Seminar on

Aseptic Processing operation

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.

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
Containe
r

Closure

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 compon

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 1000s 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)

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

Whats 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 30-35C
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

Personnel training & monitoring

Environmental monitoring
Facilities design
Media fills

Personnel Training & Monitoring

Avoiding contamination means knowing the potential sources

of contamination

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

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

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 0.3m 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 doesnt 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
30-35C for SCD, 20-25C 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) .

http://www.fda.gov/cber/gdlns/steraseptic.pdf

http://www.emedicinehealth.com/images/4453

http://pathmicro.med.sc.edu/fox/lps.jpg

http://micro.med.harvard.edu/faculty/rudner.html

ThanQ