Counting and Contamination Analysis in Fluid Power
Systems
In 1988 WearCheck introduced
particle counting to its battery of tests.
Introduction
to methods of contamination analysis
Typically, most oil
analysis companies have relied on spectrometric and
debris analysis for the detection of wear particles and
contaminants in oil lubricated components. The ICP
(inductively coupled plasma) spectrometer used by
WearCheck is limited to a maximum particle size of eight
microns that it can detect, so other techniques must be
employed to detect larger wear particles and
contaminants. The ideal situation would be to filter all
oil samples and examine any debris under a microscope;
this is highly labour intensive in terms of sample
preparation and visual analysis of the debris and only
provides a qualitative description of the debris. WearCheck uses particle
quantification as a screening test to detect the presence
of wear particles greater than eight microns. IN this
test a bulk magnetic measurement of the oil is made and a
particle quantification index is determined; depending on
the level of this index and the type of component the oil
has come from, a visual debris analysis will be made. Particle quantification,
however, also has its drawbacks. Because it is a magnetic
measurement, it only detects the presence of ferrous
particles in the oil and takes no account of other types
of contaminants in the oil, eg. Coal dust, coarse dirt,
fibrous material, etc. In 1988 WearCheck introduced
particle counting to its battery of tests. In this test,
the total number of particles, irrespective of origin,
are counted in a number of sizes, ranging from 5 to 400
microns. The results are expressed as the total number of
particles per ml of oil in the various specified size
ranges.
A brief
history of fluid power
This test is of
particular importance to clean oil systems, eg,
hydraulics, transmissions, turbines, compressors and
other fluid power systems. It has been shown that 70-85%
of hydraulic component failures are due to particulate
contamination with up to 90% of these failures due to
abrasive wear. The concept of fluid power
systems dates back to the times of Archimedes and the
invention of the screw pump. In the 15th
century, Leonardo da Vinci advanced many ideas including
that of the hydraulic press. In the 16th and
17th centuries both Galileo and Pascal were
involved in the development of hydraulic power theory.
Many consider Pascal to be the true father of hydraulic
power systems. The industrial revolution saw the
development of the hydraulic press by Joseph Bramah and
the use of hydraulic power was demonstrated to the Duke
of York in 1813 by uprooting a tree in Hyde Park. The
hydraulic power industry was finally recognized in 1925
and since that time there has been concern over
contamination and cleanliness of hydraulic fluid power
systems. Actual particle counting techniques were
developed in the late 1950’s and early 1960’s.
WearCheck uses a Hiac/Royco model
8000A automatic particle counter.
Particle
counting techniques
In this test the oil
is drawn through a membrane of known pore size and the
number of particles in a variety of size ranges is
counted by viewing the membrane under a microscope.
Although this technique is still used today, it is
tedious, time-consuming and unreproducible when compared
to other techniques. Other contaminant analysis
techniques exist, such as Patch Tests, Gravimetric
Analysis and determination of silting indices. All these
tests, while providing total contamination levels,
provide no information on the distribution of particle
size. Image analysers using
video and computer systems give accurate particle count
information. However, this method is time-consuming and
very expenxive. In the mid 1960’s automatic liquid
particle counters were developed and this is now the
preferred technique for particle counting in the
1990’s as many advancements and refinements have
been made with instrumentation in the last 30 years. Automatic liquid particle
counters operate on three general principles: electrical
resistance, fluid flow decay and light blockage. As
electrical resistance (coulter conters) devices depend on
the medium under test to conduct electricity, these
systems are rarely used in oil analysis. With fluid flow
decay devices, such as the Diagnetics Instrument, the oil
is passed through a screen of known mesh (usually ten
microns) and the time taken to plug the screen is
determined, the instrument then calculates the
distribution in other size ranges by extrapolation. The
disadvantage of using this technique is that it assumes a
predetermined size distribution without actually
measuring the number of particles in each size range. The
most common types of automatic particle counters operate
on a light blockage principle when oils are being
analysed. With this type of
instrument, a known volume of oil (usually 5 ml) is
injected through a very small sampling cell. On one side
of the cell is a beam of laser light and on the other
side, a detector. As particles pass through the cell,
they block the beam of light and thus cast a shadow on
the detector. The drop in light intensity received by the
detector is proportional to the size of the particle
blocking the light beam. In this way, both the number and
size of the particles can be measured.
Particle
counting by light blockage
The instrument that
WearCheck uses is a Hiac/Royco model 8000A automatic
particle counter and it operates on this light blockage
principle. The instrument is set up
to measure particles in five different size ranges. Those
size ranges, in microns, are as follows: 5-15, 15-25,
25-50 50-100 and greater than 100. The results are
expressed as the total number of counts (particles) per
ml of oil. With the advent of automatic particle counters
it was realised that some form of categorisation of
particle counts was needed in order to determine if an
oil was "clean" or "dirty".
WearCheck calibrates its instrument
with the ACFTD method.
During
the 1960’s a number of systems for the
classification of oil cleanliness was developed, among
them were the SAE 749D, NAS1638 and MIL1246A.While these enjoyed some popularity
in the 1960’s they were all eventually discarded,
the main problem being that all these early
classification systems assumed a fixed particle/size
distribution. Finally, in July 1972 a
system of cleanliness classification was proposed and
eventually ratified by the International Standards
Organization in September 1974. The system is known as
the ISO 4406 and is still in use today. This system
reflects the philosophy of contamination control experts
throughout the world and can be used to describe a
theoretically infinite range of contamination levels in
oil. The ISO 4406 cleanliness
rating is expressed as a two number code X/Y, where X
represents the total number of particles per ml greater
than five microns and Y represents the total number of
particles per ml greater than 15 microns. These two sizes were
selected because it was felt that the smaller size would
give and accurate assessment of the "silting"
condition of the fluid, while the population of the
particles greater than 15 microns would reflect the
prevalence of "wear" catalysts. The ISO Standard, outlined
later in this article, gives an explanation of the
relationship between the X/Y code and the actual number
of particles per ml in the chosen size ranges.
Calibration
of particle counters
For any laboratory
instrument to give meaningful and accurate results it
must first be calibrated against a precisely known
standard. Unfortunately, there are two methods for
accurately calibrating the instrument and these two
methods give different results. The first method involves
using a very clean oil and dispersing an accurately
measured mass of mono-sized latex spheres in the oil
(sometimes the spheres are made of glass). This oil is then tested in
the instrument and because the size of the particles is
very accurately known, the instrument can be calibrated
against known standards. This method is currently gaining
a lot of popularity in western Europe and North America.
The other method is to use Air Cleaner Fine Test Dust
(ACFTD) dispersed in very clean oil. The ACFTD is a
naturally occurring dust and the particle size
distribution of the dust is known very accurately. From
this size distribution an accurate calibration of the
instrument can be made. The main advantage of
using ACFTD is that the particles are typical of
contaminants and wear metals in hydraulic systems with
regard to size and shape. This is the only method of
calibration according to the International Standards
Organization (ISO 4402). The disadvantage of using this
method of calibration is that the particles are not
uniform (as is the case with a sphere) and the counter
will measure size on the basis of the largest dimension.
Treatment in the laboratory must be
standardized.
Because
of the Hydroscopic nature of the test dust it is very
difficult to prepare the calibrating fluid and it has a
limited shelf life. Most importantly, production of ACFTD
has been halted.Due
to the halt in production of ACFTD it seems likely that
the International Standards Organization will eventually
adopt the latex sphere method for ISO 4402. It has been
shown that there is a linear relationship between the two
methods so that either calibration can be adopted.
WearCheck is currently keeping abreast of any changes in
calibration techniques for automatic particle counters.
Sampling
techniques
Finally, some
thought must be given to sampling techniques both in the
field and in the laboratory. Obviously the sample
container must be scrupulously clean and any external
contamination must be avoided, these procedures are
actually laid out in the International Standards
Organization method ISO 3722. Treatment in the laboratory
must also be standardized and watched very carefully. For
example, during transport to the laboratory, most of the
contaminants will settle out so the sample must be
agitated to get them evenly dispersed in the oil. At one time it was thought
that using an ultrasonic bath to agitate the sample would
be an ideal method until it was discovered that the
ultrasound actually breaks up some of the larger
particles into smaller particles. Although automatic
particle counters are widely used and provide accurate,
repeatable and reproducible results, not all oils are
amenable to this test. Oils that are badly oxidized and
discoloured may not transmit enough light to give a
reliable result or oils that contain water give
erroneously high results because the counter
"see" the water droplets as particles. Some oils actually contain
wax particles suspended in them which will also provide a
bad result.
What of the
future?
As particle counting
becomes more accepted as an analytical technique and more
OEM’s and endusers become aware of the critical
importance of contamination control in the hydraulic
fluid power industry, the greater the emphasis will be on
keeping hydraulic fluids clean. For warranty purposes,
certain manufacturers have already laid down maximum ISO
4406 ratings for the hydraulic equipment and a number of
oil companies are concerned that their hydraulic fluid be
as clean as is practically possible when dispatched to
the customer. In certain circumstances
it has been found that some new oils do not meet the
cleanliness requirements of the OEM. This does not mean
the oil is no fit for use but the piece of equipment,
fitted with a good filtration system, is quite capable of
cleaning the oil down to very low ISO 4406 levels as the
oil is continuously circulated through the filtration
system.
References
Day, M.J. Calibration
of Automatic Particle Counters.
Fitch, E.C.; Hong,
I.T. Contamination Control in the Fluid Power
Industry.
Failure to properly measure and control relative humidity in the
cleanroom can result in lower yields, increased scrap and waste,
contaminated product inadvertently reaching consumers, customer lines
down, increased liabilities, and decreased revenues—among other
situations best avoided. Carefully monitoring and controlling the
relative humidity in a cleanroom is an absolute requirement—with no
options.
Particulate count. Temperature. Airflow. Humidity. These five words are
among the environmental factors that must be measured and controlled in
the cleanroom environment. Sometimes the ‘stickiest’ of these is
humidity. Measuring and controlling it within prescribed parameters can
be a challenge. Too little or too much RH can impact much more than the
personal comfort of cleanroom employees. Too little humidity can be
quite electrifying—creating issues of static build-up and discharge. Too
much humidity brings its own woes: encouraging the growth of bacteria
and microbes, corroding sensitive metals whether in products or
equipment, and manifesting itself in moisture condensation and water
absorption. Then there’s photolithographic degradation. Photoresist
processes are among the most sensitive to humidity, and can be among
the most costly to control for, due to their tightly required
parameters. The bottom line: any of these conditions can result in cost
overruns, scrapped products, and shortened equipment life. In short, the
diminution of cleanroom performance, which is costly in itself.
Simply put, because humidity is relative to temperature, controlling RH
within very tight tolerances or at extremely low levels can end up
costing you more money in both construction and operating budgets. It’s
important to understand that target humidity and temperature control
decisions impact costs. A cleanroom target temperature of 65 degrees
will have a lower relative humidity than a target temperature of 60
degrees. The lower your controlled temperature goes, more is required to
“dry out” the air to reach a set RH level. Driving lower moisture
content drives cost.
To
confirm that the system is working properly, it is necessary to check
the airflow at the supply vents and also to check the distribution of
airflow throughout the room. Image: Kanomax Maintaining
appropriate air velocity in the cleanroom helps ensure a clean
environment; correct system performance plays an important role. To make
certain the system functions as expected, periodic checks using the
proper instruments are recommended to measure velocity and uniformity in
the clean space.
Room performance can be affected by room size,
AHU capacity, length of duct run, as well as other factors. Methods
used to check airflow within a cleanroom vary depending on the
ventilation set-up—the two most common being laminar flow and turbulent
airflow.
Ventilation set-up
In a
laminar flow system, air flows through the cleanroom in one direction,
either horizontal flow or top to bottom. Koji Miyasaka, with Kanomax,
Andover, N.J., notes that, “To confirm that the system is working
properly, it is necessary to check the airflow at the supply vents and
also to check the distribution of airflow throughout the room. At the
supply vents or fan filters the volumetric flow should be checked by
using the following formula: Q = V x A. (V is the average or center air
velocity and A is the area of the vent or fan filter.) To determine the
total volumetric flow for the room, the procedure should be repeated at
each vent or fan filter and then summed. This number should then be
compared to the specifications for the cleanroom to find if it is in
tolerance. Many modern anemometers come with this calculation function
built in.”
In a turbulent airflow system, the room is designed
to dilute and remove contaminates based on a certain number of air
exchange rates per hour. To check this type of system, measure the
airflow at both the supply and the returns and then calculate the number
of air exchanges that occur per hour.”
Taking measure
“In
unidirectional cleanrooms the airflow velocities are typically measured
using either a thermal anemometer (mass flow devices) or an electronic
micromanometer in conjunction with a multi-point sensor array
(volumetric flow device),” says Cary Binder, ENV Services Inc.,
Hatfield, Pa. Binder points to guidance from IEST-RP-CC 006.3 Section
6.1.1b.1 which states, “Divide the plane into a grid of equal area.
Individual areas should not exceed approximately 0.4m2 (4 ft2)” while
the probe is typically placed 6-in. from the filter face or diffuser.
Binder
offers the following steps for measurement. “Calculate the effective
media area of each filter and multiply the average velocity for each
filter to determine the airflow volume (cfm). Add the calculated volumes
for all the filters and the result is the total airflow volume for the
room. Divide the total airflow volume for the room by the room volume
and multiply by 60 to obtain the ACPH for the room. When a balometer is
used, simply add the measured volumes for all the filters and the
result is the total airflow volume for the room.”
For more information
The contributors to this article have provided more detailed explanation and examples in articles that are hosted on the Controlled Environments
website, www.cemag.us. On the site, search “air velocity” to find
expanded material on the topic of airflow measurement for cleanrooms and
compounding pharmacies.
ts.
he highest possible quality of an end product, in compliance with
requirements and regulations, can be attained only if quality assurance
is not merely limited to final product testing. Rather, the entire
manufacturing process, besides incoming quality control of the raw
materials used, needs to be continuously monitored.
In the
pharmaceutical industry, risk analysis of individual manufacturing steps
is performed and the results of this analysis are used to define
in-process quality control tests. Such QC tests permit timely detection
of inconsistencies or non-conforming items and, in particular, increases
in the bioburden as they occur in manufacture so that corrective action
can be promptly initiated. Even though the risk of contamination has
been considerably reduced by GMP-compliant production, decontamination,
and sterilization of the end products, as well as by strict hygiene
standards, quality control of the final product continues to be of prime
importance.
Microbial enumeration
Quantitative
analysis of microorganisms involves counting the colony-forming units
(CFU), hence the term “microbial enumeration.” This number can be
expressed either as the total viable number of CFUs in general or of
certain product-relevant species of microorganisms. This is why
microbial limit tests are performed on various products from different
sectors, including the pharmaceutical, beverage, and waste water
industries, to ensure that defined limits are not exceeded. The accuracy
and reliability of microbial limit test results are essential as they
serve as the basis for the release of products, whether potable water or
pharmaceuticals, and the impact of undetected pathogens can be
potentially devastating on the health of consumers.
Membrane filtration
For
microbial enumeration, membrane filtration continues to be the method
of choice for reliable quantification of microorganisms in liquid
samples. The principle of this method is based on the concentration of
organisms—which are filtered out from relatively large sample volumes—on
the surface of a membrane filter and their subsequent cultivation by
incubating the filter with the retained microbes on a culture medium.
Unlike
direct incubation of a sample, membrane filtration offers the advantage
that large sample volumes can be tested without individual
microorganisms going undetected. In addition, inhibitors, such as
antibiotics or preservatives, can be removed by rinsing the membrane
with buffer so that microbial growth is not suppressed.
Microbiological tests in the pharmaceutical industry
From
a microbiological viewpoint, pharmaceuticals can be subdivided into two
categories: non-sterile and sterile products. For both categories, the
potential risk resulting from microorganisms and their toxins on
patients’ health must be eliminated or at least mitigated. At the same
time, the quality and effectiveness of such pharmaceuticals must be
retained.
Products defined as sterile, such as eye drops,
physiological saline, antibiotics, etc., need to be tested for sterility
(USP Chapter 71 and EP, Chapter 2.6.1) in order to be verified as such.
Unlike sterile products, non-sterile end products are tested for their
number of viable microbes according to the microbial limit test (USP
Chapter 61 and EP Chapter 2.6.12). Furthermore, in the pharmaceutical
industry, in-process microbiological quality control tests are carried
out on raw materials, mostly water, as well as bioburden analysis during
manufacture.
Critical steps in microbial enumeration
The
classic equipment setup for performing membrane filtration consists of a
vacuum pump, a multi-branch vacuum manifold, membrane filters, reusable
funnel-type filter holders or single-use filtration units, culture
media, and tweezers.
In this method, the filter support of a
reusable filter holder is sterilized by flaming, and a membrane filter
is subsequently placed on this support. Then the funnel is attached to
the support and a sample is poured into this funnel. Filtration begins
when the tap on the vacuum source is opened. At the end of filtration,
tweezers are used to remove the membrane filter and transfer it to an
agar culture medium.
The culture medium is incubated for a
defined time at a predetermined temperature inside an incubator. At the
end of incubation, evaluation is done by enumerating the individual CFUs
and comparing their count with the permissible microbial limits for
each particular sample.
Flaming or disinfecting the filter
support poses an added risk of contamination due to the inherent
inaccuracy in performing these sterilization procedures. In particular,
maintaining the required time of contact with the flame or disinfectant,
the choice of disinfectant (not just a bactericide, but a sporicide)
and regular changing of the disinfectant are all critical factors in
determining whether sterilization is 100% effective. Besides
representing a health hazard for lab personnel, flaming also poses the
risk that not all areas contaminated by microbes are exposed to the
hottest point of the flame long enough in order to kill off these
organisms.
Minimization of secondary contamination
A
single-use filter unit does not require any decontamination, provided
that a single-use filter base is used. As a result, the only especially
critical step that remains is transferring the membrane filter to an
agar medium, which increases the risk of secondary contamination and can
lead to false-positive results. The reason lies in the use of tweezers
to transfer the membrane. Although these tweezers are also flamed, i.e.,
sterilized, they can potentially carry over exogenous microbes when
used to grasp the membrane.
Single-use filter units increase the
safety and efficiency of microbiological quality control by eliminating
the need for disinfection or flaming of the filter support, as well as
for using tweezers to transfer a membrane to a culture medium. A system
comprised of single-use filter units and agar media dishes can increase
efficiency and reliable results.
The filter unit in this type
of system is a sterile, ready-to-use combination of a funnel, a filter
base, and a gridded membrane filter. This filter unit is connected to a
stainless steel multi-branch manifold in order to directly filter a
sample. Afterwards, the filter unit is easy to remove from the manifold
and eliminates the critical step of decontaminating the stainless steel
base of a reusable filter holder.
Agar media dishes are used for
microbial limit testing. They are pre-filled with different types of
agar medium, sterile-packaged and, when together with a single-use
filter, are ready to use immediately. In combination with a single-use
filter unit, these media dishes feature an active lid that permits
touch-free transfer of a membrane onto agar, without using any tweezers.
This active lid lifts the membrane filter from the base of the filter
unit so the filter can be safely transferred onto the pre-filled agar
dish. Once the medium dish is closed, the membrane is ready to incubate.
Solution for safe membrane transfer
The
combination of agar media dishes and filter units represents a new
membrane transfer and agar concept. As just a few steps are all it takes
to proceed from sampling to incubation, a single-use system of agar
media dishes and filter units accelerates workflows, making them
cost-efficient. At the same time, touch-free membrane transfer enables
even more reliable results to be obtained in analysis, while reducing
secondary contamination to an absolute minimum.
Companies
producing medicines and biotech products are concerned with airborne
microbial contamination. They need to ensure that products and people
are kept safe. The traditional, accepted method to test for
microorganisms at critical locations in a process is the use of active
air samplers or settling plates. Typically, 1-meter-cubed samples are
taken onto agar plates and sent to a lab for culturing. The colony
forming units (CFUs) results come back from the lab after four to ten
days. Only after this waiting period will end users know whether the
manufacturing environment was in control. Recently, the
commercialization of a technology based on laser induced fluorescence
(LIF) has made it possible to look at airborne viable microbial counts
in real time. The potential to instantly respond to an airborne
microbiological event when it happens is exciting—and beneficial.
Root cause analysis
Results
from active air samplers are important—they can inform us that there
has been a problem, possibly an excursion of some kind, and also enable
identification of the microorganism to support a root cause
investigation. However, they do not tell us when the contamination
happened, or the source of the contamination. New LIF-based
bio-detection products can provide better insight into these unknowns by
measuring airborne viable particle counts and displaying this data in
real-time. The data can be viewed via a local display or integrated
directly into a facility monitoring system (FMS).
One quality
assurance professional recently stated that their company spends
thousands of dollars each year looking for root cause of microbial
contaminations, with limited success. Positioning air samplers or settle
plates to narrow down the source of airborne microbiological
contamination is difficult and time consuming. Even when using good
scientific and risk-based approaches, there is at least a four-day wait
to know the result. And, taking periodic samples simply does not provide
enough information to find root cause. But, with real-time viable
particle counters, time-resolved data can provide valuable insights into
root cause. An immediate notification to presence of airborne viable
particles means finding the source could potentially take minutes
instead of days or weeks. Furthermore, the instrument sample probe can
be attached to sample tubing and is identical to ones used by standard
optical particle counters. The probe can be configured to beep every
time an airborne viable particle is detected, just like a Geiger
counter, enabling end users to sniff out the exact location of the
contamination source.
Another example is contamination that
comes from workers, particularly when they start a shift in a cleanroom.
By having data to support the impact of gowning practices on
cleanliness, companies can provide enhanced training programs. Once root
causes are identified, actions can be taken to rectify the issue, be it
training, ventilation, filtration, machine maintenance, or facility
adjustments.
Process improvement
Regulatory
authorities are very interested in root cause investigations, and what
preventative and corrective actions were taken to ensure the problem
will not occur again. When real-time viable particle counters are
integrated into a FMS software package, the data can become the basis of
informational reports that offer insight into processes, providing
alarms, warnings, and trending. Quality control is typically concerned
with product, alarms, excursions, and corrective actions. Quality
assurance is more concerned with process, trending, and preventative
actions. With real-time airborne microbial data, reports can be viewed
with an eye toward preventative maintenance and identifying adverse
trends before a microbial excursion occurs. (Figure 1)
Figure
1: Quality control utilizes alert and action limits for reactive and
corrective activities. In Quality assurance, attention is placed on
trending data, process improvement, and preventative actions.One
challenge in critical ISO 5 or Grade A pharmaceutical processing
environments is that they are very clean. Much cleaner than when the
cleanliness limits detailed in the cGMP Aseptic Processing guidance was
conceived. Today, Grade A isolators are continuously monitored for
airborne contamination using active air samplers, settle plates, and
traditional optical particle counting technology. When correctly
designed, these environments easily meet and exceed the airborne
cleanliness requirements as defined in the GMPs. In some cases, many
weeks, months, and even years will pass without any airborne microbial
contamination being detected using current methods. Similarly, low
numbers are seen in surrounding Grade B environments.
It is
surprising, then, that these very clean critical processes have to be
interrupted, growth media introduced or manipulated, in order to meet
the regulatory requirement for AAS environmental monitoring. The good
news is these potentially hazardous and disruptive process steps are
unnecessary with real-time airborne viable particle counting technology.
Real-time viable particle counters not only offer the potential to
monitor these very clean and well-controlled environments, but can also
provide continuous data when integrated into an FMS system.
Saving time and money
In
today’s competitive environments, facilities are always looking for
ways to save time and money. Real-time viable particle counters provide
opportunities for real savings. Similar to the example above, let’s look
at an isolator. One pharmaceutical company has calculated that they
could increase line capacity of an isolator by over 20% by reducing the
downtime required to change agar plates with active air samplers. Agar
plates have to be changed every three to four hours as they will dry out
and not support growth. By using a real-time viable particle counter,
the need for changing agar plates could potentially be eliminated, or at
least minimized, saving valuable equipment downtime and optimizing
labor. This also saves on the production time required to re-establish a
clean environment in the isolator before production can begin again.
This approach could potentially save thousands of dollars per year for
each isolator.
Room certification after construction, renovation,
and room changeover can be a lengthy and expensive process for
facilities, taking upwards of three to seven days while waiting for
incubation results to release a zone. However, with real-time viable
data in an FMS system, rooms could be released in an hour or less,
providing facilities with the opportunity to increase utilization rates
of expensive rooms and equipment.
Another operational concern is
energy. Energy is expensive. And cleanrooms, with a high number of air
changes and HEPA filtration, generally use a lot of energy. If the air
change rate could be reduced, while maintaining cleanliness levels,
facilities could potentially see significant savings. Cleanroom studies
can be performed with a real-time viable particle counter to see if air
change rates can be reduced. Of course, at no time should any
energy-saving measures take precedence over product safety.
Summary
Modern
technology continues to move forward, providing better measurements and
data. In the case of microbial detection, new LIF-based products
provide real-time viable particle counts. This data, when integrated
into a facility monitoring system, allows users to see information in
the form of reports and test results. Then, the information can be used
to develop knowledge of facilities and systems. Knowledge is a powerful
tool when looking for root causes of excursions, for process
improvements, and for opportunities to save time and money.
But
do regulators embrace this new technology and information? The answer
appears to be yes. Regulatory bodies certainly want to ensure that
medicines and biotech products are safe for consumers. To that end, they
want to be sure that root causes are identified, with corrective and
preventative actions put in place. And they want process improvements to
provide an even higher level of safety in the future. Vendors of
real-time viable particle counters have submitted a Type V Drug Master
File (DMF) with the U.S. FDA. This provides the FDA and customers with a
file that demonstrates the science behind the technology, as well as
the test results to support the measurements.
Troy Tillman is a Senior Global Marketing Manager for
Contamination Control at TSI Inc. He has spent over 20 years defining
and developing products for markets such as pharmaceutical cleanrooms,
laboratories, hospitals, and vivariums. He has been an active member in
IEST, ASHRAE, and CETA, speaking at numerous conferences. www.tsi.com;
pr@tsi.com.
This article appeared in the September 2014 issue of Controlled Environments.
With
water contributing the largest component of pharmaceutical products,
especially injected products, control of the quality of water in both
systems and finished product is paramount. The particulate burden of
finished product is a Pharmacopoeia regulation in the major standards
documents, USP<788>1 and USP<789>2, EP<2 .9.19="">3, and
JP<6 .07="">4.
Figure 1. Light obscuration particle counter schematic.Figure 2. Light scattering particle counter schematic.It
should be noted that particle counting is not a method for determining
the distribution of particles in a suspension, as here the particle
burden would saturate the optics of most commonly used particle counters
employed in contamination controls; there is an expectation that the
liquids being tested are “essentially free” from contamination and the
particle counter is looking for particles possibly caused by events in
the handling and management of the water supply that has caused an out
of control condition.
Technology
There are two primary methods for measuring the particle contamination of liquids: light obscuration and light scattering.
Light
obscuration particle counting is where a beam of light (laser) is
directed through a narrow capillary tube with a flowing stream of
liquid; any particle passing through the laser beam blocks a certain
amount of light and casts a shadow across the photo-detector. The amount
of light blocked is equivalent to the size of the particle in the
liquid; accurate sizing of the detected particles can be determined by
calibrating the particle counter with particles of known sizes suspended
in clean water.
This method of light obscuration is ideal for
measuring particles that are relatively large, 1.5 microns (1.5 µm) up
to over 150 microns (150 µm); particles greater than 150 µm are
typically within the range of those particles that are potentially
visible.
For smaller particles than the 1.5 µm lower limits of light obscuration, light scattering is used.
Light
scattering is where the laser beam is directed through a narrow
capillary tube with a flowing stream of liquid. When a particle within
that stream passes through the laser beam light is scattered off the
particle by several different interactions with the particle’s surface
(reflection, diffraction, and refraction), this scattered light is then
collected using a series of mirrors and focused onto a photodetector for
analysis. The amount of light scattered off a particle is equivalent to
its size; i.e. the bigger the particle the more light is scattered, and
accurate sizing of particles within a liquid can be determined by the
calibration of a particle counter against known size standards.
This
method of particle counting using scattering is used for volumetric
instruments down to 0.2 µm and is effective up to above 20 µm. Below the
0.2 µm threshold volumetric sensors tend to give way to non-volumetric
instruments where only a small fraction of the total liquid flow is
monitored.
Applications
There are two
primary applications for particle counters in production environments:
the primary one being the testing of finished products to those
standards identified above and a second one of monitoring the quality of
the water for injection (WFI).
Testing of finished products to
the Pharmacopoeia standards is performed against a strict test
requirement. Test samples comprise of either a pooled sample of small
injectable products, sufficient to perform testing. A minimum of 10
pooled containers should be used, or where large production volumes are
manufactured a portion of several individual containers can be used.
These differences are based upon the finished products’ normal supplied
volume being less than or greater than 100ml. The sample is drawn using a
syringe sampler through a light obscuration particle counter and the
number of particles measured are reported as either particles per
container volume, or particles per ml. The current limits for maximum
allowable concentrations are given in the table below.
Instruments
used for performing these tests must be validated to meet the
requirements of “suitably calibrated” and the regional requirements for
count standard accuracy.
The second application for particle
counters is for demonstrating control over the particle burden of the
WFI system. There are no current regulations that require monitoring be
performed; however, several facilities that have employed particle
counters on the WFI loop have been able to notice when filters are
beginning to degrade as there is a shift in the distribution of
particles remaining in the water. When filters begin to blind and the
smaller pores block, two thing occur: a rise on pressure across the
filter, and a shift in the distribution of particles where the smaller
sized particles increase relative to the overall population. It is
common to change filters based upon either a maintenance schedule of
time, or an increase in the pressure drop across the filter, where
particle counters have been employed; however, filter life can be
extended as the distribution shift is monitoring, or shortened as
increases in overall levels of particles is witnessed in the clean
supply.
Table 1. Current Pharmacopoeia Limits for Finished Product.It
also allows for the identification of when a recirculation problem may
exist within a filling tank; if the recirculation pumps on the system
begin to fade, the filters become less efficient (overall volume
filtered) and so a recovery of the system can be identified.
References
1. USP <788>. Particulate Matter in Injections, United States Pharmacopoeia 37-NF 32, May 1, 2014 2. USP <789>. Particulate Matter in Ophthalmic Solutions, United States Pharmacopoeia 37-NF 32, May 1, 2014 3. EP <2 .9.19="">. Particulate Contamination: Sub-Visible Particles, European Pharmacopoeia 5.0, January 2005. 4. JP <6 .07="">. Insoluble Particulate Matter Test for Injections, The Japanese Pharmacopoeia 16, March 2011
Mark Hallworth, Market Manager for the Life Sciences Division of
Particle Measuring Systems in Boulder, Colo., has spent over 17 years
with the company. He has over 25 years’ experience in particles,
including their transportation and measurement in many industrial and
scientific applications. He has designed several instruments for the
measurement of particles, including extreme environmental conditions and
fully integrated controls systems, which has led to software products
that meet the demands of a regulated industry.
mhallworth@pmeasuring.com; www.pmeasuring.com
This article appeared in the October 2014 issue of Controlled Environments.
Water
is utilized abundantly to process, formulate, and manufacture
pharmaceutical products. Traditional culture-based methods used to
ensure water quality, however, are ill-suited in providing a robust
assessment of risk and control. These methods are plagued by limitations
in sensitivity, episodic sampling, and retrospective results. New
technologies based on laser-induced fluorescence (LIF) detect intrinsic
fluorescence instead of growth, can operate continuously, and deliver
real-time results. As applied to pharmaceutical water quality,
LIF-based, instantaneous microbial detection technologies enable
real-time bioburden monitoring, risk reduction, and process control.
Figure
1: Emission spectra of two microorganisms and eight materials with
405nm excitation. An approximate Raman band for water and two example
detection ranges for an instantaneous microbial detection system with
two PMTs are labeled.The pharmaceutical industry
continues to recognize a need to leverage modern technologies to advance
the course of risk reduction and process control. This forward thinking
has been captured in industry relevant guidance such as the FDA’s 2004
“Guidance for Industry” document on Process Analytical Technology (PAT),
ICH Guidelines Q8, Q9, and Q10, and the FDA’s “Pharmaceutical cGMPs for
the 21st Century,” which encourage the adoption of quality by design
(QbD) principles and new technologies. More recently, working groups
composed of representatives from key pharmaceutical companies have also
joined forces to help articulate their needs in water quality assessment
and encourage the development and use of new technologies best suited
to today’s tasks.
Need for an online pharmaceutical water assessment tool
The
currently accepted and primarily practiced method for assessing water
quality throughout a pharmaceutical water loop is through samples
obtained at points-of-use (POU), utilizing traditional culture-based
methods. The goal of such testing is to ensure the quality of an entire
water system; however, POU testing can occur as infrequently as once
every two weeks at each sample point. This limited sampling frequency,
combined with the retrospective nature of culture-based methods, make a
robust and timely assessment of risk and control difficult. Furthermore,
there is the potential for sample contamination during collection (a
false positive), and for a false-negative result due to limitations in
sensitivity of culture-based methods. While growth-based methods offer
the opportunity for identification, a number of organisms go undetected,
such as viable but non-culturable organisms, due to the chosen medium
and incubation parameters. A complementary technology capable of
real-time and continuous monitoring of water system bioburden, based on a
different method of detection, could alleviate such limitations and aid
in risk reduction and process control.
Table 1: Business benefits summarized in the OWBA Business Benefit Estimates document.Online pharmaceutical water bioburden analyzer
With
an aim to improve the tools being applied to pharmaceutical waters, an
Online Water Bioburden Analyzer (OWBA) Workgroup recently outlined user
requirements, a testing protocol, and business benefits to guide the
development of an OWBA system.1,2,3,4 This workgroup,
composed of representatives from seven major pharmaceutical companies,
has a mission to aid instrumentation vendors in the creation of an
online water bioburden analyzer that satisfies both industry and
regulators. They believe, “an online water bioburden analyzer has the
potential to eliminate sampling and testing errors via reduced
manipulations while providing increased product safety and process
control through the availability of statistically significant data.”3
According to the group, such an OWBA system is not primarily designed
to eliminate compendial water testing, but should be used as a risk
reduction tool. Potential business benefits are shown in Table 1 and
include energy savings, labor reduction (resource allocations), and
increased product quality and process understanding.2
Figure
2: With a scatter detector and two fluorescence detectors (PMTs), an
instantaneous microbial detection system for water can create a
three-dimensional plot of biologic and interferent particles. Through
assessment of the three different signals and an advanced processing
algorithm, such a system offers enhanced interferent discrimination
capabilities.Technical system requirements are
provided, which include specifications for bioburden sensitivity,
calibration, chemical compatibility, operating parameters, and needed
consumables.3 Also included is a requirement for a limit of detection
(LOD) equivalent to that set forth for culture-based methods (10
CFU/100mL) and analysis modes that include continuous sampling,
time-based sampling, and daily operation at designated times. Overall,
the system should be capable of continuous and periodic monitoring of
critical control points (CCP) and POU, with sufficient sensitivity to
detect microorganisms in water and limited susceptibility to potential
interferents such as rouge, residual sanitizer, and gasket materials.
Laser-induced fluorescence
One
technique capable of satisfying the OWBA requirements is laser, or
light, induced fluorescence (LIF). LIF is a spectroscopic technique
capable of high sensitivity in the detection of compounds that
fluoresce. Fluorescence is the luminescence that occurs with the
absorption of radiation at one wavelength followed by the emission of
radiation at a different wavelength. Substances that typically fluoresce
may be referred to as fluorophores. Quinine is a familiar fluorophore
due to its presence in tonic water.
The application of LIF to
detect microorganisms has been leveraged in flow cytometry, capillary
electrophoresis, solid-phase cytometry, adenosine triphosphate
bioluminescence, and growth-based auto fluorescence. In a number of
these techniques, microorganisms are dyed to increase the measurable
fluorescence. Measuring the intrinsic fluorescence of a microorganism
removes the requirement for dyes and sample preparation, but requires an
instrument with significant sensitivity. As lasers of additional
wavelengths at higher power levels have become commercially available,
LIF has become very relevant in applications requiring detection of low
levels of microbial intrinsic fluorescence.
A light source such
as a laser is the excitation source in LIF. A laser of appropriate
wavelength and intensity is capable of inducing intrinsic fluorescence
emission from microbes due to constituent fluorophores such as
tryptophan, nicotinamide adenine dinucleotides (NADH), and flavins that
are present in microorganisms.7 The target excitation
wavelength is based on the excitation spectra of target fluorophores
such that sufficient fluorescence intensity is induced for measurement
and a greater number of non-biologic materials may be excluded. Yet,
non-biologic materials such as plastics, rubbers, and paper can also
fluoresce pointing to the importance of software discrimination
algorithms.
OWBA: Instantaneous microbial detection technology for water
An
OWBA system based on LIF enables the instantaneous detection of
microbes in water, without the need for consumables and the limitations
presented by traditional testing methods. Commercially available systems
for water employ a 405nm laser to simultaneously induce Mie scatter and
intrinsic fluorescence, on a particle-by-particle basis, as a sample
travels along a flow path and traverses this excitation source.
Detection and correlation of the Mie Scatter and fluorescence signals
provide real-time information on the presence and biologic status of
particles. Detection based on the intrinsic fluorescence of
microorganisms removes requirements for sample preparation. Furthermore,
this fundamental method of detection is inherently different from
traditional growth-based methods, and is not susceptible to the
growth-based limitations resulting from improper media selection and
incubation.
Figure
3: Representative data from the IMD-W system showing IMD-W biologic
counts as compared to colony forming unit culture results obtained using
the traditional method with TSA plates.In
LIF-based systems, intrinsic fluorescence is captured on a
photomultiplier tube (PMT), a detector highly sensitive to light. Both
one-PMT and two-PMT designs are available. In water, two-PMT designs
provide better discrimination of non-biologic fluorescing materials such
as rouge, as requested in the OWBA requirements.3 Each
material and microorganism has a different excitation and emission
spectrum. Once an excitation wavelength has been chosen, some materials
show a broad fluorescence emission and others a narrow emission
spectrum. Similarities in the emission spectra of biologic versus
non-biologic materials can be used advantageously. Figure 1 contains the
emission spectra of certain biologic and non-biologic materials with
405nm excitation. Two notional PMT detection regions have been
highlighted on either side of a Raman band in this figure. The Raman
band represents fluorescence produced from the interaction of the laser
light with water. Therefore, in order to detect particulate within the
interrogated water stream, this band must be avoided in the detection
regions utilized by the system. With 405nm excitation, the Raman band
for water has a maximum at approximately 469nm8.
With
two PMT detection regions, the differences in non-biologic versus
biologic emission spectra can be utilized to aid in the classification
of non-biologic materials as inert. As shown in Figure 2, a particle’s
scatter and fluorescence signals can be combined to create a
three-dimensional map of interferent and biologic particles. Advanced
algorithms can then be utilized to aid in the discrimination of biologic
and interferent materials.
Real-time bioburden monitoring, risk reduction, and process control
The
use of an instantaneous microbial detection system for pharmaceutical
water provides the ability to monitor bioburden continuously and in real
time, resulting in an increased potential for risk reduction and
process control. Figure 3 shows representative data from the IMD-W™, a
system designed with the OWBA requirements in mind, comparing IMD-W
biologic counts to culture plate results for three OWBA suggested
organisms. This data covers a wide dynamic range and speaks to the
potential sensitivity and ability of such systems to monitor bioburden.
The
continuous data offered by these systems creates a robust historical
dataset that is ideally suited for trending, particularly when compared
to episodic sampling with traditional methods. Sampling considerations
set forth in “USP<1231> Water for Pharmaceutical Purposes”
recommends monitoring pharmaceutical water systems at a frequency
“sufficient to ensure that the system is in control and continues to
produce water of acceptable quality.”5 The general
information chapter states it is best to operate monitoring
instrumentation in a continuous mode such that a large volume of
in-process data can be generated, and suggests the use of trend analysis
as an alert mechanism for loop maintenance.5 A combination
of historical trending data and real-time results enable users to
identify an out-of-specification event or deterioration in
microbiological control significantly earlier than with traditional
sampling methods. By continuously monitoring the state of control,
timely loop maintenance can be performed if bioburden data trends
upward, permitting further risk reduction and an increased level of loop
control. A real-time and historical knowledge of control can also be
important during a POU testing deviation.2 If POU testing is
positive for microbial contamination, knowledge and data to support a
state of control may narrow the root-cause investigation to the POU as
opposed to contamination in the entire loop.
Conclusions
Regulatory
guidance and calls from industry work groups support the need for
better tools for pharmaceutical water monitoring. New instantaneous
microbial detection systems based on LIF enable real-time bioburden
monitoring, increased risk reduction, and process control for
pharmaceutical waters. Through continuous monitoring, these systems
provide significant historical data for robust trending and assessment
of water loop bioburden levels, providing the means to monitor the level
of control and react to out-of-specification events in a much more
timely manner than with traditional methods alone. Users stand to
benefit through increased product quality and process understanding,
energy savings, and risk reduction.
References 1. Cundell, A., Luebke, M., Gordon, O., Mateffy, J., Haycocks, N., Weber, J. W., et al. (2013, May 16). On-Line Water Bioburden Analyzer Business Benefits Estimation.
Retrieved August 8, 2014, from
http://www.miclev.se/fileadmin/user_upload/jennie/Online_Water_BioBurden_Analyzer_Business_Benefits.pdf
. 2. Cundell, A., Gordon, O., Haycocks, N., Johnston, J., Luebke, M., Lewis, N., et al. (2013, May/June). Novel
Concept for Online Water Bioburden Analysis: Key Considerations,
Applications, and Business Benefits for Microbiological Risk Reduction.
American Pharmaceutical Review, 26-31. 3. Cundell, A., Luebke, M., Gordon, O., Mateffy, J., Haycocks, N., Weber, J. W., et al. (2013, March 18). On-Line Water Bioburden Analyzer User Requirement Specifications (URS). Document ID OWBA-DURS-2013-v1.3. 4. Cundell, A., Luebke, M., Gordon, O., Mateffy, J., Haycocks, N., Weber, J. W., et al. (2013, April 24). On-Line Water Bioburden Analyzer Testing Protocol. Document ID OWBA-TP-2013-v1.5. 5.
USP<1231> Water for Pharmaceutical Purposes. Pharmacopeial forum,
Vol. 32; United States Pharmacopeial Convention, Inc.: Rockville, MD,
2008. 6. Lakowicz, J. R. (2006). Principles of Fluorescence Spectroscopy (3rd ed.). New York: Springer Science & Business Media. 7. Ammor, M. S. (2007). Recent Advances in the Use of Intrinsic Fluorescence for Bacterial Identification and Characterization. Journal of Fluorescence, 17:455-459. 8. Rouessac, F., & Rouessac, A. (2013). Chemical Analysis: Modern Instrumentation Methods and Techniques (2nd ed.). West Sussex: John Wiley & Sons.
\\
Introduction
to methods of contamination analysis
This
method of particle counting using scattering is used for volumetric
instruments down to 0.2 µm and is effective up to above 20 µm. Below the
0.2 µm threshold volumetric sensors tend to give way to non-volumetric
instruments where only a small fraction of the total liquid flow is
monitored.
