bioburden validation strategy for cleaning validation
DESCRIPTION
This presentation is based on the article published 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-ByronTRANSCRIPT
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 selectioncontamination distributionsignificance 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 areaso assumes uniformity of contamination surfaceo 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…..
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.
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 inertDry 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 activeToxic to bacteria due to metal ions
release
Silicone rubber Non porous inert Less suitable for adherence than plastic
Teflon, dacron Porous inertBacteria adherence more than glass but
lesser than plasticPolyethylene, 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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