validationofa rapidsystemfor environmental monitoringand ... - biolumix ·...

VALIDATION OF ARAPID SYSTEM FORENVIRONMENTALMONITORING ANDWATER TESTING

Ruth Eden and Roger Brideau

This chapter appeared in Environmental Monitoring, Volume 6, edited by JeanneMoldenhauer. Copyright ©2012 and published by the Parenteral Drug Association(PDA) and Davis Healthcare International (DHI). All rights reserved.

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14

VALIDATION OF A RAPID SYSTEMFOR ENVIRONMENTAL MONITORING

AND WATER TESTING

Ruth Eden and Roger BrideauBioLumix Inc.Ann Arbor, MI

USA

INTRODUCTION

The FDA expects manufacturers to be in control of the environmental conditions within theirmanufacturing facility. Controlling the environmental conditions is not only a regulatoryrequirement but also part of protecting the manufactured products and assuring the productionof quality products. Environmental Monitoring (EM) of manufacturing facilities:

• provides assurance that the environment is controlled and in compliance

• provides current information on the quality of the processing environment

• prevents future microbial contamination by monitoring adverse trends

• ensures that the environment is properly controlled.

There is substantial evidence establishing a direct relationship between the level ofenvironmental control and the final quality of the product. EM serves a critical role in productsafety by ensuring that the environment is maintained under proper control.

The purpose of surface sampling as part of an overall environmental monitoring programis to track the level of surface contamination in the facility to ensure that cleaning andsanitization is effective. Swabs are often used for sampling irregular or hard-to-reach surfaces

241

and critical surfaces where contact plates are not practical. In addition, cleaning hold-timestudies are often performed using swabs. Sanitizers collected from surfaces can be neutralizedand dilutions can be done when highly contaminated areas are sampled. In general, the purposeof a microbial EM program is to:

• provide crucial information on the quality of the work process environment duringmanufacturing

• prevent future microbial contamination by detecting and reacting to adverse trends

• prevent the release of a potentially contaminated batch if the appropriate standards are notfulfilled

• prevent the risk of contamination of the product

• ensure there are environmental controls in the production areas

• provide a profile of the microbial cleanliness of the manufacturing environment.

Thus, the Microbial EM Program serves as a tool for assessing sanitation procedures.

Most EM is done by plate counting of colonies, which is simple and inexpensive. However,plate counting methods are slow, requiring two to seven days to complete, thereby causing adelay in the detection of contamination, which can lead to an increase in product loss, plantdowntime and expensive clean up. Delays can cause increasing inventory holding cost. Thedelay in obtaining results impacts reaction to contamination issues and can make investigationsvery difficult. For example, the rooms in question have typically been cleaned numerous times,so re-sampling results are almost always meaningless, and determining the root cause of thecontamination is difficult. Since real-time response is not possible, batches are jeopardized.

The plate count methodology is also labor-intensive and requires manual data entry anddocumentation. Such documentation is prone to human errors and compliance issues.

Methods are available to measure total particles in the air, including Total Organic Carbon(TOC), and Adenosine Tri-phosphate (ATP). These methods are very fast to perform but do notcorrelate well with total bacterial count or any specific group of organisms, and do not measureviable organisms (Carrick et al., 2001; Easter, 2010). Therefore, these results do not measureviable organisms in the environment or on production lines. The standard plating methodologiescan take several days for results. Rapid microbiological methods (RMM) can provide rapid andefficient solutions over traditional plating methodologies. Therefore both manufacturers andregulators are motivated to develop initiatives and help in the implementation of rapid testingmethods (FDA, 2004). The Pharmaceutical industry is slow to adopt RMM for the release offinished products, in part due to the need to file regulatory documentation to change from thetraditional sterility test to any alternative method. Any new RMM must be validated against thecurrent plate count methodology to show that it results in equivalent data results. The USPinformational chapter <1223> (2005) provides guidance for the validation of methods for useas alternatives to official compendial microbiological methods.

Environmental Monitoring242

THE BIOLUMIX® SYSTEM

Technology

The BioLumix Optical System is based upon the detection of microorganisms due to color orfluorescence changes caused by the growth and metabolic activity of microorganism in the testvials. The media reagents and/or the vial sensor change their color/fluorescence as metabolismoccurs. Changes in color or fluorescence, expressed as light intensity units, are detected by theoptical sensor, and recorded in the instrument computer 10 times per hour. Sample volumes ofup to 2 ml can be used in the ready-to-use disposable vial.

Figure 14.1 Typical curves with microbial growth and no growth

The sensitivity of the system can measure a single viable cell per sample vial. One bacterialcell can be detected within 8–18 hours, a single yeast cell is detected in 20–30 hours, and moldcells require 35–48 hours to be detected. However, the BioLumix system is a growth-basedsystem in which organisms must first grow to a level exceeding a specific detection threshold.This threshold is ~100,000 cells/ml for bacteria, and ~10,000 cells/ml for yeast and molds. Thesystem creates dynamic patterns as the microorganisms grow in the medium as shown in Figure14.1. The negative curve is flat without any significant increase in the signal and no detectiontime (DT) when organisms are not present (no growth). The second curve illustrates a positivecurve generated when the microorganism grows in the vial (microbial growth). A DT of 11hours was generated and is shown on the graph. The time to detection depends on the initialconcentration of the organisms in the sample, their generation time, and the physiological stateof the organisms. Highly contaminated samples are detected rapidly, thereby providing timelywarning of contamination.

Validation of a Rapid System for EM and Water Testing 243

Disposable vials

A critical component of the technology is the two-zone detection vial with two compartments:

• an upper incubation zone where the sample and microorganisms reside

• a lower reading zone that remains optically clear.

The upper zone tends to contain product debris and turbidity due to microbial growth. This two-zone vial eliminates interference of the optical pathway by the product and microbial turbidity.Since changes of color or fluorescence are monitored in the reading zone, results are notinfluenced by the presence of the product sample or the turbidity in media due to growingmicroorganisms. There are two types of vials used in the BioLumix system— a membrane vialand a CO2 vial.

Membrane vials

The patent-pending vial has an embedded 0.2 micron filter that separates the incubation zonefrom the reading zone.

Carbon dioxide vials

CO2 is a universal metabolite produced by all microorganisms. The transparent solid sensorlocated at the bottom of the vial detects CO2, resulting in a color change. Only gases canpenetrate into the sensor — liquids, microorganisms and particulates are blocked.

Available test vials

Total Aerobic Microbial Count (TAMC), Total Combined Mold and Yeast Count (TCMY),Enterobacterial count (bile tolerant gram negative bacteria), and a variety of vials to test forobjectionable organisms, including tests for Escherichia coli, Pseudomonas aeruginosa,Staphylococcus aureus, and Salmonella.

How microorganisms change color or fluorescence

Several types of reactions are being used in conjunction with the system.

CO2 sensor

The sensor, at the bottom of the vial, is composed of a matrix of a polymeric material which istransparent to light, and contains an indicator reagent sensitive to carbon dioxide gas generatedby the microorganisms. The matrix is configured to facilitate penetration of external light fromthe LED and monitoring the change of color of the indicator from blue–green to yellow due toCO2 production. The user introduces the sample by simply opening the screw cap and droppingthe sample into the incubation zone.


Fluorogenic enzyme substrates

The hydrolysis of fluorogenic synthetic substrates by bacterial enzymes causes an increase influorescence. Fluorogenic synthetic enzyme substrates containing 4-methylumbelliferon aremost commonly used. The E. coli assay employs 4-methylumbelliferyl-β-D-glucuronide (MUG).The enzyme β-glucuronidase (GUD) present in ~97% of E. coli strains is capable of hydrolyzingthe colorless substrate MUG to yield a bluish fluorogenic product of 4-methylumbelliferone(MU) that fluoresces under UV light (366 nm) when present in the instrument.

Change in color of a pH indicator during microbial activity

Bromocresol Purple is used as an indicator between pH 6.8 (purple) and pH 5.2 (yellow), inEnterobacteriaceae (bile-tolerant Gram-negative bacteria) media. Phenol Red is one of theindicators in the Staphylococcus media.

Modular instrument

Each BioLumix instrument has a capacity of 32 sample locations with a single incubatingtemperature. Figure 14.2 illustrates a six instrument configuration. A single personal computercan control over 30 instruments, resulting in the ability to monitor over 1,000 samplessimultaneously, and allowing the system to grow with the user’s needs. The interlocking, front-loading design allows for safe stacking of multiple instruments. The instrument collects opticaldata generated from each vial location at a rate of 10 readings per hour. Samples can berandomly introduced into the system at any time. The instrument has a temperature controlledincubator capable of either heating or cooling in order to obtain any temperature in the rangeof 15–60°C. The multiplicity of temperatures is important whenever different tests requiringdifferent temperatures are run simultaneously (e.g., total counts at 35°C and yeast and mold at28°C). Each BioLumix instrument has the structure of a drawer, enabling the user to stack unitson top of each other, thereby saving precious laboratory space.

Figure 14.2The BioLumix system


Software

A personal computer with aWindows®-based program controls the operation of the BioLumixinstrument(s). The software is cGMP, and 21 CFR Part 11 compliant, provides an audit trail,operator identification (log in and log out), password protection with various levels of security,trend analysis, and has various reports. The software allows for random access to each viallocation in starting or stopping individual tests. The data collected from the instrument areautomatically stored and processed by the computer. The results are presented as soon asdetections occur without any operator involvement. The system provides automateddetermination of Detection Time (DT), data archiving, alerts for contaminated samples, and avariety of reports. Results can be communicated over any Internet Protocol (IP) network inreal-time. Reports can be customized to fit the user’s needs. The system supports bar code entryof tests. Data are evaluated in real time to enable product release in a timely manner.

Dilute-to-spec protocol

BioLumix uses the dilute-to-specification protocol, which requires diluting the sample toproduct release specifications or in-process action levels. If growth is detected, the sample fails.If there is no detection, the sample passes (i.e., the counts are below the specification limit).For example, this protocol can be used for samples with an action level of 100 CFU/g for yeastand molds, and 500 CFU/g for total aerobic count. If the system detects growth in a 1:10sample dilution (0.1 mL of sample is added to the BioLumix vial), then the counts are >100CFU/g; if there is no detection of growth, the sample had <100 CFU/g. Different dilutions canbe used depending on the sample’s specification level (e.g., 0.2 mL of a 1:100 dilution is addedto a vial when the spec is <500 CFU/g).

Objective

The purpose of this study is to validate the BioLumix system as an alternative to the plate countmethod in detecting microbial contamination on a variety of production surfaces. Swabrecovery studies were conducted for the specific types of surfaces used in production. Thisstudy demonstrated that equivalent recovery of microorganisms from the equipment surfacewas obtained by the BioLumix method and the plate count method. The study was designed todetermine the repeatability, reproducibility, and recovery of the microorganism by the proposedswabbing procedure from the equipment surfaces.

In a second study, the equivalency in recovery of heterotrophic organisms from waterbetween the BioLumix system and the plate count methodology was demonstrated.

METHODS

Coupons (surfaces)

10 ×10 cm (4” × 4”) coupons from five different materials, as shown in Table 14.1, were usedin this study.


Table 14.1 Coupon specifications

Code Coupon Description Globe Pharma #

Steel Stainless Steel 316L SS316-#7-4X4:Alum Aluminum Alloy 6262 ALA6262 – 4X4Poly HDPE, High Density Polyethylene,White Opaque HDPE-WO-4X4Rubber Silicone Rubber,Translucent (Silastic), FDA SR-TR-FDA-4X4Plexi Lucite (Acrylic, Polymethylmethacrilate,

PMMA, Perspex, Plexiglas) LU-4X4

Test organisms

Seven different species of microorganisms were utilized in inoculation studies:

1 Bacillus spizizenii var subtilis ATCC 6633

2 Escherichia coli ATCC 8739

3 Pseudomonas aeruginosa ATCC 9027

4 Staphylococcus aureus ATCC 6538

5 Citrobacter freundii ATCC 43864

6 Candida albicans ATCC 10231

7 Aspergillis brasiliensis ATCC 16404

For total aerobic count: organisms 1–5 (listed above) were used; for combined mold and yeastorganisms 6–7 were used; and for bile tolerant Gram negative organisms 2 and 5 were used.

Coupon inoculation

The following procedure was used for the inoculation of the coupons:

• Sterilize ready-to-use coupons.

• Deposit 0.1 ml of culture on the coupon surface.

• Let the deposited culture dry for 1–2 hours.

• Ideally the concentrations should be 0.5–0.9 log above the acceptance criteria for eachorganism.

• Add negative control coupons (coupons with sterile Butterfields buffer).

• Allow the culture to dry on the coupon’s surface.


Swabbing procedure

• Wet the sterile swab (Collection and Transportation System, BD BBL Sparks, MD) withsterile Butterfields.

• Set a 5 cm × 5 cm template over the area to be swabbed.

• Swab the area while rotating swab-head in a crosshatch or zig-zag pattern across the entire25 cm2 (5*5 cm2) while rotating the swab-head move from side to side across and coverthe surface in three directions as shown in Figure 14.3.

Figure 14.3 Swabbing patterns

• After swabbing, cut the tip of the swab with sterile scissors into a sterile plastic bag.

• Add 10 ml of sterile Butterfield’s buffer.

• Mix well to remove the organisms from the swab.

• Dilutions of the content of this bag will be used to inoculate each of the vials and to eachof the plates. Dilutions were made in sterile Butterfields buffer.

Assays vials used

• TAC — Total Aerobic Count vial. The vial’s sensor detects production of CO2 generatedby microbial metabolism in the liquid medium placed above the sensor.

• YM —Yeast and Mold vial, uses the same CO2 sensor, with different growth media.

• ENT — Enterobacteriaceae vial monitors changes in color due to a pH shift asEnterobacteriaceae organisms ferment glucose in the presence of selective media.


BioLumix vial assay for swabs

Figure 14.4 shows a typical procedure utilized for the BioLumix system.

Figure 14.4 Example of a flow diagram for swab assay

• The swab was inserted into a sterile bag containing 10 ml of Butterfields buffer.

• The contents of the bag are well mixed.

• TAC testing:

– From the appropriate dilution of the bag content, aliquot 100 µL (0.100 mL) or 0.33µL (0.033 ml), or 1000 µL (1.00 mL) and add to a TAC vial for a dilution range of10–300.

– Mix the sample by inverting the vial several times.

– Place the vial in the instrument at 35°C.

– Add sample information into the computer and start the test.


• Yeast and mold testing (total combined mold and yeast testing):

– From the appropriate dilution of the bag content, aliquot 100 µL (0.100 mL) or 1000µL (1.00 mL) and add to a YM vial.

– From the appropriate dilution of the bag content, aliquot 100 µL (0.100 mL) or 1000µL (1.00 mL) and add to a YM vial.




• Gram negative bile tolerant (Enterobacteriaceae):

– From the appropriate dilution of the bag content, aliquot 100 µL (0.100 mL) or 1000µL (1.00 mL) and add to a ENT vial.




Plate count method for swabs

The following procedure was used:

• The bag containing the swab is further diluted as needed.

• Total aerobic count:

– Transfer 1.0 ml from the original bag and 1.0 ml of the 1:100 dilution into two plates.

– Add ~20 ml of molten Trypticase Soy Agar and mix well.

– Incubate the plates at 32–35°C for 48–72 hours.

– Count plates with 25–250 colonies.

• Yeast and mold count:


– Add ~20 ml of molten SDA and mix well.

– Incubate the plates at 20–25°C for 5–7 days.



• Gram negative bile tolerant (Enterobacteriaceae):


– Add ~20 ml of molten Violet Red Bile Glucose Agar and mix well.

– Incubate the plates at 32–35°C for 24 hours.


Specified levels tested for swabs

Organisms were deposited on the coupons, at levels above the desired spec. levels:

Table 14.2 Specification levels tested

Assay Specified LevelCFU/g

Total Aerobic Count <100 – <3,000Yeast and Mold <50 – <300Enterobacteriaceae <300

A number of coupons from each surface type were also tested without inoculation (cleancoupons).

BioLumix vial assay for water testing

Representative water samples were taken from both residential and manufacturing commercialsources. Water was tested directly by inoculation of BioLumix Heterotropic (HETER) vials.BioLumix Heterotropic vials have a base media similar to R2A, used for slow growingheterotrophic organisms.

• Shake the water sample well.

• Aliquot 0.1±0.01 mL from the water sample into the HETER vial.

• Mix the sample in the vial by inverting the vial several times.

• Place the vial in the instrument at 35°C.

• Add the relevant information into the computer and start the test.


Plate count method for water testing

The following procedure was used:

• Shake the water sample.

• Aliquot 0.1±0.01 mL from the water sample into each of two sterile plates.

• Promptly add to each dish 15 to 20 mL of plate count agar, previously melted and cooledto about 45°C.

• Cover the Petri dishes, mix the sample with agar by gently tilting or rotating the dishes,and allow the contents to solidify at room temperature.

• Invert Petri dishes and incubate for 48 to 72 hours at 32° to 35°C.

• Following incubation, examine the plates for growth, count the number of colonies, andexpress the average for the two plates in terms of the number of microorganisms per mLof specimen.

• If no microbial colonies are recovered from the dishes, express the results as “less than 10microorganisms per mL of specimen.”

RESULTS

Limit of detection

Table 14.3 Examples of limit of detection results

Total Count Yeast and Mold Bile Tolerant G–

# Count BioLumix Plate Count BioLumix Plate Count BioLumix Plate

1 2.3 + + 3.1 + + 2.1 + +2 2.3 + + 3.1 + + 2.1 + +3 2.3 + + 3.1 + + 2.1 + +4 2.3 + + 3.1 + + 2.1 + +5 2.3 + + 3.1 + + 2.1 + +6 2.3 + + 3.1 + + 2.1 + +7 2.3 + – 3.1 + + 2.1 + +8 2.3 + – 3.1 + + 2.1 + +9 2.3 – – 3.1 – + 2.1 + +

10 2.3 – – 3.1 – – 2.1 – –11 1.7 + + 4.3 + + 4.9 + +12 1.7 + + 4.3 + + 4.9 + +13 1.7 + + 4.3 + + 4.9 + +14 1.7 + + 4.3 + + 4.9 + +15 1.7 + + 4.3 + + 4.9 + +16 1.7 + + 4.3 + + 4.9 + +17 1.7 + – 4.3 + + 4.9 + +18 1.7 + – 4.3 + + 4.9 + +19 1.7 – – 4.3 + + 4.9 + +20 1.7 – – 4.3 – – 4.9 + +


USP <1223> (2008) states that “the limit of detection is the lowest number of microorganismsin a sample that can be detected under the stated experimental conditions”. The limit ofdetection was evaluated by inoculation with a low number of challenge microorganismsfollowed by a measurement of recovery by both the plate count and BioLumix methods. Testorganisms were diluted to achieve counts in the range of 1–10 CFU/g. Vials and plates wereinoculated with these low numbers of organisms and scored for presence or absence of theseorganisms. Statistical analyses used the Fisher’s exact test rather than Chi square because italways gives an exact P value and works well with small sample sizes.

Total count

The Fisher’s exact test yields a p=0.300, indicating that the limit of detection for the BioLumixsystem is equivalent to or slightly more sensitive (although not statistically significant) indetecting low numbers of microorganisms as directly compared to the plate count method. Thelimit of detection was approximately 1–3 organisms per sample vial.

Yeast and mold

The data showed that the limit of detection for the BioLumix system is equivalent to the platemethod in detecting low numbers of yeast or mold as directly compared to the plate countmethod. The Fisher’s exact test yields a p=1.00, indicating that there was no difference betweenthe two methods in detecting low numbers of yeast and molds.

Gram negative bile tolerant (Enterobacterial count)

The data showed that the limit of detection for the BioLumix system is equivalent to the platemethod in detecting low numbers of Enterobacteriaceae as directly compared to the plate countmethod. The Fisher’s exact test resulted in a p=1.00, indicating equivalence.

Sterile water was used to inoculate vials and plates. A total of 20 vials and plates were usedfor each assay and served as negative controls. Each of the negative control vials and plates wasabsent of growth. The conclusion from the limit of detection studies indicated that low numbersof organisms, if present, can be detected by the BioLumix system. When organisms were notpresent there were no detection times, as indicated by the automated algorithm in the system.

VALIDATION STUDY WITH SWABS

Influence of surface on organism survival

This study was conducted to validate the BioLumix system as an alternative to the plate countmethod in detecting microbial contamination on manufacturers’ production surfaces. Sterilizedcoupons representing the various surface materials that can be utilized in the manufacturingenvironment were used to test bacterial survival. The cultures of the various organisms weregrown in TSB for 24 hours at 35°C and were centrifuged three times and washed. The culturewas diluted to approximately 500,000 CFU/0.1 ml. A challenge of 0.1 ml of the culture was


deposited in the center of the coupon and spread on the coupon surface. The inoculum was leftto dry for 1–2 hours. The following total aerobic counts data values were obtained forquadruplicate coupons for the different bacteria tested:

Total count

Table 14.4 Influence of surface on the survival of total aerobic bacteria

Organism E. coli S. aureus B. subtilis P. aeruginosaSurface CFU/g CFU/g CFU/g CFU/g

Stainless steel 32,800 19,000 19,800 55,000Aluminum 32,500 88,300 10,000 39,500Rubber 97,700 153,000 110,000 39,800Plexiglas 229,000 77,900 93,700 67,000Polyethylene 190,000 170,000 477,040 52,000

In all cases there was a significant decrease in numbers of organisms recovered. This reductionmight be because of loss of microbial activity during drying, adhesion of bacteria to thesurfaces, and possibly lack of complete release of bacteria from the swabs bud. Moore andGriffith (2002) found that on using wet swabs on dry surfaces that 63% of the organisms wererecovered, and the release of organisms from the bud was found to account for approximately46% of the organisms. In this report, the metal surfaces (stainless steel and aluminum) causeda higher decline in total aerobic counts than counts recovered from plastic material such asplexiglas and polyethylene or from the rubber surface.

Yeast and molds

Table 14.5 Influence of surface on the survival of yeast and mold

Organism C. albicans A. nigerSurface CFU/g CFU/g

Stainless steel 200,000 42,500Aluminum 120,000 35,500Rubber 186,000 57,000Plexiglas 101,000 41,000Polyethylene 190,000 53,300

There was a lower decline in numbers of yeast and molds recovered from the surfacessuggesting that these cells are more resilient to these environmental conditions.


Enterobacteriaceae

Table 14.6 Influence of surface on the survival of Enterobacteriaceae

Organism E. coli C. freundiiSurface CFU/g CFU/g

Stainless steel 15,500 61,000Aluminum 25,800 42,500Rubber 7,200 5,400Plexiglas 19,600 84,800Polyethylene 26,800 15,500

There was greater than a tenfold decrease in numbers of Gram negative bile tolerant organismsrecovered when using surfaces as compared to the input inoculum. Recovery from the rubbersurface caused the largest decrease in organism numbers.

The conclusion from the inoculation of surfaces study was that there is variability in therecovery of bacterial organisms from surfaces, while recovery of yeast and mold organisms wasless variable from surface to surface. When comparing the USP methodology to a RMM, it isimportant to add enough organisms to the surfaces to allow for recovery.

TYPICAL SWAB RESULTS

Total count

In order to compare the BioLumix vial method to the plate count methods described in USP<61> and <62> (2009), the dilute to spec approach was used for the coupons. All surfaces wereinoculated to contain, after dying, counts at least 0.5 log10 above the target specified level.

Various coupons with specified levels of 300–10,000 CFU/coupon were evaluated. Fourdifferent strains of bacteria (Bacillus spizizenii var subtilisATCC 6633, Escherichia coli ATCC8739, Pseudomonas aeruginosa ATCC 9027, and Staphylococcus aureus ATCC 6538) wereinoculated onto the following surfaces: stainless steel 316L; aluminum (aluminum alloy 6262);silicon rubber (translucent); plexiglass (acrylic, polymethylmethacrilate); and high densitypolyethylene (white opaque).

Quadruplicates of either un-inoculated coupons or coupons containing counts below thespecified level were determined to yield flat curves as illustrated in Figure 14.5(a). The curvesobtained from the various bacteria on different surfaces are shown in Figure 14.5(b)–(d).



Figure14.5TypicalcurvesobtainedinTAC

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Table 14.7 summarizes a typical data set for E. coli with a specified level of <100 CFU/coupon.

Table 14.7 Comparison of plate count data to TAC vials for coupons inoculatedwith E. coli and a specified level of <100 CFU/coupon

BioLumix Plate Count

Surface DT Result CFU/coupon Result

Stainless Steel 1 11.3 + 310 +Stainless Steel 2 10.7 + 580 +Stainless Steel 3 ND – 30 –Stainless Steel 4 ND – 10 –

Aluminum 5 6.8 + 6,000 +Aluminum 6 7.4 + 4,500 +Aluminum 7 13.1 + 110 +Aluminum 8 10.8 + 140 +

Rubber 9 9.5 + 7,300 +Rubber 10 10.4 + 1,800 +Rubber 11 10.8 + 750 +Rubber 12 11.7 + 270 +

Plexiglas 13 ND – 30 –Plexiglas 14 16.1 + 90 –Plexiglas 15 ND – <10 –Plexiglas 16 ND – <10 –

Polyethylene 17 9.9 + 3,500 +Polyethylene 18 9.5 + 4,200 +Polyethylene 19 ND – 30 –Polyethylene 20 ND – 60 –

ND: no detection

Table 14.7 shows the result of 20 coupons, of which seven were <100 CFU/swab by bothmethods; 12 were >100 CFU/swab by both methods; and there was one sample (Plexiglas 14)that had a very marginal count (90 CFU/swab) and a very late detection time of 16.1 hours.

Table 14.8 shows a summary of 129 inoculated coupons, using the BioLumix TAC vialand the standard plate count method using TSA. All the swabs with plate counts above thespecified level were found to detect in the BioLumix vials. Most discrepancies between the twomethods occurred with samples that had counts very close to the specified level.


Table 14.8 Comparison of plate count data toTAC vials for all inoculated coupons

Organism Spec Total # Discrepant Results

Inoculated CFU/swab of coupons Both below Both above BL PL

E. coli 100 22 7 14 >100 90S. aureus 100 20 8 12 None NoneE. coli 3,000 20 0 20 None NoneS. aureus 3,000 21 0 20 >3,000 2,1001

B. subtilis 3,000 25 0 23 >3,000 2,700>3,000 2,800

P. aeruginosa 3,000 21 0 20 >3,000 1,2002

1 Duplicate had a count of 71,000 CFU/swab2 Average of quadruplicates was over 20,000 CFU/swab

Five coupons with counts on the TSA plates just below the specified level did detect in theBioLumix vials. One coupon that had a count of 90 CFU/swab, while technically below thespecified level, was found to detect in the vial. Three additional coupons had counts of 2,100-2,800 CFU/swab for a specified level of <3,000 CFU/g. Triplicate vials had countssignificantly above the specified level. Another positive result in the instrument was obtainedfrom a coupon that had 1,200 CFU/swab (the specified level was <3,000 CFU/swab). It isimportant to mention that the quadruplicate coupons yielded a count of 20,000 CFU/swab.Thus it is very likely that the plate count underestimated the number of CFU on the coupon.Therefore, five samples that detected in the vials had counts below the specified level. Theagreement between the methods was 96.1%.

Un-inoculated coupons or coupons with count below thespecified level

Some coupons were inoculated with organisms below the specified level. Other coupons thatwere not inoculated were also tested in this study. Over 170 swabs were tested for coupons thatdid contain counts below the specified level. None of these swabs detected in the instrument,yielding a 100% agreement between the two methods.

Yeast and mold counts

Coupons were inoculated above two specified levels: 300 CFU/swab or <50 CFU swab. Oneyeast (Candida albicans ATCC 10231) and one mold (Aspergillis brasiliensis ATCC 16404)were inoculated into the same five surfaces used for total aerobic count.

A total of 86 coupons were analyzed (Table 14.9), using the BioLumix YM vial and thestandard plate count method with SDA (Sabouraud Dextrose Agar W/ Chloramphenicol). Allthe swabs with counts on SDA plates were found to be above the specified level detected in thevials. One coupon that had a count of 40 CFU/swab, while technically below the specified


Table 14.9 Comparison of SDA plate count data toYM vials for all inoculatedcoupons



C. albicans 50 26 12 13 >50 40A. niger 50 20 0 20 None NoneC. albicans 300 20 0 20 None NoneA. brasiliensis 300 20 0 20 None None

level, is still a very marginal result since it is so close to the specified level of 50 CFU/swab,did detect in the vial. The agreement between the two methods was 98.8%.

Un-inoculated coupons or coupons below the specified level

Some coupons were inoculated with Candida or Aspergillis below the specified level. Othercoupons that were not inoculated where also tested. A total of 30 coupons below the specifiedlevel were tested for coupons that did contain counts below the specified level. None of theseswabs detected in the instrument, yielding a 100% agreement between the two methods.

Bile tolerant Gram negative (Enterobacteriaceae)

Coupons were inoculated above the specified level of 300 CFU/swab. Two Enterobacteriaceaestrains (Escherichia coli ATCC8739 and Citrobacter freundii ATCC 43864) were inoculatedinto the same five surfaces used for total aerobic count.

Table 14.10 Comparison ofVRBGA Plate count data to ENT vials for allinoculated coupons



E. coli 300 40 1 37 >300 10<300 310

C. freundii 300 35 0 35 None None

A total of 75 coupons were analyzed (Table 14.10), using the BioLumix ENT vial and thestandard plate count method with Violet Red Bile Glucose Agar (VRBGA). One swab with amarginal count of 310 CFU/swab did not detect in the vial. One coupon that had a count of 190CFU/swab did detect in the vial. The agreement between the two methods was 97.3%.


Table 14.11 Summary table

Parameter Summary of Results — Swab

All data sets A total of 550 coupons were tested, 290 were inoculated above the specifiedlevels while 260 had counts below the specified levels.There was very goodcorrelation between the BioLumix and the plate count results, with anoverall agreement for samples above spec of 97.2%.Almost all the discrepantswabs had plate counts very close to the specified level. None of the 260coupons without microorganism above the specified level detected in theBioLumix system. Consequently there was 100% agreement between thetwo methods.The overall agreement between the two methods was 98.5%

Total aerobic Coupons were inoculated with two spec. levels — <3,000 CFU/swab andcount swabs <100 CFU/swab. Four different types of bacteria were inoculated onto

five surfaces.A total of 129 swabs were analyzed, using the BioLumix TACvial and the standard plate count method with TSA.All the swabs with countabove spec detected in the vials. Five marginal samples detected in the vialsand had counts just below the specified level.The agreement betweenthe methods was 96.1%.

Yeast and mold Coupons were inoculated above two specified levels: 300 CFU/swab and<50 CFU swab. One yeast (C. albicans) and one mold (A. brasiliensis) wereinoculated into the same five surfaces used for the total aerobic count test.A total of 85 coupons were analyzed, using the BioLumix YM vial and thestandard plate count method with SDA (Sabouraud Dextrose AgarW/ Chloramphenicol).All the swabs with counts above the specified leveldetected in the vials.A few coupons with count very close to the specifiedlevel (e.g., 50–80 CFU/swab for a specified level of <50 CFU/swab) did detectin the vials. One coupon that had a count of 40 CFU/swab, while technicallyfound to be below the specified level, was a very marginal result being so close

to the specified level of 50 CFU/swab, did detect in the vial.The agreementbetween the two methods was 98.8%.

Enterobacteriaceae Coupons were inoculated above the specified levels: 300 CFU/swab wereinoculated into the same five surfaces used for Enterobacterial count.A totalof 75 coupons were analyzed, using the BioLumix ENT vial and the standardplate count method with VRBGA. One swab with a marginal count of310 CFU/swab did not detect in the vial.A few coupons with count veryclose to the specified level (e. g.300-400 CFU/swab for a specified level of<300 CFU/swab) did detect in the vials. One coupon that had a count of190 CFU/swab did detect in the vial.The agreement between the twomethods was 97.3%.

Below spec swabs Total Aerobic Microbial Count — over 170 swabs were tested for coupons thatdid contain counts below the specified level. None of these swabs detected inthe instrument, yielding a 100% agreement between the two methods.Yeast and mold — a total of 30 coupons below the specified level were testedfor coupons that did contain counts below the specified level. None of theseswabs detected in the instrument, yielding a 100% agreement between thetwo methods.Enterobacterial count — a total of 60 coupons below the specified level weretested for coupons that did contain counts below the specified level. Noneof these swabs detected in the instrument, yielding a 100% agreementbetween the two methods.


Un-inoculated coupons or coupons below the specified level

Some coupons were inoculated with E. coli or C. freundii below the specified level. Othercoupons that were not inoculated were also tested. A total of 60 coupons below the specifiedlevel were tested for coupons that did contain counts below the specified level. None of theseswabs detected in the instrument, yielding a 100% agreement between the two methods.

Summary of swab results (Table 14.11)

Above spec — 290 coupons were inoculated above the specified levels while 260 coupons hadcounts below the specified levels. There was very good correlation between the BioLumixresults and the plate count results, with an overall agreement for samples above spec of 97.2%.Almost all the discrepant swabs had plate counts very close to the specified level.

Below spec — none of the 260 coupons that did not contain microorganism above thespecified level detected. Therefore there was a 100% agreement between the two methods andno false negative results.

PROCESS WATER TESTING

Water is widely used as a raw material, ingredient, and a solvent in the processing, formulation,and manufacture of pharmaceutical products, active pharmaceutical ingredients andintermediates. As such, all water purification systems must be monitored regularly to verify thequality of the water produced. This includes chemical purity as well as microbiological quality.Microbial evaluation of water quality is an integral component of a water system monitoringprogram to assess the microbiological quality of the water produced. A well-designedmonitoring program will detect adverse trends that are potentially harmful to finished products,processes, or consumers.

The objective of water system microbial monitoring is to provide sufficient informationto control and assess the microbial quality of the water produced. An appropriate level ofcontrol may be maintained by using data trending techniques and, if necessary, limitingcontraindicated microorganisms. Consequently, it may not be necessary to detect the entiremicroorganism species present in a given sample. The monitoring program and methodologyshould indicate adverse trends and detect microorganisms that are potentially harmful to thefinished product, process, or consumer.

Monitoring of water for microbiological quality may include testing for total heterotrophicplate count, coliforms/E. coli, or by checking for the presence of other organisms suspected tobe present in a water sample. The relevant standards relating to pharmaceutical grade water areUSP <1231> Water for Pharmaceutical Purposes (1985).

The BioLumix system is capable of testing water for heterotrophic bacteria, coliform, E.coli, and pseudomonas. For levels of <1 CFU/ml the water can be inserted directly into the vial.To test for levels such as <1/100 ml the water is filtered and then the filter is added to the vial.


Figure 14.6 Heterotrophic curves of inoculated water samples

Typical heterotrophic bacteria may encompass bacterial strains of Arthrobacter, Aeromonas,Alcaligenes, Chromobacterium, Pseudomonas, Sarcina, Micrococcus, Flavobacterium,Proteus, Bacillus, Acinetobacter, Variovorax, Klebsiella, and many others. Additionally,coliforms and E. coli may also be present. Pure cultures of heterotrophic bacteria were testedin the vials and all detected in the system. Figure 14.6 shows some typical curves obtained.

An example of organisms tested is shown in Table 14.11.

Ninety-two water samples were analyzed with two specified levels (10 CFU/ml and 100CFU/ml). Sixty samples were below the specified level by both methods, while 28 sampleswere above the specified level by both methods. Four samples were below the specified levelby the BioLumix method, but above by the plate count method. All these samples had very lowcounts (1–3 colonies on the plate).There was 96.9% agreement between the two methods.

Final results were seen in the BioLumix system roughly 13 hours faster than by the platecount method using Standard Methods Agar. These particular samples were tested at specifiedlevels <10 CFU/mL and <100 CFU/mL, but the BioLumix method can detect organisms at alevel of <1 CFU/mL of water. The BioLumix system is faster, less labor-intensive, and moresensitive than the plate count method.


Table 14.11 Organisms tested in the heterotrophic medium

Organism

Acinetobacter baumannii ATCC 19606 Escherichia coli ATCC 25922Acinetobacter radioresistens Escherichia coli ATCC 8739Aeromonas hydrophila ATCC 35654 Flavobacterium spp.ATCC 51823Alcaligenes faecalis ATCC 35655 Klebsiella oxytoca ATCC 2170Arthrobacter psychrolactophilus ATCC 700733 Klebsiella pneumoniae ATCC 13883Bacillus cereus ATCC 10876 Micrococcus luteus ATCC 10240Bacillus cereus ATCC 14549 Micrococcus luteus ATCC 9341Bacillus coagulans ATCC 7050 Providencia rettgeri ATCC 2047Bacillus lentus Providencia stuartii CDC 2310Bacillus megaterium Pseudomonas fluorescens ATCC 49838Bacillus spizizenii var subtilis ATCC 6633 Pseudomonas putida ATCC 49128Burkholderia cepacia ATCC 25416 Pseudomonas stutzeri ATCC 17588Citrobacter freundii ATCC 43864 Variovorax paradoxusEnterobacter aerogenes Yersinia enterocoliticaEnterococcus feacalis ATCC 19433

CONCLUSION

Conclusions and discussion

In a case study presented at the PDA meeting (Eden and Miller, 2011) data indicated that theBioLumix system can be used as a RMM with comparable results to USP <61> (2009). It alsoindicated that the method had good specificity in detecting target organisms and excludingnon-target flora.

The data presented above indicate that the detection limit for the BioLumix system equalsor is slightly better than the limit for the plate count method. High precision or repeatabilitywas obtained for all assays tested, including total aerobic count, yeast and mold andEnterobacteriaceae.

The BioLumix system was validated as an alternative to the plate count method for EM.The study involved a total of 549 surface coupons representing five diverse types of material.These five surfaces represent those encountered in manufacturing, including metal, plasticsand rubber. Some of the coupons were inoculated with bacteria or yeast or mold. There was100% agreement between BioLumix assay and the plate count assay for the 260 coupons thatwere determined to be below the specified level by the plate count method. There was anoverall agreement of 97.2% between the two methods when swabs containing counts above thespecified level were used. In general, discrepancies in swab results between the BioLumix vialmethod and the traditional plate count method reflected marginal samples that were very closeto the specified testing level, and thus were variable.

The data for heterotrophic organisms in water showed that test results were seen in theBioLumix system approximately 13 hours before the plate count method using standardmethods agar. The BioLumix system was found to be faster, less labor-intensive, and more


sensitive than the plate count method for detection of heterotrophs. The system can also beutilized to detect coliforms and E. coli in water.

The BioLumix system generates automated EM reports that are accurate, traceable, welldocumented and timely, while eliminating transcription errors, and allow for quick review andinterrogation to the databases containing the results. In addition, the BioLumix system helpsstreamline microbial testing, the system is easy to operate, and offers an automated report ofall the assays performed that archives, thereby saving significant time, labor and money. Useof the BioLumix system is paperless and efficient, saving on disposables, time and space. Thesystem eliminates product interference, delivering accurate results. The system is versatile withcapabilities to perform all the microbiological assays for raw materials, in-process and finishedproducts with good correlation to USP <61> and USP <62> (2009).

REFERENCES

Carrick, K, Barney M, Navarro, A. and Ryder D. (2001) The Comparison of FourBioluminometers and Their Swab Kits for Instant Hygiene Monitoring and Detection ofMicroorganisms in the Brewery. J. Institute of Brewing 107, 32–37.

Easter M. (2010) A comparison of commercial ATP hygiene monitoring systems. NextGeneration Food Issue 9.

Eden, R. and Miller M.J. (2011) Case Study of a New Growth-Based Rapid MicrobiologicalMethod (RMM) that Detects the Presence of Specific Organisms and Provides anEstimation of Viable Cell Count. PDA 6th Annual Global Conference on PharmaceuticalMicrobiology, October 17–18, Bethesda, MD.

FDA (2004) Pharmaceutical cGMPs for the 21st century Dept of Health and Human Services,FDA: 1–32.

Moore, G. and Griffith, G. (2002) Factors influencing recovery of microorganisms fromsurfaces by use of traditional hygiene swabbing. Dairy, Food and EnvironmentalSanitation Vol 22, 410–421.

United States Pharmacopeia (2009) Chapter <61> Microbiological Examination of NonsterileProducts: Microbial Enumeration Tests. The National Formulary. Rockville, MD, TheUnited States Pharmaceopeial Convention.

United States Pharmacopeia (2009) Chapter <62> Microbiological Examination of NonsterileProducts: Tests for Specified Microorganisms. The National Formulary. Rockville, MD,The United States Pharmaceopeial Convention.

United States Pharmacopeia (2005) Chapter <1223> Validation of alternative microbiologicalmethods. The National Formulary. Rockville, MD, The United States PharmaceopeialConvention. 1: 681–683.

United States Pharmacopeia XXI (1985) Chapter <1231> Water for Pharmaceutical Purposes.The National Formulary. Rockville, MD, The United States Pharmaceopeial Convention.


ABOUT THE AUTHORS

Dr. Ruth (Firstenberg) Eden is the President and founder of BioLumix and has over 25 yearsof experience in new product development and commercialization of products for microbiologytesting. Dr. Eden has developed numerous methods and devices in microbiology which haveenabled the successful transfer of products from research and development to the actualproduct market.

Prior to establishing BioLumix Inc., Dr. Eden was Chief Operating Officer at BioSys, Inc.While at Centrus International she served as Chief Scientific Officer and was responsible foridentification of novel technologies, product development and transfer of technologies fromR&D into the market. She was Director of Research and Development for DIFCO Laboratoriesand senior microbiological safety manager for Campbell Soup Company.

Dr. Eden has authored more than 50 publications in scientific journals and books andgiven more than 70 presentations in a variety of venues worldwide. Dr. Eden has authoredtwelve issued U.S. patents and three European patents as well. Dr. Eden earned her Doctor ofPhilosophy degree in food microbiology and biotechnology with a bachelor degree in chemicaland food engineering from Technion Israel Institute of Technology in Israel. She has been therecipient of numerous accolades including the Michigan Leading Edge Technology Award andthe R&D 100 Award in 1993.

Roger Brideau is the senior bench microbiologist at BioLumix, Inc. He has more than 35 yearsof experience as a microbiologist having worked nearly 30 of those years as a Principal SeniorResearch Scientist at Pfizer. His research career has focused on both anti-bacterial and anti-viral approaches and he has authored more than 35 peer-reviewed manuscripts. Mr. Brideau hasserved on numerous NIH Study Sections involving anti-microbials and he has served as a GrantReviewer for the Wellcome Trust. He received his undergraduate degree from the StateUniversity of NewYork and a Master of Science degree from Oxford University, England. Mr.Brideau is a member of the American Society for Microbiology, The International Society forAntiviral Research and Sigma Xi, the National Scientific Honor Society.


CONTENTSENVIRONMENTAL MONITORING VOLUME 6

1 MICROBIAL MONITORING OF THE INTERNATIONALSPACE STATION

Duane L. Pierson, Douglas J. Botkin, Rebekah J. Bruce,Victoria A. Castro, Melanie J. Smith, Cherie M. Oubre and C. Mark Ott

2 ENVIRONMENTAL MONITORING: A PRACTICAL APPROACHTim Sandle

3 DEVELOPING AN ENVIRONMENTAL MONITORING PROGRAMBarry A. Friedman

4 PHARMA MANUFACTURING ENVIRONMENTALMONITORING IN A TRANSITIONING PARADIGM

Steve Douglas

5 ENVIRONMENTAL MONITORING FORNON-STERILE OPERATIONS

Miriam Rozo

6 RAPID MICROBIOLOGICAL MONITORINGIN PHARMACEUTICAL ENVIRONMENTS

Mark Hallworth

7 INVESTIGATION OF A FAILURE TO MEET ENDOTOXINSPECIFICATIONS IN AWATER FOR INJECTION SYSTEM

Karen Zink McCullough

8 OBJECTIONABLE MICROORGANISMS IN NON-STERILEPHARMACEUTICAL DRUG PRODUCTS: RISKASSESSMENTAND ORIGINS OF CONTAMINATION

David Roesti

9 NEUTRALIZATION OF DISINFECTANTS BY CULTUREMEDIA USED IN ENVIRONMENTAL MONITORING

Reiner Hedderich and Anne-Grit Klees

10 MICROAEROPHILICS/ANAEROBES IN ENVIRONMENTALMONITORING OF PHARMACEUTICAL MANUFACTURING

Veronica Marshall and Daniel Eshete

11 PRACTICAL AND BUSINESS-BASED APPROACHESTO MICROBIAL IDENTIFICATIONS

David Shelep

12 THE PROBLEM OF BURKHOLDERIA CEPACIAJeanne Moldenhauer

13 REALTIME CLEAN ROOM MONITORING FORTOTALANDVIABLE PARTICLES: BIOTRAK™VIABLEPARTICLE DETECTOR

Darrick Niccum and Peter Hairston

14 VALIDATION OF A RAPID SYSTEM FOR ENVIRONMENTALMONITORINGANDWATERTESTING

Ruth Eden and Roger Brideau

15 IMPLEMENTATION OFA RAPID MICROBIOLOGICALMETHODTO MONITORAND EVALUATETHEAIRBORNEPHARMACEUTICAL CLEANROOM ENVIRONMENT

Peter Noverini

16 AUTOMATION OF ENVIRONMENTAL MONITORING:ACHIEVING EFFICIENCY AND CONTROL

Susan B. Cleary and Parsa Famili

Appendix

Index

THE ENTIRE BOOK MAY BE PURCHASED AT www.PDA.org/bookstore

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