Disinfectant Qualification and Cleaning Procedures · PDF fileDisinfectant Qualification and...
Transcript of Disinfectant Qualification and Cleaning Procedures · PDF fileDisinfectant Qualification and...
Disinfectant Qualification
and Cleaning Procedures
Workshop
IVT's Cleaning Validation and Critical Cleaning Processes
in Philadelphia, PA - August 18-20, 2015
Presented by
Ziva Abraham
President
Microrite, Inc.
5019 New Trier Avenue
San Jose, CA 95136
Phone: 408-445-0507 Fax: 408-445-0507
Email: [email protected] www.microrite.com
Ziva Abraham is the President and Founder of Microrite, Inc., a California based
consulting firm providing consulting and training services to pharmaceuticals,
biotechnology, medical devices and in vitro diagnostics in the areas of quality
assurance, quality control, microbiology, and validation. Ziva has over 25 years
of academic, research, clinical and industrial experience in microbiology, and
quality assurance. Ziva has received her Master’s Degree in microbiology and has
conducted research on developing microbial insecticides using entomogenous
bacteria and fungi towards a Ph.D degree. Her career also includes founding and
managing clinical laboratories for Maccabi Medical in Israel. She has trained
personnel from various industries in microbiology techniques and methods. She
uses her extensive experience to teach why assessing risk of microbial
contamination should be in the forefront of any company that has products for
human/veterinary use. Her experience in clinical laboratories has provided her
with the framework to understand the effects of microbial contamination in
products from a patient safety perspective.
About the Speaker
Who is Microrite?
Goal Microrite’s goal is to provide practical solutions
through consulting and training
Consulting Services Microrite is a San Jose, CA based Consulting Company
helping Pharmaceuticals, Biotechnology, Medical
Devices, In-Vitro Diagnostics and Combination products
in the areas of Quality Assurance, Microbiology, Process
Development, Process Validation, Facility and Utility
Validation
Visit www.microrite.com to learn more about us
About Disinfectants, Sanitizers and Sterilants
• Definitions
• Mode of Action
• Advantages/Disadvantages
• Governing Bodies/Regulations
• Testing for Manufacturers
• Planning Disinfectant Qualification
• Methods generally used in Pharmaceuticals
Topics
• Tube Dilution Method
• Different Coupon/Hard Surface Methods
• Expiry Dating Studies
• Neutralization Study
• Method Details
• Calculations
• Disinfectant Qualification Report
Topics
About Cleaning Procedures
• Challenges in Cleaning
• Procedure for Receiving, Storage, Preparation of
Disinfectants
• Disinfecting/ Sanitizing Procedure
• Training
Topics
Plan of Attack on Planktonic Microbes
Know your enemy before you attack!
Sporicide: Chemical agent that kills all microorganisms including
bacterial and fungal spores
Disinfectant: Chemical agent that kills microorganisms but not
necessarily spores
Germicide: Chemical agent that kills microorganisms
Bactericide: Chemical agent that kills bacteria but not
necessarily spores
Fungicide: Chemical agent that kills fungi
Virucide: Chemical agent that kills viruses
Cleaning agent: Agent with surfactant
Surfactant: Agent that breaks surface tension
Sanitizer: Reduces the number of microbes
Definitions
Do not kill spores, fungal or bacterial, bactericidal and virucidal claims, leaves residue Examples: Vesphene, LpHse
Do not kill spores, fungal or bacterial, bactericidal and virucidal claims, leaves residue Example: Intercept
Kills spores, very unstable, available chlorine may vary depending upon time on shelf
3% kills vegetative bacteria, greater than 6% sporicidal, safe enough to use for food industry
H202
Bleach
QUCS
Phenolics
Disinfectant Facts
Mixture of peracetic acid and H2O2 forms a potent sporicidal agent
Formaldehyde and Gluteraldehyde potent sporicides, highly carcinogenic
Sanitizer, 30% water required to seep through cell wall barrier and disrupt cell contents, some bacteria may use it as carbon source (pseudomonads)
PAA
Aldehydes
Alcohol
Disinfectant Facts
Bioguinide compounds are used as antiseptics in hand
wash solutions and for surgical gloves
Proven to have upto 1.5 log reduction
Iodides, and other combinations are used to clean
before surgical procedures
Ability to lower the surface tension of water by
addition of surfactants makes some disinfectants good
cleaning agents
Other Antimicrobials and Surfactants
Phenolic residue
Courtesy of Veltek Associates, all rights reserved
Regulatory Bodies
• Disinfectants are classified by FDA, EPA,AAMI and CDC mainly for medical device industry
• OSHA oversees the health and safety aspects
• Disinfectants used for hard surfaces are registered with EPA
Required testing before marketing disinfectants:
• Simulated Use Testing
• Actual Use Testing
• Biocompatibity data-evaluation of residue
• Toxicity data
• Material Compatibility Data
• Chemical Indicator
Regulatory Bodies
Regulations
• Required by EPA/FDA to confirm label claims
• Disinfectants must pass the tests described in the
AOAC (Association of Official Analytical Chemists)
in order to be approved by EPA or FDA
• No harmonized consensus on methodology
• AOAC: Association of Official Analytical Chemists
• AFNOR: Associacion Francais de Normalization
• BSI: British British Standards Institution
• DGHM: Deutshe Gesellschaft fur Hygeine und Mikrobiologie
• CEN-TC 216: European Committee for Standardization
Regulatory Bodies
AOAC Methods
Disinfectants must pass the tests described in the
AOAC Manual in order to be approved by the
Environmental Protection Agency.
AOAC Tests Include:
• Spray Products
• Phenol Coefficient
• Use Dilution
• Hard Surface Carrier
• Tuberculocidal Activity
• Sporicidal Activity
• Fungicidal Activity
Testing for Manufacturers
T. mentagrophytes for fungicidal claim
EU methods require a mold A. niger and a yeast C.
albicans
AOAC for bacterial is S. cholerasuis, S. aureus and P.
aeruginosa also common for many different regions
and agencies
AOAC for sporicidal is an aerobic B. subtilis and an
anaerobic C. sporogenes
What Organisms are tested for label claim?
What Organisms are tested for label claim?
AOAC for tuberculocidal is M. bovis; M. smegmatis can
be used as a presumptive test but its resistance is
much weaker to germicides than M. bovis or M. terrae
AOAC for virucidal is not set, they recommend
following the EPA method which generally uses HBV,
HCV, Herpes simplex, HIV and influenza
FDA uses polio virus II and an enveloped virus one
selects while EU calls for polio virus I and adenovirus
V
What Organisms are tested for label claim?
Label claim testing does not address all
microorganisms found in cleanrooms
Cleanroom flora much diverse
Clinically relevant organisms such as S. aureus,
Salmonella or Trichophyton not commonly
encountered in cleanrooms
Bacterial Contamination
Bacteria
Gram Positive
Aerobic
Gram Negative
Aerobic
Cocci Rods Cocci Rods
Mainly human
borne
Staph aureus (bad
bug) (<61>, <62>)
Staph epidermidis
Staphylococcus
warneri
Enterococcus
Streptococcus
Bacillus:
Live in soil
Bad gowning and
cleanroom practices
Disinfection program
nor adequate
Facility design flawed
Concern: product
contamination (
preservative system
may not be adequate
and biofilms
B. subtilis
B. licheniformis
B. cereus (serious
problem)
Neisseria
meningitidis, N.
gonorrhoeae,
Moraxella
catarrhalis
Cocco-bacillus:
Haemophilus
influenzae,
Acinetobacter
Live in soil
Flourish in water
systems
Cause endotoxin
Detrimental to patient
Bad gowning and
cleanroom practices-
soil
Concern:
Endotoxin , Biofilm
E.Coli
Enterobacter
Pseudomonas
Fungi Found in Cleanrooms
Zygomycota
Ascomycota
Fungal Mycelia
Fungal Mycelia
The Bacterial Spore Challenge
Bacillus cereus is a serious problem
Paenibacillus has been a continuous issue once sets in
Biofilms formed by Bacillus is not uncommon in
fermentation equipment
Biofilm organisms are your planktonic ones-just
smarter
United we stand, divided we fall
Biofilms
Use Dilution Method
• Cylinders introduced into test culture for 15 minutes
• Dried for 40 minutes
• Dropped in diluted disinfectant
• Contact time achieved
• Dropped in neutralizing broth and incubated
• No growth in 10 out of 10 - Pass
• Growth in any - Fail
• Acceptance Criteria-Maximum dilution of disinfectant which
kills test organisms in a 10 minute interval
AOAC Tests
Carrier Test Method
• Fire polished borosilicate glass carriers used
• Culture suspension is sonicated and 1 mL of culture
introduced per carrier
• Carriers inverted on filter paper after 15 minutes and dried
for 40 minutes
• One carrier dropped into one tube of disinfectant every 30
seconds
• At 10 minute contact time, each carrier is removed at
interval of 30 seconds
• Excess disinfectant is removed and carriers dropped in tubes
with letheen broth
• Broth with carriers are incubated at 37° C for 48 to 54 hours
• 2 positive out of 60 is the acceptance criteria
AOAC Tests
Suitability of Methods
• AOAC Methods not suitable for Pharmaceutical
Industry
• No one method used or recommended
• Minimum guidance in Pharmacopeial Forum Vol. 28
(1) Jan-Feb 2002 <1072> Disinfectants and
Antiseptics
Methods commonly used in Industry:
• Tube Dilution Method
• Coupon Methods
Planning Disinfectant Qualification
Common factors no matter which method you choose
• Choice of organisms
• Purity of organisms
• Maintenance of organisms
• Media growth promotion and sterility for buffers performed
• Enumeration for target inoculum
• Maintenance of target inoculum
• Daily inoculum verification
• Method validation
• Recovery study and recovery loss calculation-in case of hard
surfaces
• Calculating log reduction
Planning Disinfectant Qualification
When is it applicable?
Initial screening of disinfectants
Testing disinfectants for hard to kill microorganisms
before using for routine cleaning
Tube Dilution Method
Disinfectant dilution(s) challenged with inoculum
After contact time is attained, the disinfectant with
inoculum my be filtered
Results may be qualitative or quantitative
Consider the target inoculum to avoid multiple
dilutions and filtration
Positive controls considered as target inoculum
Consider running negative controls be run per lot of
media and buffer
Tube Dilution Method
Two tubes per organism for each microorganism per
disinfectant dilution tested
Two negative controls of dilution of disinfectant to be
tested
Two positive controls of organisms to be tested-
positive controls may be prepared in water or saline
Tube Dilution Method
Planning
• Number of organisms
• Saline tubes for enumeration
• Number of plates for enumeration
• Number of disinfectants
• Number of dilutions
• Number of time points per dilution
• Number of tubes
• Number of filters
• 400mL buffer per filter
• Number of plates for filters
Tube Dilution Method
Planning - Example
Organism - S. aureus
Disinfectant tested – LpHse
1 dilution - MRC
1 time point - 10 minutes
Always test one microorganism at a time
Tube Dilution Method
Planning for Enumeration - Agar plates and saline
tubes
• Microbial Identification
• Two slants for working culture
• Two plates for purification of culture
• Two plates for purification of culture
• 8 plates for enumeration
• 4 plates for controls
• 1 saline tube for harvest
• 8 saline tubes for enumeration dilutions
• 2 saline tubes for target inoculum
• Streakers
Tube Dilution Method
Planning for Filtration Method – Efficacy – S. aureus
with LpHse for one dilution and one Contact Time
• 2 filters
• 800mL buffer for method validation of
• 800 mL for positive control
• 800mL buffer for negative control
• 2 TSA plates for challenge
• 2 filters for negative control
• 2 plates for negative control
• 2 filters for positive control
• 2 plates for positive control
Tube Dilution Method
Efficacy Data Analysis
With target inoculum of 1 X 103 results can be quantitative with
1 or two log reduction
1 log reduction- 100 CFUs can be enumerated on filter
2 log reduction 10 CFUs can be enumerated
TNTC data (>200 CFUs cannot be enumerated)
2 X 103 results may be quantitative with 1 or two log reduction
1 log reduction 200 CFUs can be enumerated
2 log reduction 20 CFUs can be enumerated
Tube Dilution Method
Efficacy Data Analysis
3 X 103 results may be quantitative with 1 or two log
reduction
1 log reduction 300 CFUs may not be enumerated
2 log reduction 30 CFUs can be enumerated
The inoculum strength falls after enumeration
The enumeration reading is not the exact value of
target inoculum
Positive control may be treated as Time “0” count
Tube Dilution Method
Efficacy Study-Quantitative
Dilutions of Disinfectants made
Each dilution to be tested in duplicate or triplicate for each contact time for each organism
Each dilution inoculated with test organism-106-8
After contact time is attained serial dilutions are filtered
The higher the target inoculum the more dilutions needed
106 - 5 dilutions to be made - last 4 filtered
107 - 6 dilutions to be made - last 4 filtered
108 - 7 dilutions to be made - last 4 filtered
Tube Dilution Method
Benefits
Less time consuming than testing on coupons
Drawbacks
Does not prove that the disinfectant is effective on
hard surfaces
Materials used for higher inoculum as expensive as
coupon study
Tube Dilution Method
This Coupon Method can be used in a manner similar to the tube
dilution study
The only difference would be to inoculate coupons instead of
directly inoculating the disinfectant dilution
Inoculum is applied to the coupon, dried and then sprayed or
flooded with the disinfectant
After the contact time is achieved the coupon may be placed in
a tube containing buffer
The tube with the coupon and the buffer is vortexed and then
filtered
Multiple dilutions may be made depending in the target inoculum
Coupon Methods 1
The difference between this method and the Coupon
Method 1 is that instead of using filtration as a way of
neutralizing the disinfectant, the coupons are placed
in a tube with neutralizing broth
Pour plate method or steak plate method may be
used
If the neutralizing broth used is in large quantities,
the recovery may not be adequate
Coupon Methods 2
Points to consider
Calculate a target inoculum whereby many dilutions
are not required yet a 3 log reduction or target log
reduction can be achieved
Ensure that the coupon sizes are small so as to avoid
using large quantities of neutralizing broth-getting
adequate dilutions to yield useable results may be
difficult
Perform method validation
Coupon Methods 1 and 2
Advantages
Coupon Method 1 is a quantitative study
Coupon Method 2 is less time consuming considering
large quantities of broth is not used
Disadvantages
Only sterilizable coupons such as stainless steel and
glass can be used for both methods
Coupons like wall material will introduce
contamination which may make reading surviving
CFUs a challenge in both methods
Coupon Method 1 and 2
Microorganism inoculated on the coupon and dried
Contact plates used to recover surviving
microorganisms from coupon
Recovery considerably lower than swab recovery
method
Coupon Method 3 (contact plates)
This study is both qualitative and quantitative depending upon
the effectiveness of the disinfectant and inoculum size
For achieving a 1-3 log kill an approximately 2 X 103 target
inoculum can be used the results can be quantified
~200 CFUs can be counted on a contact plate
Less than a log of kill will result in TNTC where the results will
be qualitative
If care is taken to adjust the inoculum strength with the
knowledge of disinfectant capability this study can save a lot of
time and cost
Due to variability in microbiological methods attaining accurate
inoculum strength is difficult
Coupon Method 3 (contact plates)
Challenges
Recovery may be inconsistent due to:
• Nature of surfaces
• Technique
If loss of recovery is close to a log it may become
difficult to prove 1-3 log reduction
Contact plates may not contain adequate neutralizers
DE Neutralizing agar which neutralizes all
disinfectants, is not used for routine EM
Coupon Method 3 (contact plates)
Other challenges
Positive control for efficacy may be difficult to read
with a 103 or 104 challenge
Plating dilutions of the target inoculum may help to
assess time “0” count
Coupon Method 3 (contact plates)
Benefits
Less time consuming among the representative Hard
Surface Studies
Drawbacks
Very inconsistent and may be able to prove a 3 log
reduction due to limited number of colonies that can
be counted on 25 cm. sq. area
Hard to enumerate fungi and spreaders
Not always quantitative (with TNTC growth, the CFUs
cannot be counted)
Coupon Method 3 (contact plates)
Coupons inoculated with target inoculum
Dried and treated with disinfectant
Recovered using swab, preferably two swabs into
tube containing buffer
Buffer with swabs vortexed and appropriate number
of dilutions made
Each dilution filtered, filters plated and incubated
Positive controls represent the recovery loss due to
drying of technique
Coupon Method 4 (swab recovery method)
Rinsing of coupons may be used instead of swabbing
Both recoveries and efficacies can me much lower than swab method
Coupon Method 5 (Rinse Method)
Using plastic bags and manual dislodging is not
practical for wall surfaces
Wall coupons cannot be steam sterilized
Mold growth may take over
Coupon Method 6 (Manual Dislodging)
Benefits
All representative hard surfaces can be tested
Complete quantitative study
Drawbacks
Extensive and time consuming
Coupon Method 4 (swab recovery method)
Benefits
All representative hard surfaces can be tested
Complete quantitative study
Drawbacks
Extensive and time consuming
Inconsistent recoveries
Coupon Method 5 (rinse method)
Benefits
All representative hard surfaces cannot be tested
Coupons that cannot be sterilized may add
contaminants
Complete quantitative study
Drawbacks
Extensive and time consuming
Low and inconsistent recoveries in dislodging method
Loss of CFUs as they can adhere to coupons and bags
Coupon Method 6 (manual dislodging method)
Planning Disinfectant Qualification
• Choice of test microorganisms
• Choice of surfaces
• Enumeration
• Calculating target inoculum for method validation and
efficacy
• Method Validation
• Efficacy
• Ensuring viability of inoculum-Time “0” count
• Calculation log reduction
• Expiry Study
• Neutralization Study
• Translation of results to cleaning and EM Procedures
Performed for:
• Undiluted and stored disinfectants
• Disinfectants that are diluted in-house and stored in
containers with an expiry date
Tests performed include:
• Efficacy: To verify no drop in efficacy on around
expiry
• Chemical Stability: Where applicable, to verify
no drop in chemical stability has occurred on or
around expiry
Expiry Dating Study
Neutralization Study
• To verify that the residue on surface does not interfere with
recovery of organisms during environmental monitoring
• The residue from disinfectants does not exert bacteriostatic /
fungistatic effect on surviving microorganisms
• To verify that the neutralizers in the contact plates are
adequate to neutralize the active ingredients in the
disinfectant residue
• The time lapse between cleaning and sampling is appropriate
for recovery of organisms
Neutralization Study
• Coupons - Stainless steel
• Disinfectants - highest concentration used for
disinfectant qualification
• Time points - 0, 2hrs, 4 hrs, 6 hrs, 12 hrs, 24 hrs
(aligned with cleaning practices)
• Organisms - Used for disinfectant qualification
Neutralization Study
• Duplicate coupons are treated with disinfectant
• At each time point coupons re sampled using
appropriate contact plates
• Contact plates are inoculated with ~ 100 CFU of
test organism
• Positive control contact plates are inoculated
with ~ CFU or test organism
• Contact plates are appropriately incubated
Acceptance criteria- The time point that yields 70% or factor
of 2 CFU count as compared to positive control is appropriate
for sampling of surfaces after disinfection / sanitization
Neutralization Study
• ATCC Strains: USP challenge organisms
• In-house Isolates: Predominant and objectionable
• Gram Positive Cocci - ATCC Strain and/or In-house
organism
• Gram Negative Baccilli - ATCC Strain and/or In-
house organism
• Gram Positive Bacilli In-house organism
(predominant)
• Yeast - ATCC Strain and/or In-house organism
• Mold - ATCC Strain and/or In-house organism
• Include objectionable and process microorganisms
Choice of Microorganisms
Choice of Microorganisms
Hard Surfaces Evaluation
• Plastic
• Glass
• Stainless Steel (316L, 316S etc.)
• Painted Surfaces
• Tile
• Aluminum
• Rubber
• Wood
• Flooring
• Latex
• Epoxy
• Vinyl
Choosing Hard Surface Coupons
• TSA settle and contact plates- tested for growth
promotion and sterility
• SDA settle and contact plates - tested for growth
promotion and sterility
• Rinse Fluids: Fluid A, Peptone Water or Phosphate Buffer
- tested for sterility
• Suspension Fluid for Inoculum: Sterile Saline - tested for
pH and sterility
• Letheen Broth or DE Neutralizing Broth - tested for
sterility
• Manifolds
Media and Supplies
• Dacron swabs
• Filters-correct size and material
• Sterile tubes
• Sterile saline
• McFarland standards
• Coupons-correct size and number
• Totes
• Disinfectants with short and extended expiry
• Water for dilution of disinfectants
• Hood
Media and Supplies
Choosing Hard Surface Coupons
• Small coupons of surfaces that may be autoclaved
may be used for immersion into broth medium ~1”
X 3”
• For efficacy tests performed using contact plates,
coupons should be appropriately ~3” X 3” in size,
up to 4” X 4” not larger - multiple coupons to be
tested under a hood-hood size is important
• Coupons for efficacy tests using swab recovery ~2 ‘
X 2” up to 4” X 4” not larger - multiple coupons to
be tested under a hood
Choosing Hard Surface Coupons
Cleaning or Sterilization of Hard Surfaces
• Hard surfaces coupons may be re-used.The coupons
need to be clean and free of micro-organisms
Choice of Sterilization Method
• Autoclave - For hard surfaces e.g. plastic, metal,
steel, etc.
• Chemical - Using a sporicidal compound for hard
surfaces that cannot be autoclaved, e.g. wall, floor
etc. followed by a wipe down with 70% IPA or sterile
water
Cleaning and Sterilization of Coupons
• Contact time: Elapsed time between application of
the disinfectant and its removal
• Test at manufacturer recommended contact time
• Understand the time disinfectants are active after
drying-study to show activity
Contact Time
Covering the entire area where inoculum is applied
may be attained by:
• Spraying
• Flooding
• Spreading without removal of inoculum
Special attention should be paid to surface tension
which prevents the disinfectant from completely
covering a hard surface
Application of Disinfectant
Swabbing
• Two swab technique
• Consistency in swabbing technique is critical
• Swabbing is accomplished by ten strokes in one
direction and additional ten strokes at 90 degrees
to the first ten. Each stroke is a full forward and
backward motion with the swab
Contact plates
• Contact plates with neutralizers are used
Sampling Method For Coupons
Sampling Method For Coupons
Rinse
Develop a method for rinsing and be consistent
Manual Dislodging
Be as consistent as possible
Sampling Method For Coupons
Disinfectant Neutralizer(s)
Gluteraldehyde Sodium Sulphite, Bisulphite
Chlorine Sodium Thiosulphate
Phenolics Tween (polysorbate)
QAC Lecithin, Polysorbate
Hydrogen peroxide Catalase
TSA and SDA contact plates contain lecithin and polysorbate 80
Letheen Broth contains lecithin and polysorbate 80
DE neutralizing agar and broth contain all above mentioned neutralizers - DE is not used in industry
Neutralization of Disinfectant
Incubation Time
• Bacteria generally up to 48 hours
• Yeast generally up to 72 hours
• Mold until profuse sporulation occurs-use
microscopy to ensure sporulation
Harvesting Organisms
• Avoid scraping agar in case of bacteria and fungi
• Lightly roll wet swab over sporulation of mold to
avoid mycelia
Enumeration
Storage of Harvest
• Bacteria and yeast stored at 2-4°C for 7 days
• Mold and spores stored at 2-4°C for 30 days
Dilutions
• Use Mc. Farland Standards equivalent suspensions
as starting point for dilutions
• 3.0 X 107-8 bacteria in 1 Mc. Farland equivalent
• Use 2 – 4 Mc. Farland Standard equivalent for mold-
determine during dry run
Enumeration
Filtration Method
• This applies to methods where swab recovery, rinse with
buffer or manual dislodging is buffer is used
• To verify that the filtration method and the amount of rinses
remove all disinfectant
• Filter the disinfectant dilutions to be tested, followed by the
number of rinses used in the method (3 X 100mL)
• Filter a suspension of ~100 CFU of test organism
• Incubate and compare to positive control
• Acceptance criteria - Within factor of 2 or 70% of positive
control
Method Validation
Neutralizing Broth
• This method can be applied only when using coupons that are
immersed in a neutralizing broth
• Neutralizing broth cannot be filtered
• Spray or apply disinfectant to coupon
• After contact time place the coupon inside the broth tube
• Inoculate with 102 microorganisms
• Positive Controls of broth without disinfectant
(enumeration may be a challenge)
• Negative control of broth only
• Incubate and compare test with positive control
Method Validation
Contact Plates
• Spray coupons with disinfectant
• Take samples using contact plate
• Inoculate contact plate with ~ 102 CFU of test
organism
• Positive controls without disinfectant
• Negative controls of media plates
Method Validation
• Stored inoculum may drop in viability
• Time ‘0’ Study verifies the inoculum count on the
day of testing
• Time ‘0’ is performed at the end of efficacy when
the same target inoculum is used over days
• Efficacy must be repeated if Time ‘0’ drops too low
(where 3 log reduction cannot be calculated)
Time ‘0’ Study or Inoculum Verification
• Each organism is tested by method of choice in
duplicate or triplicate
• Different surfaces dry or soak disinfectant at
different rates
• 15 minute time point not attainable in practice
• 10 minute time point recommended but difficult to
attain
Efficacy Study
Calculation of Target Inoculum
Calculate target inoculum in the enumeration study
by counting CFU on the plate where the count is less
than 300 CFU and multiplying with the dilution factor
Calculation of Time “0” Inoculum
Calculate Time “0” inoculum by counting CFU on the
plate where the count is less than 300 CFU and
multiplying with the dilution factor
Calculations
Calculation of Log Reduction
The log of the average CFU recovered from the
efficacy study is subtracted from the log of those
recovered on the positive control
Calculations
Test Matrix
• Disinfectants
• Dilutions
• Time points
• Surfaces
• Organisms
Disinfectant Qualification Report
• Enumeration methods summary
• Method Validation summary and results
• Microbiological efficacy results
• Expiry dating results
• Chemical stability results for efficacy and expiry
dating
• Conclusions and discussions
• Deviations
Disinfectant Qualification Report
Tabulating results for easy review Organism
• Disinfectant
o RC
o Time Point
o Hard Surface
o Target inoculum
o Time ‘0’ count
o Efficacy count
o Log of efficacy count
o Positive control
o Log of positive control
o Log reduction
Disinfectant Qualification Report
• Target inoculum un attainable for certain fungi
• Drop in inoculum
• Disinfectant does not cover organisms of hard
surface due to surface tension
• Variability within replicates
• Data does not represent disinfectant’s capability
Common errors
Challenges in Cleaning and Disinfecting
• Nature of Surface
• Smooth - Surface tension, needs multiple strokes to
cover entire surface
• Porous - Absorbs disinfectant fast, dries out fast,
needs multiple strokes to cover surface
• Soft - Absorbs disinfectant fast, dries fast, needs
multiple strokes to keep wet for contact time
• Hard - Surface tension, needs multiple strokes to
cover entire surface
Cleaning and Disinfection
Aluminum Surface
Nature of Surfaces
Courtesy of Veltek Associates, all rights reserved
Stainless Steel Surface
Nature of Surfaces
Courtesy of Veltek Associates, all rights reserved
Epoxy Coated Surface
Nature of Surfaces
Courtesy of Veltek Associates, all rights reserved
Cleanroom Curtain
Nature of Surfaces
Above picture is courtesy of Veltek Associates
Cleanroom Curtain
Nature of Surfaces
Above picture is courtesy of Veltek Associates
Vinyl Surface
Nature of Surfaces
Above picture is courtesy of Veltek Associates
Points to consider
• Cleanroom surfaces are irregular in nature
• Such surfaces trap residues and contaminants and
makes surfaces more difficult to disinfect
Nature of Surfaces
Procedure for Receiving, Storage, and Preparation
of Disinfectants
Points to consider
• Safety Considerations
• Definitions
• List of all agents used in Manufacturing Faculty
• Procurement, Receipt, and QC Release
Procedure
• Preparation and use
Cleaning Procedures
Procedure for Disinfecting/Sanitizing of
Manufacturing Facility
Points to consider
• Responsibility
• Definitions
• Safety Considerations
• Supplies and storage
• Sequence of Cleaning
• Schedule or Frequency of Cleaning
Disinfection / Sanitization Procedure
Procedure for Disinfecting/Sanitizing of
Manufacturing Facility
Points to consider
• Disinfectants/Sanitizers
• Rotation of Agents
• Cleaning Procedure
• Removal of Residue
• Documentation
• Review
• Training
Disinfection / Sanitization Procedure
Chemical Kill vs Physical Removal
Chemical Kill
• Choice of disinfectants
• Rotation of disinfectants
• Wipe down procedures
• Disinfectant qualification
Physical Removal
• Choice of mops - ergonomic and reaching hard to clean areas
• Choice of mop heads - capacity to hold and dispense
adequate disinfectant to attain contact time
• Choice of adequate wipes to attain contact time and
physically remove particulates and residue
• Disinfection needs both chemical kill and physical
removal
• Surfaces are irregular and differ in time taken to
absorb or dry
• Adequate application of disinfection without
soaking is the key
• Ability of supplies to cover all surfaces evenly
• Ability of supplies facilitate physical removal
• Is contact time a myth?
• Good coverage and maximum contact time=
reduction of contamination
Significance of contact time
Mops and Mop Handles
• Hold and deliver disinfectant
• Reach hard to clean areas
• Physically remove debris and residue
• Ergonomic
• Can be used on all cleanroom surfaces-ceilings,
walls floors, behind equipment
Wipes
Non linting
Remove debris and residue
Cannot promote microbial proliferation
Points to consider
Is contamination removed or added?
Can you access the hard to clean areas?
Can you access all the critical surfaces?
If you can access these surfaces can you apply
adequate amount of disinfectant uniformly?
Can you remove residue buildup?
Do you generate particulates by way of cleaning?
If so, do you know what is the amount of
contamination generated?
Are wipes made of organic or inert materials?
Disinfectant Qualification using Biofilms
Disinfectant Qualification using Biofilms
Disinfectant Qualification using Biofilms
Disinfectant Qualification using Biofilms
Disinfectant Qualification using Biofilms
Disinfectant Qualification using Biofilms
What is the measure of cleaning/disinfecting efficacy?
Disinfectant qualification proves the chemical efficacy
and contact time for the desired kill
Environmental monitoring data validates the chemical
kill and physical removal
Also understand the lapse time between and
monitoring
Your disinfectant qualification for (b)(4) and (b)(4) bi-spore
disinfectants documented that the log reduction criteria
(Bacteria ≥ 4, Fungi ≥ 3) was not met when challenged with
multiple organisms in a variety of surfaces. After disinfection,
you recovered Micrococcus luteus on vinyl, (b)(4), stainless
steel, glass, and wall laminate and Enterobacter cloacae,
Rhodococcus sp, Burkholderia cepacia, Pseudomonas aeruginosa,
Methylobacterium mesophilicum and, Acinetobacter lwoffi on
glass. However, your procedures for routine cleaning of the
aseptic manufacturing area continue to require the use of
unqualified disinfectants during days (b)(4) through (b)(4) of
your disinfectant program.
FDA 483 Observations
Consistent with SOP XXX, Requirements for Cleaning/Disinfecting
Procedures in Controlled Areas in that surfaces were not wiped
with a sterile wipe after disinfectant dwell time. There is no
assurance that the disinfectants applied without wiping do not
inhibit growth on RODAC surface samples. Qualification studies
used to demonstrate the ability of [redacted] to support that the
growth of microorganisms is not inhibited by potential exposure
to residual antimicrobial agents found on surfaces following
cleaning did not specify how surfaces were cleaned for the study
(disinfectants allowed to dry on or wiped with sterile wipe after
dwell).
FDA 483 Observations
“The disinfectant ___ was replaced by _____ as
cleaning agent in September 1998, however was used
to clean Laminar Airflow unit ____ in April 1999”
FDA 483 Observations
“Contact time for disinfectants not documented in
cleaning log”
“Daily cleaning failed to remove objectionable
material”
“End of process failed to remove objectionable
material”
“Operators failed to assure satisfactory room
cleanliness during filling operations”
FDA 483 Observations
Also, your environmental monitoring program does not provide
meaningful information on the quality of the aseptic processing
environment and ancillary areas used in the manufacture of [redacted] .
For example, your sampling and testing procedure does not incorporate
the use of positive or negative controls; the frequency of routine
environmental monitoring was changed from [redacted] per DCR 05-678
in February of 2006 without adequate justification or rationale or the
change; and an evaluation of environmental monitoring sampling
locations had not been conducted to assure the locations provide a true
and accurate assessment of microbial flora and in the production areas
they represent locations of microbiological risk to the product.
Furthermore, no evaluation had been performed to assure cleaning
solution residues do not negatively impact environmental monitoring
sampling or testing.
FDA 483 Observations
Disinfection, Sterilization, and Preservation; Seymor S. Block
Ed., Lippincott, Williams and Wilkins
PDA Publications
Survey Volume 51, Number 6, Nov-Dec 1997
Current Practices in the Use of Disinfectants within the
Pharmaceutical Industry; Denny and Marsik
Survey Volume 53, Number 3, May-June 1999
Elements of successful disinfection program in the
pharmaceutical environment. Denny, Kopis and Marsik
Some Suggested Reading
Questions??