Bioburden Validation Strategy for Cleaning Validation

angelsalaman@yahoo.com
BIOBURDEN RECOVERY
METHOD SUITABILITY FOR
CLEANING VALIDATION
Angel L Salamán, Ph.D.

Outline
 Cleaning Validation Process Basis
 Analytical requirements
 Sample methods
 Problem statement
 Method Suitability Test Perspective
 Surface may Influence Mortality Rate of
Bacteria
 Conclusion

Source
 This presentation is based on the article
published online by Pharmaceutical
Technology (USA) entitled “BIOBURDEN
METHOD SUITABILITY FOR
CLEANING AND SANITATION
MONITORING: HOW FAR WE HAVE
TO GO?”, Aug 2010. by Angel L. Salaman-
Byron (http://www.pharmtech.com/pharmtech/Analytics/Bioburden-Method-Suitability-for-Cleaning-and-Sani/ArticleStandard/Article/detail/683682)

Inadequate equipment cleaning procedures
may result in number of contaminants present
in the next batch manufactured on the such as:
 Active Pharmaceutical Ingredient, ingredients and product
intermediates
 The previous product or product intermediates.
 Solvents and other materials employed during the manufacturing
process.
 Airborne material
 Microorganisms and microorganisms byproducts such as toxins
and pyrogens. This is particularly the case where microbial growth
may be sustained by the product or their product ingredients.
 Cleaning agents themselves, lubricants, ancilliary material.

Validation Process
Why cleaning validation is so important?
 Pharmaceuticals can be contaminated by potentially
dangerous substances
 Essential to establish adequate cleaning procedures

Validation Process
Why cleaning validation is so important?
 “Particular attention should be accorded to the
validation of … cleaning procedures” (WHO)
 “Cleaning validation should be performed in order to
confirm the effectiveness of a cleaning procedure”
(PIC/S)
 “The data should support a conclusion that residues
have been reduced to an „acceptable‟ level” (FDA)

Validation Process
 The manufacturer needs a cleaning validation
strategy
 Assess each situation on its merits
 Scientific rationale must be developed
equipment selection
contamination distribution
significance of the contaminant
 “Visually clean” may be all that is required

Validation Process
Strategy on cleaning validation
 Define product contact surfaces
 After product changeover
 Between batches in campaigns
 Bracketing products for cleaning validation
 Periodic re-evaluation and revalidation

Validation Process
Cleaning validation Protocol
Should include :
 Objective of the validation
 Responsibility for performing and approving
validation study
 Description of equipment to be used

Validation Process
Cleaning validation protocol (cont.)
Should include:
 Interval between end of production and cleaning, and
commencement of cleaning procedure
 Cleaning procedures to be used
 Any routine monitoring equipment used
 Number of cleaning cycles performed consecutively
 Sampling procedures used and rationale
 Sampling locations (clearly defined)

Validation Process
Record of cleaning validation
Should include :
 Data on recovery studies
 Analytical methods including Limit of Detection and
Limit of Quantitation
 Acceptance criteria and rationale
 When revalidation will be required
 Must have management and QA involvement
 Management commitment and QA review

Validation Process
Results and reports
 Cleaning record signed by operator, checked by
production and reviewed by the QA Unit
 Final Validation Reports, including conclusions

Validation Process
Personnel
 Manual cleaning methods are difficult to validate
 Cannot validate people; can measure proficiency
 Good training, documented
 Must have effective supervision

Validation Process
Microbiological aspects
 Include in validation strategy
 Analyze risks of contamination
 Consider equipment storage time
 Equipment should be stored dry
 Sterilization and pyrogens contamination

Validation Process
How to sample?
 Swab/swatch
 Rinse fluid
 Placebo
 The sample transport and storage conditions should
be defined

Validation Process
Swab samples
 Direct sampling method
 Reproducibility
 Extraction efficiency
 Document swab locations
 Disadvantages
o inability to access some areas
o assumes uniformity of contamination surface
o must extrapolate sample area to whole surface

Validation Process
Rinse samples
 Indirect method
 Combine with swabs
 Useful for cleaning agent residues
 pH, conductivity, TOC
 Insufficient evidence of cleaning
 Sample very large surface areas

Validation Process
Analytical methods:
 Validate analytical method
 Must be sensitive assay procedure:
• HPLC, GC, HPTLC
• TOC
• pH
• conductivity
• UV
• ELISA

Validation Process
Analytical methods (cont.)
Check:
 Precision, linearity, selectivity
 Limit of Detection (LOD)
 Limit of Quantitation (LOQ)
 Recovery, by spiking
 Consistency of recovery

Setting limits:
 Regulatory authorities do not set limits for specific
products
 Logically based
 Limits must be practical, achievable and verifiable
 Allergenic and potent substances
 Limit setting approach needed
Validation Process

Setting limits (cont.)
 Uniform distribution of contaminants not guaranteed
 Decomposition products to be checked
 Setting limits; cleaning criteria:
 visually clean
 10ppm in another product
 0.1% of therapeutic dose
Validation Process

Setting limits: “Visually clean”
 Always first criteria
 Can be very sensitive but needs verification
 Use between same product batches of same
formulation
 Illuminate surface
 Spiking studies
Validation Process

Setting limits: “10ppm”
 Historical
 In some poisons regulations
 Pharmacopoeias limit test
 Assumes residue to be harmful as heavy metal
 Useful for materials for which no available
toxicological data
 Not for pharmacologically potent material
Validation Process

Setting limits: not more than 0.1%
 Proportion of MINIMUM daily dose of current
product carried over into MAXIMUM daily dose of
subsequent product
 Need to identify worst case
Validation Process

Residue limits…
Residue limits for cleaning, cleaning validation, and
numerous associated considerations continue to be
a confused, misinterpreted, and generally
misunderstood topic of discussion among global
validation personnel. Support for this assertion
may be found on the US Food and Drug
Administration website listing of frequent FDA-
483 observations.
Cleaning/sanitization/maintenance (Code of
Federal Regulations Title 21 Part 211.67) was
among the 10 most cited observations for drug
inspections

Other issues
 Clean-In-Place (CIP) systems
 Placebo studies
 Detergent residues; composition should be known
 Scrubbing by hand
Validation Process

Validation Process
Questions for the GMP Inspector to ask
 How is equipment cleaned?
 Are different cleaning processes required?
 How many times is a cleaning process repeated
before acceptable results are obtained?
 What is most appropriate solvent or detergent?
 At what point does system become clean?
 What does visually clean mean?

Observation 5: Written procedures for cleaning and
maintenance fail to include description in sufficient
detail of methods, equipment and materials used.
 SOP 1 indicates: "If necessary, brush the interiors and exteriors
and walls with XXX detergent." When asked when brushing is
necessary, one operator said that he "thinks" it is always
necessary to brush while another operator said that it should be
done for every major cleaning.
 SOP 2 indicate spraying or rinsing parts with XX. Operator said
that he can either spray the part with XX and wipe it with a
cloth a "little bit" damp with XX or just wipe it with the XX
damp cloth.
 The current version of SOP 3 is missing a rinse step; after
washing parts with the detergent solution, step X indicates
wiping with XX. According to the firm's officials, this step was
inadvertently left out when the current version was written.
 Andrx Pharmaceutical, Inc., 483 Inspectional Observations, Fort
Lauderdale, FL, dated 03/06/2006 - 04/18/2006

Industrias Quimicas Falcon de Mexico, Cuernavaca, Mexico,
Warning Letter from the US FDA 14th June 2011 .
The letter‟s second observation was…cleaning
validation was incomplete for non-dedicated
manufacturing equipment. …. responded that it
was committed to starting cleaning validation
activities once a validated analytical method was
available. However, the FDA commented that it
was concerned about the impact that the lack of
cleaning validation has on marketed products…..

BIOBURDEN METHOD
VALIDATION
PERSPECTIVE

Background
 Surface Microbial Bioburden monitoring
methods are described in Standard Methods
for the Examination of Dairy Products, 17th
Edition, 2004.
 Literature review showed a poor correlation
with the amount of microbial contamination
on surfaces and the recovery obtained.

Background
 Many factors may contribute to this poor
correlation, including differences in
materials used (e.g., cotton, polyester,
rayon, calcium alginate), the organisms
targeted for culture, variations in surface,
and differences in the personnel collecting
and processing samples.

Background
 It is suggested that the lack of standardization
of both the swabbing pattern and the pressure
applied to the swab during sampling,
meaning, technician-to-technician variation
in the sampling procedure may potentially
play a significant role in the recovery and
enumeration of the sampled surface.

Background
 Based on these studies it is widely accepted
that positive swab samples are indicative of
high surface concentration of microbes,
whereas negative swab samples do not
assure that microorganisms are absent from
the surface sampled

Bioburden Method Validation
Process
 Studies are performed using coupons of the representative surfaces
inoculated with the test microorganisms.
 Test microorganisms (usually known laboratory-adapted strains) are
spread onto a space that is ~ 25 cm2 and allowing to air dry.
 After air drying test microorganisms are recovered by either swab or
contact plates.
 The test samples along with positive and negative controls are treated
and/or incubated.
 Results are analyzed based on the percent of test microorganisms that
grow after recovery compared with an inoculation control

Bioburden Method using swab
 Variables affecting the accuracy of the detection
and enumeration using swabbing technique
initially include the ability of the swab to remove
the microflora from the surface as well as its
effectiveness to release removed microorganisms
from the swab and their subsequent recovery and
cultivation.

Bioburden Method using swab
 The proportion of attached microflora on surfaces that are
trapped or tenaciously bound to the interwoven fibers of a
swab head are unknown, and sampling techniques that
preserve the underlying surface as well as the viability of
the detached micro-flora, will detach only a portion of the
total population.
 Adherent bacteria on surfaces become increasingly
difficult to remove by use of swabs, especially if they
become associated with a biofilm.

Recovery is LOW
 Studies conducted under controlled conditions have demonstrated that
recovery is low.
 Kusumaningrum, et al. (2003) reported that in evaluating the survival
and recovery of Bacillus cereus, Salmonella enteriditis,
Campylobacter jejuni, and Staphylococcus aureus on stainless steel
surfaces, the direct contact method using solidified agars recovered
18% of Bacillus cereus, 23% of Salmonella enteriditis, 7% of
Campylobacter jejuni, and 46% of Staphylococcus aureus from the
initial concentration applied to the surface .
 A validation and comparative study on recovery of microorganisms
using swabs, Hygicult TPC dipslide, and contact agar plate yielded
similar results and did not differ in precision, with recoveries ranging
from 16 to 30% of the microbial load applied to the surface.

Test Method Suitability:
Problem Perspective
 The validation of surface recovery methods (i.e. chemical and microbiological)
is a pre-requisite for residual determination of cleaning effectiveness in
process validation studies.
 These methods should be challenged in the Laboratory by pilot-scale
controlled conditions in order to evaluate its suitability for its intended use.
 For this purpose validation specialists select representative surfaces identified
within the production area and potentially in contact with ingredients, product
intermediates and bulk products are commonly chosen.
 Surfaces challenged selected for method validation commonly include
Stainless Steel 316L, glass, plastic (i.e. such as Polyvinyl-chloride and
Polyethylene) and some metal alloys.
 However, surface selections for challenging studies are not justified based on
what really matters: demonstrating the effectiveness of the Monitoring
method

THE SURFACES
CHALLENGING
 There are many types of surfaces in the pharmaceutical
production areas and cGMP equipment, all with distinct
physico-chemical properties.
 Most of these surfaces are well defined. When
microorganisms are released into the manufacturing area,
they will be deposited onto these surfaces as either aerosol
particles or as liquid droplets.
 The type of surface greatly influences their ability to
survive and their possibility to contaminate other materials.

Types of Surfaces
 Porous and non-porous, inert or active,
rough or smooth, hydrophobic or
hydrophilic, etc.
 Glass and stainless steel are examples of
Non-porous inert surfaces.
 Galvanized steel, brass and copper are
example of Non-porous active surfaces.

Types of Surfaces (cont.)
 Stainless steel is the principal material of
construction of GMP equipment and it has been
extensively studied.
 Microscopically stainless steel may show grooves
and crevices that can trap bacteria while glass does
not.
 Some bacteria have been found to be able to
adhere to the stainless steel surfaces after short
contact times if the conditions are appropriate (i.e.
adequate temperature and humidity).

The Porosity Factor
 The porosity of surface is a major factor
affecting bacteria adherence.
 Highly porous surface facilitates adherence
of bacteria.
 Adherence of bacteria is depending of the
number of cells: the higher the number of
cells the higher the probability those cells
remain attached on surface after rinse.

The Porosity Factor
 Porous materials such as plastics, Teflon, Dacron
and their combination are used less often as
materials of construction in GMP equipment.
 Rijnaarts and colleagues (1996) reported bacteria
deposition on Teflon is faster than glass.
 It was reported that rubber and plastic coupons
were significantly more accessible to the bacteria
than glass coupons as revealed by the high
population of bacteria recovered from their
surfaces.

The Porosity Factor
 Porosity may prevent water evaporation.
 The lethal effect of desiccation was found to be
the most important death mechanism in bacteria.
 Similar studies performed on Teflon surface using
Escherichia coli, Acinetobacter sp., Pseudomonas
oleovorans, and Staphylococcus aureus
demonstrated that all four species survived well
during the droplet evaporation process, but died
mostly at the time when droplets were dried out at
40 to 45 mins.

The Porosity Factor
 Fabrics are porous surfaces (i.e cotton, polyester,
polyethylene, polyurethane and their combinations, etc.)
that demonstrated survival of Gram-negative and Gram-
positive microorganisms, even longer than plastics.
 It has also been observed that Gram-positive bacteria
survive a little longer than gram-negatives.
 It is recommended to rinse fabrics and other porous
surfaces in order to detach microbes from them .
 Swab and plate contact methods are not suitable for
fabrics.

The Charge Factor
 It is quite well known that charged molecules in
solution are able to kill bacteria.
 However, it has been realized more recently that
charges attached to surfaces can kill bacteria upon
contact.
 Certain surfaces such as brass, copper and
galvanized steel can be toxic to bacteria because
the presence of water and air allows the release of
metal ions from metal surface.

The Charge Factor
 Metal ions exert an antimicrobial effect by
interfering with biological pathways and enzymes.
 Copper releases Cu2+ ions, galvanized steel
releases Zn2+ ions and brass releases both Cu2+
and Zn2+ ions.
 These metal ions are in fact essential
micronutrients of bacterial cells but at very low
concentrations.

Plastics
 Poly-vinyl chloride (PVC) and poly-propylene (PP) are
two similar plastics, but have different properties.
 PP is more stable and less reactive that PVC.
 PVC surfaces show high mortality rates for bacteria while
PP surfaces show no significant levels of mortality.
 Studies with Enterococcus faecalis aerosol on PVC and PP
demonstrated that PVC had a significant effect on the
survival of bacteria due to oxidation reactions with the
walls of Gram-negative bacteria.

salamaa@wyeth.com
SURFACE TYPE
INFLUENCE MORTALITY
RATE OF BACTERIA

Survival of Bacteria on Plastic
 Wildfǜhr and Seidel (2005) reported that Pseudomonas
aeruginosa; Staphylococcus aureus and Candida albicans
survival rate on plastic was almost double (50% more) that
on stainless steel or glass coupons.
 In this study test microorganism‟s suspensions were
transferred onto stainless steel, glass and plastic coupons
and then dried. After 90 minutes it was evident that only a
very small quantity of bacteria was present on the stainless
steel and glass surface, but the quantity of viable bacteria
on plastic was still up to 120 min.

Survival of Bacteria on Plastic
 Tiller et al, 2001 reported that plastic coupons (i.e.
Polypropylene and Polystyrene) keep bacteria more viable
than aluminum, steel and glass.
 In this experiment, suspensions (106 cells per mL) of S.
aureus in distilled water were sprayed over the surface
various materials, air dried for 2 min and incubated in a
0.7% agar bacterial growth medium overnight, after which
the colonies counted.
 Bacterial adherence in the presence of oral liquid
pharmaceuticals on different coupons showed that rubber
and plastic coupons were significantly more accessible to
the bacteria than glass coupons

Microbial Adherence
 Nevertheless, studies of bacterial adhesion with laboratory
strains of bacteria (i.e. type culture collection strains),
many of which had been transferred thousands of times
and lost their ability to adhere, first indicated that very
smooth surfaces might escape bacterial colonization.
 Subsequent studies with “wild” and fully adherent bacterial
strains showed that smooth surfaces are colonized as easily
as rough surfaces and that the physical characteristics of a
surface influence bacterial adhesion to only a minor extent.
 This fact is important when selecting test microorganisms
for suitability testing.

Material Surface nature Interaction with microbes
Stainless Steel Non-porous inert
Dry conditions leads to dead.
Some bacteria have been found to be
able to adhere to the stainless steel
surfaces after short contact times if the
conditions are suitable (i.e. adequate
temperature and humidity
Glass Non-porous inert
Dry conditions leads to dead. Bacteria
are less viable than stainless steel
Brass, copper, galvanized
steel, aluminum and
aluminum alloys and other
metal alloys
Non-porous active
Toxic to bacteria due to metal ions
release
Silicone rubber Non porous inert Less suitable for adherence than plastic
Teflon, dacron Porous inert
Bacteria adherence more than glass but
lesser than plastic
Polyethylene, Polyurethane,
Polypropylene and
Polystyrene Plastic and
rubber
Porous inert
More suitable for bacteria adherence and
survival than Silicone rubber, Teflon,
Dacron, steel, brass, cooper, aluminum
and metal alloys.
Fabrics (cotton, polyester,
polyethylene, polyurethane
and their combinations)
Porous inert and
active
More suitable for bacteria adherence and
survival than plastic. Rinse water method
is advised.
Microorganism-substratum interaction for microorganism
adherence and survival

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Bioburden Validation Strategy for Cleaning Validation

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