STERILE PROCESS

VALIDATION

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 Contents
Introduction

Four pillars
Validation plan
Major steps
Basic principles
Sterilization methods
Validation of support system and facilities
Media fills
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 Introduction
Aseptic / sterile - “ A state of control attained by using an
aseptic work area and performing activities in a manner that
precludes microbiological contamination of the exposed sterile
product”
Validation of aseptic process should be designed to provide
assurance through appropriate testing that all phases and
activities of the process remain sterile and it is controlled
within the predetermined parameters.
Drug product, container, and closure are subject to sterilization
separately, and then brought together.

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 Aseptic Processing – Overview
Certain pharmaceutical products must be sterile
– injections, ophthalmic preparations,
irrigations solutions, haemodialysis solutions.
Two categories of sterile products
– those that can be sterilized in final container
– those that cannot be terminally sterilized and must be
aseptically prepared.
SAL achieved in an aseptic operation depend on aseptic
technique.
(SAL:- sterility assurance level)

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 The Four Pillars of a
Aseptic Process
 Personnel training & monitoring
 Environmental monitoring
 Facilities design & HVAC validation
 Process simulation (media fills) (It is the performance of an
aseptic manufacturing procedure using a sterile microbiological growth
medium in place of the drug solution.
PURPOSE OF VALIDATION
 Minimize reliance on end product testing.
 To build sterility into a product.
 Increase SAL to all units.
 To provide greater assurance and support the end product
of sterility testing. 5
For All Process Validation Plan
Include
IQ- specification set by mfg.

OQ-demonstration of reliability of a equipment.

Product validation- consistently meet the specification for

acceptance and it has been shown to be stable under conditions
of the process under consideration.
Process validation- process consistently produce the product
meet the specification for acceptance.

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 Aseptic Processing
Drug Sterile
Sterilizatio
Drug Product
Product n
Process

Container Sterilizatio Sterile

n Container
Process
Sterile
Final
Aseptic Produc
Sterilizatio Sterile
Closure Processin t
n Closur
Process e g

Sterile
Excipient Sterilizatio
n Excipien
Process t

Can use multiple sterilization processes each optimized for the individual component
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 Terminal Sterilization

Sterile Drug Product !

Sterilization Process must be compatible with all components !

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 Basic Principles In Validation Of
Any Process
 Written documentation

 Mfg parameters

 Testing parameters

 In-process control

 Final product testing

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 Major steps
 Select or define the desired attributes of the product.

 Determine specification.

 Select the appropriate processes and equipment.

 Develop and conduct the tests that process,

evaluate equipment and personnel.
 Examine the test procedure to ensure accuracy

and reliability.

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 D,F and Z values-
D value- Time required for 90% reduction in microbial
population.

Z value- A Z-value is defined as the number of degrees

(Celsius or Fahrenheit) required to change a D-value by
one factor of ten.

F value- It measures equivalent time that monitored article is

exposed to desired temp.
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 Sterilization Method

1. Steam sterilization

2. Dry heat sterilization

3. Radiation sterilization

4. Filtration sterilization

5. Gaseous sterilization

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 Validation of Steam Sterilization
Heat distribution studies-
In production size sterilizer, 15-20 thermocouples are used &
distributed geometrically throughout sterilizer.
Thermocouple should be placed in the exhaust drain which
is adjacent to the sensor that control vessel temperature.
Temperature deviation should not greater than +2.5
degree Celsius of the mean chamber temperature.
Heat penetration studies-
 The microorganisms most frequently used to challenge moist
heat sterilization cycles are bacillus stearothermophilus &
clostridium sporogenes.
 These studies are typically conducted concurrently
with the heat penetration studies
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 Dry Heat Sterilization

Qualification of all equipments and instrumentations.

Heat distribution and heat penetration study.

Mechanical reliability.

Biological process validation(ex-B.subtilis)

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 Gaseous Sterilization
Address the product specification and package design.
Verify calibration of all equipment and instruments.
Monitoring with thermocouples and biological indicators.
Repetitive runs with loaded EtO sterilizer.
Testing .

Radiation Sterilization
Address the product specification and package design.
Licensing agreement
Reliability and calibration of dosimeter system
Radiation source strength.
Dose rate.

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 Filtration Sterilization
Filter Validation

Filter must be validated to demonstrate ability to remove

bacteria(Bacterial challenge test).
most common method is to show that filter can retain a

microbiological challenge of 107 CFU of Brevundimonas

diminuta per cm2 of the filter surface
preferably the microbial challenge is added to the fully

formulated product which is then passed through the filter.

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 Conti……
….
if the product is bactericidal, product should be passed
through the filter first followed by modified product
containing the microbial challenge (after removing any
bactericidal activity remaining on the filter)
filter validation should be carried out under worst case
conditions e.g. maximum allowed filtration time and
maximum pressure

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 Aseptic Processing
Filter integrity
• Filters of 0.22μm or less should be used for filtration of
liquids and gasses (if applicable)
– filters for gasses that may be used for purging or
overlaying of filled containers or to release vacuum in
lyophilization chamber
• filter integrity should be verified before filtration
and confirmed after filtration
– bubble point
– pressure hold
• methods are defined by filter manufacturers and limits
determined during filter validation

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Aseptic Processing: Essential
Elements
Facility

Documentation Equipment

Aseptic
Finish Product
Processing
Process
Testing

Control &
Personnel
Verification

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Validation of Facility & Support
System
Facility  Support System

Room classification  Water system

Airflow patterns Air system

Pressure differentials Equipment sterilization

Personnel flow patterns Filtration system

Material flow patterns Clean steam system

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 Manufacturing Environment
Facility

Class (0.5μ ISO ≥ 0.5μ WHO Grade

parts/ft3) Designation particles/
m3)
100 5 3520 A
1000 6 35200 B
10000 7 352000 C
100000 8 3520000 D

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 Manufacturing Environment
Four grades of clean areas:
 Grade D (equivalent to Class 100,000, ISO 8):
• Clean area for carrying out less stages in
manufacture of aseptically prepared
critical eg.
products of components after washing.
handling

 Grade C (equivalent to Class 10,000, ISO 7):

• Clean area for carrying out less critical stages
manufacture of aseptically prepared in
products of solutions to be filtered.
preparation eg.
Grade B (equivalent to Class 1000, ISO 6):
• Background environment for Grade A zone, eg.
clean room in which laminar flow workstation is
housed.
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 Manufacturing Environment
Grade At rest In operation

maximum permitted number of particles/m3

0.5 - 5.0 µm > 5 µm 0.5 - 5.0 µm >5 µ
A 3 500 0 3 500 0
B 3 500 0 350 000 2 000
C 350 000 2 000 3 500 000 20 000
D 3 500 000 20 000 not defined not defined

“At rest” - production equipment installed

and operating
“In operation” - Installed equipment
functioning in defined operating mode and
specified number of personnel present
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 Manufacturing Environment
• Grade A (equivalent to Class 100 , ISO 5) :

– Local zone for highrisk operations eg. product filling,

stopper bowls, open vials, handling sterile materials, aseptic

transfer of partially stoppered containers to be lyophilized.
connections,

– Conditions usually provided by laminar air flow workstation.

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 Manufacturing
Environment
Environmental Monitoring - Physical
• Air Changes/Airflow patterns
– Air flow over critical areas should be uni-directional
(laminar flow) at a velocity sufficient to sweep particles
away from filling/closing area
– for B, C and D rooms at least 20 volume changes per hour
are usually required.
• Clean up time/recovery
– Particulate levels for the Grade A “at rest” state should be
achieved after a short “clean-up” period of 20 minutes after
completion of operations (guidance value)
– Particle counts for Grade A “in operation” state should be
 Manufacturing
Environment
Environmental Monitoring - Physical
• Temperature and Relative Humidity
– Ambient temperature and humidity should
not be uncomfortably high (18-22°C) & 30-60 % RH.

• Airflow velocity
– Laminar airflow workstation air speed of approx 0.45m/s

± 20% (80-120ft/min) at working position

(guidance value)

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 Manufacturing Environment
Environmental Monitoring - Physical
• Differential pressures
– Positive pressure differential of 10-15 Pascal's should
be maintained between adjacent rooms of different
classification (with door closed)+ve pressure should be
0.2-0.3 water gauge.
– Most critical area should have the highest pressure

– Pressures should be continuously monitored

frequently recorded.
and
– Alarms should sound if pressures deviate.

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 Manufacturing Environment
Personnel
• Minimum number of personnel in clean areas
– especially during aseptic processing
• Inspections and controls from outside
• Training to all including cleaning and maintenance staff
– initial and regular
– manufacturing, hygiene, microbiology
– should be formally validated and authorized to enter aseptic
area
• Special cases
– supervision in case of outside staff
– decontamination procedures (e.g. staff who worked
with animal tissue materials)
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 Manufacturing Environment
Personnel
• Outdoor clothing not in change rooms leading to Grade B
and C rooms
• Change at every working session, or once a day
• Change gloves and masks at every working session
• Frequent disinfection of gloves during operations
• Washing of garments – separate laundry facility
– No damage, and according to validated
procedures (washing and sterilization)
• Regular microbiological monitoring of operators 29
Personnel flow pattern

Area 1
gowning 1
Area 2
gowning 2
Area 3
additional gowning
Area 4

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 Personnel monitoring

Gowning process must be validated.

Done by Glove fingertips into Petri dish containing
agar media.

Material Flow
Entry through air lock system or sterilizing ovens or filters.

Contact plate testing should be done for assurance.

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 Nonviable Particulate Monitoring

HVAC Validation and

Maintenance Considerations:
Air velocity, airflow patterns and turbulence should be
validated; smoke studies to determine flow patterns during
static and dynamic conditions
High pressure drop across the filters shows blocked.
HEPA filter integrity testing
HEPA filter efficiency testing
Air pressure differentials
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 Nonviable Particulate Monitoring
Airborne cleanlinessclassifications should be met during
operations

Nonviable monitoring should occur routinely during operations

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 Validation of Support System
Water system:-
Objective-
To provide assurance that the system eliminates
endotoxins
from incoming water and prevention
of
formation. endotoxins
Validation-
1. System description including specifications.
2. Installation qualification.
3. Operational qualification.
4. To prove that system delivers the WFI.
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 Validation of Water System

To monitor water system for both microorganisms

and

endotoxins.

Validation report include description of system along with blue

print.

Report should contain a diagram showing all sampling points. 35

 Filtration system

All process gases/liquid entering into the aseptic core must be

validated as sterile and particle and pyrogen free.

The validation protocol consider not only bioburden of

incoming solutions but also effect of viscosity,pH,ionic
strength, flow rate, temperature & pressure on the ability of
the filter to remove microorganisms.

Filtration should be carried out under positive pressure.

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 Air System

HEPA filter system validated by introducing a DOP(Dioctyl

Phthalate) aerosol into duct system

The conc. Of DOP should be in 80-100mcg/lit of air per min.

The validation protocol includethe capability of filter

to remove appropriate amount of particles.

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 Equipment Sterilization
• Equipment includes tanks, centrifuges and dryers is intended to
be sterilize before use.
• The validation program should show the effectiveness
of
disinfecting program.
• It must prove that being delivered meets criteria
for
steam WFI, pyrogens,and
• bioload.
It also prove that steam is sterilizing all surfaces.
• Heat distribution study should be done to determine the
cold spots where condensate could accumulate.
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 Media Fill Trials
 Verification of medium sterility
 Aseptic filling operation
 Challenge unit incubation
 Evaluation of result
Media used:-
 Soybean casein digest media
 Fluid thioglycolate media
Media fill frequency:-
 6 months interval.
 If change in aseptic process
Acceptance criteria:-
 Historically 0.3%(as per WHO)
 0.1% as per Parentral Drug Association. (PDA)

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 Conclusion
We have seen that how the validation is being carried out for
aseptic process validation.

To achieve the high standards of purity & quality product.

The definitive concern for aseptic processing is the presence of

personnel to perform complex operations while maintaining a
sterile field.

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 References
1. Akers MJ, Anderson NR, Sterilization Validation, In: Nash RA,
Wachter AH, Pharmaceutical Process Validation, 3rd ed., USA:
Informa Healthcare , 2003, p. 83-157
2. Agalloco J, Akers JE, Validation of Aseptic Processing, In: Carleton FJ,
Agalloco J, Validation of Pharmaceutical Processes: Sterile Products, 2nd
ed., USA: Informa healthcare, p.669-702

3. Kasubick RV, Validation of Sterile APIs, In: Berry IR, Harpaz D,

Validation of Active Pharmaceutical Ingredients, 2nd ed. New York: CRC
PRESS, 2001, p. 432-449

4. Potdar MA, Pharmaceutical Quality Assurance, 2nd ed., Pune: Nirali

Prakashan , 2007, p.8.1-8.22

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 Resources
I. www.fda.gov/FDA Guidance for Industry.
II. www.validation.org
III. www.biomanufacturing.org

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TH A N K Y O U

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