Seminar onAseptic Processing operation

byRanjith Kumar kankala

MPharm (I sem)

Department of PharmaceuticsBLUE BIRDS COLLEGE OF PHARMACY

Affiliated to Kakatiya universityWarangal

2009

Schedule (contents) Introduction to aseptic processing Aseptic Processing vs Terminal Sterilization contamination

Sources and control Microbial environmental monitoring Microbiological testing of air and water Characterization of aseptic process Media and incubation conditions Conclusion References

Aseptic Processing

Aseptic Processing is the processing of drug components ( drug product containers excipients etc) in a manner that makes impossible of microbiological contamination of the final sealed product

Progression of Symptoms

FeverDecreased Blood PressureRapid Breathing and Heart RateSkin LesionsSpontaneous Blood Clotting Organ FailureDeath

ldquoSepsis is a serious medical condition characterized by a whole-body inflammatory state caused by infectionrdquo

Schedule (contents) Introduction to aseptic processing Aseptic Processing vs Terminal Sterilization contamination

Sources and control Microbial environmental monitoring Microbiological testing of air and water Characterization of aseptic process Media and incubation conditions Conclusion References

Aseptic Processing

Aseptic Processing is the processing of drug components ( drug product containers excipients etc) in a manner that makes impossible of microbiological contamination of the final sealed product

Progression of Symptoms

FeverDecreased Blood PressureRapid Breathing and Heart RateSkin LesionsSpontaneous Blood Clotting Organ FailureDeath

ldquoSepsis is a serious medical condition characterized by a whole-body inflammatory state caused by infectionrdquo

Aseptic Processing

Aseptic Processing is the processing of drug components ( drug product containers excipients etc) in a manner that makes impossible of microbiological contamination of the final sealed product

Progression of Symptoms

FeverDecreased Blood PressureRapid Breathing and Heart RateSkin LesionsSpontaneous Blood Clotting Organ FailureDeath

ldquoSepsis is a serious medical condition characterized by a whole-body inflammatory state caused by infectionrdquo

Progression of Symptoms

FeverDecreased Blood PressureRapid Breathing and Heart RateSkin LesionsSpontaneous Blood Clotting Organ FailureDeath

ldquoSepsis is a serious medical condition characterized by a whole-body inflammatory state caused by infectionrdquo

Causes of sepsis

Sterile drug manufacturers should have a keen awareness of the public health implications of distributing a non-sterile product Poor cGMP conditions at a manufacturing facility can ultimately pose a life-threatening health risk to a patientrdquo

Asepsis is the practice to reduce or eliminate contaminants (such as bacteria viruses fungi and parasites) from entering the field to prevent infection Ideally a field is sterile mdash free of contaminants mdash a situation that is difficult to attain However the goal is elimination of infection

Asepsis is the practice to reduce or eliminate contaminants (such as bacteria viruses fungi and parasites) from entering the field to prevent infection Ideally a field is sterile mdash free of contaminants mdash a situation that is difficult to attain However the goal is elimination of infection

Producing drug products by

Terminal sterilization Product containers are filled and

sealed under high-quality environmental conditions designed to minimize contamination but not to guarantee sterility

Product in its final container is subject to a sterilization process such as heat or irradiation

Aseptic processing

Drug product container and closure are subject to sterilization separately and then brought together

Because there is no process to sterilize the product in its final container it is critical that containers be filled and sealed in an extremely high ndashquality environment

Terminal Sterilization

Drug Product

Container Closure

Excipiants

Sterilization Process

Sterile Drug

Product

Aseptic Processing Drug

Product

Sterilization Process

Container

Closure

Excipient

Sterilization Process

Sterilization Process

Sterilization Process

Sterile Closure

Sterile Excipient

Aseptic Processin

g

Sterile Drug

Product

SterileContainer

Sterile Final

Product

Can use multiple sterilization processes each optimized for the individual component

Bacteria virus fungi and other viable microbes cause a serious contamination

Bacterial spores and endotoxins Non viable Particles like dust fibers or other material are

suspended in the air and may contaminate product

Contaminating agents

Humans and bacteria

Over 200 different species of bacteria are found associated with humans

Bacteria are found in the intestines eyes nares mouth hair and skin

Dry skin can have 1000rsquos of microbes mm2

Staphylococcus epidermidis Scanning EM CDC

Sources of Contamination

Personnel born contaminants Poor or improper Sanitization Procedures deficient or poorly

executed Air born contaminants Inadequate HEPA seal (over 90 vials contaminated) Velocity through HEPA Filters Variable velocities between

filters Inadequate laminar flow resulted Low or undetectable velocity at work surface

Mechanical failure of filling tank main pump failure cooling system leaks at joints

Control

1st step ndash eliminating the source of

contamination 2nd Step - Reduce the Risk of

contamination through Sterile barriersSurface monitoring Aseptic technique

Gowning (sterile barrier)

If people are a major source of contamination we avoid contaminating the product while we process it

Surface Monitoring

Touch or Contact plates - RODAC

Plates (Replicate Organism Detectionand Counting)

Swabs

Terminal Sterilization

Drug Product

Container Closure

Excipiants

Sterilization Process

Sterile Drug

Product

Aseptic Processing Drug

Product

Sterilization Process

Container

Closure

Excipient

Sterilization Process

Sterilization Process

Sterilization Process

Sterile Closure

Sterile Excipient

Aseptic Processin

g

Sterile Drug

Product

SterileContainer

Sterile Final

Product

Can use multiple sterilization processes each optimized for the individual component

Bacteria virus fungi and other viable microbes cause a serious contamination

Bacterial spores and endotoxins Non viable Particles like dust fibers or other material are

suspended in the air and may contaminate product

Contaminating agents

Humans and bacteria

Over 200 different species of bacteria are found associated with humans

Bacteria are found in the intestines eyes nares mouth hair and skin

Dry skin can have 1000rsquos of microbes mm2

Staphylococcus epidermidis Scanning EM CDC

Sources of Contamination

Personnel born contaminants Poor or improper Sanitization Procedures deficient or poorly

executed Air born contaminants Inadequate HEPA seal (over 90 vials contaminated) Velocity through HEPA Filters Variable velocities between

filters Inadequate laminar flow resulted Low or undetectable velocity at work surface

Mechanical failure of filling tank main pump failure cooling system leaks at joints

Control

1st step ndash eliminating the source of

contamination 2nd Step - Reduce the Risk of

contamination through Sterile barriersSurface monitoring Aseptic technique

Gowning (sterile barrier)

If people are a major source of contamination we avoid contaminating the product while we process it

Surface Monitoring

Touch or Contact plates - RODAC

Plates (Replicate Organism Detectionand Counting)

Swabs

Aseptic Processing Drug

Product

Sterilization Process

Container

Closure

Excipient

Sterilization Process

Sterilization Process

Sterilization Process

Sterile Closure

Sterile Excipient

Aseptic Processin

g

Sterile Drug

Product

SterileContainer

Sterile Final

Product

Can use multiple sterilization processes each optimized for the individual component

Bacteria virus fungi and other viable microbes cause a serious contamination

Bacterial spores and endotoxins Non viable Particles like dust fibers or other material are

suspended in the air and may contaminate product

Contaminating agents

Humans and bacteria

Over 200 different species of bacteria are found associated with humans

Bacteria are found in the intestines eyes nares mouth hair and skin

Dry skin can have 1000rsquos of microbes mm2

Staphylococcus epidermidis Scanning EM CDC

Sources of Contamination

Personnel born contaminants Poor or improper Sanitization Procedures deficient or poorly

executed Air born contaminants Inadequate HEPA seal (over 90 vials contaminated) Velocity through HEPA Filters Variable velocities between

filters Inadequate laminar flow resulted Low or undetectable velocity at work surface

Mechanical failure of filling tank main pump failure cooling system leaks at joints

Control

1st step ndash eliminating the source of

contamination 2nd Step - Reduce the Risk of

contamination through Sterile barriersSurface monitoring Aseptic technique

Gowning (sterile barrier)

If people are a major source of contamination we avoid contaminating the product while we process it

Surface Monitoring

Touch or Contact plates - RODAC

Plates (Replicate Organism Detectionand Counting)

Swabs

Bacteria virus fungi and other viable microbes cause a serious contamination

Bacterial spores and endotoxins Non viable Particles like dust fibers or other material are

suspended in the air and may contaminate product

Contaminating agents

Humans and bacteria

Over 200 different species of bacteria are found associated with humans

Bacteria are found in the intestines eyes nares mouth hair and skin

Dry skin can have 1000rsquos of microbes mm2

Staphylococcus epidermidis Scanning EM CDC

Sources of Contamination

Personnel born contaminants Poor or improper Sanitization Procedures deficient or poorly

executed Air born contaminants Inadequate HEPA seal (over 90 vials contaminated) Velocity through HEPA Filters Variable velocities between

filters Inadequate laminar flow resulted Low or undetectable velocity at work surface

Mechanical failure of filling tank main pump failure cooling system leaks at joints

Control

1st step ndash eliminating the source of

contamination 2nd Step - Reduce the Risk of

contamination through Sterile barriersSurface monitoring Aseptic technique

Gowning (sterile barrier)

If people are a major source of contamination we avoid contaminating the product while we process it

Surface Monitoring

Touch or Contact plates - RODAC

Plates (Replicate Organism Detectionand Counting)

Swabs

Humans and bacteria

Over 200 different species of bacteria are found associated with humans

Bacteria are found in the intestines eyes nares mouth hair and skin

Dry skin can have 1000rsquos of microbes mm2

Staphylococcus epidermidis Scanning EM CDC

Sources of Contamination

Personnel born contaminants Poor or improper Sanitization Procedures deficient or poorly

executed Air born contaminants Inadequate HEPA seal (over 90 vials contaminated) Velocity through HEPA Filters Variable velocities between

filters Inadequate laminar flow resulted Low or undetectable velocity at work surface

Mechanical failure of filling tank main pump failure cooling system leaks at joints

Control

1st step ndash eliminating the source of

contamination 2nd Step - Reduce the Risk of

contamination through Sterile barriersSurface monitoring Aseptic technique

Gowning (sterile barrier)

If people are a major source of contamination we avoid contaminating the product while we process it

Surface Monitoring

Touch or Contact plates - RODAC

Plates (Replicate Organism Detectionand Counting)

Swabs

Sources of Contamination

Personnel born contaminants Poor or improper Sanitization Procedures deficient or poorly

executed Air born contaminants Inadequate HEPA seal (over 90 vials contaminated) Velocity through HEPA Filters Variable velocities between

filters Inadequate laminar flow resulted Low or undetectable velocity at work surface

Mechanical failure of filling tank main pump failure cooling system leaks at joints

Control

1st step ndash eliminating the source of

contamination 2nd Step - Reduce the Risk of

contamination through Sterile barriersSurface monitoring Aseptic technique

Gowning (sterile barrier)

If people are a major source of contamination we avoid contaminating the product while we process it

Surface Monitoring

Touch or Contact plates - RODAC

Plates (Replicate Organism Detectionand Counting)

Swabs

Control

1st step ndash eliminating the source of

contamination 2nd Step - Reduce the Risk of

contamination through Sterile barriersSurface monitoring Aseptic technique

Gowning (sterile barrier)

If people are a major source of contamination we avoid contaminating the product while we process it

Surface Monitoring

Touch or Contact plates - RODAC

Plates (Replicate Organism Detectionand Counting)

Swabs

Gowning (sterile barrier)

If people are a major source of contamination we avoid contaminating the product while we process it

Surface Monitoring

Touch or Contact plates - RODAC

Plates (Replicate Organism Detectionand Counting)

Swabs

Surface Monitoring

Touch or Contact plates - RODAC

Plates (Replicate Organism Detectionand Counting)

Swabs

Aseptic Technique (skill)

Contact sterile materials only with sterile instruments Operators should not contact sterile products containers

closures or critical surfaces with any part of their gown or gloves

Keep the entire body out of the path of unidirectional airflow Approach a necessary manipulation in a manner that does not

compromise sterility of the product

Whatrsquos wrong with this picture

CORRECT

Horizontal airflow Vertical airflowwwworsodnihgovdspubsbscgraphicsfig3gif

Unidirectional airflow

The operator should never come between the

air source and the product

pressure differential bn critical area from external environment (175-50 Pa)

Disinfectants

ISOPROPYL ALCOHOL (70) Powerful disinfectant Effectively kills bacteria and fungi

Mode of action denatures proteins dissolves lipids and can lead to cell membrane disintegration

But does not inactivate spores eg phenols Alcohols Aldehydes etc

Sporicidal agents Glutaraldehyde Formaldehyde sodium hypochlorite Iodine and iodophors Peroxygens Ethylene oxide P- Propiolactone

Isolators Advantage

No direct contact between operator amp product

Microbial identification should extend to the species level

Routine traditional techniques phenotypic and

biochemical Genotypic techniques are

suggested for failure investigations

Microbial Environmental Monitoring Identification

Phenotypic technique

Gram Stain

Identifying Microbes

Staphylococcus xylosus

Reduction of Tetrazolium Violet

Biochemical Assays

Genotypic Methods

Use DNA sequence (often ribosomal RNA genes rDNA) to identify organism

Faster and more accurate then traditional biochemical and phenotypic techniques

QC Micro Identifying Microbes Genotype Based AssayPCR Polymerase Chain Reaction

Extremely heat stable ndash recommended conditions for inactivation are 180 0 C for 3 hours

Endotoxin a pyrogenic (fever inducing) substance (eg lipopolysaccharide) present in the bacterial cell wall Endotoxin reactions range from fever to death

Endotoxin Testing

LAL Assay (Limulus amoebocyte lysate)ENDOTOXIN LIMIT FOR WFI IS 025 EUml

Microbiological testing of water Universal solvent Used as Vehicle and used to rince and cleaning of

apparatus Water should also be tested for presence of coliforms andor

pseudomonads if appropriate (may cause biofilm) Water should be tested using R2A agar (low nutrient for the recovery

of water borne organisms) incubated for at least 5 days at 30-35degC Sampling procedures should follow those used in production

Microbiological testing of airCompressed AirNitrogenCO2

Air sampling should be done and tested for the presence of non-viables and viables by exposure to the environment

Pressure control orifices should be used to provide a steady stream of air

Fall out plate Slit sampler (slit-to-agar sampler)

Slit Sampler (New Brunswick Scientifics Model STA-230 Slit-to-Agar Air Sampler)

Characterization of aseptic process The four pillars of a robust aseptic process

Personnel training amp monitoring Environmental monitoring Facilities design Media fills

Personnel Training amp Monitoring Avoiding contamination means knowing the potential sources

of contamination Personnel Equipment Airliquids Drug product Containersclosures Outside environment

Anything Brought in contact with or in the vicinity of the product is a potential source of contamination

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Whatrsquos wrong with this picture

CORRECT

Horizontal airflow Vertical airflowwwworsodnihgovdspubsbscgraphicsfig3gif

Unidirectional airflow

The operator should never come between the

air source and the product

pressure differential bn critical area from external environment (175-50 Pa)

Disinfectants

ISOPROPYL ALCOHOL (70) Powerful disinfectant Effectively kills bacteria and fungi

Mode of action denatures proteins dissolves lipids and can lead to cell membrane disintegration

But does not inactivate spores eg phenols Alcohols Aldehydes etc

Sporicidal agents Glutaraldehyde Formaldehyde sodium hypochlorite Iodine and iodophors Peroxygens Ethylene oxide P- Propiolactone

Isolators Advantage

No direct contact between operator amp product

Microbial identification should extend to the species level

Routine traditional techniques phenotypic and

biochemical Genotypic techniques are

suggested for failure investigations

Microbial Environmental Monitoring Identification

Phenotypic technique

Gram Stain

Identifying Microbes

Staphylococcus xylosus

Reduction of Tetrazolium Violet

Biochemical Assays

Genotypic Methods

Use DNA sequence (often ribosomal RNA genes rDNA) to identify organism

Faster and more accurate then traditional biochemical and phenotypic techniques

QC Micro Identifying Microbes Genotype Based AssayPCR Polymerase Chain Reaction

Extremely heat stable ndash recommended conditions for inactivation are 180 0 C for 3 hours

Endotoxin a pyrogenic (fever inducing) substance (eg lipopolysaccharide) present in the bacterial cell wall Endotoxin reactions range from fever to death

Endotoxin Testing

LAL Assay (Limulus amoebocyte lysate)ENDOTOXIN LIMIT FOR WFI IS 025 EUml

Microbiological testing of water Universal solvent Used as Vehicle and used to rince and cleaning of

apparatus Water should also be tested for presence of coliforms andor

pseudomonads if appropriate (may cause biofilm) Water should be tested using R2A agar (low nutrient for the recovery

of water borne organisms) incubated for at least 5 days at 30-35degC Sampling procedures should follow those used in production

Microbiological testing of airCompressed AirNitrogenCO2

Air sampling should be done and tested for the presence of non-viables and viables by exposure to the environment

Pressure control orifices should be used to provide a steady stream of air

Fall out plate Slit sampler (slit-to-agar sampler)

Slit Sampler (New Brunswick Scientifics Model STA-230 Slit-to-Agar Air Sampler)

Characterization of aseptic process The four pillars of a robust aseptic process

Personnel training amp monitoring Environmental monitoring Facilities design Media fills

Personnel Training amp Monitoring Avoiding contamination means knowing the potential sources

of contamination Personnel Equipment Airliquids Drug product Containersclosures Outside environment

Anything Brought in contact with or in the vicinity of the product is a potential source of contamination

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

CORRECT

Horizontal airflow Vertical airflowwwworsodnihgovdspubsbscgraphicsfig3gif

Unidirectional airflow

The operator should never come between the

air source and the product

pressure differential bn critical area from external environment (175-50 Pa)

Disinfectants

ISOPROPYL ALCOHOL (70) Powerful disinfectant Effectively kills bacteria and fungi

Mode of action denatures proteins dissolves lipids and can lead to cell membrane disintegration

But does not inactivate spores eg phenols Alcohols Aldehydes etc

Sporicidal agents Glutaraldehyde Formaldehyde sodium hypochlorite Iodine and iodophors Peroxygens Ethylene oxide P- Propiolactone

Isolators Advantage

No direct contact between operator amp product

Microbial identification should extend to the species level

Routine traditional techniques phenotypic and

biochemical Genotypic techniques are

suggested for failure investigations

Microbial Environmental Monitoring Identification

Phenotypic technique

Gram Stain

Identifying Microbes

Staphylococcus xylosus

Reduction of Tetrazolium Violet

Biochemical Assays

Genotypic Methods

Use DNA sequence (often ribosomal RNA genes rDNA) to identify organism

Faster and more accurate then traditional biochemical and phenotypic techniques

QC Micro Identifying Microbes Genotype Based AssayPCR Polymerase Chain Reaction

Extremely heat stable ndash recommended conditions for inactivation are 180 0 C for 3 hours

Endotoxin a pyrogenic (fever inducing) substance (eg lipopolysaccharide) present in the bacterial cell wall Endotoxin reactions range from fever to death

Endotoxin Testing

LAL Assay (Limulus amoebocyte lysate)ENDOTOXIN LIMIT FOR WFI IS 025 EUml

Microbiological testing of water Universal solvent Used as Vehicle and used to rince and cleaning of

apparatus Water should also be tested for presence of coliforms andor

pseudomonads if appropriate (may cause biofilm) Water should be tested using R2A agar (low nutrient for the recovery

of water borne organisms) incubated for at least 5 days at 30-35degC Sampling procedures should follow those used in production

Microbiological testing of airCompressed AirNitrogenCO2

Air sampling should be done and tested for the presence of non-viables and viables by exposure to the environment

Pressure control orifices should be used to provide a steady stream of air

Fall out plate Slit sampler (slit-to-agar sampler)

Slit Sampler (New Brunswick Scientifics Model STA-230 Slit-to-Agar Air Sampler)

Characterization of aseptic process The four pillars of a robust aseptic process

Personnel training amp monitoring Environmental monitoring Facilities design Media fills

Personnel Training amp Monitoring Avoiding contamination means knowing the potential sources

of contamination Personnel Equipment Airliquids Drug product Containersclosures Outside environment

Anything Brought in contact with or in the vicinity of the product is a potential source of contamination

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Horizontal airflow Vertical airflowwwworsodnihgovdspubsbscgraphicsfig3gif

Unidirectional airflow

The operator should never come between the

air source and the product

pressure differential bn critical area from external environment (175-50 Pa)

Disinfectants

ISOPROPYL ALCOHOL (70) Powerful disinfectant Effectively kills bacteria and fungi

Mode of action denatures proteins dissolves lipids and can lead to cell membrane disintegration

But does not inactivate spores eg phenols Alcohols Aldehydes etc

Sporicidal agents Glutaraldehyde Formaldehyde sodium hypochlorite Iodine and iodophors Peroxygens Ethylene oxide P- Propiolactone

Isolators Advantage

No direct contact between operator amp product

Microbial identification should extend to the species level

Routine traditional techniques phenotypic and

biochemical Genotypic techniques are

suggested for failure investigations

Microbial Environmental Monitoring Identification

Phenotypic technique

Gram Stain

Identifying Microbes

Staphylococcus xylosus

Reduction of Tetrazolium Violet

Biochemical Assays

Genotypic Methods

Use DNA sequence (often ribosomal RNA genes rDNA) to identify organism

Faster and more accurate then traditional biochemical and phenotypic techniques

QC Micro Identifying Microbes Genotype Based AssayPCR Polymerase Chain Reaction

Extremely heat stable ndash recommended conditions for inactivation are 180 0 C for 3 hours

Endotoxin a pyrogenic (fever inducing) substance (eg lipopolysaccharide) present in the bacterial cell wall Endotoxin reactions range from fever to death

Endotoxin Testing

LAL Assay (Limulus amoebocyte lysate)ENDOTOXIN LIMIT FOR WFI IS 025 EUml

Microbiological testing of water Universal solvent Used as Vehicle and used to rince and cleaning of

apparatus Water should also be tested for presence of coliforms andor

pseudomonads if appropriate (may cause biofilm) Water should be tested using R2A agar (low nutrient for the recovery

of water borne organisms) incubated for at least 5 days at 30-35degC Sampling procedures should follow those used in production

Microbiological testing of airCompressed AirNitrogenCO2

Air sampling should be done and tested for the presence of non-viables and viables by exposure to the environment

Pressure control orifices should be used to provide a steady stream of air

Fall out plate Slit sampler (slit-to-agar sampler)

Slit Sampler (New Brunswick Scientifics Model STA-230 Slit-to-Agar Air Sampler)

Characterization of aseptic process The four pillars of a robust aseptic process

Personnel training amp monitoring Environmental monitoring Facilities design Media fills

Personnel Training amp Monitoring Avoiding contamination means knowing the potential sources

of contamination Personnel Equipment Airliquids Drug product Containersclosures Outside environment

Anything Brought in contact with or in the vicinity of the product is a potential source of contamination

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Disinfectants

ISOPROPYL ALCOHOL (70) Powerful disinfectant Effectively kills bacteria and fungi

Mode of action denatures proteins dissolves lipids and can lead to cell membrane disintegration

But does not inactivate spores eg phenols Alcohols Aldehydes etc

Sporicidal agents Glutaraldehyde Formaldehyde sodium hypochlorite Iodine and iodophors Peroxygens Ethylene oxide P- Propiolactone

Isolators Advantage

No direct contact between operator amp product

Microbial identification should extend to the species level

Routine traditional techniques phenotypic and

biochemical Genotypic techniques are

suggested for failure investigations

Microbial Environmental Monitoring Identification

Phenotypic technique

Gram Stain

Identifying Microbes

Staphylococcus xylosus

Reduction of Tetrazolium Violet

Biochemical Assays

Genotypic Methods

Use DNA sequence (often ribosomal RNA genes rDNA) to identify organism

Faster and more accurate then traditional biochemical and phenotypic techniques

QC Micro Identifying Microbes Genotype Based AssayPCR Polymerase Chain Reaction

Extremely heat stable ndash recommended conditions for inactivation are 180 0 C for 3 hours

Endotoxin a pyrogenic (fever inducing) substance (eg lipopolysaccharide) present in the bacterial cell wall Endotoxin reactions range from fever to death

Endotoxin Testing

LAL Assay (Limulus amoebocyte lysate)ENDOTOXIN LIMIT FOR WFI IS 025 EUml

Microbiological testing of water Universal solvent Used as Vehicle and used to rince and cleaning of

apparatus Water should also be tested for presence of coliforms andor

pseudomonads if appropriate (may cause biofilm) Water should be tested using R2A agar (low nutrient for the recovery

of water borne organisms) incubated for at least 5 days at 30-35degC Sampling procedures should follow those used in production

Microbiological testing of airCompressed AirNitrogenCO2

Air sampling should be done and tested for the presence of non-viables and viables by exposure to the environment

Pressure control orifices should be used to provide a steady stream of air

Fall out plate Slit sampler (slit-to-agar sampler)

Slit Sampler (New Brunswick Scientifics Model STA-230 Slit-to-Agar Air Sampler)

Characterization of aseptic process The four pillars of a robust aseptic process

Personnel training amp monitoring Environmental monitoring Facilities design Media fills

Personnel Training amp Monitoring Avoiding contamination means knowing the potential sources

of contamination Personnel Equipment Airliquids Drug product Containersclosures Outside environment

Anything Brought in contact with or in the vicinity of the product is a potential source of contamination

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Sporicidal agents Glutaraldehyde Formaldehyde sodium hypochlorite Iodine and iodophors Peroxygens Ethylene oxide P- Propiolactone

Isolators Advantage

No direct contact between operator amp product

Microbial identification should extend to the species level

Routine traditional techniques phenotypic and

biochemical Genotypic techniques are

suggested for failure investigations

Microbial Environmental Monitoring Identification

Phenotypic technique

Gram Stain

Identifying Microbes

Staphylococcus xylosus

Reduction of Tetrazolium Violet

Biochemical Assays

Genotypic Methods

Use DNA sequence (often ribosomal RNA genes rDNA) to identify organism

Faster and more accurate then traditional biochemical and phenotypic techniques

QC Micro Identifying Microbes Genotype Based AssayPCR Polymerase Chain Reaction

Extremely heat stable ndash recommended conditions for inactivation are 180 0 C for 3 hours

Endotoxin a pyrogenic (fever inducing) substance (eg lipopolysaccharide) present in the bacterial cell wall Endotoxin reactions range from fever to death

Endotoxin Testing

LAL Assay (Limulus amoebocyte lysate)ENDOTOXIN LIMIT FOR WFI IS 025 EUml

Microbiological testing of water Universal solvent Used as Vehicle and used to rince and cleaning of

apparatus Water should also be tested for presence of coliforms andor

pseudomonads if appropriate (may cause biofilm) Water should be tested using R2A agar (low nutrient for the recovery

of water borne organisms) incubated for at least 5 days at 30-35degC Sampling procedures should follow those used in production

Microbiological testing of airCompressed AirNitrogenCO2

Air sampling should be done and tested for the presence of non-viables and viables by exposure to the environment

Pressure control orifices should be used to provide a steady stream of air

Fall out plate Slit sampler (slit-to-agar sampler)

Slit Sampler (New Brunswick Scientifics Model STA-230 Slit-to-Agar Air Sampler)

Characterization of aseptic process The four pillars of a robust aseptic process

Personnel training amp monitoring Environmental monitoring Facilities design Media fills

Personnel Training amp Monitoring Avoiding contamination means knowing the potential sources

of contamination Personnel Equipment Airliquids Drug product Containersclosures Outside environment

Anything Brought in contact with or in the vicinity of the product is a potential source of contamination

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Isolators Advantage

No direct contact between operator amp product

Microbial identification should extend to the species level

Routine traditional techniques phenotypic and

biochemical Genotypic techniques are

suggested for failure investigations

Microbial Environmental Monitoring Identification

Phenotypic technique

Gram Stain

Identifying Microbes

Staphylococcus xylosus

Reduction of Tetrazolium Violet

Biochemical Assays

Genotypic Methods

Use DNA sequence (often ribosomal RNA genes rDNA) to identify organism

Faster and more accurate then traditional biochemical and phenotypic techniques

QC Micro Identifying Microbes Genotype Based AssayPCR Polymerase Chain Reaction

Extremely heat stable ndash recommended conditions for inactivation are 180 0 C for 3 hours

Endotoxin a pyrogenic (fever inducing) substance (eg lipopolysaccharide) present in the bacterial cell wall Endotoxin reactions range from fever to death

Endotoxin Testing

LAL Assay (Limulus amoebocyte lysate)ENDOTOXIN LIMIT FOR WFI IS 025 EUml

Microbiological testing of water Universal solvent Used as Vehicle and used to rince and cleaning of

apparatus Water should also be tested for presence of coliforms andor

pseudomonads if appropriate (may cause biofilm) Water should be tested using R2A agar (low nutrient for the recovery

of water borne organisms) incubated for at least 5 days at 30-35degC Sampling procedures should follow those used in production

Microbiological testing of airCompressed AirNitrogenCO2

Air sampling should be done and tested for the presence of non-viables and viables by exposure to the environment

Pressure control orifices should be used to provide a steady stream of air

Fall out plate Slit sampler (slit-to-agar sampler)

Slit Sampler (New Brunswick Scientifics Model STA-230 Slit-to-Agar Air Sampler)

Characterization of aseptic process The four pillars of a robust aseptic process

Personnel training amp monitoring Environmental monitoring Facilities design Media fills

Personnel Training amp Monitoring Avoiding contamination means knowing the potential sources

of contamination Personnel Equipment Airliquids Drug product Containersclosures Outside environment

Anything Brought in contact with or in the vicinity of the product is a potential source of contamination

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Microbial identification should extend to the species level

Routine traditional techniques phenotypic and

biochemical Genotypic techniques are

suggested for failure investigations

Microbial Environmental Monitoring Identification

Phenotypic technique

Gram Stain

Identifying Microbes

Staphylococcus xylosus

Reduction of Tetrazolium Violet

Biochemical Assays

Genotypic Methods

Use DNA sequence (often ribosomal RNA genes rDNA) to identify organism

Faster and more accurate then traditional biochemical and phenotypic techniques

QC Micro Identifying Microbes Genotype Based AssayPCR Polymerase Chain Reaction

Extremely heat stable ndash recommended conditions for inactivation are 180 0 C for 3 hours

Endotoxin a pyrogenic (fever inducing) substance (eg lipopolysaccharide) present in the bacterial cell wall Endotoxin reactions range from fever to death

Endotoxin Testing

LAL Assay (Limulus amoebocyte lysate)ENDOTOXIN LIMIT FOR WFI IS 025 EUml

Microbiological testing of water Universal solvent Used as Vehicle and used to rince and cleaning of

apparatus Water should also be tested for presence of coliforms andor

pseudomonads if appropriate (may cause biofilm) Water should be tested using R2A agar (low nutrient for the recovery

of water borne organisms) incubated for at least 5 days at 30-35degC Sampling procedures should follow those used in production

Microbiological testing of airCompressed AirNitrogenCO2

Air sampling should be done and tested for the presence of non-viables and viables by exposure to the environment

Pressure control orifices should be used to provide a steady stream of air

Fall out plate Slit sampler (slit-to-agar sampler)

Slit Sampler (New Brunswick Scientifics Model STA-230 Slit-to-Agar Air Sampler)

Characterization of aseptic process The four pillars of a robust aseptic process

Personnel training amp monitoring Environmental monitoring Facilities design Media fills

Personnel Training amp Monitoring Avoiding contamination means knowing the potential sources

of contamination Personnel Equipment Airliquids Drug product Containersclosures Outside environment

Anything Brought in contact with or in the vicinity of the product is a potential source of contamination

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Phenotypic technique

Gram Stain

Identifying Microbes

Staphylococcus xylosus

Reduction of Tetrazolium Violet

Biochemical Assays

Genotypic Methods

Use DNA sequence (often ribosomal RNA genes rDNA) to identify organism

Faster and more accurate then traditional biochemical and phenotypic techniques

QC Micro Identifying Microbes Genotype Based AssayPCR Polymerase Chain Reaction

Extremely heat stable ndash recommended conditions for inactivation are 180 0 C for 3 hours

Endotoxin a pyrogenic (fever inducing) substance (eg lipopolysaccharide) present in the bacterial cell wall Endotoxin reactions range from fever to death

Endotoxin Testing

LAL Assay (Limulus amoebocyte lysate)ENDOTOXIN LIMIT FOR WFI IS 025 EUml

Microbiological testing of water Universal solvent Used as Vehicle and used to rince and cleaning of

apparatus Water should also be tested for presence of coliforms andor

pseudomonads if appropriate (may cause biofilm) Water should be tested using R2A agar (low nutrient for the recovery

of water borne organisms) incubated for at least 5 days at 30-35degC Sampling procedures should follow those used in production

Microbiological testing of airCompressed AirNitrogenCO2

Air sampling should be done and tested for the presence of non-viables and viables by exposure to the environment

Pressure control orifices should be used to provide a steady stream of air

Fall out plate Slit sampler (slit-to-agar sampler)

Slit Sampler (New Brunswick Scientifics Model STA-230 Slit-to-Agar Air Sampler)

Characterization of aseptic process The four pillars of a robust aseptic process

Personnel training amp monitoring Environmental monitoring Facilities design Media fills

Personnel Training amp Monitoring Avoiding contamination means knowing the potential sources

of contamination Personnel Equipment Airliquids Drug product Containersclosures Outside environment

Anything Brought in contact with or in the vicinity of the product is a potential source of contamination

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Staphylococcus xylosus

Reduction of Tetrazolium Violet

Biochemical Assays

Genotypic Methods

Use DNA sequence (often ribosomal RNA genes rDNA) to identify organism

Faster and more accurate then traditional biochemical and phenotypic techniques

QC Micro Identifying Microbes Genotype Based AssayPCR Polymerase Chain Reaction

Extremely heat stable ndash recommended conditions for inactivation are 180 0 C for 3 hours

Endotoxin a pyrogenic (fever inducing) substance (eg lipopolysaccharide) present in the bacterial cell wall Endotoxin reactions range from fever to death

Endotoxin Testing

LAL Assay (Limulus amoebocyte lysate)ENDOTOXIN LIMIT FOR WFI IS 025 EUml

Microbiological testing of water Universal solvent Used as Vehicle and used to rince and cleaning of

apparatus Water should also be tested for presence of coliforms andor

pseudomonads if appropriate (may cause biofilm) Water should be tested using R2A agar (low nutrient for the recovery

of water borne organisms) incubated for at least 5 days at 30-35degC Sampling procedures should follow those used in production

Microbiological testing of airCompressed AirNitrogenCO2

Air sampling should be done and tested for the presence of non-viables and viables by exposure to the environment

Pressure control orifices should be used to provide a steady stream of air

Fall out plate Slit sampler (slit-to-agar sampler)

Slit Sampler (New Brunswick Scientifics Model STA-230 Slit-to-Agar Air Sampler)

Characterization of aseptic process The four pillars of a robust aseptic process

Personnel training amp monitoring Environmental monitoring Facilities design Media fills

Personnel Training amp Monitoring Avoiding contamination means knowing the potential sources

of contamination Personnel Equipment Airliquids Drug product Containersclosures Outside environment

Anything Brought in contact with or in the vicinity of the product is a potential source of contamination

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Genotypic Methods

Use DNA sequence (often ribosomal RNA genes rDNA) to identify organism

Faster and more accurate then traditional biochemical and phenotypic techniques

QC Micro Identifying Microbes Genotype Based AssayPCR Polymerase Chain Reaction

Extremely heat stable ndash recommended conditions for inactivation are 180 0 C for 3 hours

Endotoxin a pyrogenic (fever inducing) substance (eg lipopolysaccharide) present in the bacterial cell wall Endotoxin reactions range from fever to death

Endotoxin Testing

LAL Assay (Limulus amoebocyte lysate)ENDOTOXIN LIMIT FOR WFI IS 025 EUml

Microbiological testing of water Universal solvent Used as Vehicle and used to rince and cleaning of

apparatus Water should also be tested for presence of coliforms andor

pseudomonads if appropriate (may cause biofilm) Water should be tested using R2A agar (low nutrient for the recovery

of water borne organisms) incubated for at least 5 days at 30-35degC Sampling procedures should follow those used in production

Microbiological testing of airCompressed AirNitrogenCO2

Air sampling should be done and tested for the presence of non-viables and viables by exposure to the environment

Pressure control orifices should be used to provide a steady stream of air

Fall out plate Slit sampler (slit-to-agar sampler)

Slit Sampler (New Brunswick Scientifics Model STA-230 Slit-to-Agar Air Sampler)

Characterization of aseptic process The four pillars of a robust aseptic process

Personnel training amp monitoring Environmental monitoring Facilities design Media fills

Personnel Training amp Monitoring Avoiding contamination means knowing the potential sources

of contamination Personnel Equipment Airliquids Drug product Containersclosures Outside environment

Anything Brought in contact with or in the vicinity of the product is a potential source of contamination

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

QC Micro Identifying Microbes Genotype Based AssayPCR Polymerase Chain Reaction

Extremely heat stable ndash recommended conditions for inactivation are 180 0 C for 3 hours

Endotoxin a pyrogenic (fever inducing) substance (eg lipopolysaccharide) present in the bacterial cell wall Endotoxin reactions range from fever to death

Endotoxin Testing

LAL Assay (Limulus amoebocyte lysate)ENDOTOXIN LIMIT FOR WFI IS 025 EUml

Microbiological testing of water Universal solvent Used as Vehicle and used to rince and cleaning of

apparatus Water should also be tested for presence of coliforms andor

pseudomonads if appropriate (may cause biofilm) Water should be tested using R2A agar (low nutrient for the recovery

of water borne organisms) incubated for at least 5 days at 30-35degC Sampling procedures should follow those used in production

Microbiological testing of airCompressed AirNitrogenCO2

Air sampling should be done and tested for the presence of non-viables and viables by exposure to the environment

Pressure control orifices should be used to provide a steady stream of air

Fall out plate Slit sampler (slit-to-agar sampler)

Slit Sampler (New Brunswick Scientifics Model STA-230 Slit-to-Agar Air Sampler)

Characterization of aseptic process The four pillars of a robust aseptic process

Personnel training amp monitoring Environmental monitoring Facilities design Media fills

Personnel Training amp Monitoring Avoiding contamination means knowing the potential sources

of contamination Personnel Equipment Airliquids Drug product Containersclosures Outside environment

Anything Brought in contact with or in the vicinity of the product is a potential source of contamination

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Extremely heat stable ndash recommended conditions for inactivation are 180 0 C for 3 hours

Endotoxin a pyrogenic (fever inducing) substance (eg lipopolysaccharide) present in the bacterial cell wall Endotoxin reactions range from fever to death

Endotoxin Testing

LAL Assay (Limulus amoebocyte lysate)ENDOTOXIN LIMIT FOR WFI IS 025 EUml

Microbiological testing of water Universal solvent Used as Vehicle and used to rince and cleaning of

apparatus Water should also be tested for presence of coliforms andor

pseudomonads if appropriate (may cause biofilm) Water should be tested using R2A agar (low nutrient for the recovery

of water borne organisms) incubated for at least 5 days at 30-35degC Sampling procedures should follow those used in production

Microbiological testing of airCompressed AirNitrogenCO2

Air sampling should be done and tested for the presence of non-viables and viables by exposure to the environment

Pressure control orifices should be used to provide a steady stream of air

Fall out plate Slit sampler (slit-to-agar sampler)

Slit Sampler (New Brunswick Scientifics Model STA-230 Slit-to-Agar Air Sampler)

Characterization of aseptic process The four pillars of a robust aseptic process

Personnel training amp monitoring Environmental monitoring Facilities design Media fills

Personnel Training amp Monitoring Avoiding contamination means knowing the potential sources

of contamination Personnel Equipment Airliquids Drug product Containersclosures Outside environment

Anything Brought in contact with or in the vicinity of the product is a potential source of contamination

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Microbiological testing of water Universal solvent Used as Vehicle and used to rince and cleaning of

apparatus Water should also be tested for presence of coliforms andor

pseudomonads if appropriate (may cause biofilm) Water should be tested using R2A agar (low nutrient for the recovery

of water borne organisms) incubated for at least 5 days at 30-35degC Sampling procedures should follow those used in production

Microbiological testing of airCompressed AirNitrogenCO2

Air sampling should be done and tested for the presence of non-viables and viables by exposure to the environment

Pressure control orifices should be used to provide a steady stream of air

Fall out plate Slit sampler (slit-to-agar sampler)

Slit Sampler (New Brunswick Scientifics Model STA-230 Slit-to-Agar Air Sampler)

Characterization of aseptic process The four pillars of a robust aseptic process

Personnel training amp monitoring Environmental monitoring Facilities design Media fills

Personnel Training amp Monitoring Avoiding contamination means knowing the potential sources

of contamination Personnel Equipment Airliquids Drug product Containersclosures Outside environment

Anything Brought in contact with or in the vicinity of the product is a potential source of contamination

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Microbiological testing of airCompressed AirNitrogenCO2

Air sampling should be done and tested for the presence of non-viables and viables by exposure to the environment

Pressure control orifices should be used to provide a steady stream of air

Fall out plate Slit sampler (slit-to-agar sampler)

Slit Sampler (New Brunswick Scientifics Model STA-230 Slit-to-Agar Air Sampler)

Characterization of aseptic process The four pillars of a robust aseptic process

Personnel training amp monitoring Environmental monitoring Facilities design Media fills

Personnel Training amp Monitoring Avoiding contamination means knowing the potential sources

of contamination Personnel Equipment Airliquids Drug product Containersclosures Outside environment

Anything Brought in contact with or in the vicinity of the product is a potential source of contamination

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Characterization of aseptic process The four pillars of a robust aseptic process

Personnel training amp monitoring Environmental monitoring Facilities design Media fills

Personnel Training amp Monitoring Avoiding contamination means knowing the potential sources

of contamination Personnel Equipment Airliquids Drug product Containersclosures Outside environment

Anything Brought in contact with or in the vicinity of the product is a potential source of contamination

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Personnel Training amp Monitoring Avoiding contamination means knowing the potential sources

of contamination Personnel Equipment Airliquids Drug product Containersclosures Outside environment

Anything Brought in contact with or in the vicinity of the product is a potential source of contamination

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Environmental Monitoring

The goal of the environmental monitoring program is to provide meaningful information on the quality of the aseptic processing environment during production as well as environmental trends

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

1

2

3 4

5

67

8

9 10

11

13

12

Environmental Monitoring

Critical (processing) areas

Sampling

Sampling of adjacent classified areas (aseptic corridors gowning rooms etc) will provide trenddata and may help identify sources of contamination

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Facilities General Clean room Design

HEPAULPA filters on ceiling Exhaust vents on floor Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors walls and ceilings constructed of smooth hard

surfaces that can be easily cleaned Limited equipment fixtures and personnel Layout of equipment to optimize comfort and movement

of operators

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Facilities Clean room Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classificationge05um

particlesm3

Viable Microbes(cfum3)

Ave Airflow Velocity

(fpm)Air

changeshr

100000 8 3520000 100 5-10 5-48

10000 7 352000 10 10-15 60-90

1000 6 35200 7 25-40 150-240

100 5 3520 1 40-80 240-480

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Class 10000 clean room

httpwwwamericancleanroomscomamphotogallery_08html

Class 100 clean room

Facilities Clean room Classification

Facilities HEPA Filters

httppeopledeasharvardedu~joneslab_archnano_facilitieshepagif

High Efficiency Particulate Air filters

Minimum particle collection efficiency 9997 for 03microm diameter particles

Disposable

Filter made of pleated borosilicate glass

Media Fill test Used to validate the aseptic process

Use microbial growth media instead of drug product-any contamination will result in microbial growth

It doesnrsquot provide a direct relation for sterility but gives an adequate evaluation for operational processing steps

Media and Incubation conditions

Soybean casein digest medium (SCD) Fluid thioglycollate medium (FTM) for anerobes

Inoculated with lt 100 cfu challenge At least 14 days incubation 30-35degC for SCD 20-25degC for FTM temperatures should be monitored product produces suspension flocculation or deposit in

media suitable portions (2-5) should be transferred to fresh media after 14 days and incubated for a futher 7 days

Theoretical Evaluation Whyte mathematical model contamination is due to air borne microbes

d = equivalent particle diameterA= area of container opening (cm2)

t = time (sec)

Cont rate (c) = 00032d2At

PostScript (conclusion)

The challenge in aseptic processing is always personnel

1048708 As a source of microbial andParticle contamination1048708As a brake on the implementation ofImproved technology

REFERENCES

Encyclopedia of pharmtechnology

RUSSELL A D Bacterial Spores and Chemical Sporicidal Agents clinical microbiology reviews 3(2) 99-119 (1999)

httpwwwfdagovcbergdlnssterasepticpdf

httpwwwemedicinehealthcomimages4453

httppathmicromedscedufoxlpsjpg

httpmicromedharvardedufacultyrudnerhtml

Media Fill test Used to validate the aseptic process

Use microbial growth media instead of drug product-any contamination will result in microbial growth

It doesnrsquot provide a direct relation for sterility but gives an adequate evaluation for operational processing steps

Media and Incubation conditions

Soybean casein digest medium (SCD) Fluid thioglycollate medium (FTM) for anerobes

Inoculated with lt 100 cfu challenge At least 14 days incubation 30-35degC for SCD 20-25degC for FTM temperatures should be monitored product produces suspension flocculation or deposit in

media suitable portions (2-5) should be transferred to fresh media after 14 days and incubated for a futher 7 days

Theoretical Evaluation Whyte mathematical model contamination is due to air borne microbes

d = equivalent particle diameterA= area of container opening (cm2)

t = time (sec)

Cont rate (c) = 00032d2At

PostScript (conclusion)

The challenge in aseptic processing is always personnel

1048708 As a source of microbial andParticle contamination1048708As a brake on the implementation ofImproved technology

REFERENCES

Encyclopedia of pharmtechnology

RUSSELL A D Bacterial Spores and Chemical Sporicidal Agents clinical microbiology reviews 3(2) 99-119 (1999)

httpwwwfdagovcbergdlnssterasepticpdf

httpwwwemedicinehealthcomimages4453

httppathmicromedscedufoxlpsjpg

httpmicromedharvardedufacultyrudnerhtml

Media and Incubation conditions

Soybean casein digest medium (SCD) Fluid thioglycollate medium (FTM) for anerobes

Inoculated with lt 100 cfu challenge At least 14 days incubation 30-35degC for SCD 20-25degC for FTM temperatures should be monitored product produces suspension flocculation or deposit in

media suitable portions (2-5) should be transferred to fresh media after 14 days and incubated for a futher 7 days

Theoretical Evaluation Whyte mathematical model contamination is due to air borne microbes

d = equivalent particle diameterA= area of container opening (cm2)

t = time (sec)

Cont rate (c) = 00032d2At

PostScript (conclusion)

The challenge in aseptic processing is always personnel

1048708 As a source of microbial andParticle contamination1048708As a brake on the implementation ofImproved technology

REFERENCES

Encyclopedia of pharmtechnology

RUSSELL A D Bacterial Spores and Chemical Sporicidal Agents clinical microbiology reviews 3(2) 99-119 (1999)

httpwwwfdagovcbergdlnssterasepticpdf

httpwwwemedicinehealthcomimages4453

httppathmicromedscedufoxlpsjpg

httpmicromedharvardedufacultyrudnerhtml

Theoretical Evaluation Whyte mathematical model contamination is due to air borne microbes

d = equivalent particle diameterA= area of container opening (cm2)

t = time (sec)

Cont rate (c) = 00032d2At

PostScript (conclusion)

The challenge in aseptic processing is always personnel

1048708 As a source of microbial andParticle contamination1048708As a brake on the implementation ofImproved technology

REFERENCES

Encyclopedia of pharmtechnology

RUSSELL A D Bacterial Spores and Chemical Sporicidal Agents clinical microbiology reviews 3(2) 99-119 (1999)

httpwwwfdagovcbergdlnssterasepticpdf

httpwwwemedicinehealthcomimages4453

httppathmicromedscedufoxlpsjpg

httpmicromedharvardedufacultyrudnerhtml

PostScript (conclusion)

The challenge in aseptic processing is always personnel

1048708 As a source of microbial andParticle contamination1048708As a brake on the implementation ofImproved technology

REFERENCES

Encyclopedia of pharmtechnology

RUSSELL A D Bacterial Spores and Chemical Sporicidal Agents clinical microbiology reviews 3(2) 99-119 (1999)

httpwwwfdagovcbergdlnssterasepticpdf

httpwwwemedicinehealthcomimages4453

httppathmicromedscedufoxlpsjpg

httpmicromedharvardedufacultyrudnerhtml

REFERENCES

Encyclopedia of pharmtechnology

RUSSELL A D Bacterial Spores and Chemical Sporicidal Agents clinical microbiology reviews 3(2) 99-119 (1999)

httpwwwfdagovcbergdlnssterasepticpdf

httpwwwemedicinehealthcomimages4453

httppathmicromedscedufoxlpsjpg

httpmicromedharvardedufacultyrudnerhtml

ThanQ

• Schedule (contents)
• Aseptic Processing
• Slide 4
• Causes of sepsis Sterile drug manufacturers should have a kee
• Slide 6
• Producing drug products by
• Terminal Sterilization
• Aseptic Processing
• Contaminating agents
• Humans and bacteria
• Sources of Contamination
• Control 1st step ndash eliminating the source of contamination
• Gowning (sterile barrier)
• Slide 15
• Aseptic Technique (skill)
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Sporicidal agents
• Isolators
• Microbial Environmental Monitoring Identification
• Slide 24
• Slide 25
• Genotypic Methods
• Slide 27
• Slide 28
• Microbiological testing of water
• Microbiological testing of air
• Characterization of aseptic process The four pillars of a robu
• Personnel Training amp Monitoring
• Environmental Monitoring
• Slide 34
• Facilities General Clean room Design
• Facilities Clean room Classification
• Slide 37
• Facilities HEPA Filters
• Media Fill test
• Slide 40
• Theoretical Evaluation
• PostScript (conclusion)
• REFERENCES
• ThanQ