Rapid Sterility Testing Using Pallchek

17

RAPID STERILITY TESTINGUSING ATP BIOLUMINESCENCE

BASED PALLCHEK™ RAPIDMICROBIOLOGY SYSTEM

Claudio Denoya, Jennifer ReyesPfizer Global R&D

Groton, CTUSA

Maitry Ganatra and Daniel EshetePall Corporation

Port Washington, NYUSA

INTRODUCTION

The quality attributes of manufactured pharmaceutical productinclude the physical, chemical, and microbiological characteristicsof the raw materials, excipients, active pharmaceutical ingredient(API) as well as the final drug product (Table 17.1). Absence ofmicrobiological contamination is considered a critical qualityattribute due to its potential to dramatically impact, directly orindirectly, the safety and/or the efficacy of the drug product.“Sterile”, is a medieval word derived from the Latin sterilis(unfruitful), meaning, in modern terms, free from living germs ormicroorganisms. In pharmaceutical manufacturing, it is critical to

433

Table 17.1 Definitions of quality attributes

• Quality Attribute (QA)– A physical, chemical or microbiological property or characteristic

of a material

• Key Quality Attribute (KQA)– potential to impact product quality or process effectiveness– associated analytical method

• Critical Quality Attribute (CQA)– directly or indirectly impacts the safety or efficacy of a drug product

assure sterility of “sterile” products in order to release aseptic andsafe medicines to patients. Therefore, the importance of adequateand effective microbiological controls cannot be overstated enough.

Sterility testing is performed to evaluate a finished pharma-ceutical product as a batch release quality control test by followingthe requirements delineated in the compendia (USP <71>, 2011a; EPSection 2.1.6, 2010a; JP, 2006). The test is used to determine thepresence or absence of viable, multiplying microorganisms (bacteria,yeast and fungi) under standardized growth conditions. As thesterility test is a very exacting procedure, it is performed by qualifiedpersonnel under tightly controlled environmental conditions wherestrict asepsis is ensured, maintained andmonitored (USP, <71> 2011a;FDA, 2004).

Current harmonized compendial sterility test methods usingeither membrane filtration or direct inoculation require at least 14days of incubation. In cases where drug products either possess anintrinsic turbidity, or because of their formulation become opaqueor cloudy during the incubation period, identification of microbialcontamination based on visual confirmation of turbidity of growthmedia becomes difficult. In such instances, at the end of the 14-dayincubation, a portion of the sample is sub-cultured into freshmedium for an additional 4–5 days to allow detection, furtherextending the incubation period. This and similar transfer/dilution

Rapid Sterility Testing434

steps used for sterility testing of suspension products are timeconsuming and may compromise test integrity by introducingadditional risk for contamination. In general, the complexity of thetest procedure and required lengthy test period contribute toincreased cost of manufacturing.

The replacement or the supplement of the conventional sterilitytest by a rapid microbiology test will have significant benefits. Arapid method has the potential to produce test results much faster atenhanced sensitivity. In addition, a test based on current technologiescan increase throughput and allow better data handling. Theshortened time to results would allow the reduction of warehousing,a timely distribution of the drug product to the market, a betterunderstanding and control of the manufacturing process and mostimportantly, a greater assurance of product safety. Currently, anumber of alternative rapid microbiology methods that are based onvarious biophysical principles including ATP bioluminescence,nucleic acid amplification, vital dye/auto florescence, pH changeand spectroscopic detection, are available. An ideal rapid method forsterility testing of a particular group of products, in addition to beingequivalent and/or better than the traditional method, must besimple, economic and relatively easy to implement.

SELECTION OF A RAPID MICROBIOLOGICAL METHODSUITABLE FOR STERILITY TESTING

In the selection process that led to this work, the following initialquestions and supporting information were considered.

• How fast is the “rapid” method?

• How much effort is needed to implement?

• How much is the capital investment?

• Howmany samples (throughput) can be processed in one shift?

• References and previous submissions

• Vendor support

• Validation parameters.

ATP Bioluminescence-based Pallchek™ 435

A large number of alternative microbiological test platforms wereevaluated, trying to identify the best match between QC andmanufacturing process requirements and the capabilities of aparticular rapid assay. Some of the characteristics considered in therapid system selection process are summarized as follows.

• A method compatible with an initial membrane filtration steptherefore allowing flexibility in the sample volume.

• Good references from previous customers and excellent vendortechnical support.

• Relatively low maintenance and cost.

• Portable, requiring minimum laboratory bench space.

• Simple training and ease of use.

• Easy verification of proper system operation prior to use.

• Standard specific kit allowing corroboration of calibration andoperation.

• Results in units that can be reasonably correlated to ColonyForming Units (CFU) of the traditional method.

• Previous validation experience.

• Detection of viable cells, including those that cannot grow inthe compendia media or they could be injured or stressed as aresult of manipulations or sanitization procedures appliedduring manufacturing (i.e., viable but non culturable (VBNC)),assuring better QC.

• A method compatible with the addition of a short growth step(“enrichment”) prior to assay to differentiate viable andculturable cells from those VBNC.

• Simple qualitative (presence/absence) procedure.

• Sensitivity down to one CFU-equivalent (see below) withenrichment.

• Minimal sample manipulation.


Upon consideration, the Pallchek™RapidMicrobiology system (PallLife Sciences, Port Washington, NY) was selected because it matchedmost of the criteria described above. For our application, where verylow levels of microbial contamination were expected in the samplessubmitted to the sterility test, the incubation of the membrane filterin liquid growth medium (enrichment) prior to the bioluminescenceassay processing, mimics the traditional test (i.e., a growth basedmethod using similar media and incubation conditions), making thecomparability study easier to interpret. This enrichment step has theintention to allow detection down to one microbial cell or cellaggregate (i.e., the equivalent of 1 CFU detected in a conventionalagar plate method) present in the original sample in a period of timeshorter than the 14 days required in the traditional method for abroad range of microorganisms. The work detailed in the nextsections was designed to demonstrate the previous assertion and, ifverified, determine the shortest incubation time needed for detectionof very low counts of a broad range of microorganisms.

DETECTION OF MICROBIAL CONTAMINATION USINGATP BIOLUMINESCENCE

The use of adenosine triphosphate (ATP) bioluminescence in themicrobiological evaluation of pharmaceuticals is well established.The early acceptance of the technology by major regulatoryagencies has led to the use of ATP bioluminescence basedmicrobiological test applications on an increasingly diverse groupof pharmaceutical samples (Stanley, 1989; Kramer et al., 2008;Nielsen and Van Dellen, 1989; Denoya et al., 2010).

A luciferin-based bioluminescence reaction makes use of thefirefly enzyme luciferase (luciferin 4-monooxygenase, EC 1.13.12.7),which catalyzes the oxidation of D-luciferin in the presence of ATP,magnesium ions and molecular oxygen (Figure 17.1). Adenosinetriphosphate, which is the prime energy carrier in all living cells,constitutes the main driver of the bioluminescence reaction(Lehninger, 2008). The reaction yields a quantum of yellow light at564 nm for each molecule of the luciferin substrate oxidized(Chappelle and Levin, 1968; White et al., 1961). Light producedfrom the luciferin–luciferase reaction is proportional to the amount


of ATP used and can be measured in Relative Light Units (RLU) bya luminometer. In microbiological tests using bioluminescence,where microorganisms are the source of ATP in the reaction, theluminescence measured in RLU can be correlated to the number ofmicrobial cells present in a sample.

Figure 17.1The luciferin/luciferase bioluminescence reaction

[Luciferase, Mg2+]ATP + Luciferin + O2 AMP + Oxyluciferin + CO2 + PPi + Light (Bioluminescence)

PALLCHEK RAPID MICROBIOLOGY SYSTEM

The Pallchek Rapid Microbiology System selected for the applicationdescribed in this chapter consists of a compact, portable luminometerand reagent kits. The system is versatile and allows measurement ofmicrobial contamination on amembrane, in liquid samples as well asmeasurement of surface contamination using swabs (Pall LifeSciences, USTR 2358). Microbial detection by the system is based onthe luciferin-luciferase enzyme/substrate system that uses anenzyme isolated from the firefly, Photinus pyralis (White et al., 1961).

In the most common application, measurements are usuallyperformed on filtered liquid samples. After filtration of the sample,the membrane filter can then be washed to remove any componentspresent in the sample that could interfere with the ATP-basedbioluminescence measurement. In addition, the membranefiltration step and subsequent wash eliminates any free ATP thatcould be present in the sample. The latter assures that only ATPlocated internally in the potential microbial contaminants isdetected. A reagent is then directly added to the membrane torelease the intracellular ATP from microorganisms and make itavailable for a second reagent containing the luciferin–luciferasereaction mix. The resulting light is detected and measured with thePallchek Luminometer, and reported as RLU. For applications likesterility testing, where very low levels of microbial contamination isexpected, the membrane can be incubated in liquid growth medium


(enrichment step). This enrichment step allows detection down toone cell (the equivalent to one CFU as counted on a traditional agarplate) for a broad range of microorganisms.

VALIDATION OF AN ALTERNATIVE MICROBIOLOGICALMETHOD

The principal aim of the validation of an alternative microbiologicalmethod for the purpose of using it to test pharmaceutical productsis the demonstration of its equivalence or superiority to the corres-ponding compendia test method. The validation process identifiesrisk areas in the test method and challenges them appropriately toevaluate and define the operating parameters and performancecharacteristics of the test system. Guidance on validationapproaches can be found in the compendia (USP <1223>, 2011b; EPSection 5.1.6, 2010b) as well as industry guidance and vendorsupport documents (PDA, 2000; Pall Life Sciences USTR 2359).

The objective of this chapter is to discuss an initial validationparameter study of a bioluminescence-based rapid sterility testmethod and the subsequent feasibility study conducted as part ofthe development of a rapid sterility assay for a specific drugproduct. The drug products under study posses a special challengefor the traditional sterility test method on account of its turbidity.

The following sections describe the practical considerationsthat were taken into account in developing the rapid sterility testusing Pallchek Rapid Microbiology System based on membranefiltration, as well as the results of the feasibility study.

CHALLENGE MICROORGANISMS, MEDIA AND GROWTHCONDITION

Pharmacopoeia referenced strains derived from reconstitutedBioBalls (bioMérieux, Hazelwood, MO) as well as environmentalisolates from a number of manufacturing sites that represent knowncontaminants were used as challenge organisms. The range ofmicroorganisms consisted of yeast, mold, Gram positive and Gram


Table 17.2 List of microorganisms and growth conditions

Organism Description Strain Media IncubationTemp. (°C)

Escherichia coli Gram negative ATCC 8739 TSB 30–35

Aspergillus brasiliensis Mold ATCC 16404 TSB 20–25Bacillus subtilis Gram positive ATCC 6633Candida albicans Yeast ATCC 10231

Clostridium sporogenes Gram positive ATCC 11437 FTM 30–35Pseudomonas aeruginosa Gram negative ATCC 9027Staphylococcus aureus Gram positive ATCC 6538Bacillus subtilis Gram positive ATCC 6633

Acinetobacter baumannii Gram negative Environmental TSB 20–25Aspergillus fumigatus MoldMethylobacterium Gram negative

rhodesianum slow growerMicrococcus luteus Gram positivePaenibacillus macerans Gram positive

spore formerPenicillium chrysogenum MoldRalstonia pickettii Gram negative

Propionibacterium acnes Gram positive, Environmental FTM 30–35anaerobic,slow grower

Bacteroides vulgatus Gram negative,anaerobic

negative bacteria (aerobic and anaerobic) to represent a widespectrum of possible microbial contaminants. The use of referencemicroorganisms offers various advantages, including comparabilityof data and commercial availability in convenient formats. However,it is important that studies also include organisms that reflect thetypical bioburden of manufacturing environment and the testmaterial to allow proper interpretation of test data. In total 16 strainswere used in these studies (Table 17.2). The cultures were diluted to


<1, 5 or 50 CFU per 100 µL as required. Growth promotion ofnutrient media, as well as identification of challenge organisms wereperformed as appropriate.

In principle, any growth media and incubation condition maybe used and validated for a method which utilizes the PallchekRapid Microbiology System. However, it is advisable to restrictgrowth conditions to those that are well established and recognizedby regulatory authorities, unless a different growth condition offersa special advantage. The following commonly used media andrinse agents were used — Tryptone Soy Agar (TSA), SabouraudDextrose Agar (SDA), Fluid Thioglycollate Medium (FTM), Fluid Aand Sodium Chloride 0.9% w/v Solution.

Samples were filtered using GN-6 membrane (0.45 µm) (PallLife Sciences PN 4800). The use of disposable MicroFunnel™ filterfunnels allows the filtration of sample, washing of filter membraneand incubation of the membrane in growth media all in one filterunit, without the need to transfer the filter membrane. MicroFunnelfilter funnels are convenient to use and also minimize the risk ofcontamination.

COMPONENTS OF PALLCHEK RAPID MICROBIOLOGYSYSTEM

Pallchek Rapid Microbiology System consists of the following:

• Pallchek Luminometer (PN13673)

• Aluminium Test Plate for Pallchek Luminometer (PN 13679)

• High Sensitivity Bioluminescent Kit (PN 7142)

• High Sensitivity ATP Correlation Kit (PN 7150)

• Disposable Sample Holders for Liquid Samples (PN 7147)

• Spreaders (PN 7149).


BIOLUMINESCENCE ASSAY REQUIREMENTS OF TESTENVIRONMENT

Detection of light by the Pallchek Luminometer from exogenoussources may contribute to a high background signal that mightinterfere with measurement. In order to reduce background signaland reagent decay, the luminometer was operated under subduedlight. Tests were run under aseptic conditions (gloves and laminarflow cabinet) to eliminate externalmicrobial contamination, andwereperformed at room temperature (18–22 °C).Astandard suitability testperformed at the beginning of each test session verifies that theambient light is adequately controlled and that test components donot generate significant background signal. Tests were conducted asdescribed by the manufacturer (Pall Life Sciences USTR 2358).

DRUG PRODUCT SAMPLE

Product samples from a stability study program were used in thisstudy. Analytical studies conducted on the product indicated thatits chemical composition was acceptable, and samples were sterile.Drug Product (1 mL vial) is an aqueous, white and cloudysuspension.

PRESENCE–ABSENCE TEST WITH ENRICHMENT

Sample preparation included a filter pre-rinse, sample filtering ofup to 100 mL volume and a post-filtering wash with SodiumChloride 0.9% w/v Solution. Following the last wash, a volume ofsterile media (usually 10 mL) was added to the MicroFunnel filterfunnel. The container was then incubated (enriched) at anappropriate temperature for a time period that would allow thedetection of a very low count of microbial contamination by thebioluminescence assay. Negative controls were processed inparallel. Following the enrichment period, an aliquot of 8 mL wasfiltered in a final volume of up to 100 mL with sterile water. Theremnant 2 mLwas saved for microbial identification. An additional100 mL wash was occasionally performed to remove any excesscomponents of the filtrate. The filter membrane was removed fromthe MicroFunnel filter funnel aseptically and placed on a sample


holder, and bioluminescence measured as described by themanufacturer (Pall Life Sciences USTR 2358). The output in RLUwas recorded. The time it took to obtain a reading was less than oneminute including the reagent addition. The presence ofmicroorganisms is determined when the RLU value is greater thanthe background RLU value as determined, based on a predefinedthreshold value. For studies that required multiple sampling froman enrichment culture, a proportionately larger volume of sampleand media were used for incubation.

VALIDATION STRATEGY OF RAPID STERILITY TEST

The overall strategy to validate the rapid sterility method usingbioluminescence included:

• studies to determine the system suitability of the luminometer,reagents and media

• the establishment of background values for the differentexperimental conditions to be used in the protocol

• the validation tests using eitherATP standard solution, referenceand environmental microbial isolates and pharmaceuticalsamples.

SYSTEM SUITABILITY TESTING

A system suitability test is generally performed before each readingsession to ascertain that the test environment, as well as equipmentand reagents are functioning as intended. These tests include thedetermination of the level of background noise, negative andpositive control tests for reagents, and negative control for themeasurement of a filtered liquid sample. A typical suitability testresult is shown in Table 17.3.


Table 17.3 System SuitabilityTest

Sample RLU

Aluminum Plate 11 (<20)Aluminum Plate + Sample Holder 11 (<20)Aluminum Plate + Sample Holder + Pallchek Reagents 14 (<80)Enzyme activity; positive control:Aluminium plate+ sample holder +100µL ATP (10–9 M)+100µL Enzyme ≥105 (≥105)

TSB Negative Control (membrane filtrated,two washes of saline) 220 (≤ 1000)

Measurements are the average of duplicates. Acceptance criteria are shown inparenthesis

ESTABLISHMENT OF BACKGROUND VALUES

Interference from background luminescence should be minimizedas much as possible. Type of filter membrane, media and rinse fluidand amount must be investigated and selected accordingly todefine conditions that provide the lowest background. Pall GN-6Metricel® MCE membrane and saline washes generally give lowbackground signals (<100 RLU).

Once the optimal conditions that give low background wereidentified, a control sterility test was conducted, using either TSBalone or in combination with sterile pharmaceutical samplesincubated for not less than 24 hours at 35°C (true negative) todetermine the background in conditions close to those used duringactual test. These conditions were similar to those employed duringthe enrichment phase of the rapid sterility method (see below). Theestablishment of this value is critical since the “threshold value” is setbased on the background value. The binary designation of samples aspositive or negative is based on whether an RLU reading obtained isabove or below this threshold value. This value is determined byconsidering the acceptable level of risk of false positive, primarily abusiness risk, and false negative, a potential health risk. The threshold


value was established at a RLU value higher than eight times theaverage background level (Table 17.4). A value above this thresholdvalue indicates presence of microorganism.

Table 17.4 Background Luminescence value

Mean 131.4Standard Deviation 60.595% Confidence Interval 103.1–159.7Threshold RLU value 1000

Sterile TBS samples were incubated for 24hrs at 35°C, membrane filtered, washed withsterile saline and assayed.Values are expressed in RLU, n= 20.

INITIAL VALIDATION PARAMETERS OF THEQUALITATIVE RAPID METHOD

Validation parameters were identified and evaluated based on com-pendia requirements (USP <1223>, 2011b; EP Section 5.1.6, 2010b),and the recommendation of the Parenteral Drug Association (PDA,2000). The initial validation experiments were carried out using theATP standard solution provided in the Pallchek kit. These tests didnot include the use of microorganisms or pharmaceutical samples.

Linearity

The bioluminescence based sterility test as outlined here is aqualitative presence–absence test. Test for linearity is a validationparameter generally reserved for a quantitative assay (USP <1223>,2011b). However, the designation of “presence” or “absence” usingPallchek depends on an initial quantitative determination of RLUby the luminometer. It is therefore important to determine if withinthe range of the detection limits of equipment, a predictablerelationship exists between RLU and concentration of ATP. Figure17.2 shows a typical RLU/ATP correlation curve where a very goodlinear relationship is observed (R2 > 0.95).


Figure 17.2 Linearity

ATP Correlation Curve by single user

Ruggedness and robustness

The ruggedness and robustness of an ATP bioluminescence basedtest in part can be evaluated using ATP standards and generatingRLU/ATP correlation curves under different test conditions.RLU/ATP correlation curves performed by different operators ondifferent dates produced comparable results showing the test’srugged nature (Figure 17.3).

The test for robustness involves the introduction of small butdeliberate changes in test parameters to determine the effect on theassay performance. ATP standard and reagent solutions arerecommended to be kept refrigerated at all times. During a routinetest both ATP and reagent preparations are exposed to roomtemperature for various amounts of time. Figure 17.4 shows theRLU/ATP correlation curve performed using ATP solution andluciferase enzyme deliberately left out at room temperature forvaried periods of time. The robustness of the test is suggested bythe comparable correlation coefficients obtained under different testconditions (Figure 17.4). At room temperature, the diluted ATPstandard solutions, as well as the luciferase enzyme, remainedstable up to at least four hours, allowing flexibility in the set up ofthe sterility test. When properly kept refrigerated, reconstitutedreagent is stable for at least one week (not shown).


Figure 17.3 Ruggedness

ATP correlation curves by multiple users, R2 shown in parenthesis

Figure 17.4 Robustness

ATP and enzyme stability: correlation curve usingATP and enzyme preparation exposed toroom temperature for various times, R2 is shown in parenthesis


Bioluminescence undergoes decay with time. The signal is morestable at higher ATP concentration and starts to diminish after 30seconds particularly at lower ATP concentrations. The data areconsistent with those obtained by Pall (Pall Life Sciences). It istherefore very important to perform the detection within 10 secondsof the initiation of the reaction to assure accurate and repeatablemeasurements (Pall Life Sciences USTR 2359) (Figure 17.5).

Figure 17.5 Robustness: bioluminescence decay

Specificity, limit of detection and repeatability

Additional validation parameters were investigated in the presenceof reference and environmental isolates. The objective of this part ofthe study was to determine the time required to detect lowmicrobial counts by the bioluminescence assay. A typical test designand results are shown in Table 17.5 for Ralstonia pickettii. As seen inTable 17.5, the rapid sterility test method was able to detect astarting count of ≤3 cell of R. pickettii within 24 hrs.


Table 17.5 Detection of Ralstonia pickettii

Dilution Time 0 1 day 1 day 14 day 14 day VisualSpiked cell enrichment enrichment enrichment enrichment growth(1 mL) count Pallchek plate count Pallchek plate count observed

(CFU) (RLU) (CFU) (RLU) (CFU) T 1/T 14

TSB negativecontrol 0 7.3 × 102 0 3.2 × 102 0 No/No10–5 3 x 104 1.4 × 106 TNTC NP TNTC Yes/Yes10–6 3 × 103 1.3 × 106 TNTC NP TNTC Yes/Yes10–7 3 × 102 9.3 × 105 TNTC NP TNTC Yes/Yes10–8 3 × 102 9.7 × 105 TNTC NP TNTC Yes/Yes10–9 3 1.6 × 106 7.5 × 107 2.7 × 105 TNTC Yes/Yes10–10 0.3 9.2 × 102 0 3.4 × 102 0 No/No10–11 0.03 9.3 × 102 0 3.2 × 102 0 No/No

NP: Not Processed TNTC:Too Numerous To Count

The results shown in Table 17.6 correspond to a study using theAcinetobacter baumannii culture. The rapid sterility test method wasable to detect ≤5 cells of A. baumannii within 17 hrs, which wasfaster than the time to visual detection (Table 17.6). No visualgrowth was observed at 17 hrs in the enrichment sample spikedwith ≤5 cells of A. baumannii (time to visual observation of growthwas within 48 hrs). However, the sample was confirmed to bepositive by Pallchek RapidMicrobiology System at that earlier time.In summary, the Pallchek Rapid Microbiology System providedfaster sterility results for A. baumannii than the traditional sterilitymethod of visual observation for microbial growth.

Contamination by all challenge organisms was detected in allinstances (specificity) at a very low microbial inoculum level. Table17.7 summarizes the time to detection for various microorganisms.The length of incubation time (enrichment) for a starting count of1–5 cells of any of the listed microorganisms required to achieve adetection using the bioluminescence assay was ≤48 hours. Undersimilar conditions, the time to visual detection for the traditionalmethod was ≤96 hours.


Table 17.6 Detection of Acinetobacter baumannii

Dilution 0 hour 17 hr 17 hr 48 hr 48 hr VisualSpiked cell enrichment enrichment enrichment enrichment growth(1 mL) count Pallchek plate count Pallchek plate count observed

(CFU) (RLU) (CFU) (RLU) (CFU) 17 hr/48 hr

TSB negativecontrol 0 7.1 × 102 0, 0 2.4 × 102 0, 0 No/No10–6 4200 1.9 × 106 TNTC NP TNTC Yes/Yes10–7 420 1.0 ×. 107 TNTC NP TNTC No/Yes10–8 42 2.7 × 106 TNTC 4.0 x 106 TNTC No/Yes10–9 4.2 1.3 × 107 TNTC 3.2 x 106 TNTC No/Yes10–10 0.42 4.8 × 102 0, 0 2.3 × 102 0, 0 No/No10–11 0.042 7.0 × 102 0, 0 4.8 × 102 0, 0 No/No

NP: Not Processed,TNTC:Too Numerous To Count

Table 17.7Time to detection of samples spiked at time 0 with 1–5 cells

Organism Time to Time todetection (hrs) detection (hrs)Pallchek Visual

Escherichia coli 24 24Bacillus subtilis 20 20Staphylococcus aureus 20 20Pseudomonas aeruginosa 20 20Aspergillus brasiliensis 48 96Candida albicans 48 48Bacteroides vulgatus 28 28Propionibacterium acnes 48 96Paenibacillus macerans 17 48Ralstonia pickettii 24 24Micrococcus luteus 22 24Methylobacterium rhodesianum 22 48Bacillus pumilus 24 24Penicillium chrysogenum 18 48Aspergillus fumigatus 22 72Acinetobacter baumannii 17 48


The next two sections describe the feasibility studies of the rapidsterility test using pharmaceutical samples (eight commonly-usedexcipients and one drug product). Sterile samples were spiked withinocula containing a low count (<5 cells) of a number of compendialreference and environmental strains, and tested after various times ofincubation (enrichment) either by a traditional method (membranefiltration and/or plate count) or the rapidmethod (bioluminescence).

EVALUATION OF THE RAPID BIOLUMINESCENCE TESTIN THE PRESENCE OF EXCIPIENTS

Excipients are used in a wide variety of drug formulations. Theircompatibility with the test system widens the spectrum of theapplicability of the Pallchek system. The presence of any inhibitoryactivity present in commonly-used excipients that could affecteither the growth of microorganisms or the bioluminescence assayin the rapid sterility procedure was evaluated using Gram positiveand Gram negative microorganisms, yeast and mold. Microbialcultures were exposed to each excipient (1% in TSB) for 30 min atroom temperature. After incubation, samples were processed asdescribed earlier. No significant deleterious effect was observed onmicrobial growth or the bioluminescence reaction (Table 17.8).

Product specific feasibility study

Drug product was prepared by pooling samples to a total of 10 mL.An aliquot of DP was added to a sterile bottle (250 mL) containing150 mL of either FTM or TSB. The preparation was spiked with <5microbial cells and incubated for up to five days at the appropriatetemperature. A 10 mL aliquot was taken at various time points andwas analyzed using Pallchek Rapid Microbiology System.

Most of the challenge organisms were detected by the end of 48hrs. Isolates of M. luteus, and R. pickettii though could be detected at48 hrs, the peak RLU reading was reached later. A. fumigatus wasdetected at 120 hrs which was the only time point the culture wassampled after 17 hrs. However data from other experiments haveshown that the organism can be detected as early as 72 hrs (not


Table 17.8 Bioluminescence assays conducted in the presence ofexcipients (1% inTSB)

Excipient Gram Positive Gram Negative Yeast/MoldB. subtilis E. coli C. albicansS. aureus P. aeruginosa A. brasiliensis

S. enterica

Microcrystalline Cellulose � � �Lactose � � �Gelatin � � �Magnesium Stearate � � �Ethanol � � �Polypropylene Glycol � � �Mannitol � � �Starch � � �

Checkmark represents successful detection comparable to parallel controls run in theabsence of excipient

Figure 17.6 Detection of Microbial Contamination in DP1

Test samples were spiked with ≤5 cells of challenge microorganism


shown). These results were reproduced over several experiments. Inthe presence of DP, M. rhodesianum could not be detected at the endof 120 hrs of incubation (Figure 17.6). An additional attempt to detectM. rhodesianum in the presence of DP was done by running parallelsamples incubated either in TSB or in FTM. A weak value barelyabove the threshold value was obtained after 120 hours of incubationin FTM. A control culture of M. rhodesianum in TSB alone was alsorun and confirmed the identity of the isolate by colony morphologyand Gram stain. All suitability controls and confirming plate countswere within expected ranges for all of the above experiments.

SUMMARY

Release sterility testing is the critical quality control test that definesthe acceptability of a manufactured drug product as aseptically safeto be administered to a patient. This assay, therefore, must bedesigned, validated and executed following the most stringent QCguidance and aseptic techniques. Technician’s competence, theability of media used in sterility test to support microbial growth,the conformity of the test environment to the requirements of thePharmacopoeia in terms of viable microbial air and surface countsmust be demonstrated and documented. In addition, proceduresfor sampling, testing and follow-up must be defined in thevalidation procedures. The validation and implementation of arapid method must also comply with the previous requirementsand new parameters must be validated so that they relate moredirectly to the characteristics inherent to the mechanism ofdetection of the new assay. In addition, important considerationmust be assigned to demonstrate that the new procedure does nothave any chance of producing either false positives or, veryimportantly, false negatives (FDA, 2004). Based on the datapresented and discussed in this chapter, the Pallchek systemconstitutes a reliable rapid method for sterility testing that isequivalent or better than the traditional assay. Overview schematicsof a rapid sterility test using the Pallchek Microbiology System isshown in Figure 17.7.


Figure 17.7 Overview of sterility test using Pallchek RapidMicrobiology System

The results of the feasibility study on the application of the rapidmicrobiological Pallchek Rapid Microbiology System to a rapidsterility test for a selected drug product were shown to be veryencouraging. This drug product is a suspension which confersturbidity to the culture media and prevents visual inspection at theend of the 14-day incubation period. According to the traditionalprocedure, an aliquot of the incubated sample is inoculated intofresh, sterile media at day 14 and further tested for an additionalfive days. Since the inherent turbidity of the product does notinterfere with the bioluminescence assay, the use of the PallchekRapid Microbiology System allows readings in samples from theoriginal incubation container and detects contaminants in a periodof time as short as five days, instead of the required 14+5 daysprescribed in the traditional standard operating procedure (SOP)developed for this drug product. Of the 16 reference andenvironmental isolate microorganisms tested all but one (M.rhodesianum) were detected by 120 hr. One possible explanationmay be that DP1 is not amenable to growth of M. rhodesianum. Thisparticular isolate was not available at the time of the originalvalidation studies for the traditional test. Therefore, there is no data


precedent to this study. Additional studies will be required toconfirm this initial observation.

Rapid microbiological methods can be applied to a broad rangeof quality control operations, such as purified and process watertesting, raw materials and excipients testing, environmentalmonitoring, in-process monitoring, manufacturing process design,investigations and final products release testing. The latter isarguably the most critical test because of the immediate impact thatany failure or undetected contamination could have in the patientpopulation. The data discussed throughout the chapter stronglysuggest that a rapid sterility test using the Pallchek RapidMicrobiology System provides results in a timely manner that areequivalent to or better than those obtained in the traditionalmethod. The use of an incubation (enrichment) phase during aperiod of time significantly shorter that the 14 days of theconventional method contribute to an easier interpretation andcomparability of the results since both the rapid and the traditionalassays rely on growth of the microorganisms under conditionssimilar to those described in the pharmacopoeia (USP <71>, 2011a;EP Section 2.1.6, 2010a; JP, 2006).

In addition, the implementation of the rapid method describedhere potentially presents the following advantages:

• Reduced warehouse space and costs for raw materials

• Intermediates and final products– fast final-product release– shorter product release cycle times– time and labor savings in the lab, during manufacturing

• Decreased plant downtime

• Reduced cycle times– reduction of backorders– reduction or elimination of product losses– increased manufacturing capabilities

• Risk reduction in manufacturing

• Increased business and production flexibility


• Increased product development capabilities

• Robust understanding of manufacturing processes

• Proactive control: move from QC to QA procedures

• Immediate detection and correction of contamination

• Immediate cleaning validation

• Better protection of customers and company image.

In the decision making for the implementation of a rapid sterilitytest, a translation from the potential advantages delineated above toactual return of investment (ROI) dollars is essential. The ROIexercise should comprise calculations on the cost of the traditionaltest, the cost of implementing the rapid method, and a detailedanalysis of the savings generated by the RMM (Yvon, 2008; Gadaland Yvon, 2009).

The analysis should apply to a period of time from one to fiveyears and should include comparative data between conventionaland rapid methods, including:

• number of tests per year and price per test

• total testing time (hours) and labor cost (per hour)

• equipment (investment in new equipment, depreciation,calibration, qualification)

• lab space and environment test requirements

• disposal of used plates, reagents, etc.

• cleaning, preparation and downtime

• operation time

• time to results

• validation documents

• training

• maintenance contracts.


We have found that the relatively low cost and simplicity of thePallchek luminometer, along with the time and cost savings fromearly contaminant detection, significantly facilitates the requiredcalculations, showing ROI figures that can be realized in just 1–2years, depending on test volumes, cost of warehousing and costavoidances on additional investigations.

Overall, the results described in this chapter demonstrate thatthe Pallchek Rapid Microbiology System provides a significant timesaving for the detection of microorganisms within media that arevisually occluded due to the cloudy suspension characteristics ofthe drug product studied in the work reported here. Based on theencouraging results that the Pallchek Microbiology Systemprovided with this drug product, plans are being considered torunning a comparability study of this method in parallel to theharmonized compendial sterility test method at a manufacturingsite for a period of three to six months, using actual batch samplesof manufactured product.

Acknowledgments

We would like to thank Michael Baumstein, Amber Dellar, KarenBoeve and John Shabushnig for their support and insightfulinformation and advice during the development of this work. Inaddition, we are grateful to Michael Boquet for his excellenttechnical assistance during the early stage of these studies.

REFERENCES

Chappelle E.W., Levin, G.V. (1968) Use of the FireflyBioluminescence Reaction for Rapid Detection of CountingBacteria. Biochemical Medicine 2: 41–52.

Denoya, C., Reyes, J., Dawson, E., Sessoms, D., Baumstein, M.,Shabushnig, J. (2010) ARapid Microbiological Assay to Monitorthe Effectiveness of a Vaccine Injector Sanitization Following aMicrobial Challenge Procedure. American Pharmaceutical Review13(4): 54–61.


European Pharmacopoeia (EP) (2010a) Chapter 2.1.6 “Sterility”. 7thedition. Council of Europe, Strasbourg, France.

European Pharmacopeia (EP) (2010b) Chapter 5.1.6 AlternativeMethods for Control of Microbiological Quality. 7th edition.Council of Europe: Strasbourg, France.

FDA (2004) Guidance for Industry. Sterile Drug Products Producedby Aseptic Processing — Current Good Manufacturing PracticeU.S. Department of Health and Human Services, Food andDrug Administration, Rockville, MD.

Gadal, P., Yvon, P. (2009) Rapid Microbiology ROI: Calculatingscientific benefits as return on investment dollars.Pharmaquality. www.pharmaquality.com

Japanese Pharmacopoeia (JP) (2006) Chapter 4.06 “Sterility Test”.15th edition, the Ministry of Health, Labor and Welfare, Japan.

Kramer, M., Suklje-Debeljak, H., and Kmetec, V. (2008) PreservativeEfficacy Screening of Pharmaceutical Formulations using ATPBioluminescence. Drug Dev and Industrial Pharmacy, 34: 547–557.

Lehninger (2008) Principles of Biochemistry. Fifth edition Eds.David L. Nelson, Michael. M. Cox. WH Freeman Publishers.New York, NY.

Nielsen, P. and Van Dellen E. (1989) Rapid Bacteriological screeningof cosmetic raw materials by using bioluminescence. Journal ofthe Association of Analytic Chemists 72(5): 708–711.

PDA (2000) Technical Report No. 33. Evaluation, Validation andImplementation of New Microbiological Testing Methods. PDAJournal of Pharmaceutical Science and Technology. Supplement54(3): 1–39. Parenteral Drug Association, Bethesda, MD.

Stanley, P.E. (1989) A concise beginner’s guide to rapidmicrobiology using adenosine triphosphate (ATP) andluminescence. In ATP Luminescence: Rapid Methods in


Microbiology, P. E. Stanley, B. J. McCarthy, and R. Smither, Eds.,Blackwell Scientific Publications, Oxford, England, 1–11.

Pall Life Sciences. Testing Procedures and Applications for thePallchek Rapid Microbiology System, USTR 2358, Pall LifeSciences, Port Washington, New York.

Pall Life Sciences. Validation Guide for the Pallchek™ RapidMicrobiology System. USTR 2359. Pall Life Sciences, PortWashington, New York.

USP (2011a) Chapter <71> “Sterility Tests”. USP 34-NF 29. TheUnited States Pharmacopeial Convention/National Formulary,Rockville, MD.

USP (2011b) Chapter <1223> Validation of AlternativeMicrobiological Methods. USP 34-NF 29. The United StatesPharmacopeial Convention/National Formulary, Rockville, MD.

White, E.W., McCapara, F., Field, G.F., McElroy W.D. (1961) TheStructure and Synthesis of Firefly Luciferin. Journal of AmericanChemical Society 83: 2402–2403.

Yvon, P. (2008) Rapid Methods: Return of Investments. PodiumPresentation at Plenary Session 4 — Rapid Methods. PDA 3rdGlobal Conference on PharmaceuticalMicrobiology, Chicago, IL.

ABOUT THE AUTHORS

Claudio Denoya, Ph.D., is a Research Fellow and Group Leader ofthe Microbiological Technology Assessment group at Pfizer GlobalR&D. He is a co-chair of the Global RMM Steering Team. He is alsoan Adjunct Professor at the Department of Molecular and CellBiology, Univ. of Connecticut.

At Pfizer Dr. Denoya held several lead positions and led manymolecular and microbiology projects. Dr. Denoya has receivedseveral recognitions, including the Pfizer Global R&D Outstanding


Team Achievement Award, Pfizer Global R&D IndividualAchievement Award, two Pfizer Individual Performance Awards,two Supply Chain Recognition Awards, and the United StatesNational Hispanic Corporate Achiever Award. He has receiveddistinguished fellowships and visiting investigator positions fromthe Public Health Research Institute of the City of New York, theUniversity of Sao Paulo, the Autónoma University of Madrid, andthe PNUD-UNESCO.

He has authored over 250 patents, book chapters, journal articlesand technical presentations in the areas of biochemistry,microbiology, cell and molecular biology, and pharmaceuticalsciences. Dr. Denoya holds a Ph.D. in Biochemistry and MolecularGenetics ofAnimal Viruses, aM.S. in Biochemistry andMicrobiology,and a B.S. in Clinical Biochemistry from the University of BuenosAires.

Jennifer Reyes is a Microbiologist who joined Pfizer in 2006 andhas worked on the development of Rapid Microbiological Methods(RMMs) in the area of detection of microbial contaminants inpharmaceutical products. Prior to Pfizer, Jennifer worked as aMicrobiologist for Amgen and before that for Monsanto in the areaof Quality Control Assay Development. Jennifer received her B.S.and M.S. degrees in Microbiology from the University of RhodeIsland.

Maitry Ganatra is Global Product Manager with Pall Life Sciencesand is responsible for managing the process monitoring productportfolio and directing global cross functional team on newproducts. She has more than 10 years of business developmentexperience with Life Sciences products. Prior to joining Pall, she ledMicrobiology Validation Program at Claris Life Sciences. She hasauthored many scientific publications and is committee member ofPDA Task force for Revision of Technical Report No. 13 onEnvironmental Monitoring. Dr. Ganatra holds a Diploma inPharmacy, Ph.D. in Microbiology (Gujarat University) andcurrently pursuing MBA at Long Island University.


Daniel A. Eshete M,D. PhD., holds a Doctor of Medicine (AddisAbaba University), M.Sc. in Biochemistry (Addis AbabaUniversity/KI) and Ph.D. in Chemical Pathology (University ofCape Town) with Post-Doctoral studies in Microbiology/Immunology at Karoliniska Institute, Microbiology and TumourBiology Centre (Stockholm) and St. Louis University Division ofInfectious Diseases (St Louis, MO). Prior to moving to theBiomedical Industry he worked in academia teaching Biochemistry,Clinical Chemistry and Instrumentation. His research works havebeen focused on cellular signal transduction, and themolecular basisof host parasite interaction and microbial infection. Since joining theindustry his focus has mainly been on Pharmaceutical QualityControl with particular emphasis in rapid microbiological methodsdevelopment and validation. Dr. Eshete is currently a staff scientistand a member of the Process Monitoring and Pharmaceutical QCteam at Pall Life Sciences, Scientific & Laboratory Services.

Reprinted from Rapid Sterility Testing, edited by Jeanne Moldenhauer.Copyright 2011, co-published by PDA and DHI. All rights reserved.


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Rapid Sterility Testing Using Pallchek