Melbourne July 2017/R. Strebel Real Time Microbial Detection Global Pharmacopeia Acceptance of Alternative Methods
Agenda 2 1 Introduction 2 Pharmaceutical Water Monitoring and Analysis 3 Why On-line Microbial Monitoring 4 Real-Time Detection Technology 5 Pharmacopeia acceptance of Alternative Methods 6 Validation Guidelines
Our Solutions across the Value Chain 3 Laboratory Solutions Process Analytics Industrial Weighing Product Inspection Logistics Solutions Retail Weighing Solutions We offer solutions along our customers' value chain to help them: Streamline processes Enhance productivity Reach compliance with regulatory requirements Optimize cost and waste Confidential
Process Analytics Solutions Overview 4 Reliable in-line and real-time measurement of key analytical parameters to control process parameters and monitor product quality. Water Analytics TOC, Conductivity, Microbial, Ozone, Sodium, Silica Process Analytics ph, Oxygen, CO 2, Turbidity Gas Phase Analytics O 2, CO, CO 2, H 2 O, H 2 S, HCl Confidential
Company Background 5 60+ years of Process Analytical Expertise Ingold founded in 1948, acquired by Mettler Toledo in 1986 Thornton founded in 1964, acquired by Mettler Toledo in 2001 Sensor/Analyzer manufacturer for ph/orp, Resistivity/Conductivity, TOC, DO and water quality with focus on: - Pharmaceutical and Biotech - Chemical - Microelectronics - Power Dr. Werner Ingold Extensive research - Chemical and physical properties - New technologies - Product handling and asset management Active participation in ASTM, ISPE, PDA, Semi, EPRI, USP Dr. Richard Thornton
Why is Water Important? 6 Water is the most widely used excipient in Pharmaceutical Manufacturing and a significant part of all quality inspections Water is multi-functional - Raw material - Solvent - Ingredient - Reagent - Cleaning agent (hot water or steam) - Sterile/Packaged waters - Sterile Water for Irrigation - Sterile Water for Inhalation - Sterile Purified Water - Bacteriostatic Water for Injection - Water for Hemodialysis Availability of water will be a bigger challenge than climate change Stanford University Business School
Agenda 7 1 Introduction 2 Pharmaceutical Water Monitoring and Analysis 3 Why On-line Microbial Monitoring 4 Real-Time Detection Technology 5 Pharmacopeia acceptance of Alternative Methods 6 Validation Guidelines
Pharmacopeia Bacteria Regulations (USP 40) 8 The regulated parameters can now be measured in real time! Attribute USP 40* EP 8.2 JP 17 ChP 5 IP Water For Injection Total Aerobic (Microbial) (cfu/100 ml) 2 USP <1231> 10 10 10 10 10 Conductivity (ms/cm at 25C) (USP <645>) 1.3 (3 stage) 3 Same as USP 2.1 offline Same as USP Same as USP TOC (mg/l) (USP <643>) 0.5 4 0.5 0.5 0.5 0.5 Bacterial Endotoxins (EU/mL) (USP 71) 0.25 0.25 0.25 0.25 0.25 Attribute USP 40* EP 8.2 JP 17 ChP IP Purified Water Total Aerobic (microbial) (cfu/ml) 2 -USP <1231> 100 100 100 100 100 Conductivity (ms/cm at 25C) - USP <645> 1.3 (3 stage) 3 5.1 (1 stage) 2.1 offline 6 Same as EP Same as USP TOC (mg/l) - USP <643> 0.5 4 0.5 (optional) Same as USP Same as USP Same as USP * USP40-NF35 is the current approved version under which you can find USP <1231> and <1223> Microbiological testing is considered to be harmonized, with the exception noted that the EP test is written into the Production section, and the USP test is contained in a non-compendial general information chapter
Bulk Water for Injection(s) - Today 9
Bulk Purified Water - Today 10
Pharmacopeia adoption of new technologies 11 1840-1994 1994-1996 > 1996 2003 2006 Chemistry/Organic tests are: - Qualitative - Subject to bias - off-line - Carbon dioxide - Calcium - Ammonia - Chloride - Sulfate - Oxidizable substances Testing to compare on-line instrumentation versus wet chemistry Chemistry tests VS. conductivity instrument Oxidizable substances vs. TOC instrument USP accepted as compendial tests Conductivity instrument TOC instrument Adoption by EP JP ChP IP - Heavy metals
Pharmacopeia adoption of new technologies 12 1890-2017 2017 to 20?? 20?? 20?? Bacteria tests are; - Plate Count - Qualitative - Subject to bias - off-line - Accepted to be correct and accurate Traditional plate counts vs. on-line Real Time Instruments USP <1223> now encourages validation of alternative microbial methods Ph.EUR. 5.1.6. Alternative Methods for control of Microbiological Quality Accepted by USP as a compendial Test? on-line, realtime Instruments Accepted by the other Global Pharmacopeia s
Relevant Sections of the USP 13 Pharmaceutical Water Monographs - Purified Water (bulk) Sterile Water for Inhalation - Water for Injection (bulk) Sterile Water for Irrigation - Sterile Purified Water (packaged) Bacteriostatic WFI - Sterile WFI (packaged) Water for Hemodialysis - Pure Steam Test Chapters - 645 Water Conductivity 85 Endotoxins - 643 Total Organic Carbon 71 Sterility - 644 Conductivity planned for the future 791 ph General Information - 1230 Water for Health Applications - related to Water for Hemodialysis - 1231 Water for Pharmaceutical Purposes (contains microbial limits) - 1233 Instrumentation for Pharmaceutical Water planned for future? - 1644 Theory and Practice of Electrical Conductivity Measurements of Solutions - 1223 Validation of Alternative Microbiological Methods 2 nd Supplement USP 38
Agenda 14 1 Introduction 2 Pharmaceutical Water Monitoring and Analysis 3 Why On-line Microbial Monitoring 4 Real-Time Detection Technology 5 Pharmacopeia acceptance of Alternative Methods 6 Validation Guidelines
Drivers for Alternative Methods 15 OWBA - Online Water Bioburden Analysis Industry workgroup created to promote development and implementation of online bioburden analyzers Members from Merck, Novartis, Amgen, Fresenius, Baxter, P&G, Roche, Sanofi, Lilly, GSK and Pfizer Focused on reducing risk & cost by: Reduced Labor: Less sampling & lab-based testing Better product quality/process understanding: Continuous monitoring-faster response to excursion Fewer investigations Real-time release Better product safety Energy Savings: Reduced sanitization frequency
What is the Current Cost of Risk? 16 Knowing the microbiological quality prior to use and reacting quickly to out-of specification trends reduces financial loss and regulatory risk. OWBA False positives investigations Delayed product releases Release without completed microbial testing leading to risks of product recall Excessive inventory waiting for test results Product contaminations due to undetected real-positives - USP <1223>: "Studies on the recovery of microorganisms from potable and environmental waters have demonstrated that traditional plate-count methods 0.1% 1% of the actual microbial cells present in a sample"
Costs of Microbial Sampling from OWBA 17 Real-time microbial systems are faster and higher detection capability Microbial sampling and testing has been the roadblock for pharmaceutical manufacturing The costs for sampling and performing standard bacterial plate count tests is expensive and slow - approx. >$100/sample Typically, sampling frequency is done daily to a maximum of weekly at multiple locations through out the water system (avg. minimum of 20 points) The Pharma industry estimates that over 80% of positive results are false-positives caused by human error Results are not obtained for 5 to 7 days and some cultures take 21 days A Pharma facility has to decide the risk acceptance: do they use the water or wait for the test results Current method of microbial detection: Costly Time consuming Unreliable results from human errors Delay of product release
Drivers for Alternative Methods 18 FDA - Food and Drug Administration FDA encourages use of new technologies Food and Drug Administration - Rapid Microbial Methods (RMM): help meet Quality by Design (QbD) principles. - Regulatory mechanisms to implement RMMs are evolving. FDA Aseptic Processing Guidance (cgmps) FDA Process Analytical Technologies-PAT FDA Strategic Plan for Regulatory Science FDA Senior Microbiologist Supports RMMs FDA works in partnership with USP FDA liaisons work with USP Committee
Traditional Microbial Detection 19 A colony-forming unit (CFU) is a signal used to estimate the number of viable bacteria or fungal cells. Counting with CFU s requires culturing the microbes and counts only viable cells. Duration and temperature of incubation are also critical aspects of a microbiological test method. Classical methodologies using high-nutrient media are typically incubated at 30 35 for 120 168 hours. Because of the flora in certain water systems, incubation at lower temperatures (e.g., 20 25 ) for longer periods (e.g., 7 14 days) can recover higher microbial counts when compared to classical methods. Low-nutrient media are designed for these lower temperature and longer incubation conditions (sometimes as long as 14 and up to 21 days to maximize recovery of very slow-growing oligotrophs or sanitant-injured microorganisms), but even highnutrient media can sometimes increase their recovery with these longer and cooler incubation conditions.
Microbial Detection Methods 20 Manual work and visual inspection are potential sources of error The visual appearance of a colony in a cell culture requires significant growth. When counting colonies, it is uncertain if the colony arose from one cell or a group of cells. Plating and culturing bacteria can be done by a number of methods: - The Pour Plate method wherein the sample is suspended in a petri dish using molten agar cooled to approximately 40-45 C - The Spread Plate method wherein the sample (in a small volume) is spread across the surface of a nutrient agar plate and allowed to dry before incubation for counting. - Membrane Filtration wherein the sample is filtered through a membrane filter, then the filter placed on the surface of a nutrient agar All agar based methods request for an incubation period of a minimum of 5 to 7 days.
Traditional vs. Real-Time Microbial Detection 21 Current Plate Count Method can only culture viable cells in the growth phase Alternative Real-Time detection systems have the capability to detect all life phases Effective Detection Range Plate Count Real-Time Microbial Detection
Drivers for Alternative Methods 22 EMA European Medicine Agency Taking into account the speed which organisms can proliferate, the use of rapid Food and Drug Administration microbiological test methods and systems should be employed in order to improve or increase the probability of early detection and allow timely action to be taken. EMA/INS/GMP/489331/2016
Agenda 23 1 Introduction 2 Pharmaceutical Water Monitoring and Analysis 3 Why On-line Microbial Monitoring 4 Real-Time Detection Technology 5 Pharmacopeia acceptance of Alternative Methods 6 Validation Guidelines
Basic Principle of Operation 24 Combined Mie scattering and intrinsic fluorescence to detect bacteria Draw a sample through a flow cell into the interrogation zone UV laser light source is directed through the sample Scattered light is captured and collimated within a parabolic mirror - the scattering of light determines the size of the particle The intrinsic fluorescence is also captured - i.e. the amount of light emitted at a longer (than 405 nm) wavelength by a microorganism The two types of light (scattered/fluorescent) are separated and the data computed When a particle is detected at PD and PMT at the same time (within x μsec), then this is a BIOCOUNT!
Metabolites in Bacteria that will Fluoresce 25 (Hill et al, Field Ana. Chem. & Tech, 3(4-5), 221,1999)
Particle and AFU detected at same time point 26
Agenda 27 1 Introduction 2 Pharmaceutical Water Monitoring and Analysis 3 Why On-line Microbial Monitoring 4 Real-Time Detection Technology 5 Pharmacopeia acceptance of Alternative Methods 6 Validation Guidelines
USP Recommendation for Microbial Monitoring 28 Real-Time Systems enable continuous measurement according to USP recommendations USP < 1231 > Water for Pharmaceutical Purposes recommendation: - Pharmaceutical water systems should be monitored at a frequency that ensures the system is in control and continues to produce water of acceptable quality. - The general information chapter endorses operating monitoring instruments continuously in order that historical in-process data can be recorded for examination - The new USP <1223> promotes and encourages the validation and development of alternative microbial technologies
USP Guidelines for Microbial Monitoring 29 <1223> Validation of Alternative Methods: Acknowledges the limitations of the use of CFU as a standard signal for microbiological methods. " the cfu signal then is prone to underestimate the number of cells in a sample Provides guidance on selection, evaluation, and use of microbiological methods as alternatives to referee methods. Encourages the validation and development of alternative technologies "provided that proper technical and scientific attention is paid to the selection, qualification, and implementation of the method. USP <1223> states. Identify suitable alternative methodology Development of user specifications for the equipment selection Demonstration of the applicability of the method as a replacement for a standard referee method Qualification of the method in the laboratory Provides Options for demonstrating equivalence Alternative methods and/or procedures may be used if they provide advantages in terms of accuracy, sensitivity, precision, selectivity, or adaptability to automation or computerized data reduction, or in other special circumstances. General Notices & Requirements
<1223> - Chapter for Alternative Microbial Testing 30 <1223> Validation of Alternative Microbiological Methods (2nd supplement USP 38 "These chapter provides guidance on the selection, evaluation, and use of microbiological methods as alternatives to compendial methods. To properly implement alternative methods, one must consider a number of important issues before selecting the analytical technology and qualifying that method with the actual product. These issues include, but are not limited to, identification of suitable alternative methodology, development of user specifications for equipment selection, demonstration of the applicability of the method as a replacement for a standard compendial method, and qualification of the method in the laboratory. This chapter outlines: - User requirements - Instrument qualification - Validation of alternate technologies - Method suitability - The limitation of the use of CFU as a standard signal for microbiological methods - Four novel options for demonstrating equivalence "
European Pharmacopeia Alternative Methods 31 5.1.6 Alternative Methods for Control of Microbiological Quality The following chapter is published for information. 1. "General Introduction - The objective of this chapter is to facilitate the implementation and use of alternative microbiological methods where this can lead to cost-effective microbiological control and improved assurance for the quality of pharmaceutical products. These alternative methods may also find a place in environmental monitoring. - The microbiological methods described in the European Pharmacopoeia have been used for almost a century and these methods for enumerating and identifying microorganisms still serve microbiologists well. Over the years, these methods have been invaluable to help control and secure the production of microbiologically-safe pharmaceutical products. Nevertheless, conventional microbiological methods are slow and results are not available before an incubation period of typically up to 14 days. Thus the results from the conventional microbiological methods seldom enable proactive, corrective action to be taken. - Alternative methods for control of microbiological quality have been introduced in recent years, and some of these methods have shown potential for real-time or near-real-time results with the possibility of earlier corrective actions. These new methods can also offer significant improvements in the quality of testing."
The European Medicines Agency answers: 32 What testing should be employed during initial qualification and routine operation sampling? Testing should be conducted in line with Ph.Eur. Monograph 169 Water for Injections Use of rapid microbiological methods should be employed as a prerequisite to the control strategy to aid with rapid responses to deterioration of the system. Article 23 of Directive 2001/83/EC states:...the authorization holder must, in respect of the methods of manufacture and control...take account of scientific and technical progress... Quantitative microbiological test methods in line with Ph.Eur. 5.1.6 monograph Alternative Methods for control of Microbiological Quality. Due consideration should be given to employing alternate methods for the rapid quantitative determination of the contamination levels existing within the water system. The validation of such system should be in line with the above referenced monograph. Use of alternative/ rapid microbiological test methods should be employed as part of the overall control strategy for the system. Taking into account the speed at which organisms can proliferate, the use of rapid microbiological test methods and systems should be employed in order to improve or increase the probability of early detection and allow timely action to be taken.
Agenda 33 1 Introduction 2 Pharmaceutical Water Monitoring and Analysis 3 Why On-line Microbial Monitoring 4 Real-Time Detection Technology 5 Pharmacopeia acceptance of Alternative Methods 6 Validation Guidelines
Online vs Plate Counts Why the Difference? 34 USP <1223> acknowledges and explains the difference between CFU and AFU It is important to understand that the cfu has always been an estimation of microorganisms present, rather than an actual count. "The appearance of a visible colony requires significant growth of the initial cells planted at the time of counting the colonies it is not possible to determine if the colony arose from one cell or from 1'000 cells." Studies on the recovery of microorganisms have demonstrated that traditional plate-count methods reporting cell count estimates as colony-forming units (cfu) may recover 0.1% 1% of the actual microbial cells present in a sample. Most of the rapid microbiological methods are, to some extent, direct cell count methods. They, therefore, may provide a higher cell count estimate than the cfu method for a given sample. Observations of cell counts that differ from cfu results are not a concern if the different methods and their different signals of cell presence are equivalent to or are non-inferior to referee methods in terms of assessing the microbiological safety of an article. Higher cell counts must not be considered as necessarily indicative of greater risk given the inherent variability of standard growth methods.
Correlation Sample <USP 1223> 35 The CFU (Colony Formation Unit) has always been an estimation of microorganisms present, rather than an actual count colony may be from 1 cell or 1,000 cells (plate count). Rapid microbiological methods are direct cell count methods, therefore a higher count is expected. The difference between the CFU from plate count and AFU direct cell count is smaller when the number of bacteria cells in the water sample is low. However, the difference is proportionally much higher when the number of bacteria cells in the sample water is high. Comparison test per USP <1223> Bacteria cells/sample Plate count (CFU) Rapid Testing Biocount 1 0 1 10 0 10 50 25 50 100 50 100 100 80 60 40 20 0 1 2 3 4
How to set new limits 36 Alert, Action and Out-of-Specification Limits based on Statistical Analysis Establish new Alert Level at 2 Std. Dev. of the average AFUs over test period New Action Level at 3 Std. Dev. of the average AFUs over test period Breach or Out-of-Specification Level at 5 Std. Dev. of average AFUs over test period These are only recommendations customer can decide on their own Levels AFU 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 Reported AFU's Out-of-Specifications Level Action Level Alert Level 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101 106 111 116 121 126 131 136 141 146 151 156 161 166 Days
Why On-line Microbial Monitoring? 37 Conclusions "Current water sampling techniques are limited in their ability to identify changes in ongoing water system performance restricting real trend analysis as grab samples only provide a snap shot of the dynamic water system. - OWBA Any alternative microbial detection methods may be used provided it has been demonstrated they are equivalent or superior to the Compendia Pharmacopeia method. - FDA Senior Microbiologist Traditional plate count method has been performed for 126 years since1890 - recognized as only an estimate of the actual microbial cells present The current microbial limits were established as a compromise Traditional plate count methods have a relative standard deviation of 35% or more Current plate count method has a level of detection of 0.1% to 1% There is a better method Real-Time Microbial Detection
Mettler-Toledo Thornton 38 Thank You! Merci Grazie 謝謝ありがとう Danke 고맙습니다 Gracias Tak Thank You! Terima kasih Dziękuję Tack Cảm ơn bạn Cпасибо Obrigado Go raibh maith agat σας ευχαριστώ धन यव द ודה