycin, and novobiocin, but a divergent strain, S. hominis subsp. novobiosepticus (SHN) was found recently. This strain was named so because of its unique resistance to novobiocin and its failure to produce acid aerobically from trehalose and glucosamine. In addition, the 26 isolated strains of this new subspecies are resistant to nalidixic acid, penicillin G, oxacillin, kanamycin and streptomycin. They were also somewhat resistant to methicillin and gentamicin, and most strains were resistant to erythromycin, clindamycin, chloramphenicol, trimethoprim/sulfamethoxazole and ciprofloxacin, as well. In addition, S. hominis subsp. hominis is commonly found isolated from human skin, but there are no reports of the isolation of SHN from the human skin.[4]



The SHN is so similar to the original S. hominis, now called S. hominis subsp. hominis, that a MicroScan system that clinical microbiology laboratories use identified seven of 31 S. hominis subsp. novobiosepticus cultures as S. hominis subsp. hominis. The relationship between the two is currently unknown, but antibiotic-resistant isolates of S. hominis belong only to SHN. [5]



SHN strains seems to have thickened cell walls, and this tendency may be the result of a genetic background that also allows for vancomycin resistance. The thickened cell walls exist in subspecies with and without vancomycin resistance which suggests this subspecies did not originate from the acquiring of resistance genes. [6]



[edit] OriginThe combined resistance to novobiocin and oxacillin is hypothesized to have originated from a simultaneous introduction of genes controlling the resistance to the two. These genes were believed to have been acquired originally through heterologous DNA from a methicillin-resistant strain of one of the novobiocin-resistant species belonging to the S. sciuri or the S. saprophyticus groups. The larger genome size of the SHN compared to that of S. hominis subsp. hominis may be the result of the acquiring of heterologous DNA. This new, divergent strain was first described in 1998, and this microbe was first implicated in causing bactermia in 2002. Another hypothesis is the insertion of the mec A gene and its flanking sequence into the chromosome of SHN might have affected the expression of a closely linked gene, which converted the host to become novobiocin-resistant.[7]

REF: http://en.wikipedia.org/wiki/Staphylococcus_hominis Acessado em 12/06/12

Penicillium sp

O Penicillium (lat. penicillus= pincel) é um género de fungos, o comum bolor do pão, que cresce em matéria orgânica biodegradável, especialmente no solo e outros ambientes húmidos e escuros. Por contágio, contaminam frutas e sementes e chegam a invadir habitações, sendo responsáveis pelos bolores que se instalam em alimentos para consumo humano.


Natural Habitats Soil • Seed • Cereal crops


Suitable Substrates in the Indoor Environment Foods (blue mold on cereals, fruits,

vegetables, dried foods) • House dust • Fabrics • Leather • Wallpaper • Wallpaper glue

Water Activity Aw=0.78-0.86

Mode of Dissemination Wind • Insects

Allergenic Potential Type I (hay fever, asthma) • Type III (hypersensitivity)

Potential Opportunist or Pathogen Penicilliosis

Industrial Uses P. chrysogenum for the antibiotic penicillin • P. griseofulvum for the antibiotic

griseofulvin a • P. roquefortii for Roquefort cheese • P. camemberti for Camembert cheese

• Brie, Gorgonzola, and Danish Blue cheese are also the products of Penicillium • Used to cure

ham and salami • Production of organic acids such as fumaric, oxalic, gluconic, and gallic

Potential Toxins Produced Citrinin • Citreoviridin • Cyclopiazonic acid • Fumitremorgen B

• Grisiofulvin • Janthitrems • Mycophenolic acid • Paxilline • Penitrem A • Penicillic acid

• Ochratoxins • Roquefortine C • Secalonic acid D • Verruculogen • Verrucosidin

• Viomellein • Viridicatumtoxin • Xanthomegnin

Other Comments Penicillium is one of the most common genera of fungi

ref: http://www.nordichomeinspection.com/uploads/Penicillium.pdf Acesso: 11/06/12

Nigrospora spp

Nigrospora spp.


(described by Zimmerman in 1902)

Taxonomic classification

Kingdom: Fungi

Phylum: Ascomycota

Order: Trichosphaeriales

Family: Trichosphaeriaceae

Genus: Khuskia (teleomorph)


Description and Natural Habitats
Nigrospora is a filamentous dematiaceous fungus widely distributed in soil, decaying plants, and seeds. It is a common laboratory contaminant. Although it has been isolated from a few clinical samples, its pathogenicity in man remains uncertain [531, 1295, 2144, 2202].

Species



Nigrospora sphaerica is the best-known species of the genus Nigrospora.


Synonyms
See the summary of synonyms and teleomorph-anamorph relations for Nigrospora spp.

Pathogenicity and Clinical Significance


Nigrospora has been isolated from cutaneous lesions of a leukemic patient and from a case with keratitis. However, its pathogenic role as a causative agent is not well-known [1847, 2218].

Macroscopic Features
Nigrospora grows rapidly and produces woolly colonies on potato dextrose agar at 25°C. The colonies mature within 4 days. Color of the colony is white initially and then becomes gray with black areas and turns to black eventually from both front and reverse. Sporulation may take more than 3 weeks for some isolates [531, 1295, 2144, 2202].


Microscopic Features
Septate hyaline hyphae, hyaline or slightly pigmented conidiophores, and conidia are visualized. The conidiogenous cells on the conidiophores are inflated, swollen, and ampulliform in shape. They bear a single conidium (14-20 µm in diameter) at their apex. Conidia are black, solitary, unicellular, slightly flattened horizontally, and have a thin equatorial germ slit [531, 1295, 2144, 2202].


Compare to
Humicola
Nigrospora is differentiated from Humicola by its very black conidia that originate from hyaline, inflated conidiophores.
Laboratory Precautions

No special precautions other than general laboratory precautions are required.
Susceptibility
No data are available

ref: http://www.doctorfungus.org/thefungi/Nigrospora.php Acessado em 12/06/12

Kocuria rosea

Kocuria rosea


Kocuria rosea and Micrococcus spp. (gram-positive bacteria) are widespread in nature and commonly found along with coagulase-negative Staphylococcus spp. on the skin of humans and mammals.

REF: http://www.usmicro-solutions.com/referencelibrary/bacteriallibrary.html Acessado em 11/06/12