Successful Sterile Filtration of a Squalane Emulsion
Martha Folmsbee, Ph.D, Scientific and Laboratory Services, Pall Corporation, Port Washington, NY, USA
Ross Turmell, Senior Technical Specialist, Pall Corporation, Covina, CA, USA
Filtration Occurs at Many Points in a Biopharmaceutical
Process
y
p
Surface View of 0.2 µm Rated Membrane Challenged
With Bacteria1
Brevundimonas diminuta
bacteria (~0.3 – 0.4 µm
by ~0.6 – 1.0 µm)
Vent
Media
Media
Filtration
Bioreactor
Many pore openings at
membrane surface
are > 0.2 µm
Clarification
Cell harvesting
UF/
DF
Formulation
and Filling
Final Bulk Filtration
DNA/HCP
Removal
Virus Filtration
UF/DF
Chromatography Steps
(capture & purification)
Edge View of 0.2 µm Rated Membrane Challenged
With Bacteria2
Me
Cutting Line
m
Su
br
rf a a n e
ce
Brevundimonas diminuta
bacteria
Sources of Contaminants (particulate or microbial)
Depth of
Membrane
~10 µm
Contamination from external sources
Process fluids like water, solvents
or buffers, etc.
Raw materials
Air/personnel/premises
Contamination generated within
the process
Wear from moving components like pumps or valves
Undesired components as by-products of a chemical reaction
or fermentation process (possible bacterial growth)
Oxidation and chemical decomposition of fluid components
over time or temperature changes
• Cells may penetrate
some distance into
membrane depth.
• Typical membrane
thickness ~40–150 µm.
Relative Sizes of Small Contaminants
Filter Challenge Tests
Bacteria
( > 0.3 m)
Filter efficiencies of sterilizing-grade filters can be determined
very effectively using bacterial challenge tests.
Suspensions of the test organisms are prepared and pumped
or transferred by a pressure vessel through the filter to be
tested.
Any bacteria that might have penetrated the filter tested
can be detected on a downstream-analysis membrane.
This is achieved by placing the analysis membrane to an agar
plate, which supports growth of the challenge bacteria and
allows them to grow to a bacterial colony.
Silica
particle
(20 m)
Red blood
cell (7 m)
Yeast
(3 m)
Schematic Setup for Bacterial Challenge Tests
Relative Sizes of Some Microorganisms
Regulated
air inlet
Analysis
Membrane filter
Brevundimonas diminuta
Pressure vessel
with bacterial
suspension
Bacteria colonies on
an analysis disc after
incubation
on an agar
plate Drain
Titer Reduction (TR)
For microbial filters, filter efficiencies
are expressed as
Key Particle Removal Mechanisms
Two Retention mechanisms work to
ensure that particles stay in place.
Titer reduction or TR
TR is measured in bacterial challenge tests
as described in Pall validation guides
P
P D
D U
H
H W
U
V
W
V
Q
S Z
X R
V G
P V
V P
L
Q
V
L
D Q
J D
U
J
R
U
R R
U
R
F
L
U
F
P L
P
I R
I
R
75
Three Removal mechanisms work to
get the particles to interact with the
filter matrix.
Diffusional
Diffusional interception
intercept
Direct
interception
Inertial
impaction
Mechanical
retention
Adsorption
For sterilizing grade filters, the downstream count has to be zero;
therefore the titer reduction claim is expressed as > the total challenge
count.
Contact: +800.717.7255 (USA) • +41 (0)26 350 53 00 (Europe) • +65 6389 6500 (Asia/Pacific) • E-mail: biopharm@pall.com • Web: www.pall.com/biopharm
Results of a Bacterial Challenge of a 0.2 µm rated
Fluorodyne EX filter with a Squalane Emulsion at
a test pressure of 60 psid*
Flux
(mL/min/cm2)
14.0
10.7
14.7
Filter
1
2
3
Most filter validation are successful, however, emulsions
and similar fluids can lead to increased risk of bacterial
penetration (N=267)
Results of a Bacterial Challenge of a 0.2 µm rated
Fluorodyne EX filter with a Squalane Emulsion at
a test pressure of 30 psid*
6
Salts/chelator
Uncategorized
21
Challenge
Level
(CFU/cm2)
4.9 x 107
4.9 x 107
4.9 x 107
Total Bacterial
Recovery
(CFU/Filter
Effluent)
0
0
0
*Penetration through a 0.45 rated control filter was detected. All test filters passed
pre- and post Bubble Point Integrity Tests
Category of filtered fluids which resulted in reduced
bacterial retention (%)
38
Total
Bacterial
Challenge
(CFU/Filter)
6.8 x 108
6.8 x 108
6.8 x 108
Flux
(mL/min/cm2)
4.8
4.8
2.6
Filter
1
2
3
Blood products (and related)
Lipid and Lipid-like
Surfactant containing
Liposomal
Total
Bacterial
Challenge
(CFU/Filter)
5.8 x 108
5.8 x 108
5.8 x 108
Challenge
Level
(CFU/cm2)
4.2 x 107
4.2 x 107
4.2 x 107
Total Bacterial
Recovery
(CFU/Filter
Effluent)
0
0
0
*Penetration through a 0.45 rated control filter was detected. All test filters passed
pre- and post Bubble Point Integrity Tests
31
Conducting bacterial retention tests prior to finalization of
the manufacturing process may be beneficial for final-fill
applications involving sterile filtration of emulsions and
similar fluids
Area
High Risk Fluid
Benefit
• Determines likelihood of obtaining sterile product
prior to conducting Filter Validation Studies
• Minimizes laboratory re-work due to failed
bacterial retention
Process Optimization
• Helps evaluate likelihood of success for bacterial
(incl. High Risk Process)
retention when developing a new process or
changing process parameters
• Risk may be based on process parameters, filter
type, fluid, or all three
Results of a Bacterial Challenge of a 0.2 µm rated
Fluorodyne EX filter with a Squalane Emulsion at
a test pressure of 10 psid*
Flux
(mL/min/cm2)
2.4
2.1
1.7
Filter
1
2
3
Total
Bacterial
Challenge
(CFU/Filter)
7.0x 108
7.0 x 108
7.0 x 108
Challenge
Level
(CFU/cm2)
5.1 x 107
5.1 x 107
5.1 x 107
Total Bacterial
Recovery
(CFU/Filter
Effluent)
0
0
0
*Penetration through a 0.45 rated control filter was detected. All test filters passed
pre- and post Bubble Point Integrity Tests
Results of Particle Size Analysis Pre and Post-filtration
Through a 0.2 µm-rated Fluorodyne EX Filter
180
160
Challenge
filter
Acholeplasma
laidlawii
All filters showed complete retention at 10, 30 and 60 psid
There was no change in emulsion particle (the drug delivery
vesicle) size distribution post-filtration (data available for the 30
and 60 psid tests only)
Provides an example of the benefits of early screening to provide a technical solution for complex applications
This test validates that the filter produces sterile
filtrate under simulated worst-case process
conditions
Includes three test filters from three different lots
– One lot number is representative of minimum
specification
filter membrane
Typically uses Brevundimunas diminuta (ATCC 19146)
– Controlled culture conditions (ASTM F838-15)
– Minimal size (B. diminuta 0.3 x 0.8 µm)
– Monodispersed
– Penetration of 0.45 µm rated control filter assures
appropriate size of bacterial cells
– Total challenge ≥ 1 x 107 CFU/cm2
Analyzes total effluent for sterility
How a Sterilizing Grade Filter is Defined (Rated)
Contamination generated during maintenance
Debris from cleaning towels
Grease and lubricants
Manufacturing debris from newly installed components
Pencil
point
(40 m)
Process Specific Bacterial Retention Summary
2 2
Industry standards require that
sterilizing filters are challenged with
the microorganism Brevundimunas
diminuta at a minimum concentration
of 107 CFU /cm2 of effective filter
area. Regulatory and industry expectations are that filters
challenged according to this method provide a sterile effluent.
RESULTS AND SUMMARY
Example of an pre-screening bacterial challenge test with
an emulsion fluid (squalane based (minus a drug product)
0.75% Tween 80
0.75% Span 85
5.0% Squalane
MATERIALS AND METHODS
140
Particle Count (N=3)
BACKGROUND
120
100
Pre-filtration
80
30 psid
60 psid
60
40
Bacterial Retention Test
Test Solution Pre-Filter:
Supor® Grade EKV (Part
Number KA3EKVP1G)
Test Filter: 0.2 micron rated
Fluorodyne EX Grade EDF
(Part Number FTKEDF)
Test Solution: 5% Squalane
in water emulsion
Test Pressure: 10, 30 and
60 psid
20
Particle Sizing
Laser Diffraction technology
from Horiba (Horiba LA 950).
The refractive index was
1.470-0.010i and the
diluent was Deionized
water (17.1 mega ohms)
© 2015, Pall Corporation. Pall,
0
d10
d50
d90
Particle Size Range (nm)
ACKNOWLEDGEMENTS
Angel Lorenzo, Kevin Marino, Daniel Eshete, Julie Grace at
Pall Corporation. Special thanks to Dr. Yang Su, Kyle Jandrasitz,
Yuxian Zhang, and Steven Mesite at Microfluidics International
Corporation for formulating and producing the emulsion and for
all particle size measurements.
, Fluorodyne, and Supor are trademarks of Pall Corporation. ® indicates a trademark registered in the USA. 11/15, GN15.6417