Filtration Occurs at Many Points in a BiopharmaceuticalProcess
Sources of Contaminants (particulate or microbial)
Contamination from external sourcesProcess fluids like water, solvents or buffers, etc.Raw materialsAir/personnel/premises
Contamination generated within the process
Wear from moving components like pumps or valvesUndesired components as by-products of a chemical reactionor fermentation process (possible bacterial growth)Oxidation and chemical decomposition of fluid componentsover time or temperature changes
Contamination generated during maintenanceDebris from cleaning towelsGrease and lubricantsManufacturing debris from newly installed components
Relative Sizes of Small Contaminants
Relative Sizes of Some Microorganisms
Key Particle Removal Mechanisms
Surface View of 0.2 µm Rated Membrane Challenged With Bacteria1
Edge View of 0.2 µm Rated Membrane Challenged With Bacteria2
How a Sterilizing Grade Filter is Defined (Rated)
Industry standards require that sterilizing filters are challenged withthe microorganism Brevundimunasdiminuta at a minimum concentrationof 107 CFU /cm2 of effective filterarea. Regulatory and industry expectations are that filters challenged according to this method provide a sterile effluent.
Filter Challenge Tests
Filter efficiencies of sterilizing-grade filters can be determinedvery effectively using bacterial challenge tests.Suspensions of the test organisms are prepared and pumpedor transferred by a pressure vessel through the filter to betested.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 agarplate, which supports growth of the challenge bacteria and allows them to grow to a bacterial colony.
Schematic Setup for Bacterial Challenge Tests
Titer Reduction (TR)
Process Specific Bacterial Retention Summary
This test validates that the filter produces sterile filtrate under simulated worst-case process conditionsIncludes 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
Most filter validation are successful, however, emulsionsand similar fluids can lead to increased risk of bacterialpenetration (N=267)
Category of filtered fluids which resulted in reduced bacterial retention (%)
Conducting bacterial retention tests prior to finalization ofthe manufacturing process may be beneficial for final-fillapplications involving sterile filtration of emulsions andsimilar fluids
Area Benefit
High Risk Fluid • 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
Example of an pre-screening bacterial challenge test withan emulsion fluid (squalane based (minus a drug product)
0.75% Tween 800.75% Span 855.0% Squalane
All filters showed complete retention at 10, 30 and 60 psidThere was no change in emulsion particle (the drug deliveryvesicle) size distribution post-filtration (data available for the 30and 60 psid tests only)Provides an example of the benefits of early screening to pro-vide a technical solution for complex applications
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*
Total Total Bacterial Bacterial Challenge Recovery
Flux Challenge Level (CFU/Filter Filter (mL/min/cm2) (CFU/Filter) (CFU/cm2) Effluent)
1 14.0 6.8 x 108 4.9 x 107 0
2 10.7 6.8 x 108 4.9 x 107 0
3 14.7 6.8 x 108 4.9 x 107 0*Penetration through a 0.45 rated control filter was detected. All test filters passedpre- and post Bubble Point Integrity Tests
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*
Total Total Bacterial Bacterial Challenge Recovery
Flux Challenge Level (CFU/Filter Filter (mL/min/cm2) (CFU/Filter) (CFU/cm2) Effluent)
1 4.8 5.8 x 108 4.2 x 107 0
2 4.8 5.8 x 108 4.2 x 107 0
3 2.6 5.8 x 108 4.2 x 107 0*Penetration through a 0.45 rated control filter was detected. All test filters passedpre- and post Bubble Point Integrity Tests
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*
Total Total Bacterial Bacterial Challenge Recovery
Flux Challenge Level (CFU/Filter Filter (mL/min/cm2) (CFU/Filter) (CFU/cm2) Effluent)
1 2.4 7.0x 108 5.1 x 107 0
2 2.1 7.0 x 108 5.1 x 107 0
3 1.7 7.0 x 108 5.1 x 107 0*Penetration through a 0.45 rated control filter was detected. All test filters passedpre- and post Bubble Point Integrity Tests
Results of Particle Size Analysis Pre and Post-filtrationThrough a 0.2 µm-rated Fluorodyne EX Filter
Successful Sterile Filtration of a Squalane EmulsionMartha Folmsbee, Ph.D, Scientific and Laboratory Services, Pall Corporation, Port Washington, NY, USA
Ross Turmell, Senior Technical Specialist, Pall Corporation, Covina, CA, USA
BACKGROUND
y p
Media
MediaFiltration
Chromatography Steps (capture & purification)
UF/DF Virus FiltrationDNA/HCPRemovalFinal Bulk Filtration
Vent
Bioreactor
Cell harvesting
Clarification
Formulation and Filling
UF/DF
© 2015, Pall Corporation. Pall, , Fluorodyne, and Supor are trademarks of Pall Corporation. ® indicates a trademark registered in the USA. 11/15, GN15.6417Contact: +800.717.7255 (USA) • +41 (0)26 350 53 00 (Europe) • +65 6389 6500 (Asia/Pacific) • E-mail: [email protected] • Web: www.pall.com/biopharm
Pencil point (40 m)
Red blood cell (7 m)
Silica particle (20 m)
Bacteria ( > 0.3 m)
Yeast (3 m)
Brevundimonas diminuta
Acholeplasma laidlawii
laidlawiiAcholeplasma
laidlawiiAcholeplasma
Three Removal mechanisms work to get the particles to interact with the
filter matrix.
Two Retention mechanisms work to ensure that particles stay in place.
Direct interception
Diffusional interception
Inertial impaction
Mechanical retention Adsorption interception
Direct
fusional intercept fffusional interception Di
interception impaction Inertial
fusional interception
retention Mechanical
Adsorption Adsorption interception impaction Adsorption Adsorption
Brevundimonas diminutabacteria (~0.3 – 0.4 µm
by ~0.6 – 1.0 µm)
Many pore openings atmembrane surface
are > 0.2 µm
Brevundimonas diminutabacteria
• Cells may penetratesome distance into membrane depth.
• Typical membrane thickness ~40–150 µm.
Cutting LineMembraneSurface
Dep
th o
f M
embr
ane
Bacterial Retention TestTest Solution Pre-Filter:Supor® Grade EKV (PartNumber KA3EKVP1G)Test Filter: 0.2 micron ratedFluorodyne EX Grade EDF(Part Number FTKEDF)Test Solution: 5% Squalanein water emulsion
Test Pressure: 10, 30 and 60 psid
Particle SizingLaser Diffraction technologyfrom Horiba (Horiba LA 950).The refractive index was1.470-0.010i and the diluent was Deionized water (17.1 mega ohms)
ACKNOWLEDGEMENTSAngel 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 InternationalCorporation for formulating and producing the emulsion and forall particle size measurements.
Challengefilter
AnalysisMembrane filter
Drain
Pressure vesselwith bacterialsuspension
Regulated air inlet
Bacteria colonies onan analysis disc afterincubation on an agarplate
For microbial filters, filter efficiencies
are expressed as
TR is measured in bacterial challenge tests
as described in Pall validation guides
Titer reduction or TR
For sterilizing grade filters, the downstream count has to be zero;
therefore the titer reduction claim is expressed as > the total challenge
count.
2 2 6
21
31
38
Salts/chelator
Uncategorized
Blood products (and related)
Lipid and Lipid-like
Surfactant containing
Liposomal
0
20
40
60
80
100
120
140
160
180
d10 d50 d90
Part
icle
Cou
nt (N
=3)
Particle Size Range (nm)
Pre-filtration
30 psid
60 psid
RESULTS AND SUMMARY
MATERIALS AND METHODS
~10 µm
