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Real Time Microbial Detection
Melbourne
July 2017/R. Strebel
Global Pharmacopeia Acceptance of
Alternative Methods
Agenda
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
2
Our Solutions across the Value Chain
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
Laboratory
Solutions
Process
Analytics
Industrial
Weighing
Logistics
Solutions
Product
Inspection
Retail
Weighing
Solutions
3
Process Analytics Solutions Overview 4
Confidential
Water Analytics
TOC, Conductivity, Microbial,
Ozone, Sodium, Silica
Process Analytics
pH, Oxygen, CO2,
Turbidity
Gas Phase Analytics
O2, CO, CO2, H2O, H2S, HCl
Reliable in-line and
real-time measurement
of key analytical
parameters to control
process parameters
and monitor product
quality.
Company Background
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
▪ Extensive research
- Chemical and physical properties
- New technologies
- Product handling and asset management
▪ Active participation in ASTM, ISPE, PDA, Semi, EPRI, USP
5
Dr. Richard Thornton
Dr. Werner Ingold
Why is Water Important?
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
6
Agenda
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
7
Pharmacopeia Bacteria Regulations (USP 40)
The regulated parameters can now be measured in real time !
Attribute USP 40* EP 8.2 JP 17 ChP5 IPWater 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>) 0.54 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 IPPurified 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 offline6 Same as EP Same as USP
TOC (mg/L) - USP <643> 0.54 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
8
Bulk Water for Injection(s) - Today 9
Bulk Purified Water - Today 10
Pharmacopeia adoption of new technologies
▪ Chemistry/Organic tests
are:
- Qualitative
- Subject to bias
- off-line
- Carbon dioxide
- Calcium
- Ammonia
- Chloride
- Sulfate
- Oxidizable
substances
- Heavy metals
1840 - 1994 1994 - 1996 > 1996
▪ 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
2003 –
2006
▪ Adoption by
▪ EP
▪ JP
▪ ChP
▪ IP
11
Pharmacopeia adoption of new technologies
▪ 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, real-
time Instruments
▪ Accepted by the
other Global
Pharmacopeia’s
1890 - 2017 2017 to 20?? 20?? 20??
12
Relevant Sections of the USP
▪ 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 – 2nd Supplement USP 38
13
Agenda
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
14
Drivers for Alternative Methods
▪ 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
OWBA - Online Water Bioburden Analysis
15
What is the Current Cost of Risk?
“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"
16
Current method of microbial
detection:
▪ Costly
▪ Time consuming
▪ Unreliable results from
human errors
▪ Delay of product release
Costs of Microbial Sampling – from OWBA
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
17
Drivers for Alternative Methods
FDA - Food and Drug Administration
Food and Drug Administration▪ FDA encourages use of new technologies
- 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
18
Traditional Microbial Detection
▪ 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 high-
nutrient media can sometimes increase their recovery with
these longer and cooler incubation conditions.
19
Microbial Detection Methods
▪ 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.
Manual work and visual inspection are potential sources of error
20
Traditional vs. Real-Time Microbial Detection
▪ 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
Plate Count
Real-Time Microbial Detection
Effective
Detection Range
21
Drivers for Alternative Methods
EMA – European Medicine Agency
Food and Drug Administration▪ “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.”
EMA/INS/GMP/489331/2016
22
Agenda
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
23
Basic Principle of Operation
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!
24
Metabolites in Bacteria that will Fluoresce
(Hill et al, Field Ana. Chem. & Tech, 3(4-5), 221,1999)
25
Particle and AFU detected at same time point 26
Agenda
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
27
USP Recommendation for Microbial Monitoring
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
28
USP Guidelines for Microbial Monitoring
<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
29
<1223> - Chapter for Alternative Microbial Testing
<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………"
30
European Pharmacopeia Alternative Methods
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 micro-
organisms – 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 …."
31
The European Medicines Agency answers:
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.
32
Agenda
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
33
Online vs Plate Counts – Why the Difference?
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.”
34
Correlation Sample <USP 1223>
▪ 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.
35
0
20
40
60
80
100
1 2 3 4
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
How to set new limits
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
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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
AFU
Days
Reported AFU's
▪ Alert Level
▪ Action Level
▪ Out-of-Specifications Level
36
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
Why On-line Microbial Monitoring? 37
Mettler-Toledo Thornton
Thank You!
Thank You!
Merci
Grazie
謝謝
ありがとう
Danke
고맙습니다
Gracias
Tak
Terima kasih
Dziękuję
Tack
Cảm ơn bạn
Cпасибо
Obrigado
Go raibh maith agat
σας ευχαριστώ
धन्यवाद
ודה
38
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