Microbilogical control

1. Chapter 8
Microbiological Control

2. Chapter Objectives
 Explain why microbiological control is important
 Provide examples of how it is achieved and maintained
 Describe the various sources of microbial contamination, name specific
contaminants and their possible sources
 Explain the different microbiological cleanliness standards required
 Define aseptic processing
 Identify measures taken in controlled and classified environments
within clean rooms to prevent microbial contamination
 Describe the components of an effective environmental monitoring
program
 List quality control practices that are essential in the Microbiology QC
laboratory

3. Microbiological Control
Vital for two main reasons:
 the majority of biopharmaceutical medicines are
designed for parenteral administration and will bypass
the body’s natural external defense mechanisms
 biopharmaceutical drug substances are generally large,
complex molecules that are susceptible to degradation
from enzymes produced by contaminating microbes

4. Bacteria, Fungi and Mycoplasma
 Found in every environment including food, water, air
 Crucial to life as we know it
 Decomposition and recycling of elements for future generations
 Digestion of food
 Symbiotic with many organisms
 Rhizoids with plants
 Cellulase for termites and ruminants
 Important in food production
 Cheese
 Brewing
 Saurkraut

5. Impact of Bacteria, Fungi &
Mycoplasma on Production
 Contamination and or protein degradation/modification possible at all
stages of production
 Goal in cell culture is to maintain axenogenic or monoseptic (aseptic)
cultures that contains only the engineered cell of interest
 achieved through stringent controls of operating equipment and
conditions
 Contaminating microbes typically overgrow the mammalian cell cultures
to out compete for the available nutrients
 Results in loss of batch and product
 More difficult to detect in a microbial cell culture but is still of concern
due to modifications of product and possible residual contaminating
proteins

6. Mycoplasma
 Common contaminant in cell culture
 Smallest, free-living prokaryote; lack a cell wall
 0.2 – 0.3 micron in diameter
 Obligate parasite, requires cholesterol from host cell
 Can grow to very high concentrations in mammalian
cell cultures, but remain unobservable by light
microscopy
 Viewed by fluorescent staining of the nuclear material
 usually no overt signs that cultures are contaminated

7. Types of Mycoplasma
 5 common species
 M. hyorhinis
 M. arginini
 M. orale
 M. fermentans
 Acholeplasma laidlawii

8. Mycoplasma Contamination
 Contamination includes
 Changed growth characteristics of cell line
 Changed membrane antigenicity
 Changed cell metabolism
 Chromosomal aberrations
 Disrupted nucleic acid synthesis
 Altered transfection rates
 Increased viral susceptibility
 Significant safety and regulatory concerns
 Most common sources of contamination
 The production cell line itself
 Raw materials
 Production personnel
 environmental

9. Mycoplasma – Detection/Monitoring
 Detection technologies include
 Growth on special media plates; may require up to a month for adequate
growth
 Fluorescent staining of the nuclear material using Hoechst 33258 dye
 PCR, primarily of the 16S rDNA sequence
 Monitoring
 In the cell culture process – a closed system of cells in a vessel with
nutrients incubated for some period of time from days to weeks before
harvest
 Extraction and purification processes have no cells therefore Mycoplasma
(and viruses) are not considered problematic

10. Bacteria and Molds
Bacteria and molds
 Greatest concern during extraction of protein from cell
culture and purification
 Typically carried by people working with the process
Concerns :
 control of their numbers – can overwhelm the capacity
of downstream filtration processes to remove them
 metabolites/products can be harmful to therapeutic
protein or to the patient

11. Post-harvest processes
 Considered an open system of equipment and
environment
 Many opportunities for introduction of bacteria or
molds
 Some equipment / materials are difficult to sterilize
 Filter membranes
 Chromatography resins
 Generally the open environment
Bacteria & Mold Post-Harvest Considerations

12. Post-Harvest Considerations Cont’d
 Bacterial structures or products that impact the product
 Proteolytic enzymes that degrade the product
 Gram- cell walls releasing endotoxins (pyrogens)
potentially capable of being harmful to patients
 Parenteral delivery (injection)
 Majority of biopharmaceuticals
 Must remain free of viable organisms to protect
patient from infection

13.  1970’s – enterococci considered “opportunistic pathogens”
contaminated infusion fluids resulted in the deaths of a number
recipient patients
 2002 – fungal meningitis in a spinal injection killed a 77-year old
women; the source was found to be a rare fungal species in the
environment where production took place
 2012 – an repeat outbreak of fungal meningitis occurred in several
patients receiving infusions manufactured in a plant eventually shown
to not be properly designed to produce that product nor were they
properly monitored
Historical Examples

14. Prions
 Misfolded Infectious proteins
 Prion = Protein Infection
 No associated nuclear material
 Replicate by inducing other similar proteins to misfold
 Accumulation leads to neurological disorders
 Scrapie (sheep)
 Creutzfeldt-Jakob disease (CJD, humans)
 Bovine Spongioform Encephalopathy (“mad cow”)
 Encompassing classification – Transmissible Spongioform
Encephalopathies (TSE)
 Transmitted by eating nerve tissue (brain, CNS) of infected animals
 Animal feed containing ground offal

15. Prions Relevance
Number of animal-derived materials used in production
 Gelatin
 Amino acids
 Fetal Bovine Serum (FBS)
 Examples of Prion Infections
 Human Growth Hormone (HGH) derived from pituitary gland was
shown to carry TSE from donors with CJD
 Expedited the use of rDNA to produce CJD-free HGH
 Mad cow disease in the UK was determined to have been
transmitted to farm workers
 No cases have been documented as being derived from pharmaceuticals

16. Endotoxins
 What is it?
 toxin (lipopolysaccharide)
 Where does it come from?
 The cell membrane of Gram negative bacteria
 Which products are tested?
 Injectable drugs and medical devices which will contact
blood or spinal fluid
 raw materials, water and in process monitoring

17. • Potent, toxic, very stable and present in many
pharmaceutical ingredients and on surfaces that come
into contact with the product when formulated for
parenteral administration
• Water soluble, and will pass through 0.2 µm filters
• Not destroyed by autoclaving and are insoluble in
organic solvents
• Very difficult to eliminate in a final preparation
Endotoxin

18. Endotoxins Cont’d
 Released when bacterial cells are disrupted
 Extremely heat stable – conditions for
inactivation are 180°C for 3 hours
 Pyrogenic (fever- inducing)
 Lowers blood pressure
 activates inflammation and coagulation

19. Generally accepted endotoxin limit (EL) is defined as
acceptable endotoxin load that the body can generally
tolerate without experiencing the associated adverse events
 5.0 EU/kg for parenteral (intreavenous or intravenous)
drugs
 0.2 EU/kg for the intrathecal (spinal) route of
administration
Endotoxin Limits

20. Viruses
 Submicroscopic intracellular infectious agents
 First visualized by electron microscope
 Simplest forms consist of genetic material and a protective protein coat
 Only replicate within host (plant, animal, human, prokaryotes) cells,
therefore, not considered living
 Infectious within a cell culture
 Potential to alter the metabolism of the cultures cell, changing or eliminating the
desired product
 Contaminant in the product
 Numerous examples of serious outcomes due to contaminated
medicines
 Transfer to patient leads to deleterious, often lethal, infections

21. Examples of Virus Contamination
 HIV virus in pooled blood products, most notably in injections for hemophiliacs
 Before a detection test was available, as many as 10,000 hemaphiliacs were
unintentionally infected
 A smaller but significant number of blood transfusion recipients were also
infected
 A single infected donor to the pool will contaminate the entire pool
 A childhood disease vaccine contaminated with an apparently harmless circovirus
 Although not known to cause a human disease, vaccinations were
suspended until the contaminant was removed
 Not enough information or testing to determine that there was no adverse
affect due to contaminant

22. Viral Contamination
Issues associated with virus contamination
 Detection of virus
 How to test for all known viruses vs medically important viruses
 Test for the commonly known viruses of concern
 Discovery of new viruses, or mutated forms of old viruses, make it difficult to
maintain a complete battery of detection tests
 Elimination of viruses
 Stringent control of all processes and components to ensure the product is virus
free
 Where feasible, inactivation or removal of virus
 More ultrafiltration techniques are being built into biopharmaceutical processes to
eliminate contaminating viruses

23. Control During Production
Control is a continuing challenge in pharmaceutical production
 Biopharmaceutical processes use many organic materials that
may initially be contaminated before use
Critical issue because
 Majority of biopharmaceuticals are parenteral
(injectable) and must not cause infections or
inflammatory reactions
 Biopharmaceuticals are generally large, complex
proteins that are susceptible to microbial degradation

24. Microbiological Control in Biomanufacturing
 Manufacture of biopharmaceuticals
 Begins with closed system, axenic mono-culture
 Onto low bioburden purification
 Finish with aseptic finish/fill to produce a sterile dosage
 During the cycle
 Various microbial agents can enter in various ways
 Some may be tolerated, often zero tolerance to microbes
 Necessary to understand, monitor and control products and
impurities (microbes, metabolites, etc)
 Often difficult to determine the concentration or absence of
impurities
 Control at the environmental level is often preferred

25. Manufacturing Process
Typically two independent activities
 Manufacture of a drug substance
 Manufacture of a drug product
 May occur in separate facilities (or even countries)
 Each has its own specific, but different, requirements

26. Manufacture of Drug Substance
 Relatively long, discontinuous set of process steps
 Product is a solution that is allowed to have a low
bioburden; however, still need to exclude extraneous
microbes to retain quality
 Monitored with a Bioburden Control Strategy document
 Ongoing analysis and understanding of the microbes in
the air, water, surfaces and the people is required

27. Manufacture of Drug Product
 Preparation of the individual sterile dosage form units from the
drug substance
 One relatively short, continuous process
 Need to monitor facility, people and processes to achieve sterility
 Additional elements to monitor
 Excipients
 Preservatives
 Vials
 Syringes
 Stoppers
 Result: sterile product from a series of non-sterile components

28. Manufacturing Control Definitions
 Sterile: the absence of life. All drug products are required to be sterilized
once placed in their final container, this is performed to prevent the
provision of a product contaminated with microorganisms to patients
 aseptic: acting in such a way to prevent the introduction of
microorganisms; aseptic processing is used for those drug products that
must be sterile but cannot be subject to terminal sterilization due to
their heat-labile nature; effectively all biopharmaceutical products are in
this category
 axenic: freedom from foreign organisms. All biopharmaceuticals are
intended to be axenic cultures- they only contain the cell line desired,
without other foreign organisms.

29. Contamination
Contamination - presence of any unwanted substance
that will affect the purity of a drug product
 Common sources
 Air
 Surfaces
 Water
 Components used to manufacture the product
 Influenced by many factors
 Materials
 Degree of human contact
 Manufacturing environment
 Quality of tools, fixtures, facilities

30. Types of Contamination
Two types:
 Particulate/non-viable contaminants
 Consist of small bits of matter, called particles
 Categorized by size and type of particle
 Viable contaminants
 Microbial, bacteria, viruses, mycoplasma

31. Particulate – Non-viable
Contamination
 Any unwanted component
 Particulate (non-viable) contamination
 Matter (particles) of microscopic dimensions
 Figure: Comparison of one micron-size particulate to one human hair

32. Types of Particulate Contamination
 May be organic or inorganic
 In gases: aerosol or airborne
contamination
– 97% are microscopic, 3% are
“dust”
 In liquids: suspension when
floating, silt when settled
 In solids: called included
matter
Figure: Chart of relative sizes

33. Sources of Non-Viable Contamination
 Common sources of non-viable particulates
 Cellulose fiber from paper
 glass particulate from breaking glass vials during filling
 aluminum particles from capping vials
 gown fibers
 hair
 human dead skin cells (one of the most frequently encountered
particulates in the cleanroom)

34. Microbial/Viable Contamination
 Microbial (viable) contamination
 Bacteria, fungi, viruses
 Ultimately, all microorganisms are excluded to the fullest extent possible
 Issue: they reproduce, increasing the contamination problem, creating
metabolic products including waste by-products
 A single bacterium can cause significant problems
Figure: Example of bacterial reproduction

35. Bacterial Reproduction
Time of Day Number of Bacterial Cells
9:00 a.m. 1
9:20 a.m. 2
9:40 a.m. 4
10:00 a.m. 8
10:20 a.m. 16
1:00 p.m. 4,096
1:20 p.m. 8,192
1:40 p.m. 16,384
2:00 p.m. 32,768
2:20 p.m. 65,536
4:40 p.m. 8,388,608
5:00 p.m. 16,777,216

36. Types of Common Microorganisms
 Bacteria is most common, followed by molds
 Some examples of commonly encountered microorganisms in cleanrooms
Microorganism Example Source
Gram-positive cocci Staphylococcus
species
humans
Gram-positive cocci Micrococcus species humans
Gram-positive bacilli Bacillus species soil

37. Sources of Microbial Contamination
from Humans
 Sources of microbial contaminants from humans
Source Amount
nose secretion approximately 10 million microbes/gram
Spittle approximately 100 million microbes/gram
scalp approximately 1 million microbes/cm2
forehead 10,000–100,000 microbes/cm2
Armpit 1–10 million microbes/cm2
Hands 100–1,000 microbes/cm2

38. Sources of Human Contamination
 Common sources
 Humans
 Often the biggest problem, esp. in the
clean room
 Shed hair, skin particles
 Particles from under finger nails, on
hands, on clothes
 Talking, sneezing, coughing – even with
mask
 Typically, will shed 10 grams of skin
particles per day

39. Control of Contaminants - Design
 Facility design
 Clean room is any room or area where an attempt is
made to limit, control and eliminate the amount of
airborne contamination
 Properly designing the facility, controlling the air supply
for the environment, sterilizing the manufacturing
components, using aseptic gowning, following aseptic
techniques, and implementing a cleaning and
disinfection program

40. Contaminant Control in Clean Room
 Air supply – require high efficiency particulate air (HEPA) filters
 Remove 99.97% of the particles suspended in air that are 0.3
microns or larger
 Number of air changes per hour also controlled
 Pressure differentials, airlocks
 Sterilization of components and equipment
 Steam, dry heat, gamma irradiation, vaporized hydrogen
peroxide, filtration
 SIP for large vessels, preceded by CIP procedures

41. Sterility
Achieving sterility in final product is extremely important
 Complicated by the fact that biopharmaceutical
molecules cannot be subjected the most common
method of generating a sterile product: autoclaving
 Must be sterilized by using a sterilization filtration
process applied to the bulk formulation
 Filled under aseptic conditions into pre-sterilized
individual containers and capped with pre-sterilized
closures

42. Autoclaving – most common and arguably the most effective
 Equipment and containers may be autoclaved without concern
 Some pharmaceuticals may be “terminally sterilized” in their
sealed containers as a last step in processing before delivery
 Biopharmaceuticals tend to be heat-labile molecules and therefore
can not be subjected to the high heat of steam sterilization
without changing or being denatured
 Result – most products are sterile filtered as a bulk formulation
and then added to a sterile container under aseptic conditions, a
process that is highly complex and demanding, with each step one
more entry level for contaminants
Sterilization Methods

43. Control of Contamination - Sterilization
 Control through Sterilization
 the act or process, either physical or chemical, which eliminates or inactivates all forms
of life, including bacterial endospores
 Methods vary depending on type of material to be sterilized but include
 dry heat
 Wet (steam) heat – 121oC, 15 psi
 gamma irradiation
 ethylene oxide
 Vaporized Hydrogen Peroxide (VHP)
 filtration
 Sterilizers must validated, including their load pattern
 Ensures that it is functioning properly
 Ensures that every item is sterile
 Need to be re-validated on a planned schedule
 Include preventative maintenance

44. Validating a Sterilizing Process
Demonstrate that the equipment used for the sterilization process
(autoclave, dry heat oven, and VHP) is capable of operating to achieve
the desired end result
 run cycles to demonstrate actual operational conditions
 ensure that the required parameters of microbial kill (i.e., bioburden
reduction) are achieved
 Biological indicators are typically used for this purpose Biological indicators are a material that
is inoculated with a known quantity of microorganisms, typically one that serves as a worst
case scenario for the sterilization cycle by being resistant to the sterilization conditions
 continually monitor the process parameters (e.g., temperature, pressure,
etc.) during each sterilization cycle to ensure they are operating within the
validated parameters
 perform continuing validation studies periodically to ensure that the loads
are maintained in a validated state

45. Sterilization Process Validation
 Important that all sterilization processes operate with the validated
parameters to ensure the sterility of components, especially those
that cannot be individually tested for sterility
 Important to check expiration dates and appearance/integrity of
sterilized equipment and components before use; cannot use if
expired or compromised
 Autoclave must have even distribution of temperature to ensure all
items in the chamber are sterile; usually achieved by generating a
vacuum
 A cycle is only valid if the correct temperature and pressure is
maintained throughout the required period of time
 Materials that can be autoclaved include some culture media, filters,
glassware (bottles, etc), miscellaneous items such as caps, tubing,
forceps, etc.

46. Autoclaving -Advantages and Disadvantages
 Advantages
 Consistently highly effective
 Simplistic
 Short processing times
 No toxic residues
 Disadvantages
 High temperatures limit materials to those not adversely effected by heat,
moisture or pressure
 Burn hazards for operators

47. Sterilization - SIP / CIP / Dry Heat
 Steam-in-Place (SIP) / Clean-in-Place (CIP)
 SIP is used for large vessels and tanks that will not fit into an autoclave
 CIP must be completed before SIP to prevent “baked-on” residuals that would
result in cross contamination between uses
 Both processes, SIP and CIP, must be validated
 Dry Heat Sterilization
 Hot air at 160 – 170 C
 Oven-like chamber
 Circulated air for even temperature throughout chamber
 For heat stable but moisture sensitive material, including glassware
 Validated for time, temperature and load pattern

48. Dry Heat - Advantages / Disadvantages
 Dry Heat Sterilization – advantages
– Simplicity
– Heat penetration
– No toxic residues
– No corrosion
– Able to inactivate pyrogens (depyrogenate)
 Dry Heat Sterilization – disadvantages
– Long processing times
– High temperature limits materials
– Burn hazard for operators

49. Gamma Irradiation
 Gamma Irradiation
 Alternative for moisture and/or heat sensitive material
 Gamma ray sources include cobalt-60 and cesium-137
 Time sensitive dosage delivery
 High energy waves that disrupt DNA leading to death of any organisms
 No toxic or radioactive residues
 Disadvantage – relatively expensive method
 Gamma irradiators
 shielding
 Storage and disposal
 Safety issues
 Can be outsourced

50. Vaporized Hydrogen Peroxide (VHP)
 Vaporized Hydrogen Peroxide (VHP)
 Aerosolized, low temperature
 Used to kill bacteria (including endospores) on enclosed surfaces
 Isolators
 Workstations
 Safety cabinets
 Pass-through rooms
 Advantages
 Kills a wide variety of microorganisms and spores
 Low temperature
 31% H2O2 can be converted to water vapor and oxygen
 Disadvantage
 Only effective on exposed surfaces
 Toxic so must be used in an enclosed / sealed area

51. Filter Sterilization
 Removes but does not kill microorganisms
 Sterilize heat-sensitive materials
 Sterilize gas or liquid by passing through a porous
membrane
 Filters are classified by nominal pore size ratings
 Will not retain microorganisms smaller than the rated
size

52. Control with Aseptic Gowning
 Human body sheds ~ 1 billion dead cells and hair plus oils and
moisture each 24 hours
 ~1000 bacteria-containing cells per minute
 Can get into the air flow to cause contamination
 Control by wearing clean, sterilized clothing to create barrier
 Protects product and critical surfaces
 Not 100% effective
 Reduces but does not eliminate shed microorganisms
 Aseptic gowning to cover all or most of the person

53. Aseptic Gowning
 Full Aseptic Gowning
 Gloves
 Gown
 Mask
 Hood
 Goggles
 Boots
 Gloves

54. Aseptic Gowning Practices
 Good practices for aseptic gowning:
 Wash hands with soap and water prior to entering the gowning room
 Remove cosmetics
 Trim and clean fingernails
 Remove watches and jewelry
 Follow SOP for gowning process
 Make sure gowning apparel does not make contact with floor, walls or
equipment
 Disinfect gloves with 70% alcohol
 Inspect gloves and gowning apparel and replaced damaged items
 Keep talk to a minimum; avoid sneezing and coughing

55. Gowning Requirements
 Gowning requirements by area classification
ISO 14644-1 designations provide uniform particle values for cleanrooms in multiple industries.
Clean Area
Classification
(0.5 micron particles/ft3)
ISO
Designationa
Typical Area Gowning
Class 100 5 point of fill, sterility testing or other aseptic
manipulation
full aseptic gowning, to
consist of sterile gown/hood,
sterile double gloved, mask,
goggles, and boots
Class 10,000 7 background room/area for aseptic manipulation same as Class 100
Class 100,000 8 personnel and equipment airlocks leading into
the cleanroom facility
non-shedding one- or two-
piece suit gathered at wrists
and ankles; hair, beard, and
shoe covers
Controlled
Unclassified
NA access and exit to/from classified areas;
packaging areas
employer-issued uniform
plus non-shedding smock,
hair, beard, and shoe covers

56. Working in a Cleanroom
 Follow high standards of personal hygiene and cleanliness
 No respiratory or gastrointestinal infections
 No open skin lesions or other skin conditions (excess shedding)
 Ability to gown properly and operate in a slow, deliberate
manner
 Keep talk to a minimum
 avoid sneezing and coughing
 Ability to work in clean room conditions for long periods of time

57. Working in a Cleanroom Cont’d
Entering a cleanroom via an airlock requires the
following behaviors
 All equipment must be disinfected in the airlock prior to
entering an aseptic area and before an aseptically-gowned
person contacts it
 Opening and closing of airlock doors should be kept to a
minimum, they should be opened only to perform necessary
activities
 Both doors of an airlock should never be opened
simultaneously

58. Clean Room Conduct
 All equipment must be disinfected in the airlock
and before an aspetically gowned person contacts
it
 A gowned person and an un-gowned person
should never be in the airlock at the same time
 Do not open an airlock door to talk to anyone
 Never open both doors of an airlock at the same
time

59. Control through Aseptic Techniques
Control through aseptic techniques
 Focus on procedures executed with the mindset of
reducing risk and contamination to the lowest possible
level
 Operator technique is the single most important constant
in maintaining aseptic processes due to proximity to the
product or culture
 Extrinsic factors include sterile material, environment,
and equipment

60. Control of Contamination through
Aseptic Technique in BSC
Aseptic techniques
 Critical surfaces must not come in
contact with anything that is
potentially contaminated
 Laminar flow hoods and rooms are
Class 100 environments
 No body part, equipment, objects
or contaminated air should come
between the HEPA filter and the
critical site – first air rule

61. Control of Contaminants in Clean Room
Working in a laminar flow hood or room
 Verify proper operation of hood and certification
 Perform all aseptic manipulations at least six inches from
the front edge and sides of hood
 Disinfect all equipment prior to transferring to hood
 Disinfect gloves when entering hood and frequently
while working
 Do not touch critical surfaces
 Avoid excess turbulence and particulate shedding
 Minimize number of people and equipment under
hood
 Limit talking
 Work in an unhurried manner

62. Reducing the Probability of
Contamination
Environment
 The environment must be clean, with HEPA-filtered air
circulating
 The aseptic area should be cleaned before and after use;
cleaning after use reduces the risk of cross-contamination in a
multi-product suite and reduces the microbial load on
working surfaces
Equipment and materials
 Ensure that equipment and materials are cleanable and fit for
purpose, i.e., use equipment that is designed for cleanrooms;

63. Particle Generation
 Estimated particles (0.3 micron and larger) generated by various
activities
Particles Emitted Activity
100,000 motionless—sitting/standing
500,000 upper body motion
1 million upper body and minor leg motion
2.5 million sitting to standing or vice versa
5–10 million walking >2.0mph

64. Viable and Non-viable Particles
If specifications require measurement of viable and non-viable
particulates during your operations, the following experiments can be
performed:
 Viable particulates: surface monitoring plates may be used to measure viable
contaminants on personnel and equipment/surfaces and air viable devices may
be used to measure viable air contaminants
 Non-viable particulates: a particulate measuring device can also be used to
measure the total of both viable and non-viable particles

65. Contamination Control –
Cleaning and Disinfecting
Control through cleaning and disinfecting
 Cleanrooms do not have self-cleanup capabilities to offset any contamination
 Most contaminants settle to the floor or other horizontal surfaces
 Introduced into the air by air currents or activity
 A one micron anthrax spore requires ~20 minutes to settle or move laterally one meter
 Contamination needs to be removed from these surfaces by frequent cleaning
and disinfection
 Cleaning is applying a detergent (along with the physical removal of particles
and microorganisms from surfaces) by mopping, wiping, or brushing
 Disinfection is the elimination of most recognized disease-causing or harmful
microorganisms but not necessarily all microbial forms
 Methods include UV radiation, boiling water, steam or, typically, chemicals

66. Contamination Control – Cleaning
Purpose is to remove contaminants and residues that
interfere with effective disinfection
 Four step surface cleaning process
• Scrub the surface with a mop or wipe and use a detergent solution
• Rinse the surface before the surface dries
• Collect any remaining liquid on the surface by wiping or vacuuming
• Allow the surface to dry, then disinfect

67. Contamination Control – Disinfecting
Disinfection
 Chemical agent/s used on surfaces that destroy disease-causing microbes
 A disinfectant will typically contain both an antimicrobial and a detergent
 The pH of a disinfectant may limit the growth of a contaminant if <4 or >10
 No single disinfecting agent or procedure is adequate for all purposes
• Especially, endospores, which require special procedures
 Effectiveness of a disinfectant depends upon:
• Disinfectant concentration
• Length of exposure to the disinfectant
• Amount of organic matter (e.g., soil, blood) present
• Nature and amount of microorganisms on the surface
• Material to be disinfected

68. Classification of Disinfectants
 Chemical disinfectants are classified based on their antimicrobial compound
• alcohols – 70% IPA
• aldehydes
• halogens - sodium hypochlorite
• peroxygens - peracetic acid
• phenolics
• surface active agents
 Further classified by microbial target
• Bacteriocidal
• Fungicidal
• Virucidal
• Sporicidal
• Bacteriostatic
 Solutions must be sterile for use in class 100 and 10,000 areas

69. Summary of Disinfectants and Efficacy
Disinfectant
Category
Gram- Positive
Bacteria
Gram- Negative
Bacteria
Endospores Fungi
Alcohols 3 3 0 2
Aldehydes 4 4 4 4
Halogens 3 3 1 2
Peroxygens 4 4 4 4
Phenolics 3 2 1 3
Surface Active
Agents
3 1 0 2
 Summary of disinfectant categories and their efficacy
4 = most efficacious in killing
1 = least efficacious in killing
0 = not effective in killing

70. Disinfecting - Best Practices
 Best practices
 Rotate types of disinfectants
 Usually alternate between two different disinfectants each cycle
 Rationale – what resistant microbe one does not eliminate, the other will
 All equipment and containers should be disinfected in the
equipment airlock
 Using sterile 70% IPA, wipe from top to bottom and back to front
 From cleanest to most contaminated
 All components being used in a laminar flow hood should also be
wiped with 70% IPA
 Frequently apply 70% IPA to gloved hands while working in the
laminar flow hood

71. Disinfectant Usage
 Surface disinfectant usage on surfaces in various area classifications
PA: Peracetic Acid (Peroxygen)
Hypochlorite: Halogen
IPA: Isopropyl Alcohol
NA: Not Applicable
Area
Classification
Ceilings Walls Floors Plastic Curtains Critical Work
Surfaces
Class 100 phenolic and
monthly
sporicide
phenolic and
monthly
sporicide
phenolic and
monthly
sporicide
PA or
hypochlorite
and monthly
sporicide
all followed by
IPA wipe
PA or
hypochlorite
and monthly
sporicide
all followed by
IPA wipe
Class 10,000 as above as above as above as above NA
Class 100,000 phenolic phenolic phenolic NA NA

72. Frequency of Usage/Application
 Frequency of application for disinfectants
 The frequency of disinfectant use should be determined by environmental
monitoring results
Area
Classification
Ceilings Walls Floors Plastic
Curtains
Critical Work
Surfaces
Class 100 daily to
weekly
mopping
daily to
weekly
mopping or
spraying
daily
mopping
daily
mopping,
wiping, or
spraying
daily wiping
or spraying
Class 10,000 weekly
mopping
weekly
mopping or
spraying
daily
mopping
NA NA
Class 100,000 monthly
mopping
monthly
mopping or
spraying
daily
mopping
NA NA

73. Environmental Monitoring Program
 Mandated by cGMP – for facilities, personnel and process utilities
 Pre-req’s
 HEPA filter certification program
 Clean room cleaning and disinfection program
 Clean room qualification/requalification program
 Air flow pattern visualization program
 SOP’s – provide a written description of the program specifics for
monitoring and testing of classified areas, personnel and utility systems
 Also include alert and action levels
 Personnel training program – documented training!

74. Environmental Monitoring
 Include scheduled monitoring of:
 Airborne viable/microbial and non-viable particulate levels
 Microbial contamination on personnel, work surfaces, floors,
walls and equipment
 Microbial contamination of clean utilities
 Pressure differentials
 Direction of air flow
 Temperature
 humidity

75. • Water and clean steam monitoring
• Air monitoring- non-viable
• Air monitoring – viable
• Microbial surface testing using RODAC plates
• Gown and fingertip RODAC testing
Environmental monitoring includes…..

76. Testing Performed by QC
Microbiology
 Environmental monitoring
 Utility testing
 Sterility testing
 Microbial content testing
 Bioburden
 Microbial Limit
 Bacterial Endotoxin (LAL)
 Microbial identification
 Antimicrobial Effectiveness Testing
 Cleaning validation
 Media fills

77. Air Monitoring-Non Viable Contaminants
 Non-viable particulate monitoring
 Provides real time data on the environment
 Airborne particle counters
 Measure particles in a number of size ranges; particles of 0.5 microns
or greater are generally recognized as indicators of environmental
contamination
 Optical particle counting – vacuum pump pulls air into the sensor;
particles in the air sample pass through the optical detection view
where a laser light source is concentrated; particles scatter the laser
light, which is focused onto a photo diode; the photo diode detects
and converts the light signal to electrical impulses; height of impulse
is directly proportional to particle size

78. Design of Environmental
Monitoring Strategy
No monitoring can provide a high level of confidence without an
overall cleanroom systems management
 Adherence to cGMP
 Facility design control
 Effective supervision
 Sound corrective action steps
 Proper employee training
A documented sampling program that
 Describes the procedures and methods for sampling in a cleanroom
 Identifies the sampling sites, frequency and number of samples
 Describes the method of analysis and interpretation of results

79. Sample Site Selection
 Air, surface, personnel and utilities that are representative of locations that:
• Come in contact with exposed product and/or components
• Are in close proximity to exposed product
• Are areas of high personnel and equipment flow
• Contribute to the particulate and microbial levels within an area (e.g.,
personnel, equipment, etc.)
• Represent the most difficult or inaccessible areas to clean or disinfect
 Collectively represent the systems performance over time for a quality
product
 Sites may be selected based on analysis of risk, smoke studies, and or data
from the Performance Qualification

80. Sample-Site Collection Considerations
 Consideration should be allowed for extent of exposure or contact at each
location, choosing sites with the greatest opportunity for contaminating the
product
 May not be able to sample at the most critical contact sites
• Sample may increase the risk of contamination
 Routine sampling sites include non-product contact sites
• Floors
• Walls
• Doors
• Ceilings
 May not sample areas with low probability of contamination during processing

81. Recommended Critical Sampling
Locations (Laminar Flow)
Tests Test Location Rationale
Critical Sites
(Laminar Flow)
Microbial Air
Particulate Air
Sample within approximately one foot of
the critical process point or container
opening.
Sample is to monitor the air where there
is increased activity at a location that is
close to / representative of the air in
contact with the open product.
Surface Contact
Plate
Sample within approximately one foot of
the product container opening or critical
aseptic process step.
Samples are to monitor the critical
surfaces where there is increased activity
at locations that represent surfaces close
to or in contact with exposed product or
components.
Personnel Contact
Plate
Sample gown in two locations (chest,
forearm) and gloved fingertips of each
hand.
Samples are to reflect the parts of the
gowned and gloved person that are in
closest proximity to the exposed product.
Examples of critical test-site locations and rationale

82. Recommended Routine Sampling
Locations (Non-Laminar Flow)
Tests Test Location Rationale
Routine Sites
(Non-Laminar Flow)
Microbial Air
Particulate Air
Sample in a central location in the room or
otherwise representative location.
Sample is to reflect general area/room
conditions which are representative of
room conditions.
Surface Contact
Plate
Sample on a representative wall, curtain
(outside), door push plate/handle/push
button, floor surface, or other frequently-
utilized surfaces as determined by the
sampling plan.
Sample is to reflect the general
condition of the area surfaces.
Personnel Contact
Plate
Sample gown in 2 locations (chest,
forearm) and gloved fingertips of each
hand.
Routine personnel testing is to ensure
that gowning is performed properly
and that glove disinfection is effective.
Examples of routine test-site locations and rationale

83. Sampling Frequency
 Vary by room classification and nature of operation
 Per process or critical site testing
• Microbial and particulate testing accompanying manufacturing
• Process: a set of independent steps or manipulations conducted within the same
set-up or procedure
• Begins with aseptic set-up and concludes with product completion
 Routine monitoring
• Not performed during processing
• Minimum – weekly
 Ensures that operations, cleaning and HVAC continue to
operate and perform satisfactorily and consistently

84. Environmental Testing for
Class 100 areas
Example of critical site test requirements and frequency of testing
for Class 100 areas
Environmental Test Class 100 – Critical Site Testing Frequency
Microbial Air (active) once per process (minimum of 1m3 of air)
Microbial Air (passive) one settling plate per designated location
Surface Contact Plate minimum of one site per process
Product Contact Surface
Contact Plate (Sterile Filling
Only)
minimum of four product contact surface sites to be performed during
filling prior to the breakdown of a line at the end of a fill; to include all
component bowls and representative fill needles
Personnel Contact Plate the fingertips of both gloved hands and the chest and forearm of
personnel who perform aseptic operations or testing during
processing
Particulate Air minimum of once per hour per process in LFH (minimum of 1m3 of air)

85. Environmental Testing and
Response Categories
Interpretation of results and response
 Passing: within acceptable, i.e., established, levels. Failures are called
environmental test excursions and require a response
 Alert level: test excursion above the established norm for the site. Usually
based on historical statistical data and re-evaluated yearly. Set at 10% - 50% of
the action level
 Action level: result reaches or exceeds area classification as established by
industry or regulatory guidelines. Indicates a possible problem that requires a
corrective action be taken. An investigation is launched to determine the
impact on the product and the root cause (Root Cause Analysis)

86. Environmental Testing and
Classification Examples

87. Industry/Regulatory Guidance for
Environmental Monitoring
Environmental Test Class 100 Class 10,000 Class 100,000
Particulate Air
( 0.5 microns/m3)
> 3,520 > 352,000 > 3,520,000
Particulate Air
( 5 microns/m3)
> 29 > 2000 > 20,000
Active Microbial Air (Colony-Forming Unit or
CFU/m3)
≥ 1 > 10 > 100
Passive Microbial Air (CFU) ≥ 1 N/A N/A
Surface Contact Plate (CFU)  1 > 5 > 25
Floor Contact Plate (CFU) > 3 > 10 N/A
Compressed Gas Particulate ( 0.5 microns/m3) > 3,520 > 352,000 > 3,520,000
Compressed Gas Microbial (CFU/m3) ≥ 1 > 10 > 100
Example of industry/regulatory guidelines for environmental testing

88. Microbial Identification for All Alerts
Microbial Identification
 Must be determined for all alert and action level results
 Aids in evaluating impact on product, source
 Microbes deemed “objectionable”: by FDA include
• Burkholderia cepacia
• Escherichia coli – fecal contamination indicator
• Pseudomonas aeruginosa
• Salmonella species – pathogens
• Shigella species
• Staphylococcus aureus – generally, poor production hygiene
 Parenteral pharmaceuticals must be sterile, therefore, all microbes are
objectionable

89. Environmental Excursion
Investigations
Environmental excursion investigations
 Required for all action level and recurring alert level findings
• Assess environmental controls during production
• Include time, location and conditions during sampling
• Alert / action results of particulate & viable counts included in report
• Activities in progress during the sampling
• Previous cleaning disinfecting
• Any potential causal events
• The investigation should attempt to determine the probable cause
 Many other elements may go into an excursion investigation including past
maintenance and activities, trending data, data from other sites, identification
of isolated microbes, physical inspection of the site, personnel input and
corrective actions being taken

90. Corrective Actions
Corrective action examples
 Restricting activities that may increase the bioburden or the total number of
microorganisms detected in or on an article
 Increasing the area ventilation, room pressure, or quality of air delivered (e.g.,
HEPA filtration)
 Testing of process HEPA filters and correcting leaks
 Increasing or altering cleaning/disinfecting procedures and using a sporicidal
agent
 Conducting additional personnel training to reduce practices that may add
bioburden
 Increasing gowning requirements for the area
 Restricting improper personnel flow from less clean areas
 improving facility surfaces

91. Data Trending
Trending environmental monitoring test results
 There is a need to overcome the delay factor in getting viable test outcomes
 Prevent filling and package of contaminated product
 Can decrease costly activities by establishing shift and trending data for the
environment
 Hold until new test data is obtained based on previous testing
 Developed through regular evaluation of critical areas with graphical representations of results
 Shift is the tendency for the most recent six-month period to have either higher
or lower results than the previous six-month period
 Statistically significant change
 Trends only follow the most recent six-month period
 Shows upward or downward movement of test results
 Also statistically significant trend

92. Testing Methodology
Test methodology
 Airborne counters
• Non-viable particles
• Viable microbes on growth media
• Most be disinfected before use if moved between locations
• Ideally, should be dedicated to a particular space
 Direct contact plates and settle plates
• For surfaces and timed air sampling , respectively
• Microbial growth media
 Sampling is done during dynamic “in operation” activities
• Ensures environment is under control during processing
• Static sampling is usually only Performance Qualification test

93. Air Monitoring
Microbial air monitoring
 Airborne microbes have a higher potential for contamination vs surface
microbes
 Standard unit of measurement is the colony forming unit (CFU)
 Require 5 day incubation for growth
Active air monitoring
 Performed with equipment ; an air vacuum to draw a specific volume of
environmental air sample across a media plate
 Report as CFU/m3 (cubic meter) of air in 100 and 10000 level
 CFU/ft3 (cubic foot) in 100,000 level
 Air samplers must be calibrated annually

94. Passive Air Monitoring
Passive microbial air monitoring
 Uses settling, or exposure, plates
 Media plates are opened to the air and dust and microbes are allowed to settle
onto the media
 Exposure time varies but should not exceed 4 hours
 Reported as CFU/4 hours or some other time period

95. Non-viable Monitoring
Non-viable particulate monitoring
 Generally, basis of clean room classification vs viable
 Real time data, no incubation required
 Difficult to correlate particle count to viable without direct comparison of
both results from tests done at the same time
 >0.5 micron particle size indicates environment contamination
 Expressed as the number of particles >0.5microns and >5.0 microns per
cubic meter or cubic foot

96. Surface and Equipment
Microbial Testing
Surface and equipment microbial monitoring
 Methods need to be qualified using standard laboratory procedures
 Contact impression plate and swabbing techniques yield direct microbial
monitoring of surfaces
 RODAC plates
• Replicate organism detection and count
• Media extends above wall of plate base and will make direct contact with a surface
• Results are reported as CFU/plate
Figure: RODAC plate with bacterial growth colonies

97. Surface and Equipment
Microbial Monitoring (Continued)
Surface and equipment microbial monitoring
 Swabbing
• Used on irregularly-shaped surfaces not suitable for contact plates
• Pre-moisten with sterile culture media
• Sample ~4 in2 (square inches) while rolling the swab to contact all surfaces
• Swabs generally are submitted to the microbiology lab for processing
 All tested surfaces (plate or swab) must be disinfected following sampling
Microbial monitoring of gowned personnel
 People are the greatest source of contamination in a cleanroom
 Need to be trained in proper aseptic gowning and techniques
• Must pass initial qualification testing following training
 Personnel monitoring ensures both

98. Personnel
Microbial Monitoring
Microbial monitoring of gowned personnel
 Periodic gown and fingertip microbial testing on all personnel
 Contact plates used on chest, forearms and fingertips of both gloved hands
Figure: Microbial personnel / gloved-fingertip RODAC testing Figure: Microbial personnel / chest RODAC testing

99. Process Utility Monitoring
 Initial Qualification (IQ) of a process utility system ensures
that the actual performance is consistent with the required
performance
 All process utilities that have contact with the product must
be monitored
• Compressed gas
• Clean steam
• Water for injection (WFI) – produced from filtering and
distilling potable water

100. High-Purity Water Monitoring
 Same methods for clean steam
 Tap water contains up to 500 CFUs/ml – not high quality for manufacturing
 Water standards are set by the USP for water used to manufacture
pharmaceuticals and clean equipment = Water For Injection (WFI)
 Samples taken periodically from holding tank and distribution system
 Test assess microbial quality – colony count, indicator microorganisms and
endotoxin

101. Water System Sampling
 Samples need to be collected in a manner consistent with the
manufacturing process
• Sample from flush cycle if there is one, do not flush for a sample if there is no flush
cycle in the manufacturing process
• Sample from hoses rather than point of use, if appropriate
 Samples should be refrigerated at 2o – 8o C until processed
 Minimum quantity is 200 ml
 Use multiple containers for samples used in multiple tests
 Appropriate safety measures need to be in place for hot
water sampling

102. Water System Monitoring
 Microbial colony counts performed by filtration of the water sample
through 0.45 micron filter and incubation of the filter on low-nutrient
agar for 5 days at 30o – 35oC
 If counts reach alert or action level, microbes must be identified
 Selective media is used to screen for particular objectionable
organism
• Pseudomonas aeruginosa
• Burkholderia cepacia
• Coliforms
 Any WFI test fails must be investigated to determine product quality
• Possible quarantine or product discard

103. Environmental Monitoring – Media Fills
 Simulation of an aseptic process that uses microbial growth-promoting culture
media in the place of actual product
 To demonstrate the capability of a specific aseptic process to produce sterile
drug products
 To qualify or certify aseptic processing personnel
 To comply with regulatory requirements
 Should be performed under actual full normal conditions for a production
process
 Containers are incubated for 14 days
 Contamination leads to investigation and ID of isolated microorganisms
 Must be able to achieve successful media fills before processing product

104. Microbial Monitoring and
Identification of Microorganisms
 Start with a pure culture isolated by streak culture
 Gram stain for classification and morphology
 Phenotypic methods
• Observable physical properties
• Metabolic reactions
 Genotypic methods
• Some DNA characterization
• Often 16S ribosome gene sequence
• May use RNA characterization
 ID both genus and species level for isolates from the 100 and 10,000 level areas
 Mold is also identified at genus and species level by macro- and microscopic
methods
 Changes in cleanroom flora should be investigated for breaches

105. Environmental Monitoring and Data
Utilization
Utilization of information derived from environmental monitoring
 Can generating massive amounts of data weekly, based on size of facility
 Interpreted by the QC department
 Gathered and converted into “knowledge” which needs to managed and
communicated
• To personnel at various levels within the organization
• To the right personnel at the right level of detail to endure productive actions
• To determine if the facility is in control or has regained control if once lost
 Patterns are based on all data collected from all aspects of the processes,
personnel, utilities, facilities and any other sources
 Knowledge Management (KM) involves acquiring, analyzing, storing, and
disseminating information

106. Knowledge Management Methodology
Utilization of information derived from environmental monitoring
Figure: Knowledge Management Methodology

107. Data Utilization and ICH Q10
Utilization of information derived from environmental monitoring
 ICH Q10 (International Committee on Harmonization)
• “Product and process knowledge should be managed from development through
the commercial life of the product up to and including product discontinuation. For
example, development activities using scientific approaches provide knowledge for
product and process understanding.”
• KM is “...a systematic approach to acquiring, analyzing, storing, and disseminating
information related to products, manufacturing processes, and components. Sources
of knowledge include, but are not limited to, prior knowledge (public domain or
internally documented); pharmaceutical development studies; technology transfer
activities; process validation studies over the product lifecycle; manufacturing
experience; innovation; continual improvement; and change management
activities.”

108. Data Utilization Considerations
Utilization of information derived from environmental monitoring
 Questions to be considered for effective Knowledge management
• What are the data?
• What do the data mean (i.e., what knowledge can we obtain from the data)?
• How is this knowledge owned by the area management?
• How are those who work in the area kept informed of the requisite knowledge?
• How does one monitor the effectiveness of the actions undertaken and have
effective investigations in this area?

109. Quality Control, Microbial
Quality Control Practices in the Microbiology Laboratory
• Plan to ensure reliable, reproducible and accurate test results
• Guidelines are available from government agencies and professional
organizations
• Includes end-user assessment of commercially obtained material
Quality Control of Microbiological Culture Media
• Culture media has the potential to impact every aspect of laboratory
operations
• Three primary aspects
 the control of the culture media preparation and storage conditions
 the physical and chemical characterization of culture media
 growth promotion testing

110. Control of Media Preparation –
In-house Prep
Control of the culture media preparation and storage conditions
 Areas of QC for media prepared in-house
• Raw material storage conditions and expiration dates
• Compounding of the media to prevent errors
 Weighing
 Water measurement
 Improper mixing of ingredients
• Heating to melt agar before sterilization without damaging heat-labile components
• Validated autoclave cycle (15 psi, 121oC, 15 minutes minimum)
 Need to be able to audit each stage of preparation

111. Control of Media Preparation –
Commercially Prepared
Control of the culture media preparation and storage
 Areas of QC for commercially prepared media
• Little control of QC
• Check for physical and chemical properties and growth promotion
• Store under controlled conditions through the expiration date
 Temperature
 Humidity
 Light exposure where applicable
 Dehydration
• Steps to prevent dehydration of prepared media
• Discard if the media becomes dehydrated

112. Quality Control of Media
Control of physical and chemical characteristics
 Screen for acceptability before more labor intense testing
 Examine visually for clarity and color
• Turbidity could result from precipitating components
• OK to use if the precipitate re-dissolves at incubation temperature
• Failure should result in discarding the media
 Check containers for cracks or leaks
Growth promoting testing
 Test with compendial and other stock microorganisms to ensure it supports
growth
 Miles and Misra (surface variable count) method

113. Quality Control –
Maintenance of Stock Cultures
 Stock maintained to provide known strains of microbes for compendial tests
 Confirmation of identity of all microorganisms received must be the first step in
building a stock culture supply, even if received from a reputable source
 Ensure pure cultures (Homogeneity) by streak isolation plating of the culture
 Stocks should include representatives of
• Typical morphology
• Physiological and biochemical characteristics
• Typical isolates found within the facility
 Provide culture media to maintaining stable and viable stocks for long periods
• No excessive growth or metabolic activity
 Storage at proper temperature
 Complex methods such as lyophilization (freeze drying) for indefinite storage

114. Quality Control – Barrier Isolation
 Intrinsic need to separate people (high contamination risk) from aseptic
processing
 Barrier isolators are 100 level, airtight enclosures of impervious material
 Operations are performed from the outside
• By remote controlled machinery
• Individuals wearing half body suits of rubber arm length gloves
 Increasing popularity within the aseptic processing industries