Seminar on
Aseptic Processing operation
by
Ranjith Kumar kankala.
M.Pharm (I sem)
Department of Pharmaceutics
BLUE BIRDS COLLEGE OF PHARMACY
Affiliated to Kakatiya university
Warangal
2009
Schedule (contents)
 Introduction to aseptic processing,
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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.
“Sepsis is a serious medical condition characterized by a
whole-body inflammatory state caused by infection.”
Progression of Symptoms
Fever
Decreased Blood Pressure
Rapid Breathing and Heart Rate
Skin Lesions
Spontaneous Blood Clotting
Organ Failure
Death
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
patient.”
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" — free of contaminants — a
situation that is difficult to attain. However, the goal is
elimination of infection.
Producing drug products by
Terminal sterilization
Aseptic processing
 Product containers are filled and
 Drug product, container, 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.
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 –quality
environment.
Terminal Sterilization
Drug
Product
Container /
Closure
Excipiants
Sterilization
Process
Sterile
Drug
Product !
Aseptic Processing
Drug
Produc
t
Sterilization
Process
Container
Sterilization
Process
Sterile
Drug
Product
Sterile
Container
Closur
e
Sterilization
Process
Sterile
Closure
Excipient
Sterilization
Process
Sterile
Excipient
Aseptic
Processin
g
Sterile
Final
Product
Can use multiple sterilization processes each optimized for the individual component
Contaminating agents
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.
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 1000’s 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 – eliminating the source of
contamination !
2nd Step - Reduce the Risk of
contamination through:
 Sterile barriers
 Surface 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
Detection
and Counting)
Swabs
Aseptic Technique (skill)
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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
What’s wrong with this picture?
CORRECT
Unidirectional airflow
The operator should
never come between the
air source and the product.
pressure differential b/n
critical area from external
environment (17.5-50 Pa)
www.ors.od.nih.gov/ds/pubs/bsc/graphics/fig3.gif
Horizontal airflow
Vertical airflow
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!
e.g., 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 &
product.
Microbial Environmental Monitoring:
Identification
 Microbial identification should
extend to the species level.
 Routine traditional techniques
phenotypic and
biochemical.
 Genotypic techniques are
suggested for failure
investigations.
Identifying Microbes
Phenotypic technique
Gram Stain
Biochemical Assays
Reduction of
Tetrazolium Violet
Staphylococcus xylosus
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 Assay:
PCR: Polymerase Chain Reaction
Endotoxin Testing
Endotoxin: a pyrogenic (fever inducing) substance (e.g. lipopolysaccharide)
present in the bacterial cell wall. Endotoxin reactions range from fever to death.
LAL Assay (Limulus amoebocyte lysate)
ENDOTOXIN LIMIT FOR WFI IS
0.25 EU/ml
Extremely heat stable – recommended conditions for
inactivation are 180 0 C for 3 hours.
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 and/or
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 30-35°C
 Sampling procedures should follow those used in production
Microbiological testing of air
Compressed Air/Nitrogen/CO2
 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
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Personnel training & monitoring
Environmental monitoring
Facilities design
Media fills
Personnel Training & Monitoring
 Avoiding contamination means knowing the potential sources
of contamination
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Personnel
Equipment
Air/liquids
Drug product
Containers/closures
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.
Environmental Monitoring
Sampling
7.
Critical (processing) areas
6.
8.
Sampling of adjacent classified
areas (aseptic corridors, gowning
rooms, etc) will provide trend
data and may help identify
sources of contamination.
4.
3.
5.
13.
10.
9.
12
.
11.
2.
1.
Facilities: General Clean room Design
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HEPA/ULPA 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
ISO 14644-1
Cleanroom
Cleanroom
classification classification
≥0.5um
particles/m3
Viable
Microbes
(cfu/m3)
Ave Airflow
Velocity
(fpm)
Air
changes/hr
100,000
8
3,520,000
100
5-10
5-48
10,000
7
352,000
10
10-15
60-90
1000
6
35,200
7
25-40
150-240
100
5
3,520
1
40-80
240-480
Facilities: Clean room Classification
Class 10,000 clean room
Class 100 clean room
http://www.americancleanrooms.com/am/photogallery_08.html
Facilities: HEPA Filters
High Efficiency Particulate Air filters
Minimum particle collection efficiency:
99.97% for 0.3µm diameter particles.
Disposable
Filter made of pleated borosilicate glass
http://people.deas.harvard.edu/~jones/lab_arch/nano_facilities/hepa.gif
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 doesn’t 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 < 100 cfu challenge
 At least 14 days incubation
 30-35°C for SCD, 20-25°C 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
Cont rate (c) = 0.0032.d2.A.t
d = equivalent particle diameter
A= area of container opening (cm2)
t = time (sec)
PostScript (conclusion)
The challenge in aseptic processing is always
personnel:
􀂄 As a source of microbial and
Particle contamination.
􀂄As a brake on the implementation of
Improved technology.
REFERENCES

Encyclopedia of pharm.technology

RUSSELL A. D.. Bacterial Spores and Chemical Sporicidal Agents.
clinical microbiology reviews. 3(2): 99-119 (1999) .
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http://www.fda.gov/cber/gdlns/steraseptic.pdf
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http://www.emedicinehealth.com/images/4453
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http://pathmicro.med.sc.edu/fox/lps.jpg
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http://micro.med.harvard.edu/faculty/rudner.html
ThanQ