VALIDATION GUIDE. for WaterSep Hollow Fiber Cartridges. Doc. No. VGWA2012REV1.0

Transcription

1 VALIDATION GUIDE for WaterSep Hollow Fiber Cartridges Doc. No. VGWA2012EV1.0

2 Terms of Sale WaterSep sells all goods and services per the terms and conditions of sale as specified by the WaterSep sales agreement. To receive a copy of these terms and conditions, or to comment on our products, contact us at: WaterSep Technology Corporation 420 Maple Street, Suite 1 Marlborough, MA USA Telephone: , extension 204 Fax: Contact@WaterSep.net All third party trademarks are the property of their respective owners by WaterSep Bioseparations. All rights reserved. 2 WaterSep Validation Guide

3 CONTENTS List of Tables 5 List of Figures 5 Chapter 1 Introduction How this Validation Guide Can Help You 6 Intended Audience 6 Getting Help 7 What is Process Validation? 7 Chapter 2 Product Information Lab-, Pilot- and Production-Scale Hollow Fiber Cartridges 9 Intended Applications 9 Cartridge Design 10 Cartridge Labeling and Catalog Numbers 11 Materials of Construction 14 Packaging 14 Product Specifications 15 Void Volume of Hollow Fiber Cartridges 15 Membrane Performance 20 Cartridge Performance 20 Storage of New and Used Cartridges 22 Preparing Your Cartridge for Use 22 Chapter 3 Validation Information Membrane Water Flux 23 Membrane Solute ejection 24 Cartridge Integrity Test 25 Non-Destructive Testing 25 Integrity test - Pressure Hold Test 25 eference values for WaterSep Factory Membrane difusion test. 27 Cartridge Water Flux 27 Cartridge Crossflow 30 Cartridge Chemical Compatibility Study 33 Cartridge insing Study 36 Hollow Fiber Cartridge Scalability Studies 37 WaterSep Validation Guide 3

4 Chapter 4 Product Safety Biocompatibility Studies 41 Chapter 5 Quality Assurance Information Certificate of Compliance 42 Appendices Appendix I Additional Documentation 43 Appendix II Test Procedures and eports 44 Index Index 45 4 WaterSep Validation Guide

5 LIST OF TABLES Table 1. Overview of the hollow fiber cartridges described in this validation guide 9 Table 2. Physical characteristics of MiniDiscover, Discovery, and Explorer HF Cartridges 16 Table 3. Physical characteristics of Investigator and BioProducer cartridges 17 Table 4. Void volumes of WaterSep hollow fiber cartridges 18 Table 5. Air flow specifications for hollow fiber cartridges 21 Table 6. Water permeability of WaterSep membranes 23 Table 7. esults of solute passage through WaterSep membrane (% P percent passage) 24 Table 8. esults of membrane integrity tests for Investigator12 Cartridges 27 Table 9. Water flux results for an Investigator12 hollow fiber cartridges 28 Table 10. Chemical compatibility list for WaterSep cartridges ( = recommended, L = limited exposure, N = not recommended, U = unknown) 33 Table 11. esults from the E. Coli lysate clarification scalability study 39 Table 12. Helpful information available at 43 Table 13. Test reports available from 44 LIST OF FIGUES Figure 1. Main parts of a WaterSep hollow fiber cartridge 10 Figure 2. Cartridge showing hollow fibers encapsulated in the hollow fiber housing 11 Figure 3. Typical hollow fiber cartridge label show information helpful to users 12 Figure 4. Identifying cartridge properties by catalog number 13 Figure 5. Clean water flux for an Investigator12, 30K MWCO, 1.0 mm ID 28 Figure 6. Clean Water flux for an Investigator12, 300K MWCO, 1.0 mm ID 29 Figure 7. Clean Water flux for an Investigator12, 0.2 um, 1.0 mm ID 29 Figure 8. Delta P versus crossflow with water for Producer12/24/41 cartridges 31 Figure 9. Delta P versus crossflow with water for Investigator12/24/41 cartridges 32 Figure 10. Comparison of water permeability (NWP) and retention of the membrane before and after 10 caustic cycles 35 Figure 110. eults of Extractables Level vs Volumetric Thoughput of Purified Water (Liters per m2) 37 Figure 12. Pressure and permeate flux profiles for the Explorer12 cartridge 38 Figure 13. Pressure and permeate flux profiles for the Explorer24 cartridge 39 Figure 14. Optimization results of scalability study using Explorer12 and Investigator12 cartridges 40 Figure 15. Example of a cartridge certificate of compliance 42 WaterSep Validation Guide 5

6 Introduction CHAPTE 1 OVEVIEW HOW THIS VALIDATION GUIDE CAN HELP YOU WaterSep created this validation guide to help scientists and engineers use our hollow fiber cartridges properly and efficiently. You can benefit many ways from reading and understanding the information in this validation guide: You can save considerable time when setting up and using your hollow fiber cartridge. You can obtain consistent results and extend the service life of your cartridge. You can find the information you need to help validate your hollow fiber cartridge system to meet FDA regulations. WHAT YOU WILL LEAN Designing and validating a hollow fiber cartridge system to meet FDA requirements involves applying technical knowledge in an organized fashion. While the path to system design and validation can take many directions, the performance and specifications of the hollow fiber cartridge remain constant. You will need the following information to validate your WaterSep cartridges within your application: Product labeling, materials of construction, cartridge physical characteristics, and product performance specifications esults of our cartridge integrity, performance, compatibility, and scalability studies esults of extractable, USP, hemolysis, and cytotoxicity tests performed by independent testing laboratories Operational, quality control, and regulatory support documents INTENDED AUDIENCE This validation guide was written for scientists and engineers who have laboratory operation and process engineering skills. If you need assistance, or do not fully understand the information in this guide, contact WaterSep Bioseparations for support. 6 WaterSep Validation Guide

7 Introduction GETTING HELP WaterSep engineers are membrane filtration experts. If you have questions or need specific product or application information, please contact our technical support team. WaterSep Technology Corporation 420 Maple Street, Suite 1 Marlborough, MA USA Telephone: x204 Fax: Contact@WaterSep.net WHAT IS POCESS VALIDATION? If you are new to process validation, the FDA offers the following introduction as quoted from CPG Sec Process Validation equirements for Drug Products and Active Pharmaceutical Ingredients Subject to Pre-Market Approval. "Validation of manufacturing processes is a requirement of the Current Good Manufacturing Practice (CGMP) regulations for finished pharmaceuticals (21 CF and ), and is considered an enforceable element of current good manufacturing practice for active pharmaceutical ingredients (APIs) under the broader statutory CGMP provisions of section 501(a)(2)(B) of the Federal Food, Drug, and Cosmetic Act. A validated manufacturing process has a high level of scientific assurance that it will reliably produce acceptable product. The proof of validation is obtained through rational experimental design and the evaluation of data, preferably beginning from the process development phase and continuing through the commercial production phase. efer also to the Guideline of General Principles of Process Validation (May 1987, originally published by CDE, CBE, and CDH and presently recognized by CDE, CBE, and CVM)1. (Note: The guideline is under revision as of the date of this CPG.) Before commercial distribution begins, a manufacturer is expected to have accumulated enough data and knowledge about the commercial production process to support post-approval product distribution. Normally, this is achieved after satisfactory product and process development, scale-up studies, equipment and system qualification, and the successful completion of the initial conformance batches. Conformance batches (sometimes referred to as "validation" batches and demonstration batches) are prepared to demonstrate that, under normal conditions and defined ranges of operating parameters, the commercial scale process appears to make acceptable product. WaterSep Validation Guide 7

8 Introduction Prior to the manufacture of the conformance batches the manufacturer should have identified and controlled all critical sources of variability." Guidelines for validation of biological systems and processes can be found in publications from the FDA. This validation guide provides you with information that can facilitate validation of your WaterSep products and systems. In addition, WaterSep can provide you with additional membrane and product information specific to your particular needs. For assistance, contact WaterSep at , extension 204. As you begin to validate your system, it is helpful to know that you must accomplish three qualifications: 1 An installation qualification (IQ) that verifies that the hollow fiber membrane process and supporting equipment can consistently operate within established limits and specifications. 2 An operating qualification (OQ) that verifies that the process can consistently reproduce operating results and that the process is effective. 3 A performance qualification (PQ) that verifies, through operational testing and data, that the finished product produced by the specific process meets and conforms to the product release specifications for functionality and safety. 8 WaterSep Validation Guide

9 Product Information CHAPTE 2 PODUCT INFOMATION LAB-, PILOT- AND PODUCTION-SCALE HOLLOW FIBE CATIDGES WaterSep manufactures hollow fiber cartridges for lab-, pilot- and production-scale separation operations (Table 1). The cartridges are designed for linear scale-up, offering consistency in design elements such as materials of construction, fiber configuration, and fiber length. Other product benefits include low fouling performance and easy cleaning. Table 1. Overview of the hollow fiber cartridges described in this validation guide Cartridge Type Applications ange of Surface Areas ange of Sample Volumes/Cartridge Lengths (Inches) MiniDiscovery Lab ft 2 ( cm 2 ) ml 12 and 24 Discover Lab ft 2 ( cm 2 ) ml 12 and 24 Explorer Lab ft 2 ( cm 2 ) ml 12, 24, and 41 Investigator Pilot ft 2 ( m 2 ) 1 25 L 12, 24, and 41 BioProducer Production ft 2 ( m 2 ) L 12, 24, and 41 Maximizer Production ft 2 ( m 2 ) > 300 L 24 and 41 INTENDED APPLICATIONS The intended applications in the biopharmaceutical/bioindustrial sectors include crossflow microfiltration and ultrafiltration for: Primary recovery/clarification Vaccine purification and concentration Cell washing Cell harvesting Bacteria separation in fermentation broths Debris removal post-centrifugation Primary recovery of recombinant biopharmaceuticals Macromolecule concentration and diafiltration (enzymes, antibodies, proteins, and viruses) WaterSep Validation Guide 9

10 Product Information CATIDGE DESIGN Hollow fiber cartridges consist of a housing with an inlet port for feed, an outlet port for retentate, and two outlet ports for the permeate (Figure 1). The housings contains hollow fiber membrane made from an antifouling modified polyethersulfone (PES) composition (Figure 2). Fluid flows into the feed port, through the lumens of the hollow fibers and out of the retentate port, returning to the feed tank. Fluid and solutes that pass through the walls of the hollow fiber membrane are flow out the two permeate ports. Cartridge size, port size, port type, and the number and size of hollow fibers vary according to the cartridge type. Figure 1. Main parts of a WaterSep hollow fiber cartridge Outlet (retentate) Housing Outlets (permeate) Inlet (Feed) 10 WaterSep Validation Guide

11 Product Information Figure 2. Cartridge showing hollow fibers encapsulated in the hollow fiber housing Encapsulated fibers Housing CATIDGE LABELING AND CATALOG NUMBES WaterSep hollow fiber cartridges include labels that provide useful information (Figure 3). Cartridge labels include the following information: Company name Cartridge name Membrane material and pore size Inside diameter of the fibers Membrane surface area Cartridge nominal dimensions Bar code Maximum operating temperature Maximum operating pressure Catalog (part) number Lot number WaterSep Validation Guide 11

12 Product Information When ordering cartridges, the catalog number enables you to identify the cartridge properties (Figure 4). The bar code enables you to identify each cartridge using a universal bar code scanner. The lot number is also unique to each cartridge to ensure traceability. Figure 3. Typical hollow fiber cartridge label show information helpful to users 12 WaterSep Validation Guide

13 Product Information Figure 4. Identifying cartridge properties by catalog number WA DIS 12 LL Catalog number NMWC 001 = 1 K 003 = 3 K 005 = 5 K 010 = 10 K 030 = 30 K 050 = 50 K 100 = 100 K 300 = 300 K 500 = 500 K 750 = 750 K 910 = 0.1 µm 920 = 0.2 µm 945 = 0.45 µm 965 = 0.65 um Cartridge Type DIS = Discover EXP = Explorer INV = Investigator PO = BioProducer MAX = Maximizer Fiber Length 12 = 12 inches 24 = 24 inches 41 = 41 inches Product Code Designator WA = Modified PES Membrane BA = Modified PES Membrane Fiber Lumen Diameter (mm) 20 = 2 mm (special production) 10 = 1 mm 05 = 0.5 mm Type of Connector ML = Luer Lok (Mini Discover) LL = Luer Lok (Discovery) SO = 3/4-inch TC (Explorer) SO = 1-inch TC (Investigator) SG = 1.5-inch TC (BioProducer) SH= 2-inch TC (Maximizer SK = 3-inch TC (BioProducer) WaterSep Validation Guide 13

14 Product Information MATEIALS OF CONSTUCTION The materials of construction (and wetted components) of WaterSep hollow fiber cartridges include these: Membrane modified polyethersulfone (PES) composition Encapsulant FDA-compliant urethane Housing white polysulfone Polypropylene Screen made from 100% virgin polypropylene resin. PACKAGING New hollow fiber cartridges are packaged in a sealed polyethylene bag. The feed, retentate, and permeate ports are double capped to retain moisture and prevent contamination from external sources. Depending on the cartridge size, packaging consists of different types of foam and cardboard boxes. A label similar to the label affixed to the cartridge is affixed to the upper-left side of each box. The finished goods package also includes a certificate of analysis, operating instructions, and an application guide. 14 WaterSep Validation Guide

15 Product Information PODUCT SPECIFICATIONS You can purchase WaterSep hollow fiber cartridges in a variety of sizes from lab scale to production scale (Table 2 and Table 3). By design, WaterSep hollow fiber cartridges provide a linear and predictive scale-up process from laboratory to pilot-scale to manufacturing scale by using matching materials, fluid-path length, and performance characteristics. A typical scale-up path includes these products and volumes: PODUCT MiniDiscover/Discover Explorer Investigator BioProducer Maximizer VOLUME ml ml 1 25 L > 10 L > 300 L For additional scale-up information, contact WaterSep at , extension 204. VOID VOLUME OF HOLLOW FIBE CATIDGES The design of WaterSep hollow fiber cartridges maximizes surface area and minimizes hold-up volume and system working volume. The design elements increase efficiency and enable the highest possible concentration factors. Void volumes and other product characteristics for MiniDiscover/Discover, Explorer, Investigator, BioProducer and Maximizer hollow fiber cartridges are presented in Table 2, Table 3, and Table 4. WaterSep Validation Guide 15

16 Product Information Table 2. Physical characteristics of MiniDiscover, Discovery, and Explorer HF Cartridges MiniDiscover HF Cartridges Characteristic 12 Inch 24 Inch Dimensions in inches (mm) 3/8 x 12 (9.4 x 30) 3/8 x 24 (9.4 x 60) Membrane surface area in ft 2 (cm 2 ) (17.3) (35.6) ecommended batch volume per cartridge (ml) ecommended permeate flow rate (ml/hr) Up to 70 Up to140 Membrane cut-off (k) NMWC 1, 3, 5, 10, 30, 50, 100, 300, 500, 750 Membrane pore size (µm) 0.1, 0.2, 0.45, 0.65 Feed/retentate connectors Permeate connector Luer Lok Luer Lok Discover HF Cartridges Characteristic 12 Inch 24 Inch Dimensions in inches (mm) 3/8 x 12 (9.4 x 30) 3/8 x 24 (9.4 x 60) Membrane surface area in ft 2 (cm 2 ) (51.8) (106.9) ecommended batch volume per cartridge (ml) ecommended permeate flow rate (ml/hr) Up to 200 Up to 400 Membrane cut-off (k) NMWC 1, 3, 5, 10, 30, 50, 100, 300, 500, 750 Membrane pore size (µm) 0.1, 0.2, 0.45, 0.65 Feed/retentate connectors Permeate connector Luer Lok Luer Lok Explorer HF Cartridges Characteristic 12 Inch 24 Inch 41 Inch Dimensions in inches (mm) 0.5 x 12.3 (13 x 312) 0.5 x 23.8 (13 x 605) 0.5 x 41.8 (13 x 1062) Membrane surface area in ft 2 (cm 2 ) 0.16 (155) 0.34 (320) 0.62 (580) ecommended batch volume per cartridge (ml) , ,000 ecommended permeate flow rate (ml/hr) Up to 600 Up to 1,280 Up to 2,300 Membrane cut-off (k) NMWC 1, 3, 5, 10, 30, 50, 100, 300, 500, 750 Membrane pore size (µm) 0.1, 0.2, 0.45, 0.65 Feed/retentate connectors Permeate connector ½-inch TC Barbed hose 16 WaterSep Validation Guide

17 Product Information Table 3. Physical characteristics of Investigator and BioProducer cartridges Investigator HF Cartridges Characteristic 12 Inch 24 Inch 41 Inch Dimensions in inches (mm) 1.3 x 12 (33.4 x 305) 1.3 x 23.5 (33.4 x 597) 1.3 x 41 (33.4 x 1054) Membrane surface area in ft 2 (m 2 ) 1.4 (0.13) 2.3 (0.27) 5.5 (0.50) ecommended batch volume per cartridge (L) ecommended permeate flow rate (L/hr) Up to 5.2 Up to 11 Up to 20 Membrane cut-off (k) NMWC 1, 3, 5, 10, 30, 50, 100, 300, 500, 750 Membrane pore size (µm) 0.1, 0.2, 0.45, 0.65 Feed/retentate connectors Permeate connector 1-inch TC ½-inch TC BioProducer HF Cartridges Characteristic 12 Inch 24 Inch 41 Inch Dimensions in inches (mm) 3.5 x 15.0 (89 x 381) 3.5 x 13.5 (89 x 344) 3.5 x 26.5 (89 x 673) 3.5 x 25.0 (89 x 636) 3.5 x 44.5 (89 x 1130) 3.5 x 43.0 (89 x 1093) Membrane surface area in ft 2 (m 2 ) 13.5 (1.25) 29.3 (2.72) 54.0 (5.0) ecommended batch volume per cartridge (L) ecommended permeate flow rate (L/hr) Up to 50 Up to 110 Up to 200 Membrane cut-off (k) NMWC 1, 3, 5, 10, 30, 50, 100, 300, 500, 750 Membrane pore size (µm) 0.1, 0.2, 0.45, 0.65 Feed/retentate connectors Permeate connector Maximizer HF Cartridges 1.5-inch TC or 3-inch TC 1-inch TC Characteristic 24 Inch 41 Inch Dimensions in inches (mm) 4.62 x 28.5 (117.3 x723.9) 4.62 x 46.5 (117.3 x ) Membrane surface area in ft 2 (m 2 ) 54.0 (5.0) 110 (10) ecommended batch volume per cartridge (L) > 300 > 300 ecommended permeate flow rate (L/hr) Up 200 Up 400 Membrane cut-off (k) NMWC 1, 3, 5, 10, 30, 50, 100, 300, 500, 750 Membrane pore size (µm) 0.1, 0.2, 0.45, 0.65 Feed/retentate connectors Permeate connector 2-inch TC 1-inch TC WaterSep Validation Guide 17

18 Product Information Table 4. Void volumes of WaterSep hollow fiber cartridges Performance Properties Hold Up Volume Product Fiber ID (mm) No. of Fibers Surface Area (cm 2 ) Feed Side Holdup Volume (ml) Permeate Side Holdup Volume (ml) MiniDiscover12 MiniDiscover24 MiniDiscover41 Discover12 Discover24 Discover41 Explorer12 Explorer24 Explorer41 Investigator12 Investigator24 Investigator , , , WaterSep Validation Guide

19 Product Information Performance Properties Hold Up Volume Product Fiber ID (mm) No. of Fibers Surface Area (cm 2 ) Feed Side Holdup Volume (ml) Permeate Side Holdup Volume (ml) BioProducer12 BioProducer24 BioProducer41 Maximizer24 Maximizer , , , ,183 1, , ,194 3,126 51, ,937 2, ,868 3,425 1, ,768 5,701 97, ,086 4, ,837 5,722 1,769 WaterSep Validation Guide 19

20 Product Information MEMBANE PEFOMANCE The WaterSep hollow fiber membranes are manufactured using a unique combination of modified polyethersulfone (PES) and a proprietary spinning process that results in an asymmetric, void free low binding hollow fiber membrane with narrow pore size distribution and excellent antifouling and process flow properties. CATIDGE PEFOMANCE The exceptional uniformity of WaterSep hollow fiber membrane and the precision of the hollow fibers cartridge assembly process results in hollow fiber cartridges that perform consistently and show high lot-to-lot consistency. INHEENTLY SUPEIO CHAACTEISTICS WaterSep hollow fiber cartridges are characterized by: Antifouling modified polyethersulfone (m-pes) composition for regulatory satisfaction Void-free structure for sustained reliable performance Optimization for specific applications POVEN PEFOMANCE ADVANTAGES Low fouling and anti-dead spot design High flow rate and total capacity Easy and effective cleaning Long service life and better economics 20 WaterSep Validation Guide

21 Product Information Table 5. Air flow specifications for hollow fiber cartridges Product Name Membrane NMWC Air Flow per Cartridge ml/min/7 barg (ml/min/10 psig) MiniDiscover12 < 0.2 MiniDiscover24 < 0.4 Discover12 < 0.6 Discover24 < 1.1 Explorer12 < 1.6 Explorer24 < 3.4 Explorer41 1, 3, 5, 10, 30, < 6.2 Investigator12 50, 100, 300, 500, 750 < 14 Investigator24 KD < 30 Investigator41 < 55 BioProducer12 < 135 BioProducer24 < 293 BioProducer41 < 540 Maximizer24 < 540 Maximizer41 < 750 Product Name MiniDiscover12 Membrane Air Flow per Cartridge ml/min/0.35 barg (ml/min/5 psig) < 0.2 MiniDiscover24 < 0.4 Discover12 < 0.6 Discover24 < 1.1 Explorer12 < 1.6 Explorer24 < 3.4 Explorer41 < 6.2 Investigator12 0.1, 0.2, 0.45, 0.65 µm < 14 Investigator24 < 30 Investigator41 < 55 BioProducer12 < 135 BioProducer24 < 293 BioProducer41 < 540 Maximizer24 < 540 Maximizer41 < 750 WaterSep Validation Guide 21

22 Product Information STOAGE OF NEW AND USED CATIDGES NEW CATIDGES Store new hollow fiber cartridges unopened in their original packaging in a protected location and out of direct sunlight. The temperature of the storage area should remain between 4 C to 37 C (39 F to 99 F). Under these conditions, a new cartridge can be stored for 24 months without influencing performance or leading to product alterations. USED CATIDGES In most cases, you can clean and reuse WaterSep membrane cartridges until they reach the end of their service life as determined by integrity or flux decay testing. After cleaning your cartridge using the WaterSep Hollow Fiber Cleaning and Storage Procedure, you can store the cartridge for reuse at a later time. The maximum storage time depends on the process solutions and conditions to which the cartridge was exposed. PEPAING YOU CATIDGE FO USE You must pre-condition hollow fiber cartridges before use to: emove storage solutions Sanitize the cartridge and filtration system Verify the performance of the cartridge Obtain a clean membrane water flux at a specific TMP Condition the cartridge to the operating conditions (compatible buffer and ph) Verify the integrity of the cartridge You can learn about preparing cartridges for use by reading the WaterSep publication HF SOP-Preconditioning that you can download from our website WaterSep Validation Guide

23 Validation Information CHAPTE 3 VALIDATION INFOMATION The various product lines of WaterSep hollow fiber cartridges are constructed of the same materials and use identical design elements and fluid path length. Hence hollow fiber cartridge performance is scalable, and validating a cartridge at one size normally suffices for validating cartridges of other sizes. MEMBANE WATE FLUX A common way to measure clean membrane performance and determine the level membrane recovery after cleaning is to measure the water permeability of the membrane under controlled conditions. [However, membrane water flux does not necessarily have a direct correlation to process flux in a biological fluid stream.] The water permeability of WaterSep membranes, normalized to water viscosity at the testing temperature, are provided in Table 6. Table 6. Water permeability of WaterSep membranes Ultrafiltration Membrane NMWL Normalized Water Permeability (LMH/psi) 1 3 K K K K K K K K K Microfiltration Membrane 0.1 um > um > um > These numbers are based on Explorer-size quality control cartridges. WaterSep Validation Guide 23

24 Validation Information MEMBANE SOLUTE EJECTION WaterSep s hollow fiber membranes have very well defined and narrow retention/passage specifications. Every manufacturing batch is tested for quality with known solutes and must conform to highly-defined limits for rejection and passage. Each membrane cut-off is tested with two to three solutes (membrane markers). Table 7 lists the retention passage data for different membrane cut-offs. A narrow pore size distribution, guarantees consistent membranes batches over time and minimizes membrane batch-to-batch variations. Note that there are no overlap in retention/passage specifications, for various cut-offs/pore sizes. If a membrane does not have a clear cut-off point between particle retention and passage, the membrane may pass your product of interest or provide low product yield. There will be also significant performance variations between membrane batches (wide lot-to-lot variations). By testing the retention and passage of known solutes through the membrane under controlled conditions, and measuring the concentration of the solute in the retentate and permeate in comparison to the concentration of the original sample, you can determine the performance of the membrane. Table 7. esults of solute passage through WaterSep membrane (% P percent passage) PVP- K15 PVP- K30 PVP- K60S PVP-K90 Blue Dextran Bubble Point MW = 9700 MW = 66,800 MW = 396,000 MW = 1,570,000 MW = 2,000,000 IPA H2O NMWL %P %P %P %P %P psig 3 K < 20 5 K K < K > 70 < K > K > < K > 70 < K < K < K > < 10 > um > um > um > WaterSep Validation Guide

25 Validation Information CATIDGE INTEGITY TEST NON-DESTUCTIVE TESTING Non-destructive integrity testing can be completed on hollow fiber cartridges preand post-operation in order to prevent process failure and product loss. Detecting a failed hollow fiber cartridge can eliminate process delays and allow for rapid re-processing of the batch. There are two types of non-destructive testing: the bubble point test and the pressure hold/diffusion test. WaterSep recommends using the pressure hold test for both UF and MF membranes. (1K 750K and 0.1 um 0.65 um) Pressure hold, forward flow, and pressure decay are variations of the diffusion test. INTEGITY TEST - PESSUE HOLD TEST The pressure hold test, also known as pressure decay or pressure drop test, is an alternative to the diffusion test and is based on similar principles. In the pressure hold test, an accurate pressure gauge is used to monitor upstream pressure changes due to gas diffusion through the wetted hollow fiber membrane. The pressure hold value is dependent on the diffusional flow and upstream volume. It can be calculated using the following equation: Where: D = diffusion rate (cc/min) T = time (minutes) Pa = atmosphere pressure (1 Atm. or 14.7 psi) Vh = upstream volume of apparatus (cc) DP = pressure drop (bar or psi) WaterSep Validation Guide 25

26 Validation Information POCEDUE 1. Circulate water through the hollow fiber cartridge for 10 minutes. Ensure fluid flows from both permeate ports. 2. Attach an external perssure source to the feed/retentate side of the hollow fiber system. 3. Close the permeate side and open feed and retentate ports. 4. Pressurize the system slowly to the specified pressure. 10 psi for UF membranes, 5 psi for MF membranes. 5. Drain the system of any liquid upstream. 6. Close the retentate valve 7. Open the permeate valve. 8. Let the system equilibrate for 5 minutes, while any remaining liquid will pass through the HF cartridge. 9. Close the feed port, and monitor any pressure decay in the system. 10. The system/cartridge is integral if the pressure gauge reads a positive upstream pressure after 1 minute. Average Integrity Test esults Delta 0.7 barg (10 psig) Membrane NMWC (KD) Batch 1 Batch 2 Batch < 10 < 10 < 10 Average Integrity Test esults Delta P/min at 0.35 barg (5 psig) Membrane NMWC (µm) Batch 1 Batch 2 Batch < 5 < 5 < 5 26 WaterSep Validation Guide

27 Validation Information EFEENCE VALUES FO WATESEP FACTOY MEMBANE DIFUSION TEST. Table 8. esults of membrane integrity tests for Investigator12 Cartridges Average Airflow Integrity Test esults cc/min/ft 0.7 barg (10 psig) Membrane NMWC (KD) Batch 1 Batch 2 Batch Average Airflow Integrity Test esults ml/min/ft 2 at 0.35 barg (5 psig) Membrane NMWC (µm) Batch 1 Batch 2 Batch CATIDGE WATE FLUX Cartridge water flux for a specific cartridge and molecular weight cut off can provide valuable information when it is compared to membrane water flux data. The correlation factor for water flux between a membrane and a specific hollow fiber cartridge can also be used to correlate water flux for other sizes of hollow fiber cartridges. A cartridge water flux study was conducted on membranes with different molecular weight cut off values. Included in the study were multiple cartridges for each molecular weight cut-off. The goal was to determine the average water flux values for the cartridge. Table 9 shows the average water flux for Investigator12 cartridges with different molecular weight cut off values (membrane pore size). All the cartridges had 1,300-cm 2 of membrane surface area. WaterSep Validation Guide 27

28 Validation Information The cartridge water flux results are normalized to surface area and reported as LMH (liter/m²/hour)/psig (Figure 5, Figure 6, and Figure 7). Table 9. Water flux results for an Investigator12 hollow fiber cartridges Membrane Type Average Water Flux LMH/psi at 0.68 barg (10 psig) of TMP m-pes 30K MWCO 17.4 m-pes 300K MWCO 36.3 M-PES 0.2 um Figure 5. Clean water flux for an Investigator12, 30K MWCO, 1.0 mm ID 28 WaterSep Validation Guide

29 Validation Information Figure 6. Clean Water flux for an Investigator12, 300K MWCO, 1.0 mm ID Figure 7. Clean Water flux for an Investigator12, 0.2 um, 1.0 mm ID WaterSep Validation Guide 29

30 Validation Information CATIDGE COSSFLOW The crossflow rate is a critical process parameter for crossflow filtration processes. An optimized crossflow rate minimizes gel layer formation on the membrane surface, leading to optimum process flux and maximum transmission of ions and low molecular weight substances. Optimized crossflow, in combination with low transmembrane pressure (TMP), is particularly important for cell clarification processes where transmission of proteins, antibodies, and other high-molecular-weight target products is crucial. Insufficient crossflow can lead to increased gel layer formation. The result is a decrease in process flux and an increase in the rejection of low molecular solutes, both of which lower yield. The crossflow rate of a hollow fiber cartridge is proportional to the differential pressure (delta P) along the cartridge (i.e. the pressure difference between the feed and retentate port). The optimum crossflow rate is a function of the number of fibers in a hollow fiber cartridge, the inside diameter of the fibers, and the fluid stream characteristics. While the optimum cross flow rate for a family of hollow fiber cartridges does not vary with path length, the delta P needed to achieve the optimum cross flow rate can vary. For example, the optimum crossflow for a BioProducer12/24/41 is the same, although the delta P to achieve that flow rate varies as a function of the path length of the hollow fiber cartridge. WaterSep hollow fiber cartridges are designed for intra-cartridge consistency for delta P and the crossflow rate, and this design feature ensure consistent performance. Intracartridge inconsistency between delta P and crossflow results in process variances and potential product yield decay. Figure 8 and Figure 9 show the measured crossflow vs. delta P forbioproducer12/24/41 and Investigator12/24/41 HF cartridges. 30 WaterSep Validation Guide

31 Validation Information Figure 8. Delta P versus crossflow with water for Producer12/24/41 cartridges Delta P vs. Cross Flow for Producer12 Delta P (psi) Delta P Cross Flow L/min Delta P vs. Cross Flow for Producer24 Delta P (psi) Delta P Cross Flow L/min Delta P vs. Cross Flow for Producer41 Delta P (psi) Delta P Cross Flow L/min WaterSep Validation Guide 31

32 Validation Information Figure 9. Delta P versus crossflow with water for Investigator12/24/41 cartridges Delta P vs. Cross Flow for Investigator12 Delta P (psi) Delta P Cross Flow L/min Delta P vs. Cross Flow for Investigator24 Delta P (psi) Delta P Cross Flow L/min Delta P vs. Cross Flow for Investigator41 Delta P (psi) Delta P Cross Flow L/min 32 WaterSep Validation Guide

33 Validation Information CATIDGE CHEMICAL COMPATIBILITY STUDY GENEAL CONSIDEATIONS Certain chemicals can damage and degrade the membrane structure, or ultimately dissolve the membrane polymer. It is therefore important to be aware of the membrane s chemical compatibility and use compatible process and cleaning chemicals. Under normal operating conditions, WaterSep cartridges are resistant to commonly-used aqueous solutions within ph 1 14, bio-chemicals and most solvents, with the exception of aromatic compounds. Certain operating conditions and elevated temperature, can affect the membrane s compatibility. WaterSep advises you to complete your own chemical compatibility studies under you specific operating conditions and temperatures (Table 10). Table 10. Chemical compatibility list for WaterSep cartridges ( = recommended, L = limited exposure, N = not recommended, U = unknown) Chemical Compatibility Chemical Compatibility Acetic acid (diluted-5%) Acetic acid (med conc- 25%) Acetic acid (glacial) Acetone Acetonitrile Alconox (1%) Ammonium hydroxide Amyl acetate Amyl alcohol Aniline Benzene Benzyl alcohol Boric acid Brine Butyl acetate Butyl alcohol Butylaldehyde Carbon tetrachloride Chloroacetic acid Chloroform Chromic acid Citric acid (2%) Cresol L N N N N L N N N N N N N N N N Cyclohexane L Cyclohexanone N Diacetone alcohol N Dichloromethane L Dimethyl formamide N Dimethylsulfoxide (50%) L 1,4 Dioxane L Ethers N Ethyl acetate N Ethyl Alcohol Ethyl alcohol (15%) Ethyl alcohol (95%) L Ethylene dichloride N Ethylene glycol Ethylene oxide Formaldehyde (2%) Formaldehyde (30%) Formic acid (25%) Formic Acid (50%) Freon Gasoline L Glycerine / Glycerol Hexane Hexanol WaterSep Validation Guide 33

34 Validation Information Chemical Compatibility Chemical Compatibility Hydrochloric acid (diluted-5%) Hydrochloric acid (conc- 25%) Hydrochloric acid (conc- 37%) Perchloric acid (25%) Perchloroethylene Petroleum based oils Petroleum ether Phenol (0.5%) N N Hydrofluoric acid (25%) L Phenol (10%) L Hydrogen peroxide (30%) L Phosphoric acid (25%) L Iodine solutions N Potassium hydroxide (1N) Isobutyl alcohol Isopropanol Isopropyl acetate Isopropyl alcohol / Isopropanol Isopropyl ether Lactic acid Methyl acetate Methyl alcohol Methyl alcohol (98%) Methyl cellosolve Methyl chloride N N L N Potassium hydroxide (25%) Potassium hydroxide (50%) Propanol Pyridine Silicone oil Sodium hydroxide (0.1N) Sodium hydroxide (diluted-5%) Sodium hydroxide (25%) Sodium hydroxide (conc- 50%) N Methyl ethyl ketone N Sodium Hydroxide (conc) Methyl formate N Sodium hypochlorite Methyl isobutyl ketone N Sulfuric acid (diluted-5%) Methylene chloride N-methyl-2-pyrrolidone Mineral spirits Monochlorobenzene NALCON 7647 (<1%) NALCON 7678 (<1%) NALCON 7330 (<1%) Nitric acid (diluted-5%) Nitric acid (med conc- 25%) Nitric acid (6N) Nitric acid (conc-70%) Nitric acid (concentrated) Nitrobenzene Nitropropane Pentane Peracetic acid (0.1N) N N N L N L N N Sulfuric acid (med conc- 25%) Sulfuric acid (6N) Sulfuric Acid (conc) Tetrahydrofuran Toluene Trichloroacetic acid (25%) Trichlorobenzene Trichloroethane Trichloroethylene Triethylamine Turpentine Urea Urea (6N) Water Xylene L N N N L N N N N 34 WaterSep Validation Guide

35 Validation Information HOLLOW FIBE CAUSTIC STABILITY STUDY The performance of WaterSep hollow fiber cartridges after exposure to caustic was evaluated as caustic is used to clean and sanitize WaterSep s hollow fiber cartridges for re-use. After ten 1-hour caustic cycles of 0.5N NaOH at C, the permeability and retention properties remained stable. The membrane permeability (NWP) measurements showed a maximum decrease of 19% between the initial and final test cycle. This change is well within the acceptable standard deviation of the membrane specification. The retention properties as measured by this intermediate marker increased 18% indicating a minor shift towards a tighter NMWL of the membrane. This observed retention change for a marker on the steep selectivity slope denotes less than a 2% shift of the membrane 90 value. This is well within the membrane specification range. WaterSep m-pes hollow fiber membranes showed stable permeability and retention properties after repeated exposures to hot caustic cycles (Figure 10). See the appendix for a link to the complete study report. Figure 10. Comparison of water permeability (NWP) and retention of the membrane before and after 10 caustic cycles WaterSep Validation Guide 35

36 Validation Information CATIDGE INSING STUDY Prior to use, hollow fiber cartridges need to be pre flushed with WFI or purified water or buffer to remove any storage solution. New WaterSep hollow fiber cartridges contain a water/glycerin storage solution. If a cartridge is being reused, the typical storage solution is M NaOH. The amount of rinse water required to reach a baseline of < 1 ppm of extractables depends on the storage solution and rinsing agent. Hence, a study was performed to determine the rinse down volume required for reaching a baseline of extractables of 1 ppm. An Investigator24, with 2,700 cm 2 of membrane was used in the study. A total of 2,000 ml of purified water was first flushed through the cartridge at a delta P of 0.2 barg (3 psig) through the retentate port to drain with the permeate port closed. The retentate port was then closed, and 15 liters of purified water were flushed through the permeate port at 0.33 barg (5 psig) of TMP. Throughout the rinsing, water samples were collected and analyzed for extractables in ppm. esults indicate that a minimum of 11 liters of purified water (40 L/m 2 of membrane) was required for rinsing the hollow fiber lab cartridge (Figure 110). This rinsing volume should be proportionally adjusted for larger/smaller cartridges. The minimum rinsing volume 5 liters for 0.11 m 2 (1.2 ft 2 ) cartridges should be proportionally increased when larger cartridges with more membrane surface areas are used ( L/m 2 of membrane). 36 WaterSep Validation Guide

37 Validation Information Figure 110. eults of Extractables Level vs Volumetric Thoughput of Purified Water (Liters per m2) HOLLOW FIBE CATIDGE SCALABILITY STUDIES A key benefit of WaterSep hollow fiber cartridges is the capability to linearly and consistently scale your process up or down from lab-scale to pilot-scale to productionscale. Linear and consistent scaling reduces the costs and complexity of product development and troubleshooting. The similarity in flow geometry between the different product groups enables this capability. WaterSep performed these two studies to demonstrate the scalability characteristics of our cartridges: 1. An E. Coli lysate clarification study with Explorer12 and Explorer24 cartridges 2. A protein concentration study with Explorer12 and Investigator12 cartridges WaterSep Validation Guide 37

38 Validation Information E.COLI STUDY WITH EXPLOE12 AND EXPLOE24 CATIDGES This study demonstrates cartridge scalability and provides data to assist with scalability. The E. Coli lysate clarification study was conducted using Explorer12 and Explorer24 cartridges. The results of the study are illustrated in Figure 12, Figure 13, and Table 11. CONCLUSIONS The results indicate that TMP and flux data for production Explorer12 and Explorer24 have a scalability factor close to 1.0 in this application. Figure 12. Pressure and permeate flux profiles for the Explorer12 cartridge 38 WaterSep Validation Guide

39 Validation Information Figure 13. Pressure and permeate flux profiles for the Explorer24 cartridge Table 11. esults from the E. Coli lysate clarification scalability study Process Parameter Explorer12 NMWL = 750 KD Fiber Diameter = 1 mm Explorer24 NMWL = 750 KD Fiber Diameter = 1 mm Fluid path length (inches) Membrane surface area (cm 2 ) P feed (psi) P retentate (psi) TMP (psi) Flux clarification (LMH) Flux diafiltration (LMH) WaterSep Validation Guide 39

40 Validation Information POTEIN CONCENTATION STUDY WITH EXPLOE12 AND INVESTIGATO12 CATIDGES This study demonstrates cartridge scalability and provides data to assist with scalability. A protein concentration study was conducted using Explorer12 and Investigator12 cartridges. The results of the study are illustrated in Figure 14. Figure 14. Optimization results of scalability study using Explorer12 and Investigator12 cartridges CONCLUSIONS The Flux vs. TMP curves for an Explorer12 and an Investigator12 are almost identical and suggest that the scalability factor is close to 1.0 in this application. 40 WaterSep Validation Guide

41 Safety Information CHAPTE 4 PODUCT SAFETY BIOCOMPATIBILITY STUDIES WaterSep submitted samples of hollow fiber cartridges to Toxikon for biocompatibility analyses. The following summarizes the results of the testing. USP Class VI Test GLP Compliance Study Summary: Therefore, the test article, WaterSep hollow fiber cartridge, meets the requirements of USP guidelines, for Class VI Plastics 70 C. USP Physicochemical Test for Plastics GLP Compliance Study Summary: The purified water extract of the test article, WaterSep hollow fiber cartridge, meets the test criteria described in the USP Physicochemical Test for Plastics guidelines. You can review the Toxikon reports cited above by clicking on the links in Table 12. WaterSep Validation Guide 41

42 Quality Assurance Information CHAPTE 5 QUALITY ASSUANCE INFOMATION CETIFICATE OF COMPLIANCE WaterSep supplies a Certificate of Analysis with each cartridge. The Certificate of Analysis verifies that your new cartridge was tested and complied with WaterSep s quality assurance standards (Figure 15). Figure 15. Example of a cartridge certificate of compliance 42 WaterSep Validation Guide

43 Appendices APPENDICES APPENDIX I ADDITIONAL DOCUMENTATION You can learn more about WaterSep s standard technology and obtain additional information such as operating procedures and product sheets by visiting the WaterSep website or by clicking a link below (Table 12). Table 12. Helpful information available at Standard Operating Procedures Hollow Fiber Cleaning and Storage Procedure Preconditioning for Use Procedure Product Sheets MiniDiscover 12 MiniDiscover 24 Discover 12 Discover 24 Explorer12 Explorer24 Explorer 41 Investigator 12 Investigator 24 Investigator 41 BioProducer 12 BioProducer 24 BioProducer 41 WaterSep Validation Guide 43

44 Appendices APPENDIX II TEST POCEDUES AND EPOTS You can obtain the reports cited in this validation guide from the WaterSep website by clicking on the links below (Table 13). Table 13. Test reports available from Title of eport WaterSep Membrane and Cartridge egulatory and Bio-Compatibility Summary ev. 0 - AH Hollow Fiber Caustic Stability Study Toxikon Final GLP eport: G1 Class VI Test USP Toxikon Final GLP eport: G2 Physicochemical Test for Plastics USP 44 WaterSep Validation Guide

45 Index INDEX INDEX Appendices, 43 Applications, 9 Audience for this guide, 6 Biocompatibility, 41 Biocompatibility studies, 41 Cartridge additional information, 43 biocompatibility, 41 caustic stability study, 35 certificate of compliance, 42 chemical compatibility study, 33 cross-flow, 30 design, 10 information, 9 inlets and outlets, 10 integrity test, 25 materials of construction, 14 packaging, 14 quality assurance, 42 rinsing study, 36 storage, 22 Test procedures and reports, 44 water flux, 27 Cartridge scalability study, 37 Cartridges Protein concentration, 40 scalablity, 37 Certificate of compliance, 42 Chemical compatibility, 33 Contacting WaterSep, 7 Cross-flow rate, 30 Customer support, 7 Documentation additional information, 43 ing WaterSep, 7 Extractables, 41 GLP compliance, 41 Help, 7 List of figures, 5 List of tables, 5 Materials of construction, 14 Membrane solute rejection, 24 Membrane water flux, 23 Non-destructive testing, 25 Packaging, 14 Performance advantages, 20 Performance characteristics, 20 Preconditioning, 22 Pressure and permeate flux profiles, 38 Pressure hold test, 25 Process validation, 7 Protein concentration, 40 Quality assurance, 42 insing ph and conductivity reduction, 36 insing cartridges, 36 Scalability, 37 Specifications, 15 materialls of construction, 14 physical properties, 15 water flux, 23 Storage, 22 Support, 7 Test procedures and reports, 44 Test reports, 44 USP Class VI Test, 41 USP Physicochemical Test for Plastics, 41 Validation, 7 Validation guide purpose, 6 Validation information, 23 Void volume, 15 Water flux, 27 Water permeability, 23 Website, 7 WaterSep Validation Guide 45

46 WaterSep Technology Corporation 420 Maple Street, Suite 1 Marlborough, MA USA Telephone: x204 Fax: Contact@WaterSep.net