Filtration Occurs at Many Points in a Biopharmaceutical Process Sources of Contaminants (particulate or microbial) 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 Contamination generated during maintenance Debris from cleaning towels Grease and lubricants Manufacturing 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 Bacteria 1 Edge View of 0.2 μm Rated Membrane Challenged With Bacteria 2 How a Sterilizing Grade Filter is Defined (Rated) Industry standards require that sterilizing filters are challenged with the microorganism Brevundimunas diminuta at a minimum concentration of 10 7 CFU /cm 2 of effective filter area. 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 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. 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 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 10 7 CFU/cm 2 Analyzes total effluent for sterility Most filter validation are successful, however, emulsions and similar fluids can lead to increased risk of bacterial penetration (N=267) Category of filtered fluids which resulted in reduced bacterial retention (%) 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 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 with an emulsion fluid (squalane based (minus a drug product) 0.75% Tween 80 0.75% Span 85 5.0% Squalane 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 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/cm 2 ) (CFU/Filter) (CFU/cm 2 ) Effluent) 1 14.0 6.8 x 10 8 4.9 x 10 7 0 2 10.7 6.8 x 10 8 4.9 x 10 7 0 3 14.7 6.8 x 10 8 4.9 x 10 7 0 *Penetration through a 0.45 rated control filter was detected. All test filters passed pre- 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/cm 2 ) (CFU/Filter) (CFU/cm 2 ) Effluent) 1 4.8 5.8 x 10 8 4.2 x 10 7 0 2 4.8 5.8 x 10 8 4.2 x 10 7 0 3 2.6 5.8 x 10 8 4.2 x 10 7 0 *Penetration through a 0.45 rated control filter was detected. All test filters passed pre- 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/cm 2 ) (CFU/Filter) (CFU/cm 2 ) Effluent) 1 2.4 7.0x 10 8 5.1 x 10 7 0 2 2.1 7.0 x 10 8 5.1 x 10 7 0 3 1.7 7.0 x 10 8 5.1 x 10 7 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 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 BACKGROUND Media Media Filtration Chromatography Steps (capture & purification) UF/DF Virus Filtration DNA/HCP Removal Final 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, GN15.6417 Contact: +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 laidlawii Acholeplasma laidlawii Acholeplasma 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 f ff Di interception impaction Inertial fusional interception retention Mechanical Adsorption Adsorption interception impaction Adsorption Adsorption Brevundimonas diminuta bacteria (~0.3 – 0.4 µm by ~0.6 – 1.0 µm) Many pore openings at membrane surface are > 0.2 µm Brevundimonas diminuta bacteria • Cells may penetrate some distance into membrane depth. • Typical membrane thickness ~40–150 µm. Cutting Line Membrane Surface Depth of Membrane 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 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) 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. Challenge filter Analysis Membrane filter Drain Pressure vessel with bacterial suspension Regulated air inlet Bacteria colonies on an analysis disc after incubation on an agar plate For microbial filters, filter efficiencies are expressed as T R is measured in bacterial challenge tests as described in Pall validation guides Titer reduction or T R GRZQVWUHDPRUJDQLVPVPLFURRIXSVWUHDPRUJDQLVPVPLFURRI75 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 Particle Count (N=3) Particle Size Range (nm) Pre-filtration 30 psid 60 psid RESULTS AND SUMMARY MATERIALS AND METHODS ~10 µm