Aseptic Processing. The Community College of Baltimor

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Aseptic Processing

• The Community College of Baltimore County
  Continuing Education Economic Development
  
• The Northeast Biomanufacturing Center
  Collaborative (NBC2 )
• Tom Burkett, Ph.D.
• tburkett_at_ccbcmd.edu

2
Schedule Friday, October 26th

• 730 930
• Introduction to aseptic processing,
  contamination type and sources, environmental
  monitoring
• 945-1145 Aseptic Processing vs. Terminal
  Sterilization
• 1145-1215 Lunch
• 1215-215 Maintaining an Aseptic Environment,
  Filter sterilization, Media fills, sterility
  testing, principals of sanitary design

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What is Aseptic Processing?
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• Asepsis- 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
• Guidance for industry Sterile Drug Products
  Produced by Aseptic Processing-Current Good
  Manufacturing Practice. FDA, September 2004

5

• Asepsis is the practice to reduce or eliminate
  contaminants (such as bacteria, viruses, fungi,
  and parasites) from entering the operative field
  in surgery or medicine 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, not sterility.
• http//en.wikipedia.org/wiki/Asepsis

6

• Aseptic Processing is the processing of drug
  components ( drug product, containers,
  excipients, etc.) in a manner that precludes
  microbiological contamination of the final sealed
  product.

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Sterile drug manufacturers should have a keen
awareness of the public health implications of
distributing a nonsterile product. Poor CGMP
conditions at a manufacturing facility can
ultimately pose a life-threatening health risk to
a patient.
FDA Guidance Sterile Drug Products Produced by
Aseptic Processing-Current Good Manufacturing
Practice 2004.
8
Sepsis is a serious medical condition
characterized by a whole-body inflammatory state
caused by infection.
http//en.wikipedia.org/wiki/Sepsis
Progression of Symptoms
Fever Decreased Blood Pressure Rapid Breathing
and Heart Rate Skin Lesions Spontaneous Blood
Clotting Organ Failure Death
http//www.emedicinehealth.com/images/4453/4453-44
82-12996-21147.jpg
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Recalls Lack of Sterility Assurance
Number of Recalls
Fiscal Year

• Lack of Sterility Assurance is the 1 reason
  for drug recalls in last 5 years
• Nearly all drugs recalled due to Lack of
  Sterility Assurance
• in last 20 years were produced via aseptic
  processing

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Producing sterile drug products

• Aseptic processing

• Terminal sterilization

• 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 quality environment.

• 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.

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Terminal Sterilization
Sterile Drug Product !
Sterilization Process must be compatible with all
components !
12
Aseptic Processing
Drug Product
Sterilization Process
Sterile Drug Product
Sterile Final Product
Container
Sterilization Process
Sterile Container
Aseptic Processing
Closure
Sterilization Process
Sterile Closure
Excipient
Sterilization Process
Sterile Excipient
Can use multiple sterilization processes each
optimized for the individual component
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Global Scene European Agency for the Evaluation
of Medicinal Products (EMEA)

• Terminal Sterilization
• Fo gt 15 minutes

• Adjunct Processing
• Fo gt 8 minutes, and
• PNSU gt 1 in 106

Aseptic Processing

• From Decision Trees for the Selection of
  Sterilization Methods (10/1999)

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Terms

• PNSU - Probability of a Non-Sterile Unit
• The probability of a unit (product container)
  being non-sterile after the application of a
  lethal agent.
• PNSU of 1 in 106 -- the probability that a unit
  is non-sterile is one in a million
• FO - Sterilization Process Equivalent Time
• The equivalent number of minutes at 121.1C
  delivered to a unit by a sterilization process.
• FO 8 minutes -- the cycle delivered a microbial
  lethality equivalent to 8 minutes at 121.1C

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Probability of a Non-Sterile Unit (PNSU)

• Aseptic Processing
• Impossible to scientifically determine a PNSU
• Many critical systems involved
• Processing room
• Equipment
• Personnel
• Contamination Rate assessed with media fill.
• Simulated production run with media that promotes
  growth of microbial organisms.
• PNSU - Probability of a Non-Sterile Unit The
  probability of a unit (product container) being
  non-sterile after the application of a lethal
  agent. PNSU of 1 in 106 -- the probability that
  a unit is non-sterile is one in a million

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The four pillars of a robust aseptic process

• Personnel training monitoring
• Environmental monitoring
• Facilities design HVAC validation
• Process simulation (media fills)

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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!

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Types of Contamination
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Viable Nonviable particles

• Particles of dust, fibers, or other material are
  suspended in the air and may contaminate product.
  These particles may, or may not, contain living
  organisms (bacteria and their spores).
• The more particles in the air surrounding the
  product the more likely the product will be
  contaminated with those particles.
• Standards for particulate contamination were
  initially developed by NASA for moon exploration,
  those same standards were later adopted by the
  pharmaceutical and semiconductor industry.

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Sneezing produces 100s of thousands of aerosol
droplets that can then attach to dust particles.
In the absence of any filtration system these
particles which may contain bacterial spores, or
viruses may be present in the air for weeks.
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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.
22
Sepsis is a serious medical condition
characterized by a whole-body inflammatory state
caused by infection.
http//en.wikipedia.org/wiki/Sepsis
Progression of Symptoms
Fever Decreased Blood Pressure Rapid Breathing
and Heart Rate Skin Lesions Spontaneous Blood
Clotting Organ Failure Death
http//www.emedicinehealth.com/images/4453/4453-44
82-12996-21147.jpg
23
Endotoxins
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Endotoxin a pyrogenic (fever inducing)
substance (e.g. lipopolysaccharide) present in
the bacterial cell wall. Endotoxin reactions
range from fever to death.
http//pathmicro.med.sc.edu/fox/lps.jpg
Extremely heat stable recommended conditions
for inactivation are 180 0 C for 3 hours.
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Endotoxin effects
MACROPHAGE
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• What are SPORES
• Why are they a MAJOR CHALLENGE!!!!

http//micro.med.harvard.edu/faculty/rudner.html
http//www.samedanltd.com/members/archives/PMPS/Sp
ring2003/graphics/f1_p12.gif
Heat alone will not inactivate spores!
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Viral Contamination

• Viruses are small (nm) non-living entities that
  hijack the machinery of a host cell

http//www.scq.ubc.ca/.../2006/08/viralreplication
.jpg
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• Sources of Contamination

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Would you let these people into your processing
area ?
http//www.rit.edu/
http//www.imi.org.uk/
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Sick people arent the only source of
contamination!

• "The skin is home to a virtual zoo of bacteria"
  Martin J. Blaser New York University Medical
  Center

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

www.cellgenix.com/rundgang/pix/rg_7b.jpg
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1st step eliminate the source of contamination
!

• A Well designed, maintained, and operated
  aseptic process minimizes personnel
  intervention. As operator activities increase in
  an aseptic processing operation, the risk to
  finished product sterility also increases.

FDA Guidance Sterile Drug Products Produced
byAseptic Processing-Current Good Manufacturing
Practice 2004.
33

• 2nd Step- Reduce the Risk of contamination
  through
• Sterile barriers
• Aseptic technique
• Environmental monitoring

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Gowning
http//www.coleparmer.com/techinfo/techinfo.asp?ht
mlfileCleanroomGarments.htmID63
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Personnel Gloves
When are gloves worn? What compromises
gloves? How often should gloves be examined and
sanitized?
http//www.engenderhealth.org/IP/surgical/sum4.htm
l
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Qualifying Personnel

• Assess after gowning/gloving
• Microbiological surface sampling of several
  locations
• Glove fingers
• Facemask
• Forearm
• Chest
• Periodic requalification is necessary

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Gowning Qualification

• Written ( photographic) procedures describing
  methods used to don each gown component in an
  aseptic manner and the creation of barriers by
  overlapping gown components.
• Initial training and periodic assessment. Annual
  requalification in the case of automated
  operations and environmental control.

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Environmental Monitoring Surface
Monitoring
Touch or Contact plates - RODAC Plates
http//www.blood.co.uk/hospitals/services/Micro/Ba
ct2.htm
Swabs
www.esa.int
39
Personnel Behavior
Minimize movement Work slowly and purposefully
Note Light/heavy movement refer to partial body
movements (motioning with arm, tapping toes,
etc.). Change of position refers to whole body
motion (standing up, sitting down, etc.).
Austin Contamination IndexSource Encyclopedia of
Clean Rooms, Bio-Cleanrooms and Aseptic Areas,
Dr. Philip Austin, PE, 2000
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Aseptic Technique

• Contact sterile materials only with sterile
  instruments
• Sterile instruments should be held under Class
  100 conditions between uses and placed in sterile
  containers
• Operators should not contact sterile products,
  containers, closures, or critical surfaces with
  any part of their gown or gloves

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Whats wrong with this picture?
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CORRECT
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Aseptic Technique

• Keep the entire body out of the path of
  unidirectional airflow
• Unidirectional airflow design is used to protect
  sterile equipment surfaces, container-closures,
  and product. Disruption of the path of
  unidirectional flow in the critical area can pose
  a risk to product sterility.

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Unidirectional airflow The operator should never
come between the air source and the product.
Horizontal airflow
www.ors.od.nih.gov/ds/pubs/bsc/graphics/fig3.gif
Vertical airflow
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Aseptic Technique

• Approach a necessary manipulation in a manner
  that does not compromise sterility of the product
• Proper aseptic manipulations should be approached
  from the side and not above the product (in
  vertical unidirectional flow operations).
• Operators should refrain from speaking when in
  direct proximity to the critical area.

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Whats wrong with this picture?
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CORRECT
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Whats wrong with this picture?
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CORRECT
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Personnel Hygiene

• Avoid cleanrooms when ill
• Frequent bathing and shampooing
• Avoid getting sunburned
• Avoid cosmetics such as face powder, hair sprays,
  perfumes and aftershave
• Clothing should be clean, nonfrayed and
  nonlinting
• Avoid smoking

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FOREHEAD
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• ISOPROPYL ALCOHOL (70)
• Powerful disinfectant and antiseptic
• Mode of action denatures proteins, dissolves
  lipids and can lead to cell membrane
  disintegration
• Effectively kills bacteria and fungi
• But does not inactivate spores!

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Disinfection efficacy

• Suitability, efficacy limitations of
  disinfectant agents and procedures should be
  assessed.
• The disinfection program should include the use
  of a sporicidal agent used according to a written
  schedule and when environmental data suggests
  presence of spore forming agents (Baccilus spp.).

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• Endospores
• Mycobacteria
• Fungal Spores
• Small Non-enveloped viruses (polio, rotavirus,
  rabies)
• Vegetative Fungal Cells
• Enveloped Viruses (Herpes, Hepatitis B, Hepatitis
  C, HIV)
• Vegetative Bacteria

Most Resistant
Least Resistant
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Sporicidal agents

• Glutaraldehyde
• Formaldehyde
• Other aldehydes
• Chlorine-releasing agents
• Iodine and iodophors
• Peroxygens
• Ethylene oxide
• P-Propiolactone
• A. D. RUSSELL, 1999. Bacterial Spores and
  Chemical Sporicidal Agents. CLINICAL MICROBIOLOGY
  REVIEWS Vol. 3, No. 2 p. 99-119 .

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Equipment and bacteria

• Even seemingly smooth surfaces can harbor
  bacteria !

Scanning electron micrograph of Listeria
monocytogenes forming a biofilm in soy on a
stainless steel chip. Courtesy of Professor Amy
Wong.
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Critical Surfaces

• Critical surfaces Surfaces that may come in
  contact with or directly affect a sterilized
  product or its containers or closures. Critical
  surfaces are rendered sterile prior to the start
  of the manufacturing operation, and sterility is
  maintained throughout processing.

Guidance for Industry Sterile Drug Products
Produced by Aseptic Processing -Current Good
Manufacturing Practice, FDA, September 2004
58
Isolators

• Advantage
• No direct contact between operator product
• Critical that meticulous aseptic practices be
  followed including the use of sterile tools for
  manipulations

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Isolators

• Gloves, half suits, seals, gaskets and transfer
  systems should be covered by P.M. program
• Justified replacement frequency

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Isolators

• Gloves/half suits
• Choice of material P.M. program important
• Visually inspected every use.
• Physical integrity test routinely performed
• Recommend that a second pair of inner gloves be
  routinely used.

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Open Isolators Require laminar airflow over
critical areas Use pressure differential to
insure separation of critical area from external
environment (17.5-50 Pa 0.07-0.20 water
gauge) Local protection of opening to guard
against turbulent airflow and pressure waves
that could compromise critical area
Vertical airflow
www.ors.od.nih.gov/ds/pubs/bsc/graphics/fig3.gif
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Isolator Decontamination

• Vaporized agents often used
• Glutaraldehyde, Formaldehyde, etc
• Use indicator organisms to demonstrate
  effectiveness of decontamination
• Should be able to achieve a 4-6 log reduction in
  titer
• Biological indicator should be placed in
  multiple, justified locations throughout the
  isolator
• Hard to reach areas (between fingers on gloves)
  should be addressed
• Must show that defined concentration of
  decontamination agent is uniformly reached in
  validation studies

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ENVIRONMENTAL MONITORING

• In aseptic processing, one of the most
  important laboratory controls is the
  environmental monitoring program

Guidance for Industry Sterile Drug Products
Produced by Aseptic Processing -Current Good
Manufacturing Practice, FDA, September 2004
64
Aseptic Processing

• The key is a high quality environment

65
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
  and historical data.

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Environmental Monitoring

• What should an environmental monitoring program
  cover and how?
• All production shifts
• Air, floors, walls, equipment surfaces including
  critical surfaces.
• Critical surface sampling should take place at
  the conclusion of the aseptic processing
  operation.
• The location of surfaces to be samples, timing,
  and frequency of sampling should be specified in
  writing.
• Sufficient detail to allow reproducible sampling

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Environmental Monitoring
Where do we sample?
Critical (processing) areas
68
Environmental Monitoring
Where do we sample?
Critical (processing) areas
Sampling of adjacent classified areas (aseptic
corridors, gowning rooms, etc) will provide
trend data and may help identify sources of
contamination.
69
Items to include in environmental monitoring SOPs

• Frequency of sampling
• When the samples are taken (during or at
  conclusion of aseptic operations)
• Duration of sampling
• Sample size (surface area, air volume)
• Specific sampling equipment and techniques
• Alert and action levels
• Appropriate response to deviations from alert or
  action levels

70
Environmental Monitoring Particulate Air
Monitoring
Use of remote systems recommended in laminar flow
areas
71
Environmental Monitoring Surface
Monitoring
Touch or Contact plates - RODAC Plates
http//www.blood.co.uk/hospitals/services/Micro/Ba
ct2.htm
Swabs
www.esa.int
72
Environmental Monitoring Viable Microbial
Air Monitoring
Active Air Monitoring RCS Plus Passive Air
Monitoring Settling Plates
73
Media must be qualified to support growth of USP
indicator organisms

• To support growth of bacteria and fungi
• Inoculated with lt 100 cfu challenge
• Upper and lower limits for incubation 20-350 C,
  and not less than 14 days (if two temps 7 days at
  each temp)

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Typical media

• Soybean casein digest medium
• fluid thioglycollate medium (anerobes)

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Environmental Monitoring Trending Data

• Why is it important?
• Evaluate the disinfection efficiency
• Are certain microbes migrating into the aseptic
  processing area from a lesser controller area?
• Long and short term
• Data by
• Room
• Shift
• Operator
• Product
• Container type
• Filling line
• Sampling
• Testing personnel
• Isolate

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Environmental Monitoring Trending Data

• Averages of data can be misleading and mask
  unacceptable localized conditions.
• Alert and action levels should be set for each
  sample site
• Individual sample results should be evaluated
  against the action and alert levels

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Environmental Monitoring Identification

• Microbial identification should extend to the
  species level.
• Routine identification can be done using
  traditional phenotypic and biochemical
  techniques.
• Genotypic techniques are suggested for failure
  investigations

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QC Micro Identifying Microbes
Gram Stain
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Environmental Monitoring Identification

http//www.arches.uga.edu/kristenc/cellwall.html
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QC Micro Identifying Microbes
Metabolic Based Assays
Vitek
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Reduction of Tetrazolium Violet
Staphylococcus xylosus
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Genotypic Methods

• Use DNA sequence (often ribosomal RNA genes rDNA)
  to identify organism
• Faster, and more accurate then traditional
  biochemical and phenotypic techniques

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PCR Polymerase Chain Reaction
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DNA SEQUENCE DATA
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Endotoxin Testing
Endotoxin a pyrogenic (fever inducing)
substance (e.g. lipopolysaccharide) present in
the bacterial cell wall. Endotoxin reactions
range from fever to death.
http//pathmicro.med.sc.edu/fox/lps.jpg
Extremely heat stable recommended conditions
for inactivation are 180 0 C for 3 hours.
90
QC Micro LAL Assay (Limulus amebocyte lysate)
ENDOTOXIN LIMIT FOR WFI IS 0.25EU/ml
91
LAL Assay

• Sensitivities down to .005 EU/ml (Lonza)
• http//www.lonzabioscience.com/Content/Documents/B
  ioscience/Endotoxin20limits.pdf

92
Endotoxin a pyrogenic (fever inducing)
substance (e.g. lipopolysaccharide) present in
the bacterial cell wall. Endotoxin reactions
range from fever to death.
http//pathmicro.med.sc.edu/fox/lps.jpg
Extremely heat stable recommended conditions
for inactivation are 180 0 C for 3 hours.
93
The Lysate
QC Micro LAL Assay
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QC Micro LAL Assay

• Types of LAL assays
• Gel Clot a clot forms and stays intact at the
  bottom of the assay tube.
• Turbidity An increase in turbidity (cloudiness)
  is seen.
• Chromogenic a color indicator is used to signal
  a positive result.
• Endpoint - Measure turbidity/absorbance after a
  definitive time period.
• Kinetic - Measure rate of increased
  turbidity/absorbance.

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Facilities

• Establishing and Maintaining an aseptic
  environment
• Use clean-rooms of various classes to establish
  an aseptic area
• Clean rooms use combinations of filtration, air
  exchange, and positive pressure to maintain
  clean environment
• Lower quality clean areas should not be placed
  next to high quality areas

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Facility Design Clean Area Classification
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Facilities General Cleanroom Design

• HEPA/ULPA filters on ceiling
• Exhaust vents on floor
• Drains in aseptic processing areas are
  inappropriate
• 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

98
Facilities HEPA Filters
High Efficiency Particulate Air 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/nan
o_facilities/hepa.gif
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Facilities Cleanroom Classification
Class 10,000 cleanroom
Class 100 cleanroom
http//www.americancleanrooms.com/am/photogallery_
08.html
100
Facilities Cleanroom Classification
101
Facilities Pressure Differentials

• Used to maintain airflow in the direction of
  higher cleanliness to adjacent less clean areas
• A minimum of 10-15 Pascals should be maintained
  between the aseptic area and an adjacent rooms
  with differing cleanroom classifications (doors
  open)

102
Facilities Air Lock
Permits the passage of objects and people into a
cleanroom. Consists of two airtight doors in
series which do not open simultaneously. Spray
down materials with 70 IPA before placing in the
airlock
http//news.thomasnet.com/images/large/451/451402.
jpg
103
Facilities Material NOT permitted in a
Cleanroom

• Fiber-shedding materials such as cardboard and
  paper
• Cardboard packaging must be removed and items
  placed into non-cardboard containers.
• Wood (i.e. wooden pallets)
• Undesignated charts

104
Facilities Cleaning
1. Vacuum all accessible surfaces
3. Mop floors using a lint free polyester mops
attached to stainless steel handles
2. Wipe surfaces with a cleaning solution
Water should be changed FREQUENTLY
105
Isolators

• The use of isolators prevents direct contact with
  product
• However, the use of isolators can lead to
  relaxation of aseptic practices!

106
Sterile Filtration

• Sterilizing grade filters (0.22µm pore size or
  smaller)
• Use of redundant sterilizing filters should be
  considered
• Filters should be validated including the use of
  microbial challenge

107
Dead End (Perpendicular Flow) Filters

• Traditional, dead end (perpendicular)
  filtration remains the method of choice for
  sterile filtration of process gasses, media and
  product liquids.
• Dead end filters may be purchased pre-sterilized
  or steamed in place are generally used once and
  discarded.
• Membrane types
• Cartridges
• Loose membranes

108
Dead End (Perpendicular Flow) Filters

• Plate Frame

• Depth

109
Materials

• polyvinylidenedifluoride (PVDF)
• Polytetrafluoroethylene(PTFE)
• Polyethersulfone (PES)
• Nylon 6,6 (N66)
• Choice depends on properties (i.e. hydrophobic or
  hydrophilic, and compatibility with process
  materials)

110
Filter Validation

• Factors that affect filter performance
• Viscosity surface tension of material to be
  filtered
• pH
• Filter membrane compatability
• Pressures
• Flow rates
• Temperature
• Osmolality
• Hydraulic shock

111
Filtration microbial challenge

• Should reflect worst case scenario based on
  product bioburden profile
• Use of Brevundimonas diminuta (ATCC 19146)
  commonly used (Acholeplasma laidlawii (0.10 µm )
  and Serratia marcescens (0.45 µm ) also used)
• Small size (0.3 µm) allows testing of sterile
  filtration units (0.2 µm)
• Challenge should be at least 107 organisms / cm2
  of filtration area

112
Filtration microbial challenge

• Direct inoculation of challenge organism into
  drug product should be considered
• Must consider nature of drug product
  (bactericidal and oil based formulations may
  compromise results)
• Can simulate by first processing drug product
  under worst case conditions followed by
  microbial challenge using drug product lacking
  bactericidal agent as vehicle

113
Number of uses

• Sterile filters should be routinely discarded
  after the processing of a single lot
• If repeated use can be justified the validation
  plan should include the maximum number of lots to
  be processed before replacement

114
Filtration Time Limit

• An established maximum time limit to achieve
  filtration should be established and adhered to.
• The established maximum time limit should be
  based on upstream bioburden and endotoxin load

115
Integrity Testing

• Filter integrity is essential to filter
  performance. Integrity testing can be performed
  prior to processing but must be performed after
  filtration.
• Integrity testing methods
• Forward Flow
• Bubble point
• Based on airflow through a given filter at a
  specific pressure

116
Media Fills

• Used to validate the aseptic process
• Use microbial growth media instead of drug
  product-any contamination will result in
  microbial growth

117
Media Fill Study design

• The design of a media fill study should address
  the following factors
• Any factors associated with longest run that can
  pose contamination risk
• Representative interventions that occur during a
  normal run as well as any nonroutine
  interventions
• Lyophilization (if applicable)
• Aseptic assembly of equipment
• Number of personnel and their activities
• A representative number of aseptic additions
• Shift change, breaks, gown changes
• Aseptic equipment connections/disconnection
• Aseptic sample collection
• Line speed and configuration
• Weight checks
• Container closure system

118
Media Fill Study design

• A minimum of three separate and consecutive media
  fills should be done for the initial
  qualification.
• Subsequent semi-annual requalification
• All personnel authorized to enter the aseptic
  processing room during manufacturing should
  participate in a media fill at least once a year
  and their participation should reflect the extent
  of their duties in production.

119
Media Fill Study design

• Size, duration, and speed of production runs
  should be mimicked in media fill studies
• When aseptic processing employs manual filling or
  closing, or extensive manual manipulations, the
  duration of the process should be generally no
  less than the length of the actual manufacturing
  process
• Number of units filled during the media fill
  should be based on the contamination risk
• 5,000 to 10,000 units a good starting point
• If run size is less than 5,000 units then media
  fill should be at least equal the maximum batch
  size.

120
Media Fill Study design

• Size, duration, and speed of production runs
  should be mimicked in media fill studies
• Media fill studies should address the range of
  line speeds used and each study should evaluate
  one speed

121
Media Fill Study design

• Environmental conditions
• Should be representative of actual manufacturing
  operations
• Maximum number of individuals and elevated
  activity levels allowed under SOPs

122
Media Fill Study design

• Media fills should be observed by the QC unit
• Video recording should be considered

123
Media Fill Study design

• Modern aseptic processing operations in suitably
  designed facilities have demonstrated a
  capability of meeting contamination levels
  approaching zero
• When filling lt5,000 units, no contaminated units
  should be detected. One (1) contaminated unit is
  considered cause for revalidation, following an
  investigation.
• When filling 5,000 to 10,000 units, One (1)
  contaminated unit should result in an
  investigation, including consideration of a
  repeat media fill. Two (2) contaminated units are
  considered cause for revalidation, following
  investigation
• When filling gt10,000 units , One (1) contaminated
  unit should result in an investigation. Two (2)
  contaminated units are considered cause for
  revalidation, following investigation

124
Revalidation

• At least semi-annually
• After any significant change
• Facility and equipment modifications, line
  configuration changes, significant changes in
  personnel, environmental testing anomalies,
  container closure systems, extended shutdowns, or
  end product sterility testing failure, etc.

125
Change Control

• A written change control process should be in
  place
• Any change to the product or production line
  should be evaluated using this process

126
Sterility Testing

• 21 CFR 211.167 For each batch of drug product
  purporting to be sterile and/or pyrogen-free,
  there shall be appropriate laboratory testing to
  determine conformance to such requirements.

127
Which test method should I use?

• USP lt71gt Sterility Tests is principal source
  of sterility test methods including procedures
  and methods
• Methods validation must address issue of false
  negatives!

128
Sterility Testing

• Be aware that sterility testing has serious
  limitations due to small sample sizes typically
  used.
• Therefore the FDA considers any sterility test
  failures to be serious CGMP issues that should
  be thoroughly investigated.

129
Sterility Testing

• Microbial growth in sterility test is grounds for
  considering entire lot non-sterile.
• Only if the growth can unequivocally be assigned
  to laboratory contamination can test result be
  considered invalid.

130
Sterility Testing Investigations

• In the investigation stemming from a negative
  sterility test consideration should be given to
• Speciation of the organism
• Record of laboratory results and deviations
• Environmental monitoring of production
  environment
• Monitoring personnel
• Product Presterilization bioburden
• Production record review
• Manufacturing history

131
Sterility Testing

• Any isolates from sterility testing should be
  identified to species level.
• The use of genotyping techniques is encouraged.
• Identical methodologies should be employed in
  species identification in sterility test and
  environmental monitoring program.

132
Thank You

• For further information see
• Guidance for Sterile Drug Products Produced by
  Aseptic Processing
• http//www.fda.gov/cber/gdlns/steraseptic.pdf
• Guideline for Validation of Limulus Amebocyte
  Lysate Test as an End Product Endotoxin Test for
  Human and Animal Parenteral Drugs, Biological
  Products, and Medical Devices
• http//www.fda.gov/cber/gdlns/lal.pdf

133

• Guidance for submission of Documentation for
  Sterilization Process Validation in applications
  for Human and Veterinary Drug Products
• http//www.fda.gov/cder/guidance/cmc2.pdf
• Guide to Inspections of Microbiological
  Pharmaceutical Quality Control Laboratories
• http//www.fda.gov/ora/Inspect_ref/igs/micro.html
• Guide to Inspections of Sterile Drug Substance
  Manufacturers
• http//www.fda.gov/ora/inspect_ref/igs/subst.html