10/31/2014. Automated and Rapid Microbiological Methods: Selection and Validation. Microbiology: Past. Microbiology: Today

Transcription

1 Automated and Rapid Microbiological Methods: Selection and Validation Michael J. Miller, Ph.D. President Microbiology: Past Anton van Leeuwenhoek observes bacteria Pasteur disproves spontaneous generation Koch defines pure culture and colony Fanny Angelina Hesse introduces agar-agar Hans Christian Joachim Gram develops the Gram stain Julius Petri invents glass plates for bacterial growth 1 Microbiology: Today Still using 19 th -Century methods counting colonies on agar plates and Gram staining 2 1

2 Microbiology: Today Classical methods are limited by slow growth rates Variability of microbes in their response to culturing Most microorganisms in the manufacturing environment, in-process samples and raw materials are starved, stressed or injured, and current media and incubation conditions are not optimal for the resuscitation and growth of these microorganisms Many times we will observe zero colony forming units (CFU) on agar plates when in fact, viable microorganisms are present 3 Opportunities Significant opportunities exist for improving the efficiency of manufacturing and quality assurance through the application of modern process analytical tools There are regulatory initiatives that are recommending changes in the way we approach microbiology testing These include Rapid Microbiological Methods (RMMs) and automated technologies 4 Rapid Microbiological Methods Novel technologies that provide microbial detection, quantification and identification results much faster than conventional methods Increased accuracy, reproducibility and sensitivity Automated, miniaturized and high-throughput processing Improved sampling, data handling and trend analysis For some technologies, results in real-time 5 2

3 Rapid Microbiological Methods Many RMMs do not require microbial growth Enhanced detection of single cells, stressed or injured microorganisms Healthy or stressed viable but non-culturable (VBNC) cells Cells induced into dormancy at the beginning of the stationary phase following environmental stress Improved microbial identification and strain differentiation 6 Rapid Microbiological Methods Applications Bioburden (raw materials, in-process, finished product) Sterility testing Environmental monitoring Process water testing Microbial identification Mycoplasma Microbial Limits Testing Enumeration and presence/absence 7 Agenda Overview of currently available RMMs that can be used for Microbial Limits Testing Enumeration Presence/absence testing Technology Workflow Case studies Validation strategies PDA Technical Report #33 8 3

4 RMM Technologies It is important to understand what technology platforms are available, in order to appropriately match the RMM with its intended application Consider the technical or method requirements Do you need to detect, enumerate and/or identify microorganisms? Is the RMM compatible with your samples or product? Do you need to detect different type of microorganisms? What is the required level of sensitivity or limit of detection/quantification? What sample sizes are required? Data management requirements? Operator qualification requirements? 9 RMM Technologies RMMs can provide qualitative, quantitative and/or microbial identification data Qualitative Information on the presence or absence of all microorganisms or the presence of specific microbial species Quantification The number of microorganisms present in a sample Microbial identification The identity of at the Genus, species and/or strain level We will not discuss ID systems today 10 RMM Technologies Based on a wide variety of detection principles The use of viability stains and laser excitation for the detection and enumeration of microorganisms without requiring cell growth The detection of cellular components or markers (e.g., ATP and endotoxin) Optical spectroscopy, such as light scattering The amplification of nucleic acids and detection of specific genetic sequences (e.g., PCR) The use of fluorescence techniques to rapidly detect the growth of microorganisms on conventional media Micro-Electro-Mechanical Systems (MEMS), such as microarrays, biosensors, Lab-On-A-Chip and nanotechnology 11 4

5 Disclaimer No endorsements during this presentation Order of RMMs discussed is random More than 60 different RMMs have been implemented or reviewed by various industries; we will review some of them For an in-depth review of RMM technologies, workflow, and other relevant information, see the RMM Product Matrix at rapidmicromethods.com 12 Enumeration 13 Rapid Micro Biosystems Growth Direct Digital imaging technology that enumerates micro-colonies in onehalf the time to visualize colonies The sample is filtered and the filter is placed onto a flat agar medium cassette with an optically clear lid A light emitting diode (LED) excites micro-colonies to autofluoresce, which are enumerated by a CCD imaging system 14 5

6 Rapid Micro Biosystems Growth Direct Cells fluoresce in the yellow-green spectral region when illuminated with blue light due to oxidized flavins Photosensitive pixels in the CCD camera chip detect autofluorescing micro-colonies 15 Rapid Micro Biosystems Growth Direct The system automatically incubates and analyzes each cassette over time Particles that do not grow in size over time are ignored Non-destructive can continue to incubate media to obtain colonies for microbial identification Considered an automated version of the existing compendial method Bioburden and environmental monitoring One or two temperatures Capacity: up to 350 plates 16 Rapid Micro Biosystems Growth Direct 0 hr 6 hr 7 hr 8 hr 9 hr 10 hr 11 hr 12 hr 13 hr 17 6

7 EMD Millipore Milliflex Quantum Fluorescent staining and laser excitation of microcolonies on a membrane Applicable for all filterable samples, including water, inprocess and finished product Non-destructive can continue to incubate media to obtain colonies for microbial identification 18 EMD Millipore Milliflex Quantum Filter the sample, place the membrane onto an agar cassette and remove the funnel Incubate for an appropriate time period 19 EMD Millipore Milliflex Quantum Saturate the staining cassette with a non-fluorescent substrate, remove the agar cassette from the incubator, place the membrane onto the staining cassette and incubate for 30 minutes at 32.5 C 20 7

8 EMD Millipore Milliflex Quantum Microorganisms retained on the membrane will take up the non-fluorescent substrate Within viable and culturable cells, the nonfluorescent substrate is enzymatically cleaved The cleaved substrate liberates free fluorochrome into the microorganism cytoplasm As fluorochrome accumulates inside the cells, the signal is naturally amplified 21 EMD Millipore Milliflex Quantum Following incubation, the membrane is placed into the reader and exposed to the excitation wavelength of the dye Fluorescent micro-colonies can then be counted in the instrument window or on a computer via a camera 22 EMD Millipore Milliflex Quantum Following staining and counting of micro-colonies, the membrane can be placed onto the agar cassette and re-incubated to allow larger colonies to form which can then be used for microbial identification (non-destructive method) 23 8

9 ATP Bioluminescence Bioluminescence is the generation of light by a biological process 1947: William McElroy discovered the mechanism by which bioluminescence occurs Observed in the tails of the American firefly Photinus pyralis Specific enzyme reaction catalyzing the consumption of ATP (Adenosine Triphosphate) 24 ATP Bioluminescence In the presence of the substrate luciferin, the enzyme luciferase will use the energy from ATP to oxidize luciferin and produce photons (hv; light at a wavelength of 562nm) Luciferase Luciferin + ATP + O 2 AMP + PPi + CO 2 + Oxyluciferin + Mg++ 25 ATP Bioluminescence Because all living cells store energy in the form of ATP, it can be used as a measure of organism viability Capture microorganisms, release ATP from within the cells, and measure the amount of bioluminescence generated Instruments utilize a luminometer equipped with a photomultiplier tube to detect the photons 26 9

10 ATP Bioluminescence The concentration of ATP required for measurement is about 200 attomoles, which is equivalent to one yeast or mold cell or approximately 100 bacterial cells, depending on their metabolic state. May require up to 1000 bacterial cells When low numbers of cells are expected, an enrichment step in media is required to allow the cells to multiply and produce a sufficient level ATP for detection 27 Pall Pallchek Handheld system that measures ATP on directly on surfaces, on a membrane filter or liquid samples If sufficient cells (and ATP) are present, ATP measurements are obtained within minutes When low counts are expected, incubate sample/membrane in liquid media (18-24 hrs). The media is then filtered 28 Pall Pallchek Add luciferin and luciferase reagents to the membrane or surface Place instrument over sample Results are provided as relative light units (RLU), which can be correlated with an estimation of cell count Appropriate for presence/absence testing and an estimation of cell counts 29 10

11 Case Study for Microbial Limits GSK received FDA approval to use the Pallchek system for the early release of a non-sterile prescription nasal spray product (up to four days earlier than conventional methods) They were the first pharmaceutical company to obtain an approval under the FDA PAT initiative The firm used a comparability protocol and implemented the technology under a CBE-0 Filtered the product, enriched overnight and tested the filter They used a 2-tiered approach for product release 30 Case Study 31 Millipore Milliflex Rapid Microbiology Detection System Utilizes a filter membrane to capture individual cells, allow them to grow into micro-colonies and provide an actual cell count Pass sample through 0.45 micron PVDF membrane Can rinse filter to reduce bioluminescence inhibition or interference 32 11

12 Millipore Milliflex Rapid Microbiology Detection System For bacterial detection, incubate on appropriate medium to form micro-colonies (e.g., 18 hrs) Growth is not required for yeast or vegetative mold The filter is then placed into the AutoSpray station, where ATP releasing agent and bioluminescence reagents are applied 33 Millipore Milliflex Rapid Microbiology Detection System The filter is then transferred to the Detection Tower The detection tower intensifies bioluminescence from each cell (or micro-colony) thousands of times 34 Millipore Milliflex Rapid Microbiology Detection System Light signals are captured with a CCD camera; an image processor analyzes the signal and provides a cell count Each image theoretically arises from a single cell May be non-destructive (continue to grow into a CFU) 35 12

13 Viability-based Technologies Because viability-based RMMs do not rely on microbial growth, microorganisms that are stressed, starved, difficult to culture, or viable but non-culturable (VBNC) may be detected and enumerated Could result in a higher count compared with conventional methods In these cases, a correlation between the RMM counts and the conventional counts can be developed The RMM counts can then be used to set new acceptance or specification levels 36 Flow Cytometry Counting individual cells as they pass through a laser beam in a very narrow flow cell Microorganisms are labeled with a viability stain and then passed through a laser The laser causes the stain to fluoresce Low sample volumes (1 ml or less) Sensitivity is cells Bioburden testing of liquids and nonfilterable material 37 BD Biosciences FACSMicroCount Automated enumeration of bacteria, yeast, mycoplasma and spores (bacterial and mold) as early as 4 minutes Accurate detection between organisms per ml Fully automated, robotic arm processes samples Up to 42 samples can be analyzed automatically 38 13

14 BD Biosciences FACSMicroCount Nucleic acid dye labels live & dead cells; BRAG3 labels live cells The labeled organisms pass through the flow cell and a 635 nm red diode laser 39 BD Biosciences FACSMicroCount The scatter and fluorescence intensity for each individual microorganism are displayed, as well as counts per ml 40 Solid Phase Cytometry Counting individual microorganisms that have been captured onto a filter membrane Microorganisms are stained and exposed to a laser The laser will cause the viability stain to fluoresce Sample volumes are higher than those used in flow cytometry (e.g., > 100 ml), but sample must be filterable Sensitivity down to a single cell Appropriate for bioburden testing, environmental monitoring and sterility testing 41 14

15 AES Chemunex ScanRDI Organisms are stained with a non-fluorescent substrate Within the cytoplasm of metabolically active cells, the substrate is enzymatically cleaved (by esterase) to release a fluorochrome The fluorochrome will fluoresce when excited by a laser Cells with intact membranes will retain the fluorescent label Viability substrate Enzyme Free fluorochrome 42 AES Chemunex ScanRDI All viable bacteria, yeast and spores (bacterial and mold) are detected within 2 hours, with single cell sensitivity Accurate detection between bacteria and for yeast and spores 43 AES Chemunex ScanRDI Filter the sample through a 0.4 µm polyester membrane Label with viability substrate, incubate Place membrane into laser scanning chamber 44 15

16 AES Chemunex ScanRDI The membrane is scanned by an argon laser at 488 nm Scan lines are 2.2µm apart to ensure overlap from previous scan Photo-multiplier tubes detect emitted fluorescent light in 3 min Algorithms and discrimination processes determine if the fluorescent signals originate from labeled viable microorganisms or from an auto-fluorescent particle 45 AES Chemunex ScanRDI Auto-fluorescent particles, membrane fluorescence and background noise are rejected and a total viable count is displayed 46 Case Study Bausch & Lomb Purified Water Testing 47 16

17 Presence/Absence 48 BioLumix Detects target microorganisms by monitoring changes in color or fluorescence in selective media, and/or by monitoring the generation of CO 2 49 BioLumix Each vial contains a broth medium and/or other reagents specific for the target organism with unique dyes in which target microorganisms grow and are detected by changes in color or fluorescence These changes, expressed as light intensity units, are detected by an optical sensor 50 17

18 BioLumix Disposable two-zone vials contain an incubation zone (top of vial) for the sample and microorganism, and a reading zone (bottom of vial) The two-zones eliminates masking of the optical pathway by the product and by microbial turbidity 51 BioLumix One bacterial cell is usually detected within 8-18 hours, a single yeast cell is detected in hours, and mold requires hours The threshold for bacteria is 100,000 cells/ml and the threshold for yeast/mold is 10,000 cells/ml The time to detection depends on the initial concentration of organisms in the product sample 52 BioLumix Detection of specified microorganisms Tests include total aerobic count, yeast & mold, coliforms, E. coli, lactic acid bacteria, Enterobacteriaceae, Salmonella, Pseudomonas, and Staphylococcus 53 18

19 BioLumix The system can be used to screen for an estimation of organisms in a test sample that are above or below a certain quantitative specification Dilute the test sample to a level that represents the specification level (e.g., 1:100 dilution for a spec of not more than 100 cfu) No response means that there were less than 100 cfu in the sample A positive response means that there were greater than 100 cfu in the sample 54 PCR for Presence/Absence DNA is extracted and heated to separate the double strands Heat to 90 C DNA primers (short, synthetic sequences) are added, which bind to unique target sequences on the template DNA, if they are present Heat-stable DNA polymerase and nucleotide bases (A,T,G,C) are added. The primer is elongated, producing two new complete copies of the template DNA strands Lower heat to 55 C Raise heat to 70 C Repeating the process results in millions of copies of target DNA; a probe is used to detect the DNA sequence 55 Taqman Probe to Detect Sequence Heat to denature DNA; anneal primer At the same time, a probe anneals to another region The probe contains a fluorescent reporter dye at one end and a quencher dye at the other end. There is little fluorescence when the probe is intact. As the primer extends, the probe is cleaved, the two dyes separate and the fluorescent signal increases Fluorescent signal increases with each PCR cycle 56 19

20 Taqman Probe-based Assay 57 Pall GeneDisc qpcr to simultaneously detect multiple organisms in the same sample Different primers and probes/dyes for each organism/dna sequence Taqman probe technology 58 Pall GeneDisc Samples are filtered, media is added to the filter cartridge, and the membrane is incubated for 6-16 hours Required to eliminate false positives from residual DNA Organisms on the membrane are lysed (sonicated for 8 min) and heated (100 C for 19 min) to release the DNA 59 20

21 Pall GeneDisc The purified DNA and a Master Mix (polymerase and deoxynucleotides) are added to the upper hub of a GeneDisc plate The plate is inserted into the instrument, and the disk rotates through 4 different heating and cooling sections during the PCR amplification and detection process 60 Pall GeneDisc Primers will amplify specific DNA sequences, if present Fluorescent signals increase during DNA amplification 61 Pall GeneDisc Up to 6, 9 or 12 test samples may be assayed per disc Depends on disc; includes positive and negative controls 8 modular system units have the capability of testing up to 96 samples per run (~1 hour) Plate for Compendial Specified Microorganisms Escherichia coli, Salmonella spp., Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, Aspergillus brasiliensis (A. niger) Food Testing and Environmental Testing STEC, non-stec and E. coli O157, Salmonella, Listeria Legionella, Pseudomonas, Enteroccocus, Cyanobacteria, E. coli 62 21

22 Combination Technologies New technologies are being developed that combine both enumeration and presence/absence testing 63 Rap.ID Bio Particle Explorer Viability staining and automated image analysis using dark field illumination Confocal Raman laser beam (532 nm) is then automatically aligned with the viable particle locations 64 Rap.ID Bio Particle Explorer Particles 500nm are examined for shape and size Spectral signatures from viable particles are generated and compared with a library of known microorganisms Rapid enumeration and ID with single cell sensitivity 65 22

23 Rap.ID Bio Particle Explorer Particles are collected on metal foil using impaction or filtration methods for airborne or liquid samples Viability staining and particle enumeration in 4 minutes Identification of a single viable cell is 1-5 seconds 150 bacteria and spore entries in database, customizable individual ID s per hour >150 samples per 8 hours Non-destructive for further analysis 66 Q&A Source: 67 Validation 68 23

24 Validation Guidance USP <1223>, Validation of alternative microbiological methods (UNDER REVISION) Ph. Eur , Alternative methods for control of microbiological quality (UNDER REVISION) PDA Technical Report #33, Evaluation, Validation and Implementation of Alternative and Rapid Microbiological Methods (REVISED 2013) 69 Pre-Validation Activities Proof of Concept (POC) or feasibility testing Assessment of supplier capabilities / supplier audit Review business benefits; conduct Return on Investment analysis 70 TR33 - Validation Validation of the Equipment, Software and Method Responsibilities Risk Assessment Validation Planning User Requirements Specification (URS) Design Qualification (DQ) Functional Design Specification (FDS) Requirements Traceability Matrix (RTM) SOPs and Technology Training System Integration, IT, LIMS Installation Qualification (IQ) Operational Qualification (OQ), computer system validation 71 24

25 TR33 - Validation Validation of the Equipment, Software and Method Performance Qualification (PQ) Method validation and suitability testing Ongoing Maintenance and Periodic Reviews Preventive maintenance, calibration, software updates Method Validation Quantitative and qualitative methods Standardized cultures Actual product or samples (equivalence/comparative testing) Testing procedures, acceptance criteria and recommended statistical analyses 72 TR33 - Validation Method Validation Criteria Accuracy Precision; repeatability Specificity, stressed organisms and mixed cultures Limit of Detection Limit of Quantification Linearity Range Ruggedness; intermediate precision, reproducibility Robustness Equivalence/comparative testing 73 TR33 - Validation Suitability Testing False positive and false negative testing Recommended procedures, acceptance criteria and statistical analyses Validation Additional Considerations Automated methods; extensions of compendial tests Unique Methods; Additional or Modified Validation Strategies Guidance on changing existing acceptance criteria Technology transfer 74 25

26 A Note About the Regulators FDA, EMA, Australian TGA, Japanese PMDA, WHO all accept RMMs and encourage their use Policies have been implemented that provide a framework for validating and implementing RMMs RMMs have been approved for use, even for sterility testing of finished pharmaceuticals 75 The Path Forward Rapid methods continue to gain momentum Regulators encourage their use Companies have validated and implemented RMMs RMMs have provided quality and efficiency benefits New technologies continue to be introduced and they are getting better Resources are at your fingertips 76 Additional Resources Many papers and publications PDA Encyclopedia of Rapid Microbiological Methods Dedicated RMM seminars, training and conferences Discussion forums, e.g. Rapid Micro Methods LinkedIn Group Websites, e.g., rapidmicromethods.com Lists of RMM references Technology and application matrix RMM news blogs Guidance on regulatory acceptance, validation and ROI 77 26

27 Thank You! Michael J. Miller, Ph.D. Web: microbiologyconsultants.com LinkedIn: phone: (RAPID-RAPID) 78 Q&A Source: