Integrity testing

Hollow fiber cartridges for membrane

separations
User Manual

1 Introduction
About this manual
This manual describes three tests you can use to verify the integrity of hollow fiber
cartridge filters. The tests include: bubble point, air diffusion, and pressure hold tests.
The amount of time you need to verify filter integrity can vary from a few minutes to
several hours, depending upon the techniques and equipment you use. Therefore, the
secondary goal of this manual is to provide you with ideas and techniques to help you
save time.
The manual itself helps speed the learning process—the streamlined content and format
allow easy access and fast comprehension of the information.
This manual is designed for online viewing and navigation using Adobe Acrobat. Print the
Acrobat file to obtain a hard copy. You can download this manual from the Cytiva
website.

Target audience
This manual is intended for experienced users of microfiltration and ultrafiltration
membrane filter cartridges. Specifically, the manual addresses the needs of scientists,
process engineers, and technicians who operate research, laboratory, pilot, and produc-
tion filtration systems.

cytiva.com 18117173 AD
 To use this manual, you should know how to complete basic technical procedures. For
example, you should be qualified to set up laboratory and pilot equipment; measure
temperatures, pressures, and flows; and understand filtration applications. If you do not
have these capabilities now, call Cytiva for assistance. See Getting help, on page 2 for
contact information.

Where to find more information

This manual is a supplement to the Hollow fiber cartridges for membrane separations
Operating Handbook (CY28744). In addition, Cytiva publishes easy-to-use handbooks
related to its cross flow filtration equipment. You can view and download these hand-
books from our website, cytiva.com.

Getting help
To find local contact information for support, visit cytiva.com/contact.

Safety
When using any laboratory, pilot scale, or production filtration equipment, the potential
exists for personal injury unless you follow established safety procedures. When using
Cytiva test procedures and products, you should follow OSHA, federal, state, and local
safety mandates and regulations. You should follow your company’s safety procedures
and the safety instruction provided in this manual.
This manual provides tips with safety information and expert advice in the following
format:
Tip: An expert advice tip flag provides information to use your hollow fiber
membrane cartridge and system efficiently to achieve the best results.

Specific advice to help you work safely

Two primary risks of working with cartridge filtration systems include:
• The unexpected rupture of a filter system component due to overpressurizing the
system.
• The unexpected release of wetting agents, such as alcohol, from the filtration or test
system due to overpressurizing or leaks.
To avoid the unexpected rupture of a filter system or test stand due to over pressuriza-
tion you should know—and not exceed—the maximum operating pressure of your
system components. The Hollow fiber cartridges for membrane separations Operating
Handbook (CY28744) describes the maximum operating pressure of the cartridge filters.
To avoid exceeding the maximum operating pressure, Cytiva recommends that you
install pressure gauges on the system as described in this manual.

2 Integrity testing User Manual 18117173 AD

 To avoid the unexpected release of wetting agents due to overpressurization or leaks, do
not overpressurize the system. Isolate and depressurize the cartridge filters before
disassembling them. Inspect the filtration system’s gaskets, seals, and connections for
wear. Wear the appropriate personal protection devices when testing the integrity of the
cartridge.

Return authorization
To return a cartridge purchased from a distributor, contact the distributor for instruc-
tions. To return a cartridge purchased from Cytiva, contact Cytiva to obtain a return
authorization number. See Getting help, on page 2 for more information.

Integrity testing User Manual 18117173 AD 3

2 Which test should you use?
2.1 Filter integrity tests
Integrity testing background
For the processing of biopharmaceuticals, it is desirable to determine the integrity of a
filter prior to or following the processing of a product through a filter. Although not
driven by any specific regulatory requirement, the reasons from an operational perspec-
tive are overwhelmingly compelling to conduct an in-process test to make sure the filter
performs its desired role within the manufacturing process. These reasons include:
• Verification that the filter was not damaged during any sterilization/sanitization cycle
or installation steps prior to processing product.
• Verification that the filter was installed properly.
• Elimination of the economical impact of product losses associated with installing and
using a non-integral filter in the process.
• Optimizing the manufacturing capacity by not having to run additional product
because a non-integral filter had been used in the process.
• Significant time savings associated with quality assurance (QA) and operational
personnel not having to conduct root cause analysis for low yields, product loss, or
poor product quality associated with using a non-integral filter.
The choice of what type of integrity test that is conducted on a filter should be carefully
considered. It should be a non-destructive test that takes into consideration the time
that is required to set up and conduct the integrity test, the operational (pressure) limita-
tions of the filter, the purpose of the test itself, and other quality control (QC) tests that
are used within the process. A good in-process integrity test, when used in conjunction
with a water flux test, provides evidence that the cartridge has performed or will perform
as expected. The integrity test should be reliable, repeatable, and simple to set up and
conduct either in situ or offline. Multiple integrity test protocols are available to accom-
plish the same purpose, and that is to provide the operator with information that the
filter and the seals between the upstream and downstream side of the filter are integral.
The most common integrity tests include a diffusion test, a bubble point test, or a pres-
sure hold test.
For most microfiltration (MF) applications, and for all ultrafiltration (UF) applications,
conducting a bubble point test is not practical nor is it necessary. For all UF membranes,
and most MF membranes, the water bubble point value will exceed the pressure rating
limitation of the filter, and for UF membranes the water/alcohol bubble point value will
exceed the maximum transmembrane pressure (TMP) specification for the cartridge.
Thus, there is an inherent risk that the integrity of the filter is breached by exceeding the
operating pressure limitations of the cartridge when trying to conduct a bubble point

4 Integrity testing User Manual 18117173 AD

 integrity test to ascertain a filter’s integrity. An alcohol/water solution may be used to
reduce the wetting agents surface tension, thus reducing the bubble point value to
below the TMP limitations of the filter. However, alcohol is often incompatible with the
end product and poses additional health and safety risks for most facilities, thus it does
not make a feasible alternative in most sites.
Additionally, unlike a sterilizing grade filter that is used in an aseptic final filling opera-
tion, in upstream cross flow applications there is no regulatory requirement for the
manufacturer to demonstrate a correlation between the integrity test used and the
retention performance of the filter.
A final point of consideration is that the integrity test is only one in-process test that is
used in conjunction with a battery of other in-process and final QC release testing, that
will demonstrate that the manufacturing process is operating correctly and that the
product manufactured will meet the specifications and release requirements set forth by
the manufacturer.

Integrity testing theory

As mentioned in the introduction, there are three basic integrity tests that one may use
when integrity testing cross flow devices — diffusion, pressure hold, or bubble point test.
All of these tests rely on the cartridges being fully wet out. The wetting solution is
retained in the membrane pore matrix by capillary forces. If the cartridge is pressurized
from the upstream side of the cartridge with a test gas, a plot can be made of gas flow
rate across the wetted membrane versus the test gas pressure. Illustrated below is a
representation of the gas flow diffusion curve, where the curve can be segmented into
three distinct regions. These include flow rate due to diffusion, bubble point — the pres-
sure at which the test gas pressure is greater than the capillary forces of the largest
pores in the cartridge, and bulk flow — the point at which the wetting solution has been
pushed out of the membrane pore matrix so that the test gas is not impeded from
passing through the membrane pore matrix.

Increasing gas flowrate

Bulk

Diffusive
Bubble point

Increasing pressure

Figure 2.1: Gas flow diffusion curve

Integrity testing User Manual 18117173 AD 5

Bubble point test
Bubble point testing plays a critical role in ascertaining membrane pore size. Specifically,
it is a method for estimating the largest pore(s) of the membrane in a given filtration
device, and provides an indirect means of measuring them. The bubble point value from a
theoretical perspective could be estimated based on the bubble point equation:
4kλ cos θ
Q=
d
Where:
Q = flow rate of the gas
k = correction factor for the shape of the pores
λ = surface tension of the wetting solution
cosϴ = the contact angle between the wetting solution and the membrane surface
d = pore diameter
As can be seen from the above equation, the key characteristics and parameters that will
impact the bubble point value are the pore size in the membrane, surface tension of the
wetting solution, and the surface chemistry (contact angle) between the wetting solu-
tion and the membrane surface. A low surface tension wetting solution will result in a
lower bubble point value. Additionally, residual material, such as surfactants, that are
adsorbed by the membrane may also alter and reduce the measured bubble point value,
by altering the surface chemistry between the membrane and the wetting solution.
Because the pores contained in membranes are not cylindrical in nature but rather are a
torturous path, a constant is added to the equation to correct for the shape of the pores.
It is important to note that the bubble point value is inversely proportional to the pore
size diameter, so as the pore size decreases the bubble point value will increase.
As more surface area is added to a system and diffusive flow through the membrane
increases, it may be more difficult to distinguish between the transition phase of diffu-
sive flow and the rapid bubbling associated with the bubble point value. For larger area
systems where the distinction between gas flow due to diffusion and gas flow due to
reaching the bubble point value is not clear, a diffusion test or pressure hold test is
recommended.
To complete the bubble point test, you wet the filter and subject it to increasing gas pres-
sure. You observe the pressure at which a stream of bubbles emerges from the
membrane. You then use the pressure reading to determine the largest pore in the filter
membrane.
Once you build a bubble point test stand, you can complete the bubble point test quickly,
enabling you to routinely check new and used cartridges.

6 Integrity testing User Manual 18117173 AD

Air diffusion test
The diffusion test is one of the most common tests for determining a filter’s integrity. It is
simple, easily understood, and quantitative. The diffusion test relies on the principles of
diffusion. In very simple terms, a system is separated by a semipermeable barrier and
one side is pressurized with a test gas creating a system that is out of equilibrium.
Because there is a semipermeable barrier separating the area of high gas concentration
from the low gas concentration, the system tires to equilibrate. The gas moves from the
area of high concentration and diffuses across the semipermeable barrier to the area of
low gas concentration. The illustration below is a depiction of diffusion across a wetted
membrane that acts as a semipermeable barrier.

High gas concentration

Diffusion

Fully wetted membrane

Low gas concentration

Figure 2.2: Diffusion across a wetted membrane

In quantitative terms, diffusion follows the laws of diffusion and can be expressed by the
equation:
DHP
N= φ
L
Where:
N = diffusive flux of the test gas
D = diffusivity of the test gas through the wetting solution
H = solubility coefficient of the test gas in the wetting solution
P = pressure applied to the upstream side of the cartridge
φ = overall porosity of the membrane
L = the thickness of the layer that the test gas must diffuse through (the thickness of the
membrane assuming it is fully wet out)
As can be seen from the above equation, the factors that impact the diffusion rate for a
given cartridge include the test gas used and solubility of that gas in the wetting solution,
the path length that the test gas must travel or diffuse through, the pressure at which
the test is conducted, and the porosity of the membrane being tested.

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 Because the molecular diffusive flux is being measured at a set of fixed atmospheric
pressure and temperature conditions, the molecules of gas can be converted into a volu-
metric flow rate (mL/min, cc/min). Furthermore, by holding the filter thickness,
membrane porosity, surface area, wetting fluid, and test gas constant, the equation for
diffusive flow can be simplified to a linear expression of:
F = HP
Where:
F = diffusive flow (cc/min, mL/min)
H = solubility coefficient of the test gas in the wetting solution
P = test pressure of the test gas
This linear expression is seen in Fig. 2.1, on page 5 for gas flow (diffusion) below the
bubble point value. Above the bubble point pressure, bulk flow occurs in addition to the
diffusive flow through the wetted portion of the membrane.
To complete the air diffusion test, you wet out the filter and pressurize it. You then
measure the volume of air that diffuses from the filter and compare the diffusion rate
against the specification for the cartridge.
The air diffusion test is quantitative and can be performed quickly. Hence, you can
routinely test new and used cartridges.

Pressure hold test

The pressure hold test determines the integrity of the membrane and the cartridge. The
pressure hold test is similar to the air diffusion test, but rather than measuring diffusion
on the downstream side of the cartridge, the pressure drop on the upstream side of the
cartridge is monitored. Any pressure drop that occurs that is not related to diffusive flow
through the cartridge is either due to a damaged filter, damaged seal between the
upstream and downstream side of the cartridge, an improperly installed cartridge, or a
leak on the upstream side of the cartridge itself, either from a damaged valve or seal.
The pressure drop (decay) is directly proportional to diffusive flow and may be calculated
as follows:
Pa TD
PD =
Vh
Where:
PD = the pressure decay that occurs as a result of diffusive flow through the cartridge
Pa = atmospheric pressure
(14.7 psig or 1 barg)
T = test time (minutes)
D = diffusion rate
Vh = upstream housing (system) volume

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 As can be seen from the above equation, the same factors that impact diffusion will also
impact the pressure decay or pressure hold test. In addition, a change in the upstream
housing that is not accounted for will also have an impact on the pressure drop value, as
will the test time.
To complete the pressure hold test, you wet out the filter and pressurize it. You then
observe the pressure on the upstream side of the filter to insure that the pressure is
maintained. In addition, the permeate line should be immersed in water to observe for
rapid bubbling exiting the permeate side of the filter.
Often it is possible to complete the pressure hold test without removing the cartridge
from your filtration system.

2.2 Automated integrity testers

Automated integrity testers are commonplace when testing sterilizing grade filters in an
aseptic operation. Increasingly, they are being employed in upstream applications to
test cross flow cassettes and filters. The greatest benefit to using an automated tester is
that the filter can be tested from the upstream side of the cartridge, thus not compro-
mising sterility on the downstream side of the system. There are two basic technologies
that automated testers employ to measure diffusion and bubble point values. The first
uses pressure transducers and measures any pressure changes that occur during the
course of the test. Each manufacturer has its own proprietary software algorithm that
takes these measurements and calculates the diffusive flow or bubble point. In very
simplistic terms, the parameters that are critical to determine the diffusive flow for a
given system are the same parameters that are included in the pressure decay equation
(i.e., pressure drop, time period that the pressure drop occurs in an upstream housing
volume). The software algorithm for each instrument is written specifically for the hard-
ware it is designed to work with. Each program follows specific steps taking measure-
ments at critical intervals throughout the test program. The measurements are then
used to determine, very accurately, the diffusion rate and the bubble point values for the
cartridge(s).
The second type of integrity testers employs the use of flowmeters and will measure the
gas flow directly.
Common steps for the various systems on the market include a pressure ramp up, a
stabilization time, and a test time. For customers who use a pressure transducer-based
system, an upstream housing volume has to be determined automatically as part of the
test routine or manually calculated and programmed into the test. As can be seen from
the pressure decay equation, this is a critical value that will determine the accuracy of
the test results.
The benefit to an automated sizing routine is that regardless of any changes made to the
system hardware or the size of the cartridge installed, an accurate upstream housing
volume will be used for each test. If the upstream housing size is sized manually and
programmed into the test then care must be taken to make sure that the same
upstream volume is used for each of the tests. The benefit to preprogramming an
upstream housing volume is that it can save several minutes on each test.

Integrity testing User Manual 18117173 AD 9

 For a bubble point determination the pressure is increased in small increments (~1 psig)
and held for a specified period of time. The upstream side of the filter is monitored for
larger than expected pressure drops or flow rates. Once these pressure drops or flow
rates are observed, a bubble point determination is made based on the limits set by the
manufacturer’s algorithm.
Some of the benefits for using an automated tester include the following:
• Standard, consistent test
• No operator to operator variation
• Minimizes the potential for operator error
• Printed test results that can be included in the batch records
While there are significant benefits to using automated testers there are also some
drawbacks.
• Longer test time (10 to 30 minutes/test)
• Must have all equipment and seals in optimum condition (any leaks on the upstream
side of the system will cause false failures)
• Susceptible to any thermal changes occurring during the test which may cause either
false failures or false passes. The temperature changes may be a result of a user trying
to test a filter that is not equilibrated to the environmental temperatures (such as
right after a steam sterilization cycle) or the result of a system in a room with fluctu-
ating temperature or right below a vent, next to an autoclave, or near a door causing
the system to cool down or heat up during a test.
• If a tester does not automatically size the upstream volume of the system, then the
system must be sized manually. As mentioned above, accurate measurements are
critical to achieve accurate results.

2.3 When to apply the tests

Table 2.1: Guidelines for integrity testing

Type of filter Recommended test Frequency

Microfiltration Bubble point When integrity is suspect
Microfiltration or ultra- Air diffusion A. When new
filtration
B. After cleaning
C. When integrity is suspect
Microfiltration or ultra- Pressure hold Quality control between runs
filtration

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3 Streamlining the tests
3.1 Introduction
In many cross flow filtration applications—particularly in the biopharmaceutical and
biotechnology fields—you must ensure the integrity of the cartridge filter each time you
use it. Demonstrating the integrity of cartridge filters can require a significant amount of
time unless you develop the techniques and equipment to streamline the process.
Therefore, the goal of this chapter is to provide you with ideas and techniques to help you
save time.

3.2 General recommendations

From an operational standpoint, here are some steps you can take to streamline the
integrity testing process:

Construct well-made test stands

Constructing and maintaining test stands independent of your process equipment
enables you to install and test cartridges quickly, without disturbing your process
system. Each procedure in this manual includes instructions and a diagram to help you
construct a test stand. Build your test stand using high quality components and good
technique to ensure reliability and ease of use.

When the application allows, use the

pressure hold test for quality control
between runs
The pressure hold test may be the preferred means of quality control testing between
runs. The pressure hold test can save a considerable amount of time because with the
appropriate connections, you can test the cartridge in place, with minimal adjustments
of process lines.

Maintain supplies of wetting solution

and water
You will need volumes of wetting solution and water to test your cartridge filters.
Streamline the testing procedure by keeping quantities of wetting solution in stock.

Install cartridges properly

Prior to installation, inspect all O-rings and gaskets for any signs of wear or damage.
When installing cartridges with O-rings, wet the O-rings to minimize any potential for the
O-rings pinching during the installation step.

Integrity testing User Manual 18117173 AD 11

Always make sure air is vented
completely during the wetting step
Liquid should be introduced from the low point of the system slowly and air vented from
the top.

Using an automated tester

Carefully inspect all sealing surfaces, O-rings, and gaskets for any damage or wear. Make
sure that all valves that are used on the upstream side of the system are integral. Make
sure that the system or tester is not located near a vent, door, autoclave, or any other
area where temperature changes may occur. Make sure the cartridge and hardware are
equilibrated to room temperature so that temperature changes will not occur during a
test.

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4 Bubble point test
4.1 Review test procedure
The bubble point test determines the largest pore in a cartridge filter. Using the bubble
point test, you can determine the presence of failed areas in the hollow fiber membrane
and leaks in the cartridge assembly.
To complete the bubble point test, you wet the filter and subject it to increasing gas pres-
sure. You observe the pressure at which a stream of bubbles emerges from the
membrane. You use the pressure reading that this occurs at to determine the integrity of
the cartridge.

4.2 Gather materials

Gather the following materials:
• Microfiltration cartridge you want to test
• A volume of wetting solution—100% isopropyl alcohol or a 50:50 mix of ethanol and
water. Use distilled water, water-for injection (WFI), water permeate from a 5000 or
10 000 nominal molecular weight cutoff (NMWC) membrane, or water permeate from
a reverse osmosis membrane.
• Bubble point test stand (see Section 4.4 Build a bubble point test stand, on page 17)

4.3 Bubble point procedure

You can analyze the bubble point procedure in two stages:
• Wetting the cartridge.
• Determining the bubble point.
The following sections describe each stage.

Stage 1—wetting the cartridge

Follow these steps to wet the cartridge before performing the bubble point test:

Step Action

1 Verify the composition of the wetting solution by density measurement. You

quickly can verify the composition of a 50:50 ethanol/water mix using a 0—100
Tralle alcohol hydrometer.

2 Install the filter cartridge in the bubble point test stand. (To learn how to build
a bubble point test stand, see Section 4.4 Build a bubble point test stand,
on page 17.) Open valve V4, open valve V2 to position 1, and open valve V3 to
position 2.

Integrity testing User Manual 18117173 AD 13

 Step Action

3 Start the metering pump. Wait about 15 seconds and then close valve V4. Let
the pump continue to run until the prefilter cartridge fills with wetting solu-
tion.

4 Switch valve V2 to position 2 and turn on the pump to a very low flow rate and
fill the test cartridge with wetting solution (it should take about two minutes
to fill the cartridge with wetting solution). When clear wetting solution flows
through the flowmeter, switch valve V3 to position 1. Continue to pump the
wetting solution at a low flow rate until clear wetting solution from the shell
(permeate) side of the test cartridge is flowing through the flowmeter.

5 Restrict the flow of the wetting solution by using either a pinch clamp or
diaphragm valve downstream of the flowmeter. Continue wetting out the
cartridge for about two minutes while the flow is restricted.
Result:
This will allow all the residual air entrapped within the pore matrix of the
membrane to dissolve into the fluid stream and be flushed from the system.

6 Stop the metering pump and switch valve V2 to position 1. Keep the cartridge
in the test stand and go to Stage 2.

Stage 2—determining the bubble point

Follow these steps to determine the bubble point of the wetted filter cartridge:

Step Action

1 Using the pressure regulator, adjust the air inlet pressure to 3 psig or less
(< 0.2 barg) and open valve V1.

14 Integrity testing User Manual 18117173 AD

 Step Action

2 After initial bubbling subsides, gradually increase the air pressure over the
next 15 seconds to approximately 70% of the minimum bubble point pressure
rating for the test cartridge (Table 2). Additional bubbles may appear at this
point. Wait about 15 seconds for the bubbling to equilibrate. As you increase
the test pressure, the diffusive flow of the test gas through the wetted
membrane will increase. As the surface area of the cartridge increases so will
the diffusion rate. Once the diffusion rate has stabilized resume the test.

Table 4.1: Bubble point specifications for microfiltration cartridges. Minimum bubble
point in psig (barg)

Pore size (μm) 50/50 EtOH and water Isopropyl alcohol

psig (barg) psig (barg)
0.1 35 (2.4) 24 (1.6)
0.2 18 (1.2) 12 (0.8)
0.45 12 (0.8) 8 (0.5)
0.65 6 (0.4) 4 (0.27)

3 Continue to increase air pressure gradually, this time at a much slower rate—
about 1 psig to 2 psig (~0.07 barg to 0.1 barg) per 15-second interval—while
carefully inspecting cartridge for rapid, continuous bubbling.
If the cartridge is contained in a stainless steel housing and you cannot see
the bubbles, attach a short length of 0.25-inch ID tubing to the feed side
permeate port. Immerse the other end of the tubing in a small beaker half full
with water, and watch for bubbling to occur.

4 When you observe rapid, continuous bubbles, record and release the pres-
sure. Compare the measured bubble point pressure to those listed in Table
4.1, on page 15.

5 If the bubble point reading is less than the minimum specification (Table 4.1,
on page 15), repeat the test. If you are testing a cartridge installed in a stain-
less steel housing, check the seating of the O-rings before repeating the test.
Contact Cytiva’s technical support team, if needed.
Tip:
When conducting a bubble point test, a determination will have to be made
between diffusive flow through the cartridge and rapid, continuous bubbling
associated with a failed cartridge or with the bulk flow, once the bubble point
value has been exceeded.

Integrity testing User Manual 18117173 AD 15

 Step Action

Tip:
When you first apply air pressure, air trapped on the permeate side may
appear in the permeate stream as bubbles. If the bubbles dissipate in a few
seconds where they appear only at intermittent time intervals, continue with
the test. If bubbles continue to appear and quickly increase to rapid, contin-
uous bubbling, the cartridge may be damaged or insufficiently wet out. Stop
the test, rewet the cartridge following Stage 1 steps on Stage 1—wetting the
cartridge, on page 13, and repeat step 1 on Stage 2—determining the bubble
point, on page 14. If rapid, continuous bubbling is still observed on the
permeate side of the filter, stop the test and call the technical support team at
Cytiva.
Tip:
Care should be used when selecting pressure gauges. Gauges should be
selected based on accuracy requirements and reliability.
Tip:
When making up wetting solution, make sure the correct solution concentra-
tions are used.
Tip:
Proper training should be provided to operators to ensure consistent, accurate
results between operators.
Tip:
Perform bubble point testing at ambient temperature (25°C ± 5°C) to ensure
consistent results.
Tip:
You should follow the timing requirements in the bubble point test. For
example, once 70% of the minimum expected bubble point pressure is
reached, increase the pressure slowly in small increments so that you will not
overshoot the pressure at which you observe rapid, continuous bubbling. This
would result in an indicated bubble point that is higher than the actual bubble
point.
Diffusion will occur below the bubble point. As the surface area of the device
increases, so will the diffusion rate. Care must be taken to note the difference
between intermittent bubbling associated with diffusion and rapid, continuous
bubbling associated with reaching the bubble point value of the cartridge.

16 Integrity testing User Manual 18117173 AD

 Step Action

Tip:
Cytiva hollow fibers have a highly-uniform pore size distribution matrix. Thus,
rather than a single stream of bubbles, you will most likely observe rapid
continuous bubbling where the wetting solution is expelled from a large popu-
lation of the membrane pores simultaneously.

4.4 Build a bubble point test stand

Gather materials
Gather the following materials to construct a bubble point test stand:
Pressure gauges, valves, and regulators:
• Pressure regulator—air and gas
• Pressure gauge—air and gas, 0 to 60 psig
• Pressure gauge—liquid, 0 to 60 psig
• 3-way valve (V2)
• 3-way valve (V3)
• Diaphragm valve or pinch clamp
• Check valve—air and gas
• On/off valve—air/gas (V1)
• On/off valve—liquid (V4)
Other components:
• Test solution reservoir
• Metering pump—liquid
• Flexible tubing
• Flowmeter
• Cap—cartridge permeate port
• Prefilter—Cytiva 0.1 μm cartridge

Assemble the bubble point test stand

Assemble the bubble point test stand using the parts list and standard laboratory tech-
niques (Fig. 4.1, on page 18).

Integrity testing User Manual 18117173 AD 17

 18
R 17
16
M 13 15
12-1 12-2 14
12
11
3
10
2 4 9
F 5 8
6-1 6-2 7
6

Figure 4.1: Assemble the bubble point test stand

Part Description
1 Feeding reservoir with wetting solution
2 Metering pump
3 On/off liquid valve (V4)
4 Pressure gauge
5 Prefilter 0.1 μm
6 3-way valve (V2). 6-1 valve position 1, 6-2 valve position 2.
7 Retentate port
8 Permeate port with cap
9 Cytiva microfiltration cartridge
10 Permeate port
11 Feed port
12 3-way valve (V3). 12-1 valve position 1, 12-2 valve position 2.
13 Flowmeter
14 On/off valve (air/gas valve V1)
15 Check valve (air/gas)

18 Integrity testing User Manual 18117173 AD

 Part Description
16 Pressure gauge (air/gas)
17 Pressure regulator (air/gas)
18 Air or nitrogen supply

Integrity testing User Manual 18117173 AD 19

5 Air diffusion test
5.1 Review test procedure
The air diffusion test determines the integrity of the ultrafiltration membrane and the
cartridge. To complete the air diffusion test, you wet out the filter and pressurize it. You
then measure the volume of air that diffuses from the filter and compare the diffusion
rate against a diffusion specification for the cartridge.

5.2 Gather materials

Gather the following materials:
• Ultrafiltration cartridge you want to test
• A volume of wetting solution—100% ethanol or water. Use distilled water, water-for-
injection, water permeate from a 5000 or 10 000 NMWC membrane, or water
permeate from a reverse osmosis membrane.
• Air diffusion test stand (see Section 5.4 Build an air diffusion test stand, on page 22)

5.3 Air diffusion procedure

You can separate the air diffusion procedure into two stages:
• Preparing the cartridge and test stand.
• Determining the air diffusion rate.
The following section describes each stage.

Stage 1—preparing the cartridge and

test stand
Follow these steps to prepare the cartridge for the air diffusion test:

Step Action

1 Flush the preservative from a new cartridge, or clean and flush a used
cartridge. See the Hollow fiber cartridges for membrane separations Oper-
ating Handbook (CY28744) to learn how to flush and clean cartridges.

2 Wet the cartridge out by recirculating water through the cartridge at an

average transmembrane pressure of 20 psig (1.3 barg).

3 If necessary, a high pressure intrusion wetting cycle or ethanol alcohol soak

for one hour may be required to fully wet out the cartridge.

4 High pressure intrusion wetting cycle:

20 Integrity testing User Manual 18117173 AD

 Step Action

a. Start the pump so that water is flowing through the feed side of the
cartridge in a recirculation mode.
Note:
At this time the permeate port should be closed and the retentate port
should be fully open.
b. Slowly restrict the retentate port to about 10-20% of the flow.
c. Slowly open the permeate port diaphragm valve or loosen the pinch clamp
on the permeate port tubing. The flow restriction through both the
permeate port and retentate port should be maintained. Hold the pres-
sure in the system between 25 psig and 30 psig. There should be no more
than a 2 psig difference between the feed pressure, retentate pressure,
and permeate pressure. Maintain this pressure and wetting step for two to
five minutes.
Result:
This allows all of the residual gas entrapped within the membrane pore
matrix to dissolve into the wetting solution.

5 If necessary, you can also wet the cartridge out by soaking it in ethanol for one
hour and then flushing the ethanol out of the cartridge with clean water, such
as WFI.

6 Drain excess liquid from the cartridge.

7 Install the filter cartridge in the air diffusion test setup (Fig. 5.1, on page 23).
Tip:
To learn how to build an air diffusion test stand, see Section 5.4 Build an air
diffusion test stand, on page 22.

8 Use blanks or valves to block the retentate port and the retentate-side
permeate port.

9 Use flexible tubing to connect the feed-side permeate port with the inverted
graduated cylinder. Go to Stage 2.

Table 5.1: Recommended graduated cylinder capacity

Cartridge membrane area Graduated cylinder

capacity
< 1 ft2 < 0.1 m2 10 cc
1 to 10 ft2 0.1 to 1 m2 50 cc

Integrity testing User Manual 18117173 AD 21

 Step Action

Cartridge membrane area Graduated cylinder

capacity
> 10 ft2 > 1 m2 125 cc

Stage 2—measuring the air diffusion

rate
Measure the air diffusion rate following these steps:

Step Action

1 Determine the correct diffusion specification for the cartridge based on the
surface area being tested.

2 Adjust the air (or nitrogen) inlet regulator to about 3 psig (0.2 barg) and open
valve V1.

3 After initial stream of bubbling subsides, gradually increase the air pressure
over the next 15 seconds to about 15 psig (1 barg). An additional rapid steady
stream of bubbles may appear at this point. Wait a few seconds for the
bubbling to stabilize and resume the test.

4 Place the end of the flexible tubing into the inverted graduated cylinder that is
filled with water, and using a stopwatch, measure the air diffusion rate in
cc/min.

5 Compare the results with the specifications:

Result:
Air diffusion rate for all Cytiva ultrafiltration membranes should be
< 3 cc/min/ft2 (< 32.3 cc/min/m2) at 30 psig (2.1 barg).

5.4 Build an air diffusion test stand

Gather materials
Gather the following materials to construct an air diffusion test stand:
• Pressure regulator—air and gas
• Pressure gauge—air and gas, 0 to 60 psig (0 to 4 barg)
• On/off valve—air/gas (V1)
• Permeate port diaphragm valve or pinch clamp for permeate tubing

22 Integrity testing User Manual 18117173 AD

 • Flexible tubing
• Cap for permeate port
• Cap for retentate port
• Water reservoir
• Graduated cylinder
• Pressurized, filtered air or nitrogen supply

Assemble the air diffusion test stand

Assemble the air diffusion test stand using the parts list and standard laboratory techni-
ques (Fig. 5.1, on page 23).
11
10
F

1
9
R 2

8
3 6 6

4
5 7
Figure 5.1: Flow diagram for the air diffusion test

Part Description
1 Air or nitrogen inlet
2 Pressure regulator
3 Pressure gauge
4 Valve 1 on/off valve
5 Feed port
6 Permeate port
7 Retentate port
8 Cap
9 Container filled with water
10 Optional mass flow meter
11 Inverted graduated cylinder filled with water

Integrity testing User Manual 18117173 AD 23

 Tip: When you first apply air pressure, air trapped on the permeate side may
appear as a continuous stream of bubbles. If the bubbles dissipate in a few
seconds, continue with the test. If a rapid, continuous stream of bubbles
continue to appear, the cartridge may be insufficiently wet or damaged. Stop
the test, repeat Stage 1 steps 2 through 6, and Stage 2 step 1 (see Stage 1—
preparing the cartridge and test stand, on page 20 and Stage 2—measuring
the air diffusion rate, on page 22). If bubbles still appear in the sight glass, stop
the test and call the technical support team at Cytiva.
Tip: Install a low point drain valve to the permeate port to readily drain residual
wetting solution from the permeate line. Any residual water in the permeate
line may impede gas flow through the line and lead to artificially low diffusion
values.
Tip: Use reliable and accurate pressure gauges that are routinely calibrated, and
accurate graduated cylinders.
Tip: All operators should be properly trained to ensure accurate and consistent
results between operators.
Tip: You should perform air diffusion testing at ambient temperature (25°C ± 5°C)
to assure consistent results.
Tip: Cytiva performs air diffusion tests at 30 psig (2 barg) or higher. Diffusion
testing may be performed at lower pressures, however the diffusion specifi-
cation must be normalized down to match the test pressure. There is a direct
linear relationship between diffusion and test pressure. Testing at half the
test pressure will result in half the diffusive flow, therefore the diffusion speci-
fication must be divided by two also.

24 Integrity testing User Manual 18117173 AD

6 Pressure hold test
6.1 Review test procedure
The pressure hold test determines the integrity of ultrafiltration and microfiltration
membranes and cartridges. The pressure hold test is similar to the air diffusion test, but
rather than measuring diffusive flow on the downstream side of the cartridge, the pres-
sure drop is monitored from the upstream side of the cartridge. Provided that the
upstream side of the filter is a closed system, any pressure decay should be a result of
diffusive flow through the cartridge. This allows one to test the integrity of the cartridge
without compromising any of the aseptic connections.

6.2 Gather materials

Gather the following materials:
• Ultrafiltration or microfiltration cartridge you want to test
• A volume of wetting solution—water or 100% ethanol. Use distilled water, water-for-
injection, permeate from a 5000 or 10 000 NMWC membrane, or permeate from a
reverse osmosis membrane.
• Pressure hold test stand (see Section 6.4 Build a pressure hold test stand,
on page 30)

6.3 Pressure hold test procedure

You can separate the pressure hold test into two stages:
• Preparing the cartridge and test stand.
• Running the pressure hold test.
The following sections describe each stage.

Stage 1—preparing the cartridge and

test stand
Follow these steps to prepare the cartridge for the pressure hold test:

Step Action

1 Determine the upstream volume of the closed system that will be tested. This
includes the feed/retentate side of the cartridge and any plumbing between
valve V1 and valve V2. You may consult Cytiva technical service for the
upstream volume for the cartridge itself.

2 Flush the preservative from a new cartridge, or clean and flush a used
cartridge. See the Hollow fiber cartridges for membrane separations Oper-
ating Handbook (CY28744) to learn how to flush and clean cartridges.

Integrity testing User Manual 18117173 AD 25

 Step Action

3 Wet the cartridge out by recirculating water through the cartridge at an

average transmembrane pressure of 20 psig (1.3 barg).

4 If necessary, a high pressure intrusion wetting cycle or ethanol alcohol soak

for one hour may be required to fully wet out the cartridge.

5 High pressure intrusion wetting cycle:

a. Start the pump so that water is flowing through the feed side of the
cartridge in a recirculation mode.
Result:
At this time the permeate port should be closed and the retentate port
should be fully open.
b. Slowly restrict the retentate port to about 10% to 20% of the flow.
c. Slowly open the permeate port diaphragm valve or loosen the pinch clamp
on the permeate port tubing. The flow restriction through both the
permeate port and retentate port should be maintained. Hold the pres-
sure in the system between 25 psig and 30 psig. There should be no more
than a 2 psig difference between the feed pressure, retentate pressures,
and permeate pressure. Maintain this pressure and wetting step for two to
five minutes.
Result:
This will allow all of the residual gas entrapped within the membrane pore
matrix to dissolve into the wetting solution.

6 If necessary, you can also wet the cartridge out by soaking it in ethanol for one
hour and then flushing the ethanol out of the cartridge with clean water, such
as WFI.

7 Drain excess liquid from the cartridge.

8 Install the filter cartridge in the pressure hold test stand (Fig. 6.1,
on page 29).
Tip:
To learn how to build a pressure hold test stand, see Section 6.4 Build a pres-
sure hold test stand, on page 30.

9 Use a blank-off cap to block the unused permeate port and close the reten-
tate-side valve. Go to Stage 2.

26 Integrity testing User Manual 18117173 AD

Stage 2A—running the qualitative
pressure hold test
The qualitative pressure hold test determines the integrity of ultrafiltration and microfil-
tration membranes and cartridges. The pressure hold test is similar to the air diffusion
test, but is qualitative rather than quantitative. To complete the pressure hold test, you
wet out the filter and pressurize it. You then observe for rapid, continuous bubbling
exiting the permeate that would indicate non-integrity of the filter.
Follow the procedure as outlined above.
Run the pressure hold test following these steps:

Step Action

1 Adjust the air (or nitrogen) inlet regulator to about 3 psig (0.2 barg) and open
valve V1.

2 After initial bubbling subsides, gradually increase the air pressure to about
5 psig (0.3 barg). Additional rapid bubbling may appear at this point. Wait a few
seconds for the bubbling to slow and resume the test.

3 Maintain the air pressure at 5 psig (0.3 barg). If only a small stream of bubbles
appear (from air diffusing through the membrane), membrane and cartridge
integrity are acceptable.

Stage 2B—running the quantitative

pressure hold test
Run the pressure hold test following these steps:

Step Action

1 The downstream permeate port should be open and the feed and retentate
valves should be closed. With the regulator completely closed, open valve V1
and adjust the air (or nitrogen) inlet regulator to about 3 psig (0.2 barg). Allow
the system to stabilize at this pressure for about 30 seconds.

2 Close valve V1 and monitor the pressure in the system. If the pressure drops
rapidly over a 15-second period it may be an indication that the filter is not
fully wet, a valve is open on the upstream side, or the seals in the system are
not seated properly. Check the system for leaks and repressurize the system
to 3 psig and allow the system to stabilize for another 20 to 30 seconds.

3 After the system has stabilized, gradually increase the air pressure to the test
pressure and allow the system to stabilize for about one minute at this pres-
sure.

Integrity testing User Manual 18117173 AD 27

 Step Action

4 Close valve V1 and measure the pressure drop over a set time period, (typi-
cally a 30-second or one-minute time interval).

5 Determine the maximum allowable pressure drop for the cartridge. This is
based on the diffusion specification and upstream housing volume:
Pa TD
PD =
Vh
Where:
PD = the pressure decay that occurs as a result of diffusive flow through the
cartridge
Pa = atmospheric pressure (14.7 psig or 1 barg)
T = test time (minutes)
D = diffusion rate
Vh = upstream housing (system) volume

For example, for a cartridge that has a maximum diffusion rate specification of
30 mL/min at a test pressure of 15 psig in a system with an upstream housing volume of
1000 mL, the maximum allowable pressure decay is as follows:
Pa = atmospheric pressure (14.7 psig)
T = test time (in this case, one minute)
D = diffusion specification (30 mL/min in this example)
Vh = upstream housing (system) volume (in this example [1000 mL])
Maximum allowable PD = {(14.7 psig)*(1 minute)*(30 mL/min)}/ (1000 mL)
PD = 0.44 psig over a one-minute period

Compare the pressure decay value obtained with that of the maximum allowable pres-
sure decay based on the diffusion specification for the cartridge.

28 Integrity testing User Manual 18117173 AD

 1

R 2

8
6 6
3

4
5 7

Figure 6.1: Flow diagram for the pressure hold test

Part Description
1 Air or nitrogen inlet
2 Pressure regulator
3 Pressure gauge
4 Valve 1 on/off valve
5 Feed port
6 Permeate port
7 Retentate port
8 Cap

Tip: Use reliable and accurate pressure gauges that are routinely calibrated, and
accurate graduated cylinders.
Tip: All operators should be properly trained to ensure accurate and consistent
results between operators.
Tip: You should perform the pressure hold testing at ambient temperature
(25°C ± 5°C) to assure consistent results.
Tip: Cytiva performs air diffusion tests at 30 psig (2 barg) or higher. A pressure
hold or pressure decay test may be performed at lower pressures. For some
MF cartridges this is a requirement, as at higher pressures you may either
exceed the bubble point of the cartridge or the maximum TMP specification
for the cartridge.
Tip: Please note that there is a direct linear relationship between diffusion and
test pressure. Testing at half the test pressure will result in half the diffusive
flow, therefore the diffusion specification and the maximum pressure hold
specification must be adjusted down to account for any changes in test pres-
sure that are different from those specified.

Integrity testing User Manual 18117173 AD 29

6.4 Build a pressure hold test stand
Gather materials
Gather the following materials to construct a pressure hold test stand:
• Pressure regulator—air and gas
• Pressure gauge—air and gas, 0 to 60 psig (0 to 4 barg)
• On/off valve—air/gas (V1)
• Sterilizing grade gas filter (phobic filter)
• Permeate port diaphragm valve or pinch clamp for permeate port
• Flexible tubing
• Cap for permeate port
• Cap for retentate port
• Pressurized, filtered air or nitrogen supply

Assemble the pressure hold test stand

Assemble the pressure hold test stand using the parts list and standard laboratory tech-
niques (Fig. 6.1, on page 29).

Using an automated tester

Carefully inspect all sealing surfaces, O-rings, and gaskets for any damage or wear. Make
sure that all valves that are used on the upstream side of the system are integral. Make
sure that the system or tester is not located near a vent, door, autoclave, or any other
area where temperature changes may occur. Make sure the cartridge and hardware are
equilibrated to room temperature so that temperature changes will not occur during a
test.
Tip: When you first apply air pressure, some stabilization time will be required. A
30-second period will allow the system to both physically and thermally reach
a state of equilibrium. If after this equilibration period a pressure drop is still
observed, stop the test, repeat Stage 1 steps 2 through 5 and Stage 2A step 1
(see Stage 1—preparing the cartridge and test stand, on page 25 and Stage
2A—running the qualitative pressure hold test, on page 27). If the system still
fails to equilibrate and hold pressure, call the technical support team at
Cytiva.

30 Integrity testing User Manual 18117173 AD

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18117173 AD V:5 12/2024