asepting procesing

BySUNILBOREDDY

M.Pharmacy

Certain pharmaceutical products must be sterile◦ injections, ophthalmic preparations, irrigations

solutions, haemodialysis solutions

Two categories of sterile products◦ those that can be sterilized in final container

(terminally sterilized)◦ those that cannot be terminally sterilized and

must be aseptically prepared

Aseptic processing Objective is to maintain the sterility of a

product, assembled from sterile components Operating conditions so as to prevent

microbial contamination

Objective To review specific issues relating to the

manufacture of aseptically prepared products:◦ Manufacturing environment

Clean areas Personnel

◦ Preparation and filtration of solutions◦ Pre-filtration bioburden◦ Filter integrity/validation◦ Equipment/container preparation and

sterilization◦ Filling Process ◦ Validation of aseptic processes◦ Specific issues relating to Isolators, BFS and Bulk

Classification of Clean Areas◦ Comparison of classifications

WHO GMP US 209E US Customary ISO/TC (209) ISO 14644

EEC GMP

Grade A M 3.5 Class 100 ISO 5 Grade A Grade B M 3.5 Class 100 ISO 5 Grade B Grade C M 5.5 Class 10 000 ISO 7 Grade C Grade D M 6.5 Class 100 000 ISO 8 Grade D

Table 1

Classification of Clean Areas◦ Classified in terms of airborne particles (Table 2)

Grade At rest In operation

maximum permitted number of particles/m3

0.5 - 5.0 µm > 5 µm 0.5 - 5.0 µm > 5 µ

A 3 500 0 3 500 0

B 3 500 0 350 000 2 000

C 350 000 2 000 3 500 000 20 000

D 3 500 000 20 000 not defined not defined

“At rest” - production equipment installed and operating

“In operation” - Installed equipment functioning in defined operating mode and specified number of personnel present

Four grades of clean areas: Grade D (equivalent to Class 100,000, ISO 8):

◦ Clean area for carrying out less critical stages in manufacture of aseptically prepared products eg. handling of components after washing.

Grade C (equivalent to Class 10,000, ISO 7):◦ Clean area for carrying out less critical stages in

manufacture of aseptically prepared products eg. preparation of solutions to be filtered.

Grade B (equivalent to Class 100, ISO 5): ◦ Background environment for Grade A zone, eg.

cleanroom in which laminar flow workstation is housed.

Grade A (equivalent to Class 100 (US Federal Standard 209E), ISO 5 (ISO 14644-1):◦ Local zone for high risk operations eg. product filling,

stopper bowls, open vials, handling sterile materials, aseptic connections, transfer of partially stoppered containers to be lyophilized.

◦ Conditions usually provided by laminar air flow workstation.

Each grade of cleanroom has specifications for viable and non-viable particles ◦ Non-viable particles are defined by the air classification

(See Table 2)

Limits for viable particles (microbiological contamination)

Grade Air sample (CFU/m3)

Settle plates (90mm diameter)

(CFU/4hours)

Contact plates (55mm

diameter) (CFU/plate)

Glove print (5 fingers)

(CFU/glove)

A < 3 < 3 < 3 < 3 B 10 5 5 5 C 100 50 25 - D 200 100 50 -

Table 3– These are average values– Individual settle plates may be exposed for less than 4 hours

• Values are for guidance only - not intended to represent specifications• Levels (limits) of detection of microbiological contamination should be established for alert and action purposes and for monitoring trends of air quality in the facility

Environmental Monitoring Physical

◦ Particulate matter

◦ Differential pressures

◦ Air changes, airflow patterns

◦ Clean up time/recovery

◦ Temperature and relative humidity

◦ Airflow velocity

Environmental Monitoring - Physical Particulate matter

◦ Particles significant because they can contaminate and also carry organisms

◦ Critical environment should be measured not more than 30cm from worksite, within airflow and during filling/closing operations

◦ Preferably a remote probe that monitors continuously◦ Difficulties when process itself generates particles (e.g.

powder filling)◦ Appropriate alert and action limits should be set and

corrective actions defined if limits exceeded

Environmental Monitoring - Physical Differential pressures

◦ Positive pressure differential of 10-15 Pascals should be maintained between adjacent rooms of different classification (with door closed)

◦ Most critical area should have the highest pressure◦ Pressures should be continuously monitored and

frequently recorded.◦ Alarms should sound if pressures deviate◦ Any deviations should be investigated and effect on

environmental quality determined

Environmental Monitoring - Physical Air Changes/Airflow patterns

◦ Air flow over critical areas should be uni-directional (laminar flow) at a velocity sufficient to sweep particles away from filling/closing area

◦ for B, C and D rooms at least 20 changes per hour are ususally required

Clean up time/recovery◦ Particulate levels for the Grade A “at rest” state

should be achieved after a short “clean-up” period of 20 minutes after completion of operations (guidance value)

◦ Particle counts for Grade A “in operation” state should be maintained whenever product or open container is exposed

Environmental Monitoring - Physical Temperature and Relative Humidity

◦ Ambient temperature and humidity should not be uncomfortably high (could cause operators to generate particles) (18°C)

Airflow velocity◦ Laminar airflow workstation air speed of approx

0.45m/s ± 20% at working position (guidance value)

Personnel Minimum number of personnel in clean areas

◦ especially during aseptic processing Inspections and controls from outside Training to all including cleaning and

maintenance staff◦ initial and regular◦ manufacturing, hygiene, microbiology◦ should be formally validated and authorized to enter

aseptic area Special cases

◦ supervision in case of outside staff◦ decontamination procedures (e.g. staff who worked

with animal tissue materials)

Personnel (2) High standards of hygiene and cleanliness

◦ should not enter clean rooms if ill or with open wounds

Periodic health checks No shedding of particles, movement slow and

controlled No introduction of microbiological hazards No outdoor clothing brought into clean areas,

should be clad in factory clothing Changing and washing procedure No watches, jewellery and cosmetics Eye checks if involved in visual inspection

Personnel (3) Clothing of appropriate quality:

◦ Grade D hair, beard, moustache covered protective clothing and shoes

◦ Grade C hair, beard, moustache covered single or 2-piece suit (covering wrists, high

neck), shoes/overshoes no fibres/particles to be shed

◦ Grade A and B headgear, beard and moustache covered,

masks, gloves not shedding fibres, and retain particles shed

by operators

Personnel (4) Outdoor clothing not in change rooms leading to

Grade B and C rooms Change at every working session, or once a day (if

supportive data) Change gloves and masks at every working session Frequent disinfection of gloves during operations Washing of garments – separate laundry facility

◦ No damage, and according to validated procedures (washing and sterilization)

Regular microbiological monitoring of operators

In aseptic processing, each component is individually sterilised, or several components are combined with the resulting mixture sterilized.◦ Most common is preparation of a solution which is

filtered through a sterilizing filter then filled into sterile containers (e.g active and excipients dissolved in Water for Injection)

◦ May involve aseptic compounding of previously sterilized components which is filled into sterile containers

◦ May involve filling of previously sterilized powder sterilized by dry heat/irradiation produced from a sterile filtered solution which is then

aseptically crystallized and precipitated requires more handling and manipulation with higher potential

for contamination during processing

Preparation and Filtration of Solutions Solutions to be sterile filtered prepared in a Grade

C environment If not to be filtered, preparation should be

prepared in a Grade A environment with Grade B background (e.g. ointments, creams, suspensions and emulsions)

Prepared solutions filtered through a sterile 0.22μm (or less) membrane filter into a previously sterilized container◦ filters remove bacteria and moulds◦ do not remove all viruses or mycoplasmas

filtration should be carried out under positive pressure

Preparation and Filtration of Solutions (2) consideration should be given to complementing

filtration process with some form of heat treatment Double filter or second filter at point of fill

advisable Fitlers should not shed particles, asbestos

containing filters should not be used Same filter should not be used for more than one

day unless validated If bulk product is stored in sealed vessels, pressure

release outlets should have hydrophobic microbial retentive air filters

Preparation and Filtration of Solutions (3) Time limits should be established for each phase of

processing, e.g.◦ maximum period between start of bulk product

compounding and sterilization (filtration)◦ maximum permitted holding time of bulk if held after

filtration prior to filling◦ product exposure on processing line◦ storage of sterilized containers/components◦ total time for product filtration to prevent organisms from

penetrating filter◦ maximum time for upstream filters used for clarification

or particle removal (can support microbial attachment)

Preparation and Filtration of Solutions (4) Filling of solution may be followed by lyophilization

(freeze drying)◦ stoppers partially seated, product transferred to

lyophilizer (Grade A/B conditions)◦ Release of air/nitrogen into lyophilizer chamber at

completion of process should be through sterilizing filter

Prefiltration Bioburden (natural microbial load) Limits should be stated and testing should be carried

out on each batch Frequency may be reduced after satisfactory history is

established◦ and biobuden testing performed on components

Should include action and alert limits (usually differ by a factor of 10) and action taken if limits are exceeded

Limits should reasonably reflect bioburden routinely achieved

Prefiltation Bioburden (2) No defined “maximum” limit but the limit should not

exceed the validated retention capability of the filter Bioburden controls should also be included in “in-

process” controls ◦ particularly when product supports microbial growth

and/or manufacturing process involves use of culture media

Excessive bioburden can have adverse effect on the quality of the product and cause excessive levels of endotoxins/pyrogens

Filter integrity Filters of 0.22μm or less should be used for filtration

of liquids and gasses (if applicable)◦ filters for gasses that may be used for purging or

overlaying of filled containers or to release vacuum in lyphilization chamber

filter intergrity shoud be verified before filtration and confirmed after filtration◦ bubble point◦ pressure hold◦ forward flow

methods are defined by filter manufacturers and limits determined during filter validation

Equipment/container preparation and sterilization

All equipment (including lyophilizers) and product containers/closures should be sterilized using validated cycles◦ same requirements apply for equipment sterilization

that apply to terminally sterilized product◦ particular attention to stoppers - should not be tightly

packed as may clump together and affect air removal during vacuum stage of sterilization process

◦ equipment wrapped and loaded to facilitate air removal

◦ particular attention to filters, housings and tubing

Equipment/container preparation and sterilization (2)

CIP/SIP processes◦ particular attention to deadlegs - different orientation

requirements for CIP and SIP heat tunnels often used for

sterilization/depyrogenation of glass vials/bottles◦ usually high temperature for short period of time◦ need to consider speed of conveyor◦ validation of depyrogenation (3 logs endotoxin units)

worst case locations◦ tunnel supplied with HEPA filtered air

Equipment/container preparation and sterilization (2)

equipment should be designed to be easily assembled and disassembled, cleaned, sanitised and/or sterilized◦ equipment should be appropriately cleaned - O-rings and gaskets

should be removed to prevent build up of dirt or residues rinse water should be WFI grade equipment should be left dry unless sterilized immediately

after cleaning (to prevent build up of pyrogens) washing of glass containers and rubber stoppers should be

validated for endotoxin removal should be defined storage period between sterilization and

use (period should be justified)

Additional issues specific to Isolator and BFS Technologies

Isolators◦ Decontamination process requires a 4-6 log

reduction of appropriate Biological Indicator (BI)◦ Minimum 6 log reduction of BI if surface is to be free

of viable organisms◦ Significant focus on glove integrity - daily checks,

second pair of gloves inside isolator glove◦ Traditional aseptic vigilance should be maintained

Blow-Fill-Seal (BFS)◦ Located in a Grade D environment◦ Critial zone should meet Grade A (microbiological)

requirements (particle count requirements may be difficult to meet in operation)

◦ Operators meet Grade C garment requirements◦ Validation of extrusion process should demonstrate

destruction of endotoxin and spore challenges in the polymeric material

◦ Final inspection should be capable of detecting leakers

Issues relating to Aseptic Bulk Processing• Applies to products which can not be filtered at point of

fill and require aseptic processing throughout entire manufacturing process.

• Entire aseptic process should be subject to process simulation studies under worst case conditions (maximum duration of "open" operations, maximum no of operators)

• Process simulations should incorporate storage and transport of bulk.

• Multiple uses of the same bulk with storage in between should also be included in process simulations

• Assurance of bulk vessel integrity for specified holding times.

Bulk Processing (2)• Process simulation for formulation stage should be

performed at least twice per year.◦ Cellular therapies, cell derived products etc

products released before results of sterility tests known (also TPNs, radioactive preps, cytotoxics)

should be manufactured in a closed system Additional testing

sterility testing of intermediates microscopic examination (e.g. gram stain) endotoxin testing

Environmental Monitoring Environmental Monitoring ConsiderationsConsiderations

Airborne nonviable particulate monitoring Airborne viable contaminant monitoring Viable contaminant monitoring of surfaces Viable contaminant monitoring of personnel Temperature and humidity monitoring Pressure differential monitoring

Environmental Monitoring Environmental Monitoring ComponentsComponents

Water monitoring:◦ Total organic carbon◦ Conductivity◦ Microbial Contaminants◦ Endotoxin

Environmental Monitoring Environmental Monitoring ComponentsComponents

Monitoring frequencies and strategies◦ Establishment of a meaningful and manageable

program Sampling and testing procedures Establishment of effective alert and action

limits Trending of results

General Environmental General Environmental Monitoring ConsiderationsMonitoring Considerations

Investigation and evaluation of trends as well as excursions from alert and action limits

Corrective actions to be implemented in response to environmental monitoring excursions

Personnel training - sampling, testing, investigating excursions, aseptic technique

General Environmental General Environmental Monitoring ConsiderationsMonitoring Considerations

Should include monitoring of all environments where products and their components are manufactured◦ All areas where there is a risk of product

contamination Should include monitoring of all water used

for product manufacturing as well as feed water to the final water purification system (WFI System)

Scope of Environmental Scope of Environmental Monitoring ProgramMonitoring Program

CFR GMP regulations FDA Guidance Documents USP Informational Chapter

Regulatory Basis for Regulatory Basis for Environmental Monitoring Environmental Monitoring ProgramProgram

Aseptic processing areas:◦ Easy to clean and maintain◦ Temperature and humidity controlled◦ HEPA filtered air◦ Environmental monitoring system◦ Cleaning and disinfecting procedures◦ Scheduled equipment maintenance and

calibration

21 CFR 211.4221 CFR 211.42

Ventilation, air filtration, air heating and cooling:◦ Adequate control over microorganisms, dust,

humidity and temperature.◦ Air filtration systems including prefilters and

particulate matter air filters for air supplies to production areas.

21 CFR 211.4621 CFR 211.46

Defines critical and controlled manufacturing areas

Recommends airborne nonviable and viable contaminant limits

Provides some guidance on monitoring frequencies for critical areas

Guideline on Sterile Drug Guideline on Sterile Drug Products Produced by Aseptic Products Produced by Aseptic ProcessingProcessing

Recommendations for air pressure differentials

Includes guidance on aseptic media fills Note: This guidance document was written

in 1987 and is in need of revision

Guideline on Sterile Drug Guideline on Sterile Drug Products Produced by Aseptic Products Produced by Aseptic ProcessingProcessing

USP General Information Chapter <1116> Establishment of clean room classifications

◦ Federal Standard 209E Importance of EM program Personnel training in aseptic processing Establishment of sampling plans and sites

◦ suggested sampling frequencies

Microbial Evaluation and Microbial Evaluation and Classification of Clean Rooms and Classification of Clean Rooms and Clean ZonesClean Zones

Establishment of alert and action limits Suggests limits for airborne, surface and

personnel contaminant levels. Methods and equipment for sampling Identification of isolates Aseptic media fills Emerging technologies - barrier; isolator

Microbial Evaluation and Microbial Evaluation and Classification of Clean Rooms and Classification of Clean Rooms and Clean ZonesClean Zones

“Airborne Particulate Cleanliness Classes in Clean Rooms and Clean Zones

Approved by the GSA for use by all Federal Agencies

Frequently referenced for controlled environment particulate requirements: Classes 100, 10,000 and 100,000 (based on particles > 0.5µ)

Federal Standard 209EFederal Standard 209E

Scope limited to final drug product manufacturing and data required for application submission (NDA, BLA)

Requests information on:◦ Buildings and facilities◦ Manufacturing operations for drug product Filter validation Validation of hold times

Guidance for Industry for Sterile Guidance for Industry for Sterile Validation Process Validation in Validation Process Validation in Applications for Human and Veterinary Applications for Human and Veterinary Drug ProductsDrug Products

Requests information on:◦ Sterilization and depyrogenation◦ Media fills and actions taken when they fail◦ Microbiological monitoring of the environment

Airborne microorganisms, personnel, surfaces, water system, product component bioburden

◦ Yeasts, molds, anaerobes◦ Exceeded EM limits

Guidance for Industry for Sterile Guidance for Industry for Sterile Validation Process Validation in Validation Process Validation in Applications for Human and Veterinary Applications for Human and Veterinary Drug ProductsDrug Products

Viable and Nonviable Viable and Nonviable Contaminant LimitsContaminant Limits

Classifi-cation

Nonviable (>0.5µ) Viable

ft3 m3 ft3 m3

Class 100

100 3,530 0.1 3.5

Class 10,000

10,000 353,000 0.5 18

Class 100,000

100,000 3,530,000 2.5 88

Preparation or manufacturing area where nonsterile product, in-process materials and product-contact equipment surfaces, containers and closures are exposed to the environment

Control nonviable and viable contaminants to reduce product /process bioburden

Class 100,000 or Class 10,000

Controlled AreaControlled Area

Capping areas are now considered controlled manufacturing areas◦ Should be supplied with HEPA filtered air◦ Should meet class 100,000 conditions during

static conditions

Controlled AreaControlled Area

Aseptic processing area where sterile products, components or in-process products are exposed to the environment and no further processing will occur.

Air quality must be Class 100 during processing

Local Class 100 areas are often utilized during open processing steps during drug substance manufacture.

Critical AreaCritical Area

The area just preceding the sterile core should be one classification higher than the core.

Critical AreaCritical Area

Airborne cleanliness classifications should be met during operations

Nonviable monitoring should occur routinely during operations

Monitoring during static conditions is done as part of HVAC qualification and may be done periodically after that to insure area meets acceptable conditions before use or following cleaning

Nonviable Particulate Nonviable Particulate MonitoringMonitoring

Locations for monitoring should be established during performance qualification; probes placed close to work surface

Monitoring frequencies vary:◦ For aseptic processing areas, during each use◦ For other, controlled areas, varies from each use

to weekly or less depending on use of area


HVAC Validation and Maintenance Considerations:◦ Air velocity, airflow patterns and turbulence

should be validated; smoke studies to determine flow patterns during static and dynamic conditions

◦ HEPA filter integrity testing ◦ HEPA filter efficiency testing◦ Air pressure differentials


Airborne viable contaminants Surface contaminants

◦ walls◦ equipment surfaces◦ countertops◦ floors

Personnel contaminants

Microbial MonitoringMicrobial Monitoring

Monitoring methods should be capable of detecting molds and yeasts

Should also be able to detect anaerobes◦ Most often, this is an issue associated with

products filled anaerobically (with nitrogen overlay)

All lots of media for EM sampling should be growth promotion tested


Routine microbial monitoring should take place during operations (for airborne contaminants) and immediately following operations (for surfaces and personnel).

Airborne monitoring frequencies:◦ Each use for aseptic processing areas◦ Varies from daily to weekly to less frequently for

controlled areas depending on use


Personnel and surface monitoring frequencies vary:◦ Aseptic processing - after every fill◦ Other controlled areas - varies from daily to

weekly or less for surfaces ◦ Personnel monitoring often restricted to aseptic

area personnel and personnel working in Class 100 hoods performing tasks such as inoculation


Monitoring of surfaces and airborne contaminants during rest periods (following cleaning)◦ Important for confirming adequacy of cleaning

procedures◦ Indicates whether HVAC system is operating

properly◦ NOTE: Disinfectant effectiveness studies also

required for cleaning agents used in the facility


Monitoring frequencies and procedures are influenced by a number of factors:◦ Stage of manufacturing ◦ “Open” or “closed” manufacturing step◦ Single or multiple product manufacturing


Establishment of monitoring locations should be based on performance qualification studies during dynamic conditions◦ gridding study to determine worst case

locations/most meaningful locations Should also establish common flora - will aid

in investigations


Action limits (for the most part) have been established in a variety of guidance documents

Alert limits ◦ Lower than action limits ◦ Reflect actual historical results under normal

processing conditions

Setting Alert and Action Setting Alert and Action LimitsLimits

Alert limits are designed to provide some warning that environmental quality is approaching action limit and allow you time to correct.

Exceeding alert limit triggers a warning response - i.e., alert affected area personnel

Exceeding multiple alerts - triggers action level response

Exceeding LimitsExceeding Limits

Action limit excursions require investigations◦ Speciation of organism(s)◦ Review batch records from date of excursion◦ Review other recent EM data (trends)◦ Review cleaning records◦ Interview personnel◦ Product impact - must quarantine until

determined


Excursions from action limits require corrective actions that may include:◦ More rigorous or additional monitoring◦ More rigorous cleaning◦ Retraining of personnel◦ Procedural changes - change to or addition of

disinfection procedures, for example◦ HVAC maintenance


The investigation procedures to be followed should be pre-established and included in SOPs

Depending on the outcome of the investigation, corrective actions should be pre-established to the extent possible

Investigations and Corrective Investigations and Corrective ActionsActions

Imperative that EM results be linked to product release so that affected products are not released until investigation completed

Material Review Board or equivalent should be consulted prior to releasing product that was potentially affected by adverse environmental conditions

Investigations and Corrective Investigations and Corrective ActionsActions

Should trend monitoring results (environmental and water) ◦ Periodic (quarterly or monthly) review by QA and

others◦ Re-evaluation of action and alert limits on an

annual basis◦ This trending information is generally included in

the Annual Product Review

TrendingTrending

Control of temperature and humidity required for aseptic processing areas ◦ 21 CFR 211.42(c)(10)(ii)

Generally 65°F and 35-50% humidity are average◦ Too high - Increases personnel shedding◦ Too low - Increase static electricity

Temperature and HumidityTemperature and Humidity

Temperature should be controlled throughout all manufacturing areas

Temperature and humidity should be monitored and controlled in warehouse areas where temperature/humidity sensitive raw materials are stored◦ If not able to control humidity, need procedure to

follow if humidity exceeds limit

Temperature and HumidityTemperature and Humidity

Water RequirementsWater Requirements

Test Potable Water

Purified Water

WFI

TOC none 500 ppb 500 ppb

Conduc-tivity

none See USP Table

Micro. Purity

500 CFU/ml

100 CFU/ml

10 CFU/ 100 ml

Endo- Toxin

none none 0.25 EU/ml

Water purified by distillation or reverse osmosis

Prepared from water complying with the U.S. EPA National Primary Drinking Water Regulations

Contains no added substance

Water For InjectionWater For Injection

Obtained by a suitable process, usually one of the following:◦ deionization◦ reverse osmosis◦ combination

Purified WaterPurified Water

Meets National Drinking Water Regulations 40 CFR Part 141 Periodic monitoring in-house as well as

periodic certificates from municipality (if applicable)

Potable WaterPotable Water

WFI Systems◦ Microbial quality and endotoxin

Daily system monitoring Each use point at least weekly

◦ TOC and Conductivity Weekly system monitoring can be taken from worst case point (end of loop,

return to tank)

Water System MonitoringWater System Monitoring

Purified Water Systems◦ Weekly monitoring of system for: microbial quality TOC conductivity

Water System MonitoringWater System Monitoring

WFI ◦ Solvent for preparation of parenteral solutions◦ Formulation of mammalian cell culture media◦ Formulation of purification buffers◦ Final product formulation◦ Vial and stopper washing ◦ Final rinse for product equipment

Water UseWater Use

Purified Water◦ Preparation of terminally sterilized microbiological

media◦ Initial rinsing/cleaning◦ Laboratory use◦ Feed for WFI system

Water UseWater Use

Potable Water◦ Non-product contact uses◦ Feed for purified water system

Water UseWater Use

Slit-to-Agar (STA) - Powered by vacuum, air taken in through a slit below which is a slowly revolving plate.

Sieve impactor - Vacuum draws in air through perforated cover which is impacted onto petri dish containing nutrient agar

Microbial Monitoring Microbial Monitoring DevicesDevices

Centrifugal Sampler - consists of a propeller that pulls a known volume of air into the unit and then propels the air outward to impact on a nutrient agar strip

Sterilizable Microbiological Atrium (SMA)- similar to sieve impactor; cover contains uniformly spaced orifices; vacuum draws in air which is impacted on agar plate


Surface Air System Sampler - An integrated unit containing an entry section with an agar contact plate; behind is a motor and turbine that pulls air in through the perforated cover and exhausts it beyond the motor.

Settle plates - qualitative; may be useful in worst case locations


Surface contaminant monitoring devices:◦ Contact Plates - plates filled with nutrient agar;

for regular surfaces◦ Swabs - useful for hard to reach or irregular

surfaces; swab placed in suitable diluent and inoculated onto microbiological plate


Remote sampling probes - validate use of tubing

Must sample adequate quantity of air to be statistically meaningful.◦ 80-100 ft3/min

Must validate growth promotion after exposure of settle plates (or other plates) for prolonged time periods.

Monitoring ConsiderationsMonitoring Considerations

Contamination Control

Methods to Achieve Methods to Achieve CleanlinessCleanliness Positive Pressure / Airflow

◦ Keeps contamination out of the work area◦ Depends on clean air input

Filtration◦ Development of effective filtration revolutionized industry◦ HEPA (High Efficiency Particulate Air) and ULPA (Ultra Low

Particulate Air) Filters Materials Selection User Protocols Cleaning

Facility DesignFacility Design

Complete cleanroom created with centralized air handling or fan filter units

Keeps entire room clean Requires complete gowning, careful

materials and equipment selection to maintain class

Costly, often unnecessary

Facility DesignFacility Design

Can use localized clean areas Clean Benches: Horizontal and Vertical

Laminar Flow (HLF on left, VLF on right)

Facility designFacility design

Isolators, Glove boxes provide better protection from outside contamination

Contamination Control by Contamination Control by LayoutLayout Isolation between processes

prevents cross contamination; separate rooms, air showers, door interlocks

“Onion” concept: cleanest areas are inside, have to pass through successively cleaner areas to reach these areas

Air Flow & TurbulenceAir Flow & Turbulence

Most airflow is turbulent—no clear relation between velocity vectors at different points

•Particles can be trapped in eddies for long time•Not optimal for contamination control!! Long path length for contamination to leave the room

Laminar (Unidirectional) Laminar (Unidirectional) Air FlowAir Flow

Concept of laminar airflow

In cleanrooms, often called uni-directional flow (UDF)

• Ideal for contamination control—shortest path to sweep particles out of clean areas; complete room air change in shortest period of time

High level cleanrooms designed for laminar flow in most areasCost means that for most, clean areas are some combination of laminar and turbulent flowNot always a simple tradeoff—with turbulent flow, require higher air velocities, which require larger air handlers.

UDF More Important for UDF More Important for Cleaner AreasCleaner Areas

Practical Considerations Practical Considerations for UDFfor UDF Any objects in path of laminar flow will

deflect airflow—this usually results in turbulence; USER BEHAVIOR HAS LARGE IMPACT•Most critical for laminar flow benches situated in non-clean areas; not as critical if located in larger clean area

Types of ContaminationTypes of Contamination

Particulate—encompasses most contamination

Chemical—films, vapors, etc. Biological—bacteria, viruses, etc.; for our

purposes, treat as particles Similar concerns for rooms & equipment

as for substrates

Airborne ContaminationAirborne Contamination

From Cleanrooms Magazine, 2000

Invisible to naked eye below ~50um without special illumination

Particulate Particulate ContaminationContamination Biggest concern for LCI cleanroom users Basis for classification of cleanrooms Does include biological contamination as

a subset of total particulates Many sources: personnel, equipment, etc.

Microbial ContaminationMicrobial Contamination

Outer layer of human skin can host up to 1 million microorganisms per square cm

Human saliva up to 1 billion per mL Bacteria is usually primary concern, but

foreign organic matter, viruses, fungi, algae are all included here

Cross contamination can be a big problem.

Contamination Contamination MeasurementMeasurement Particulate contamination typically

measured with laser particle counter Microbial contamination can be

measured in several ways◦ Centrifugal sampler◦ Settle plate method◦ Contact plate method◦ Swabbing

Usage of MeasurementsUsage of Measurements

Complementary to yield tracking Can use measurements to isolate

problem areas Regular measurements can help to

track changes, which can then be tied back to protocol, personnel, or material changes◦ Don’t depend upon room to maintain itself.

RealityReality

In a perfect world, could monitor many points on a very regular basis

In reality, this is usually not practical, due to personnel time and financial constraints

Important to identify a realistic test & measurement program

Contamination Control Contamination Control and Its Relationships and Its Relationships All sources of contamination and control

are interrelated

CleaningCleaning

Critical to remove contaminants that cannot be removed by air handling

Important to follow procedures appropriate to your application

What is appropriate for one industry may not be appropriate for another

Most important thing is to develop standard procedures and FOLLOW THEM

Surfaces are importantSurfaces are important

The efficiency of these cleaning methods depends on the surface being cleaned

Rough or pitted surfaces are more difficult to clean

Sharp corners are difficult to clean That’s why inside surfaces of clean rooms

are smooth.

VacuumingVacuuming

Dry and wet◦ Dry has low (<25% ) efficiency for particles

smaller than 10 microns (about .0005 inches)◦ wet uses liquids which result in greater force

on the particles and hence better cleaning

Wet wipingWet wiping

Can be very efficient Liquid breaks some bonds between

surface and particles and allows particles to float off

Those adhering on surface can be rubbed off and retained in wiper.

Must be careful not to redeposit particles Efficiency varies

Tacky rollersTacky rollers

Efficiency depends of tackiness of roller, cleanliness of tacky surface and softness of roller are also very important

Cleaning liquidsCleaning liquids

No ideal cleaning liquid Most facilities use DI water or isopropyl

alcohol with disinfectant Water with surfactant and disinfectant

may be used as well as alcohol-water solutions

The choice depends on what works, cost, history, etc.

Materials SelectionMaterials Selection

Choice of materials for supplies, equipment, gowning, etc. is important

“Clean” materials can become dirty!! Look for easy-to-clean materials Triboelectricity can cause static

problems, as can low humidity—this exacerbates contamination problems

Biofilms!!

General RequirementsGeneral Requirements

Minimize sources of contaminants◦ No smoking◦ No cosmetics◦ Avoid high particulate clothing, such as wool

sweaters◦ Cover up! Uncovered skin can lead to more

contamination

asepting procesing

Education