BioNetwork Bioprocessing Center 1800G North Greene Street Technology Enterprise Center (TEC)...

BioNetwork Bioprocessing Center1800G North Greene Street

Technology Enterprise Center (TEC)Greenville, NC 27834

252.757.3759252.757.3745 fax

www.bionetwork.org

Staff:

•William T. (Bill) Cooper, [email protected]

•Greg Smith, PhD, Curriculum [email protected]

•Vallere Shelton, Administrative [email protected]

What is the Bioprocessing Center?

Pitt Community College has established a Bioprocessing Center in conjunction with the statewide BioNetwork initiative. It is one of six specialized biotechnology centers developed by the North Carolina Community College System. This Center is a resource center created to promote biotechnology workforce development. The ultimate goal is to facilitate the growth of biotechnology firms in North Carolina and the surrounding region. Start-up funding for these centers was provided by Golden LEAF.

Why Biotechnology?

•Biotechnology is the fastest growing sector of the economy in the State of North Carolina, a state where traditional jobs, such as textiles and tobacco, are by and large extinct. •Biotechnology provides products made from living cells that are useful to all of us.•Biotechnology is the wave of the future as scientific and technological discoveries are made daily.•Biotechnology jobs pay well.

R ank State 1 C al i fo r ni a 2 M as s e c hus e t ts 3 N o r th C ar o l i na 4 M ar yl and

Ranking of U.S. States in the Number of Biotechnology Jobs

State Impact:

Growth in Protein Technology:

Illustration of the Growth in Biotechnology using theGrowth in the Number of Structures Deposited and

Available in the Protein Data Bank

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How does Industrial Systems fit in?

Because the nature of products created through biotechnology is so valuable to industrial interests, down-time can cost companies millions of dollars in lost profits. Machinery that keeps this industry running needs to be serviced, calibrated, maintained, and cleaned on a regular, expeditious basis.

Technicians trained in the culture of biotechnology are more valuable to a company than those that are not. They understand the needs of the industry and have the specialized training to keep the down-time to a minimum. Additionally, they have the expertise to be able to service the equipment without causing undue contamination to the product and the environment.

What is Contamination?

•The introduction of an unwanted substance to a particular process or environment, either intentionally or accidentally; that which has been introduced to a controlled environment. •Types of contaminants:

•Water•Soil•Air•Radiological


Sources of contamination:•Water (Pseudomonas Aeruginosa)•Soil (Bacillus Subtilis)•Air (any aerosolized form of contamination)•Radiological (any unwanted electromagnetic radiation)


Prevention of contamination:•Water (filtration or irradiation)•Soil (sanitization and/or disinfection)•Air (high efficiency particulate air filters- ultrafiltration)•Radiological (shielding or non-use)


Particles are measured in microns. If you were to slice a 1-inch cube into 25,400 equal size pieces, each slice would be 1 micron thick (so thin that you could not see it).

Since cleanroom facilities contain invisible particles that can cause defects, we need to have a way to determine their sizes and possible sources.


These particles cannot be seen with the naked eye and yet may cause serious loss to our client and to their ability to manufacture devices without defects.

We measure the amount of particles in the air with a device known as a particle counter. This helps us to determine when to perform or restrict certain activities.


To combat the tremendous amount of particulate released by the human body, cleanroom personnel are asked to wear special garments that are restrictive and may be uncomfortable if the working conditions (temperature, humidity) are not carefully controlled.


Typical Pollutant Sizes That Cause Down Time

Smoke .01 to 1.0 Microns

Bacteria .25 to 10 Microns

Human Skin Flakes 0.4 to 10 Microns

Household Dust 0.5 to 100 Microns

Mold 2.0 to 20 Microns

Human Hair 70 to 100 Microns

Pollen 5 to 100 Microns


People are dressed in booties, gloves, helmets, hoods and special facemasks that are not part of a normal experience. You are also asked to move slowly, to clean up dirt you cannot see, and to follow practices and procedures that may not immediately make sense.

The approach to cleanrooms is designed in steps so as to ensure that every effort has been made not to introduce contaminants into the cleanroom.


"Humans are walking, breathing, waste pits from a microbiological perspective and there is no indication we are going to evolve into anything cleaner."

Human dust combined with sebum will tenaciously adhere to and contaminate surfaces, creating contamination problems that are simultaneously bacterial, chemical and particulate.


There is an additional complicating factor - normal human activity. Humans breathe, sneeze, cough, talk and move around. Body and breath temperatures cause heat turbulence in addition to air turbulence.


People slough off huge numbers of particles a half micron and larger and these are propelled around by air and body movement. Some examples of how many 0.5 micron and larger particles can be released by simple movement are shown below. Vast amounts of contamination need to be controlled in a cleanroom environment.


Activity 0.3µm Particles Released

Seated 100,000 particles per minute

Head, arm, neck, and leg motion 500,000 particles per minute

All of the above with foot motion 1,000,000 particles per minute

Standing to sitting position 2,500,000 particles per minute

Moving 2 miles per hour 5 million particles per minute

Moving 3.5 miles per hour 7.5 million particles per minute

Moving 5 miles per hour 10 million particles per minute


Humans also release elemental chemicals that can cause contamination: * Spittle (saliva): potassium, chloride, phosphorus, magnesium, and sodium* Dandruff: calcium, chloride, carbon, and nitrogen* Perspiration: sodium, potassium, chloride, sulfur, aluminum, carbon, and nitrogen* Fingerprints: sodium, potassium, chloride, and phosphorusConsequently, a critical component of cleanroom management is strict adherence to protocol. Cosmetics are prohibited because in addition to their gross particle generation, cosmetics release iron, aluminum, silicone, carbon, titanium, magnesium, potassium, sulfur, and calcium.

What is Contamination Control?

Just as humans are the greatest potential contamination risk, they are also the greatest resource for contamination control. A thorough, comprehensive training program detailing all aspects of cleanroom management will empower the cleanroom operators to control the degree of contamination during the production process.


Just as all operators must be thoroughly trained in occupational skills, knowledge of the product, and all aspects of the production process, they must also be trained in the unique practices and behaviors required for working in the cleanroom environment. Testing and certification of cleanroom operators and subsequent observation of operators after training assures that the training has been implemented and is effective. Training is mandatory for all personnel (including Management), regardless of frequency of entry or job classification.


Personal health and hygiene begins at home with daily bathing or showering, shaving, brushing of teeth and hair, and application of silicone-free skin moisturizers to reduce skin flakes. Make-up, hair gels, hair sprays, aromatic after-shave lotions or body lotions are not cleanroom-compatible. While at work, all employees must wash hands after eating and/or using the toilet. Cleanroom-compatible hand cream may be applied prior to gowning.


As mentioned earlier, any activity by the cleanroom operator generates millions of viable and non-viable particles. Consequently, it is imperative to limit talking and actions in the cleanroom to only those required for the manufacture of the product. Running, horseplay, and other non-professional activities are not permitted.


Other behavioral requirements include, but are not limited to, the following:

•Smoking is not allowed inside the manufacturing facility including all cleanroom areas. Smokers release particles for at least one-half hour after smoking one cigarette.


Nothing is allowed inside the cleanroom complex which is not required in the cleanroom manufacturing process. This includes personal items such as jewelry or keys, cosmetics, tobacco or matches in any form, and food or drink in any form.

Hair may not be combed in the cleanroom gowning area.


•Only cleanroom compatible ball-point pens are allowed inside the cleanroom for recording data on cleanroom compatible paper and clipboards.

•While working in the cleanroom, mannerisms such as scratching or hand-rubbing. Cleanroom personnel may not access the inside of the cleanroom uniform.


The use of facial tissues is prohibited in the cleanroom. If one must use a cleanroom compatible non-linting tissue, it must be used only in the gowning area and disposed appropriately in waste receptacle.

All doors must remain closed when not entering or exiting. Emergency doors may be alarmed with a visual and audible alarm to enforce compliance.


An integral piece of the cleanroom management program is cleaning of the cleanroom. It is important for all personnel who may work in a cleanroom to understand the critical aspects of maintaining this clean environment.


Below is a partial list of some of the commonly known contaminants that can cause problems in some cleanroom environments. It has been found that many of these contaminants are generated from five basic sources. The facilities, people, tools, fluids and the product being manufactured can all contribute to contamination.


•Facilities•Walls, floors and ceilings•Paint, caulk and coatings•Construction material (sheet rock, wood, saw dust etc.)•Air conditioning debris•Room air and vapors•Spills and leaks•Rust


•People•Skin flakes and oil•Perspiration•Cosmetics and perfume•Spit or saliva•Coughing or sneezing•Clothing debris (lint, fibers etc.)•Hair•Gum, Cough Drops•Food or drink


•Tool Generated•Friction and wear particles•Lubricants and emissions•Vibrations•Brooms, mops and dusters•Paper•Cardboard•Duct tape•Permanent Markers•Non-clean room pens


•Fluids•Particulates floating in air•Bacteria, organics and moisture•Floor finishes or coatings•Thinners or solvents•Cleaning chemicals•Plasticizers (outgases)•Deionized water


•Product generated•Silicon chips•Quartz flakes•Cleanroom debris•Aluminum particles•Packing material•Aerosols and smoke

Monitoring Contamination?

CLASS Number of Particles per Cubic Meter by Micrometer Size

0.1 um 0.2 um 0.3 um 0.5 um 1 um 5 um

ISO 1 10 2

ISO 2 100 24 10 4

ISO 3 1,000 237 102 35 8

ISO 4 10,000 2,370 1,020 352 83

ISO 5 100,000 23,700 10,200 3,520 832 29

ISO 6 1,000,000 237,000 102,000 35,200 8,320 293

ISO 7 352,000 83,200 2,930

ISO 8 3,520,000 832,000 29,300

ISO 9 35,200,000 8,320,000 293,000

Airborne Particulate Cleanliness Classes (by cubic meter)


Air Monitoring

Class Areas Colony forming units per volume

Frequency

Class 100 and Class 1000

Critical processing areas for product and container-closures

<0.1 cfu per ft3 of air or 3 cfu per m3

Each Shift

Class 10,000 Less critical processing areas for product and container-closures

<0.5 cfu per ft3 of air or 20 cfu per m3

Daily

Class 100,000 Controlled Support Areas

2.5 cfu per ft3 of air or 100 cfu per m3

Twice/week (product/component contact areas) and once/week (other support areas)


Surface Monitoring



3 per 2 inch square (25 - 30 square cm.) RODAC Plate

Each Shift


-5 per 2 inch square (25-30 square cm.) RODAC Plate-10 per 2 inch square (25-30 square cm.) RODAC Plate for the floor

Daily


Not Applicable Not Applicable


Personnel Monitoring



-3 Gloves (25 – 30 square cm.)-5 Gloves (25 – 30 square cm.)

Each Shift


-10 Gloves (25 – 30 square cm.)-20 Gloves (25 – 30 square cm.)

Daily


Not Applicable Not Applicable

Safety in Controlled Environments:

Cleanroom safety is as important (if notmore) than any other facility havinglaboratories. Workers in these criticalenvironments must be conscientiousabout not only their own safety, but thesafety of the people benefiting from theproducts produced in the environment.


Everything one learns about safety in ageneral safety course is applicable incleanrooms and the responses wouldbe the same. However, in cleanroomsthe product is treated with much morerespect and the way one behaves ismore critical than anywhere else.


Exposure to chemicals and biologicsis a risk in cleanrooms just as it is inany laboratory setting, but less likelyto occur in controlled environmentsbecause of the likelihood of operationsceasing when maintenance orcalibration procedures are beingperformed.

Design Considerations and Operation:

The method most easily understood and universally applied is the one suggested by the Federal Standard 209E in which the number of particles equal to or greater than 0.5 microns measured in a cubic foot of air designates the class number. For example, a class 100,000 cleanroom limits the concentration of airborne particles equal to or greater than 0.5 microns to 100,000 particles in a cubic foot of air.


Cleanrooms have evolved into two major types which are differentiated by their method of ventilation - turbulent airflow and laminar airflow cleanrooms. The general method of ventilation used in turbulent airflow cleanrooms is similar to that found in buildings such as offices, schools, malls, manufacturing plants, auditoriums, shops, etc. The air is supplied by an air conditioning system through diffusers in the ceiling.


A cleanroom differs from an ordinary ventilated room in three ways:

•increased air supply•the use of high efficiency filters•room pressurization


Increased air supply:

The increased air supply is an important aspect of particle control. A typical turbulent airflow cleanroom would have at least 10 air changes per hour and likely have between 20 and 60. This additional air supply is mainly provided to dilute to an acceptable concentration the contamination produced in the room.


High efficiency filters:

High efficiency filters are used to filter the supply air into a cleanroom to ensure the removal of small particles. The high efficiency filters used in cleanrooms are installed at the point of air discharge into the room.


Room Pressurization:

Room pressurization is mainly provided to ensure that untreated air does not pass from dirtier adjacent areas into the cleanroom. The cleanroom is positively pressurized with respect to these dirtier areas. This is done by extracting less air from the room than is supplied to it.


Laminar airflow is used when low airborne concentrations of particles or bacteria are required. This airflow pattern is in one direction, usually horizontal or vertical at a uniform speed of between 60 to 90 ft/min. and throughout the entire space. The air velocity is sufficient to remove relatively large particles before they settle onto surfaces. Any contaminant released into the air can therefore be immediately removed by this laminar flow of air, whereas the turbulent airflow ventilated system relies on mixing and dilution to remove contamination.


Because airflow is such an important aspect of particle control, the design of a cleanroom requires careful consideration of air motion and airflow patterns. Depending on the degree of cleanliness required, it is common for air systems to deliver considerably more air than would be needed solely to meet temperature and humidity design.


Airborne particles can be organic or inorganic. Most contamination control problems concern the total contamination within the air. Particles of different sizes behave differently as air moves through a room. For example, in an eight-foot high room, a particle in the 50 micron range might take 60 seconds to settle, while a 1 micron particle might take 15 hours to settle.


Before any methods of contaminationcontrol of airborne particles can beapplied, a decision must be made as tohow critical this particulate matter is tothe process or product. The quantity ofthe particles of a given size that might bepresent within the area must beconsidered. The source of thecontamination is divided into externalsources and internal sources.


For any given space, there exists the externalinfluence of gross atmospheric contamination.These sources include air pollution in generaland dust storms. External contamination isbrought in primarily through the airconditioning system. Also, externalcontamination can infiltrate through buildingdoors, windows and cracks. The externalcontamination is controlled primarily by thetype of filtration used and spacepressurization.


People and the production process aresome of the greatest sources of internalcontamination. People in the workspacegenerate particles in the form of skinflakes, lint, cosmetics, and respiratoryemissions. Industry generates particlesfrom combustion processes, chemicalvapors, soldering fumes, and cleaningagents.


Other sources of internal contamination are generatedthrough the activity of manufacturing equipment.Although airflow design is critical, it alone does notguarantee that cleanroom conditions will be met.Construction finishes, personnel and garments,materials and equipment, and building entrances andexits are other sources of particulate contamination thatmust also be controlled. Room construction andmaterial finishes are an important part of cleanroomdesign. Room construction is important to provide anenclosure that will house a process to exclude outsidecontaminants and that the material finishes will notcontribute to particle generation in the space.


Walls, floors, ceiling tiles, lightingfixtures, doors, and windows areconstruction materials that must becarefully selected to meet cleanroomstandards. People must wear garments tominimize the release of particles into thespace.


The types of garments depend on the level ofcleanliness required by a process. Smocks,coveralls, gloves, and head and shoe coversare clothing accessories commonly used inclean spaces. Materials and equipment mustbe cleaned before entering the cleanroom.Room entrances such as air locks and passtrough’s are used to maintain pressuredifferentials and reduce contaminants. Also,air showers are used to remove contaminantsfrom personnel before entering the cleanspace.


The ability of a filter to removeparticles from the air is reflected by itsefficiency rating. The AmericanSociety of Heating, Refrigerating, andAir Conditioning Engineers (ASHRAE)have developed a standard formeasuring filter effectiveness. Thestandard describes test procedures toclassify filters in terms of arrestanceand efficiency.


Arrestance is the amount of dustremoved by the filter, usuallyrepresented as a percentage. Sincelarge particles make up most of theweight in an air sample, a filter couldremove a fairly high percentage ofthose particles while having no effecton the numerous small particles in thesample. Thus, filters with anarrestance of 90 percent have littleapplication in cleanrooms.


Efficiency measures the ability of thefilter to remove the fine particles.ASHRAE efficiencies of between 10percent and 40 percent should remove20 percent to 40 percent of the 1micron particles in the air, but hardlyany of the 0.3 to 0.5-micron particles.ASHRAE efficiencies of 80 percent to95 percent can remove 50 percent to70 percent of the 0.3-micron particles.


A HEPA filter, i.e., high efficiencyparticulate air filter, is defined by itsparticle removal efficiency and itsairflow rate. A HEPA filter is rated byits efficiency in removing smallparticles from air and has a minimumefficiency of 99.97 percent. These highefficiency filters are usually designedto remove particles of 0.3 microns andlarger.


An ULPA filter, i.e., ultra lowpenetration air, is a filter that hasefficiencies higher than those of astandard HEPA filter. An ULPA filterwill have efficiency greater than 99.99percent. These filters are constructedand will function the same way as aHEPA filters. They differ in that thefilter medium that is used has a higherproportion of smaller fibers and ishence more efficient.


Most cleanrooms require year-round coolingas a result of the fan energy associated withhigh cleanroom airflow as well as the heatgenerated by the process, people, and lightingwithin the facility. Temperature control isrequired to provide stable conditions formaterials, instruments, and personnel comfort.Human comfort requirements typically call fortemperatures in the range of 72F to 75F, sinceworkers frequently wear cleanroom garmentsover street clothes.


Humidity control is necessary toprevent corrosion, condensation onwork surfaces, eliminate staticelectricity, and provide personnelcomfort. The human comfort zone isgenerally in the range of 30 percent to70 percent relative humidity.


A cleanroom facility may consist of multiple roomswith different requirements for contaminationcontrol. Rooms in a clean facility should bemaintained at static pressures higher thanatmospheric to prevent infiltration by wind. Positivedifferential pressures should be maintainedbetween the rooms to ensure airflows from thecleanest space to the least clean space. The onlyexception to using a positive differential pressure iswhen dealing with specific hazardous materialswhere governmental agencies require the room tobe at a negative pressure. Pressure differentialsmust be 0.05 in WC higher in an adjacent clean areafrom the less clean area. This makes airflow critical.


Ventilation and makeup air volumes aredictated by the amount required to maintainindoor air quality, replace process exhaust andfor building pressurization. This providesassurance that carbon dioxide and oxygenremain in balance, that formaldehyde and othervapors given off by building materials andfurniture are diluted, and that air changes occurwith sufficient frequency to minimize thechance for high concentration of airbornepollutants within the building. Typically,makeup air must be 15% of all air per airchange.

Hygiene and Specialized Attire:

Cleanroom environments require peopleand they also require that those peopleare clean.• Shower or bath- All employees should

shower or bath before coming to work. This help to reduce the amount of dead skin cells that are shed when the employee arrives to work.

• Clean hair is important for reducing flaking from the scalp


• Clean shaven faces will reduce the potential of contamination from hair that may be exposed in some cases.

• Smoking before entering a cleanroom will create contamination and should not be done at least thirty minutes before entering.

• Clothing should be newly cleaned and free of lint. Efforts should be made to reduce contact with animals while wearing clothing that will be worn to work.


• Hands should be clean and disinfected per the company protocol.

• Shoes and socks must always be worn. Never wear sandals or open-toed shoes.

Preparation to Enter a Cleanroom:

•Personal Considerations•Hygiene•Illness•Clean Shaven•Smoking•Jewelry•Make-up

•Material Considerations•Material of Construction•Minimal Amounts of Materials

Preparation to Enter a Cleanroom:

•Disinfection•Tools and Supplies•Dedicated Tools•Hand Washing

•Material Transfers•Flow (separate)•Disinfection•Multiple Coverings

Gowning (Supplies and Technique):

Hand Disinfectant Safety Glasses

Alcohol Beard Cover

Gown Surgical Mask

Hair Net Sterile Sleeves and Gloves

Hood Dedicated Cleanroom Shoes or Boots

Goggles Shoe Covers

Gowning (Supplies and Technique):

•Always follow protocols•Disinfect hands prior to gowning•Obtain gowning supplies according to protocol•Don cleanroom attire from head to toe (literally) except

footwear•Disinfect hands•Aseptically don sterile gloves•Obtain footwear•Don while crossing over bench•Disinfect gloves with alcohol•View gowned self in mirror•Obtain any other garments before entering (such as sterile

sleeves)

Cleanroom Behavior:

Hygiene- Good basic hygiene is a necessity when working in cleanrooms. Shedding skin cells from unclean workers is the most common contaminant. Bacterial contaminants can also thrive on shed skin cells. Workers who have exudative lesions on their bodies (open wounds with blood or pus) must exclude themselves from cleanroom environments or appropriately and completely cover the sores.

Cleanroom Behavior:

Coughing or sneezing sometimes cannot be avoided, but it is important to exclude oneself from working in a cleanroom if the cough or sneeze is from and illness or allergy. When coughing or sneezing, always turn away from the area where cleanliness is desired most. Above all, always wear protective masks.

Cleanroom Behavior:

Facial hair can be shed easily and shouldbe completely covered when working incleanrooms. Beard covers can be utilizedto accomplish this with beards andmustaches. All masks and beard coversare porous and can still allowcontaminants to pass through.

Cleanroom Behavior:

Motion- All behaviors in cleanroom environments should be performed at a moderately slow pace. Quick movements in the cleanroom or any sterile area stir up the particle counts where they can be easily detected and frequently out-of-specification. Slow, deliberate movements are always best to avoid the turbulence that is created that will stir up particles. Scratching oneself through the cleanroom garments, running, jumping, and even handshakes, are all forbidden practices. It goes without saying that eating, drinking, or smoking is also forbidden.

Cleanroom Behavior:

Contact- Contacting surfaces is an easy way totransmit bacteria, viruses, yeast, and molds. Iftouching any area within a cleanroom bynecessity, be sure that you evaluate what you mayhave “picked-up” and transferred to another area.For example, you may open a door knob or handleand then touch a stopper on a vial that will intheory contain a sterile product. A good practice

isto regularly and frequently disinfect your gloveswith sterile alcohol between processes. Even aroutine disinfection of gloves is appropriate.

Cleanroom Behavior:

Processes- Obviously, contamination can begenerated by the work we do. For techniciansWho are maintaining or repairing equipment,Every mechanical operation performed createsa potential for contamination. Sawing,grinding, fastening screws, hammering,cleaning, drilling, all are sources ofcontamination. Flames used in cleanrooms,whether being used for sterilization or not,create turbulence which in turn stirs upparticles. Even the removal of tools to performThis work will create high particle counts.

De-gowning:

Reverse the order of gowning and refer to protocol for reuse or disposal of materials.

Documentation:

•Laboratory Notebooks- Any procedure or protocol that is not part of a standard operating procedure should be documented in a laboratory notebook so that it can be referenced at a later time what activities took place and the results that were obtained.

Documentation:

•Data Collection Forms- Provided an SOP is in place that describes the environmental monitoring procedures employed, a data collection form can be used. This form should be referenced in the SOP and designed in an orderly format that allows the manual recording of data and activities that occur during the monitoring procedures

Documentation:

•Electronic Data Collection and Part 11 Compliance- Any electronic data that is obtained as part of the monitoring process must be Part 11 complaint to ensure that data integrity is maintained. Written documentation should be double-checked for accuracy and completeness. All printed materials must be intact and maintained so that they can be archived for future reference. Printed data on thermal paper must be photocopied to preserve the original data that will fade otherwise.

Questions?

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