OPUS 5, cm Columns OPUS 45R, OPUS 60R Columns REGULATORY SUPPORT FILE

Transcription

1 OPUS 5, cm Columns OPUS 45R, OPUS 60R Columns REGULATORY SUPPORT FILE

2 The information contained in this document is subject to change without notice. Repligen Corporation makes no warranty of any kind with regard to this material, including, but not limited to, the implied warranties of merchantability and fitness for a particular purpose. Repligen Corporation shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance, or use of this material. No part of this document may be photocopied, reproduced, or translated to another language without the prior written consent of Repligen Corporation. For further information, please contact Repligen Corporation at Repligen Corporation. All rights reserved. The trademarks mentioned herein are the property of Repligen Corporation and/or its affiliate(s) or their respective owners. Customer Support sales@repligen.com (option 2) Technical Support (option 3) technical.support@repligen.com Repligen Corporation 41 Seyon Street Building #1, Suite 100 Waltham, MA

3 Contents 1. Introduction Product Information Product Overview Product Design Certificate of Analysis (CoA) Examples Pre-GMP OPUS Columns Certificate of Analysis (CoA) GMP Run Ready OPUS Columns Certificate of Analysis (CoA) Certificate of Analysis: Chromatograms Column Manufacturing and Packing Materials of Construction: Product Contact OPUS Physical Specifications Column Stability Warranty Material Safety Data Sheet Manufacturing Information Introduction Manufacturing Quality Assurance Standards and Policy Manufacturing Facilities Manufacturing Controls and SOPs OPUS Column Manufacturing Bioburden and Endotoxin Release Testing Shipping Qualification Summary Introduction Method: ISTA 2A and 2B Tests ISTA Test Methods Column Efficiency Test Methods Results: ISTA and Column Efficiency Tests Chromatographic Performance Test Results Conclusion Extractables and Leachables Introduction and Background OPUS Extractables Strategy USP<88> Biological Reactivity Tests, In Vivo CFR 21 Part 177 Indirect food Additives: Polymers USP General Chapters 661: Polypropylene Containers Animal Free and EMEA 410/ Extractable Test Protocol and Results Extruded PP: OPUS cm Columns Only... 46

4 5.4.2 Machined Compression Molded PP: OPUS 10 60/R cm Columns PP Mesh: OPUS 10 60/R cm Columns P/ E-Glass Composite: OPUS 45/R 60/R cm Columns Only Platinum Cured Silicone O-Ring: OPUS 10 60/R cm Columns Platinum Cured Silicone Braided Tubing: OPUS 10 60/R cm Columns Conclusions Leachables: OPUS Column Strategy Considerations for Leachables Testing References Appendix OPUS 5, 10-60/R cm ID Material Certificates (available upon request) Engineering Drawings OPUS 5 cm Column OPUS 10 cm Column OPUS 14 cm ID Column OPUS 20 cm Column OPUS 25 cm Column OPUS 30 cm Column OPUS 45 cm and 45R Column OPUS 60 cm and 60R Column Packaging OPUS 5 cm Column Packaging OPUS cm Column Packaging OPUS cm Column Packaging OPUS 45 cm Column Packaging OPUS 60 cm Column Packaging... 67

5 List of Tables Table 2.1 OPUS Pre-GMP and GMP Applications... 8 Table 2.2 Product Contact Materials Table 2.3 Physical Specifications Summary on OPUS cm Columns Table 2.4 Physical Specifications Summary on OPUS 45R - 60R Columns Table 2.5 Column Mass (Approximate Weight of Packed Columns) Table 2.6 Stability Study Schedule and Acceptance Criteria Table 2.7 Mechanical Stability Test Results Table 2.8 Performance Stability Study Results Table 2.9 Results of Bioburden and Endotoxin Testing Table 3.1 Repligen Water Specifications Compared with ASTM, USP Purified Water and Water for Injection Table 3.2 Parts Cleaning Specifications Table 4.1 ISTA 2A and 2B Testing Conditions (OPUS 5, cm Columns) Table 4.2 OPUS 10 cm, 20 cm and 45 cm Column Performance Data Table 5.1 Summary of USP <88> Plastics Classes Table 5.2 Polymer List and Regulatory Information List of Figures Figure 2.1 Pre-GMP OPUS Column Certificate of Analysis... 9 Figure 2.2 GMP Run Ready OPUS Column Certificate of Analysis Figure 2.3 OPUS Certificate of Analysis Testing Conditions and Chromatograms Figure 2.4 Example of OPUS cm ID Column Label Figure 2.5 OPUS Part Number Format Figure 2.6 Lifting Handles and Input/Output Ports on an OPUS 20 cm Column Figure 2.7 Castor Detail on an OPUS 45 cm Column Figure 2.8 Materials of Construction on OPUS 5 cm Column Figure 2.9 Materials of Construction on OPUS cm Columns Figure 2.10 Materials of Construction on OPUS cm Columns Figure 2.11 Materials of Construction on OPUS 45R - 60R Columns Figure 2.12 OPUS R Port Design and Materials of Construction Figure 3.1 OPUS Work Order Example (blank) Figure 4.1 OPUS 5 cm Column Shipping Container Design Figure 4.2 OPUS cm Column Shipping Container Design Figure 4.3 OPUS 45/R - 60/R Column Chipping Container Design Figure 4.4 OPUS 5 cm Column Compression and Drop Testing Figure 4.5 OPUS 20 cm Column Vibrational and Compression Testing Figure 4.6 OPUS 60/R Column Vibrational, Incline Impact and Drop Testing... 39

6 1. Introduction The (RSF) for OPUS (Open platform, User-specified) Pre-Packed Columns is intended to be used as: A guide for appropriate application use in process development, clinical, and commercial purification processes A guide to validation in manufacturing processes A support reference for CMC submissions for regulatory license approval A guide for supplier audits In place of a Drug Master File submission This regulatory support file covers all column IDs including OPUS 5, 10, 14, 20, 25, 30, 45, 60, 45R, 60R Columns. Unless otherwise specified all information applicable to OPUS 45 and 60 designs is also applicable to OPUS 45R and 60R designs. Repligen is committed to providing all relevant technical, manufacturing, and quality information, however, only non-confidential information is presented in this document. Confidential details may be made available upon request through a formal confidentiality agreement or as part of a supplier audit. Repligen s Quality Policy Copies of Repligen s quality policy, quality manual, and ISO certificate can be found on the following website, Safety Notices Follow all local regulations for safe disposal For laboratory and manufacturing production only Responsible Official The individual designated responsible for quality and regulatory affairs for Repligen, and to whom all correspondence or requests for audits should be addressed. Senior Director of Quality Telephone: cmcgrath@repligen.com 6

7 2. Product Information 2.1 Product Overview OPUS Pre-packed Columns (Open platform, User-specified ) columns are designed to perform chromatographic purification of biological molecules as part of the production process in either GMP or non-gmp applications. The OPUS Pre-packed Column platform offers an alternative to traditional self-pack glass columns, and can be reliably packed with nearly any bioprocessing resin. To accommodate a wide range of biopharmaceutical applications, OPUS columns are configurable for bed height and available in a range of industry standard internal diameters. 2.2 Product Design The OPUS 5, 10 60/R cm Column platform is designed to meet the requirements of GMP manufacturing in the pharmaceutical and biopharmaceutical industries for campaign-use applications. Platformable OPUS Columns are configurable to accommodate a wide range of purification applications for vaccines, monoclonal antibodies, and recombinant proteins. For example, an end user can configure an OPUS Column by choosing a diameter and specifying: Column bed height Chromatography resin Repligen recommended release criteria, or customized release criteria (ex: plate count, asymmetry, storage solution, etc.) Quality Standards In order to meet the needs of GMP manufacturing, OPUS 5, and 10 60/R cm ID columns are manufactured in the U.S.A. under the following quality standards: Repligen maintains an ISO 9001 compliant Quality Management System that is currently certified by BSI Americas. A copy of the current ISO certification can be downloaded from the following web address, All materials in direct fluid contact path meet USP Class VI, and USP <88> requirements for In Vivo Biological Reactivity All columns are packed in a controlled, classified clean room that meets ISO Class 7 Non-Viable Particulate (NVP) standards All fluid contact components are free from materials of animal origin or compliant with EMEA 410/01 Rev 3 In order to provide the most flexibility to end users, OPUS Pre-Packed columns are available in two levels for non-gmp applications and GMP applications. See Table 2.1 for more detail. In general, OPUS Pre-GMP and GMP Run Ready columns are identical except as noted in Table 2.1. GMP Run Ready columns come with a resin side sample for incoming resin identity testing at an end-users facility. OPUS Pre-GMP columns do not come with a resin side sample. 7

8 Table 2.1 OPUS Pre-GMP and GMP Applications Certificate of Analysis Component Pre-GMP 10 60/R cm ID s GMP Run Ready 5, 10 60/R cm ID s GMP Level X X Catalog Number X X Lot Number X X Serial Number X X Column Diameter X X Final/Achieved Bed Height X X Chromatography Resin Type X X Chromatography Resin Lot Number X X Shipping/Storage Solution X X Date of Manufacture X X Customer Property Number X X Asymmetry Specification and Result X X Theoretical plate count (N/m) Specification and X X Result Chromatography Release Test Chromatogram X X ISO 9001 Compliance Statement X X Product Contact Materials Compliance Statement (animal free, EMEA 410/01, USP Class VI) Packing Environment Statement X X Chromatography Resin Control Statement Copy of Resin Certificate of Analysis X X Endotoxin Specification and Test Result (pass/fail) Bioburden Specification Test Result (pass/fail) X X X X X 8

9 2.3 Certificate of Analysis (CoA) Examples Each OPUS Pre-packed Column is delivered with a Certificate of Analysis (CoA) with the content noted in Table Pre-GMP OPUS Columns Certificate of Analysis (CoA) Each Pre-GMP column produced is issued a Certificates of Analysis (CoA). Content is derived from the template information (Figure 2.1) as well as customer required specifications as detailed on the OPUS Work Order. The CoA package also includes the resin manufacturer s CoA which is attached to the OPUS Column CoA. Figure 2.1 Pre-GMP OPUS Column Certificate of Analysis 9

10 2.3.2 GMP Run Ready OPUS Columns Certificate of Analysis (CoA) Each GMP Run Ready column produced is issued a Certificates of Analysis (CoA). Content is derived from the template information (Figure 2.2) as well as customer required specifications as detailed on the OPUS Work Order. The CoA package also includes the resin manufacturer s CoA which is attached to the OPUS Column CoA. In addition, all GMP Run Ready columns are provided with a chromatography resin side sample which is shipped with the column and resin lot traceability. Figure 2.2 GMP Run Ready OPUS Column Certificate of Analysis 10

11 2.3.3 Certificate of Analysis: Chromatograms For both Pre-GMP, and GMP Run Ready columns, the Certificate of Analysis comes with a second page detailing the testing conditions and chromatogram. See example below of the second page of the OPUS GMP Run Ready CoA. Figure 2.3 OPUS Certificate of Analysis Testing Conditions and Chromatograms Labeling Each individual OPUS 10 60/R cm ID Column is labeled with the following information: 1. Catalog number 2. Resin type 3. Column Internal Diameter 4. Column Bed Height 5. Date of Manufacture 6. Storage temperature ( ambient or 2-8 degrees Celsius ) 7. Lot number 8. Serial number 9. Pressure Rating 11

12 Figure 2.4 Example of OPUS cm ID Column Label Each individual 5cm ID GMP Run Ready column is labeled with the following 1. Catalog Number 2. Resin description 3. Lot number 4. Serial number OPUS Column Part Numbers OPUS Column part numbers are structured so basic column configuration details can be deciphered. The OPUS part number system, which is governed by QA-FM-10107, is not unique for all user specified inputs, and therefore an OPUS Work Order is generated for every new purchase order even if the same column/part number is re-ordered. See Figure 2.5 for the OPUS part number format. Figure 2.5 OPUS Part Number Format 12

13 If for example a customer were to order a 45 x 15 cm GMP Run Ready OPUS R Column packed with Repligen-sourced CaptivA resin, the part number would be, BC-450-CPRI-150-GR. The part number code works as follows: The first 2 digits ( BC ) show the resin is Repligen sourced (vs. customer supplied) The next 3 digits show the nominal column diameter in millimeters ( 450 ) The subsequent 4 digits show the unique code for resin being packed ( CPRI ) The next 3 digits show the target/specified bed height in millimeters ( 150 ), The last 1 or 2 digits reference the GMP level (Pre-GMP or GMP Run Ready) as well as if the column configuration includes an OPUS R port ( GR ) 2.4 Column Manufacturing and Packing Obtaining User Specified Column Inputs All OPUS columns are manufactured using input from the OPUS quote request form ( which documents user specified configurable product aspects such as, Column details: internal diameter, bed height, resin source, GMP Level Acceptance criteria (if specified): plate count, asymmetry, pressure vs. flow Application specific (if specified): maximum working flow rate, maximum working pressure, preferred storage solution All fields on the OPUS Quote Request form are filled in with input from the end user and/or a Repligen sales professional. The form is then submitted for review prior to the formal quoting process. Once a purchase order has been submitted and accepted against a formal quotation, an OPUS Work Order is drafted by Repligen to formalize the user specified inputs contained in the quote request form. The Work Order is then sent to the customer for review and approval. Upon written approval from the customer, the OPUS Work Order (QA-FM-10016) is finalized and the column/s is added to the manufacturing schedule. See Section 3 for more information on the OPUS Work Order. Resin Supply When ordering an OPUS column, the resin may be supplied by the customer, drop shipped from the resin manufacturer, or procured through Repligen. If the customer decides to source the resin and supply it to Repligen for column packing, then the Health and Safety Declaration for Accepting Customer Supplied Resin form (QA-FM ) must be completed. Overview of OPUS Column Production Processes* Column Components: All incoming raw materials and custom components are subject to an incoming inspection procedure. Only the parts in compliance with the criteria set forward in the approved Raw Materials and Component Specifications documents are released for column assembly and packing. Resins: For all resins, Repligen maintains strict material controls including assignment of internal lot numbers to enable full traceability within batch records consistent with good documentation practices. The SOPs which govern these procedures are SOP-1094 (Control of Incoming Materials), 13

14 SOP (Control of Incoming Components) and SOP (Customer Property). In addition, all resins are tested for identity (FTIR) prior to release for column packing. Manufacturing Qualification: The column assembly processes are qualified and documented with appropriate SOPs and batch records as specified in Repligen s ISO 9001 Quality Management System. Preparation of Column Components: Column parts are washed and cleaned prior to assembly. Washing and cleaning procedures are qualified and validated. Column Packing: OPUS 5, 10 60/R cm ID columns are packed in a controlled, classified clean room that meets ISO Class 7 standards. Quality Control: Each pre-packed column is individually tested according to the specifications detailed on the OPUS Work Order. The specifications and the results of the release tests are documented on the Certificate of Analysis. Packaging: Columns are secured by foam packaging material, and shipped in qualified shipping containers. The OPUS 5, and cm columns are sealed in polyolefin bags in a controlled, classified clean room that meets ISO Class 7 NVP standards and then packaged in a cardboard box with foam packaging material. For OPUS cm columns, the box is strapped and shipped on a plastic pallet. See Sections 4 and 6 for more information. The OPUS 45R 60R cm columns are not bagged; however, the inlet and outlet ports are both capped and secured with SaniSure clamps. All OPUS 45R 60R cm columns are shipped in a custom made wooden crate. All crates are heat treated and stamped with heat treated assurance. OPUS 45 and 45R columns ship in the same qualified crate design, and OPUS 60 and 60R ship in the same qualified crate design. See Sections 4 and 6 for more information. The box/crate and internal packaging for the OPUS 5, and 10-60/R cm ID columns have been qualified with standard International Safe Transit Authority (ISTA) tests. For more information on shipping qualification, please refer to Section 4 for Shipping Qualifications. *Note: See Section 3 for more detailed information on the OPUS Column production process. Reference Documentation Quality Documentation: Industry standards for document control are followed as per Repligen ISO 9001 Quality Management System. Repligen Quality management documents can be found on the following website, Technical Specifications: External column dimensions are provided in the user guide and in Section 2.5 (Table 2.3 and 2.4) of this. All other column packing specifications and user specified criteria are documented in the OPUS Work Order. User Guide: A user guide for OPUS 5, 10-60/R columns is available on Repligen s website, as well as an instruction guide for unpacking resin using the OPUS R design. Please visit Un-packaging Guide: For OPUS 45-60/R columns, an uncrating guide is available on Repligen s website. Please visit, 14

15 Technical Documents: A variety of supporting technical documents, presentations, and videos can be found on Repligen s website by visiting CE-Certificate: EG directive 97/23/EG is not applicable for OPUS columns, and therefore no columns will be CE-signed. Performance OPUS columns are designed to provide chromatographic performance equivalent to or better than traditional self-packed columns. The performance is attained through a semi-automated packing process and the design of the flow distribution system. Optimal flow distribution is achieved through a flow distributor design with minimized dead space volume. Rigorous Computational Fluid Dynamics (CFD) models validated by experiments have provided evidence this flow distributor design is optimal for uniform radial distribution. Mathematical modeling and performance testing have verified that the flow distributor design provides chromatographic performance consistent with traditional columns. Multi-cycle performance tests have shown that OPUS columns deliver robust and reproducible chromatographic results Note: More information on column performance can be found on at Ease of Use The OPUS 5, 10 60/R cm ID platform design incorporates ease of use features such as lifting handles, castors, and industry standard sanitary tri-clamp inlet and outlet connections (see Table for more information). Inlet/Outlet Port Locations The OPUS 10-60/R column inlet and outlet ports are both located on the top side of the column. The column inlet and outlet are secured with a tri-clamp fitting designed by SaniSure to be both leak-proof and tamper-evident. The OPUS 5 cm column design has inlet and outlet ports located at either end of the tube like a typical lab scale self-pack column design. 15

16 Lifting Handles OPUS columns with cm IDs are designed with handles as shown below. The OPUS 5, 10, 45/R, and 60/R Columns have no lifting handles. Columns with internal diameters of 5 cm through 30 cm may be lifted safely by one or two operators. Figure 2.6 Lifting Handles and Input/Output Ports on an OPUS 20 cm Column Castors OPUS 45/R 60/R cm ID columns are equipped with twin wheel swivel castors shown below for enhanced maneuverability. Each castor has a manually engaged wheel lock and is rated to hold 100kg. The bottom caps for OPUS 45/R and 60/R Columns are designed with grooves to allow lifting of the columns with straps. Figure 2.7 Castor Detail on an OPUS 45 cm Column 16

17 2.5 Materials of Construction: Product Contact OPUS 5 and 10-60/R cm ID columns are designed using plastics which are best suited for downstream processing applications. These plastics have been chosen for performance characteristics including: Fit for Purpose Suitability for pharmaceutical use with process fluid contact USP Class VI compatibility Low levels of non-toxic extractables Free from materials of animal origin and/or compliant with EMEA 410/01 Mechanical Strength Recommended maximum 4 bar operating pressure for 5, and cm IDs, and maximum 3 bar operating pressure for 45/R 60/R cm ID columns. All columns were designed with a 2x Factor of Safety (FOS) in operating pressure. Thermal Stability: Column construction can support a working temperature range of 2 C to 40 C based on the general properties of polypropylene as well as OPUS Column ISTA shipping tests (see Section 4). Figure 2.8 Materials of Construction: OPUS 5 cm Column 17

18 Figure 2.9 Materials of Construction: OPUS cm Columns Figure 2.10 Materials of Construction: OPUS cm Columns 18

19 Figure 2.11 Materials of Construction: OPUS 45R - 60R Columns Figure 2.12 OPUS R Port Design and Materials of Construction 19

20 Materials of Construction: Product Contact OPUS 5, and 10 60/R cm ID columns are made of polymeric materials chosen to provide optimum biological and chemical compatibility with a vast range of biopharmaceutical manufacturing strategies. The elegant design of 10 60/R cm ID OPUS columns employ only two product contact materials, polypropylene and platinum cured silicone. The 5 cm ID columns employ only polypropylene. All plastics have been certified to comply with United States Pharmacopeia (USP) Class VI requirements as defined in USP <88> Biological Reactivity Tests and USP <661> Physicochemical Tests, In Vivo as well as FDA CFR Title 21 Part 177. Furthermore all product contact plastics are animal free and/or compliant with EMEA 410/01 guidance. Product Contact Materials Summary OPUS 5, and cm ID Columns o The same medical grade polypropylene homo-polymer formulation is used for the column tube, flow distributors, inlet, outlet, and bed support screens o The polypropylene bed support screens are made from a medical grade polypropylene formulation Note: the polypropylene bed support screen is ultrasonically welded to the flow distributor. o The flow distributor O-rings are made from platinum-cured medical grade silicone o The return line (10-30cm columns only) is made from platinum-cured, medical grade silicone, reinforced with a strong polyester fiber OPUS 45/R 60/R cm Columns o Column tubes are made from a 70% w/w E-glass/Polypropylene engineered composite structure assuring a polypropylene resin rich internal surface. This material exhibits a high strength-to-weight ratio, excellent toughness, and chemical resistance. o Flow distributors, inlet ports, outlet ports, OPUS R plug, and OPUS R inside port are made from the same medical grade polypropylene homo-polymer formulation o The polypropylene bed support screens are made from a medical grade polypropylene formulation Note: the polypropylene bed support screen is ultrasonically welded to the flow distributor. o The flow distributor O-rings, OPUS R plug O-ring, and the inner/outer OPUS R gaskets are made from the same platinum-cured, medical grade silicone o The return line is made from platinum-cured medical grade silicone, reinforced with a strong polyester fiber 20

21 Table 2.2 Product Contact Materials Component Material USP <88> Class CFR Animal Origin Column Tube (5, cm) Column Tube (45/R 60/R) Polypropylene Class VI Animal Free 70% w/w E-Glass / PP engineered composite structure Class VI Animal Free Flow Distributors Polypropylene Class VI Animal Free Inlet and Outlet Ports Bed Support Screens Flow Distributor O- Rings Return Line Polypropylene Class VI Animal Free Polypropylene Class VI EMEA 410/01 Platinum-cured medical grade silicone Class VI Animal Free Platinum-cured medical grade silicone, reinforced with strong polyester fiber Class VI Animal Free OPUS R Plug, and Inside Port OPUS R Inner/ Outer Gaskets, and Plug O-ring Polypropylene Class VI Animal Free Platinum-cured medical grade silicone Class VI Animal Free Non-Product Contact Materials Summary Top and bottom caps: Acrylonitrile Butadiene Styrene (ABS) co-polymer or High Density Polyethylene (HDPE) Side guard: ABS (blue) Inlet and outlet port grommets: silicone (blue) Handles: polyurethane (blue) Castors: Polyamide casing with non-marking grey polyurethane tires OPUS R outer nut and washer: ABS OPUS R BioClamp: glass filled nylon 21

22 2.6 OPUS Physical Specifications Table 2.3 Physical Specifications Summary on OPUS cm Columns Column Diameter Physical Attributes 5 cm 10 cm 14 cm 20 cm 25 cm 30 cm Internal cross section Column Body Pressure Rating 20.4 cm cm cm cm cm cm 2 4 Bar 4 Bar 4 Bar 4 Bar 4 Bar 4 Bar Bed height range 5 30 cm 5 30 cm 5 30 cm 5 30 cm 5 30 cm 5 30 cm Column Volumes 10 cm bed height 0.2L 0.8L 1.5 L 3.1 L 4.9 L 7.1 L 20 cm bed height 0.4L 1.6L 3.1 L 6.3 L 9.8 L 14.1 L 30 cm bed height 0.6L 2.4L 4.6 L 9.4 L 14.7 L 21.2 L Assembled Column Height (cm) ~20 + bed height ~20 + bed height ~30 + bed height ~30 + bed height ~33 + bed height ~35 + bed height Outer Diameter (including caps) 6.3 cm 16 cm 21 cm 27 cm 33 cm 38 cm Inlet/Outlet Flow Path Internal Diameter 3.18 mm mm mm mm mm mm Inlet and Outlet Port Connectors ¾ mini triclamp ¾ mini triclamp ¾ mini triclamp ¾ mini triclamp ¾ mini triclamp ¾ mini triclamp Table 2.4 Physical Specifications Summary on OPUS 45R - 60R Columns Column Diameter Column Diameter Physical Attributes 45.7 cm (includes R design) 59.9 cm (includes R design) Internal cross section 1,640 cm 2 2,817 cm 2 Column Body Pressure Rating 3 Bar 3 bar Bed height range 5 30 cm 5 30 cm 10 cm bed height CV 20 cm bed height CV 30 cm bed height CV 16 L 33 L 49 L 28 L 56 L 84 L Assembled Column Height (cm) ~ 90 cm ~92 cm Outer Diameter (including caps) 54 cm 61 cm Inlet/Outlet Flow Path Internal Diameter mm 0.43 inches mm 0.75 inches Inlet and Outlet Port Connectors Per ASME BPE Standards, Current Edition ¾ mini tri-clamp 1 tri-clamp 22

23 Table 2.5 Column Mass (Approximate Weight of Packed Columns) Column Diameter Bed Height 5 cm 10 cm 14 cm 20 cm 25 cm 30 cm 45 cm 60 cm 5 cm 1 kg 2 kg 4 kg 6 kg 10 kg 14 kg 69 kg 117 kg 10 cm 1.2 kg 2.5 kg 5 kg 8 kg 13 kg 18 kg 77 kg 131 kg 15 cm 1.4 kg 3 kg 6 kg 10 kg 16 kg 22 kg 86 kg 145 kg 20 cm 1.5 kg 3.5 kg 7 kg 12 kg 19 kg 26 kg 94 kg 159 kg 30 cm 1.9 kg 4 kg 9 kg 16 kg 24 kg 34 kg 110 kg 187 kg 2.7 Column Stability Repligen conducted a study to assess shelf life stability for OPUS columns when stored according to instructions in the user guide. Two different studies ran in parallel over an 18 month period: Mechanical stability study: This study looked at the structural integrity of the column assembly at pre-determined time points over the course of 18 months. Study design: Three columns of different internal diameters (10 cm, 20 cm, and 30 cm ID) initially filled with 18.5% EtOH solution up to a 20 cm bed height were pressurized to 4 bar and the pressure decay was monitored for 60 minutes. During the course of this pressure hold period, the column was visually inspected for gross failure such as leakage from ports and/or threads, and general structural integrity. See Table 2.6 for the mechanical stability test schedule and acceptance criteria. Results are summarized in Table 2.7. Chromatographic performance stability study: This study looked at the chromatographic performance of the large scale columns over an 18 month period when stored at 2-8 Celsius. The goal of this study was to show no significant performance degradation will occur if a customer purchases a column and places it on the shelf for up to 18 months. Cold storage was deemed as worst case due to the polypropylene materials becoming more brittle in cold temperatures. Study Design: Four 20 cm columns were packed with agarose resin to a 15 cm bed height. Acceptance criteria for the columns is summarized in Table 2.6 at T=0. One column was retested at each of the time intervals and compared with the performance of that specific column at time zero. The results are summarized in Table 2.8. Table 2.6 Stability Study Schedule and Acceptance Criteria Time Interval T = 0 Mechanical Stability Criteria (Empty Columns) Time at Pressure: 60 minutes Pressure: 4 bar Pressure decay: < 10% Visual: No gross defects or leaks Performance Stability Criteria (Packed Columns) Asymmetry: Plate Count: >1500 N/m (100cm/hr) P vs. F: record baseline (10 psi) Endotoxin: 1.0 EU/mL Bioburden: 100 CFU/mL 23

24 T = 3 months T = 6 months T = 12 months T = 18 months Time at Pressure: 60 minutes Pressure: 4 bar Pressure decay: < 10% Visual: No gross defects or leaks Time at Pressure: 60 minutes Pressure: 4 bar Pressure decay: < 10% Visual: No gross defects or leaks Time at Pressure: 60 minutes Pressure: 4 bar Pressure decay: < 10% Visual: No gross defects or leaks Time at Pressure: 60 minutes Pressure: 4 bar Pressure decay: < 10% Visual: No gross defects or leaks Asymmetry: Plate Count: >1500 N/m (100cm/hr) P vs. F: record baseline (10 psi) Endotoxin: 1.0 EU/mL Bioburden: 100 CFU/mL Asymmetry: Plate Count: >1500 N/m (100cm/hr) P vs. F: record baseline (10 psi) Endotoxin: 1.0 EU/mL Bioburden: 100 CFU/mL Asymmetry: Plate Count: >1500 N/m (100cm/hr) P vs. F: record baseline (10 psi) Endotoxin: 1.0 EU/mL Bioburden: 100 CFU/mL Asymmetry: Plate Count: >1500 N/m (100cm/hr) P vs. F: record baseline (10 psi) Endotoxin: 1.0 EU/mL Bioburden: 100 CFU/mL All columns passed acceptance criteria at each interim time point, as well as at the end of 18 months. The data is summarized in Tables 2.7 and 2.8 below: Table 2.7 Mechanical Stability Test Results Column Configuration 3 months 6 months 12 months 18 months 10 x 20 cm Pass Pass Pass Pass 20 x 20 cm Pass Pass Pass Pass 30 x 20 cm Pass Pass Pass Pass Table 2.8 Performance Stability Study Results Column S/N 3 months 6 months 12 months 18 months N/m Δ = < 15% As Δ = < 15% N/m Δ = < 15% As Δ = < 15% N/m Δ = < 15% As Δ = < 15% N/m Δ = < 15% As Δ = < 15% N/m Δ = < 15% As Δ = < 15% N/m Δ = < 15% As Δ = < 15% N/m Δ = < 15% As Δ = < 15% 24

25 At the end of 18 months, bioburden for all 4 packed columns (serial numbers ) was 0 CFU/ml and endotoxin for all 4 packed columns (serial numbers ) was <0.1 EU/ml. Cleanability OPUS Columns are used for purification of biological products which have specific regulatory requirements for bioburden and endotoxin levels. Therefore, a quantitative cleaning investigation was performed to demonstrate the effectiveness of sanitization using sodium hydroxide as a cleaning agent. Method: Several different OPUS Columns were packed with Sepharose 6FF in 20 cm bed heights. Each column was loaded with 1 column volume of E. coli bacteria at a concentration of 0.1 OD (optical density) at 600 nm. The columns were left to incubate at ambient temperature overnight, and then flushed with reverse osmosis deionized (RODI) water for 2-3 column volumes in downflow. Samples were collected to measure bioburden and endotoxin. A sanitization procedure was subsequently used as followed: Sanitization procedure: Flush with 1 M sodium hydroxide in up-flow at 100 cm/h for 30 minutes Flush with 1 M sodium hydroxide in down-flow at 100 cm/h for 30 minutes Recirculation of 1 M sodium hydroxide for 2 hours in up-flow at 100 cm/h Incubation of the column in 1 M sodium hydroxide for 1 hour (static sanitization for compete removal of endotoxins) Flush with RODI water at 100 cm/h until neutral ph is achieved Each column was then left to incubate at ambient temperature overnight prior to pulling samples for bioburden and endotoxin testing. Results: Results for bioburden and endotoxin levels from the microbial challenge are outlined in the table below, which shows the sanitization procedure completely removed bioburden from millions of CFU to zero CFU in the post-sanitization water rinse. In addition, endotoxin levels were brought below the limit of detection (0.25 EU/mL) for the assay. Table 2.9 Results of Bioburden and Endotoxin Testing Column Diameter Cleaning Data 20 cm 30 cm 45 cm 45R cm 60 cm 60R 0.1 M NaCl effluent after overnight E.coli incubation TNTC (CFU/Ml) >0.25 EU/mL TNTC (CFU/Ml) >0.25 EU/mL TNTC (CFU/Ml) >0.25 EU/mL TNTC (CFU/Ml) >0.25 EU/mL TNTC (CFU/Ml) >0.25 EU/mL TNTC (CFU/Ml) >0.25 EU/mL 0.1 M NaCl post sanitization effluent 0 CFU/mL <0.25 EU/mL 0 CFU/mL <0.25 EU/mL 0 CFU/mL <0.25 EU/mL 0 CFU/mL <0.25 EU/mL 0 CFU/mL <0.25 EU/mL 0 CFU/mL <0.25 EU/mL 25

26 Conclusions: In order to test effectiveness of sanitization on an OPUS column, a worst case scenario was devised where the column was loaded with an excess of E. coli culture (a gramnegative, endotoxin producing bacteria). The results of the sanitization protocol demonstrate the effective removal of bioburden and endotoxin contamination Warranty Repligen aspires to complete customer satisfaction, and has implemented the following warranty policy for OPUS Columns. If the column arrives at the customer site in damaged condition, Repligen will accept the damage risk and issue a replacement at no charge. If the column fails passing specifications as agreed to on the OPUS Work Order, Repligen will conduct troubleshooting efforts and if unsuccessful, will ask for the column to be returned for further evaluation. If the root cause of the failed test is determined to be a compromised packed bed, Repligen will pack a replacement at no charge. If the column s packed bed is compromised (i.e. a channel develops) and the root cause is packing, then Repligen will replace the column at no charge. 2.8 Material Safety Data Sheet OPUS columns are made from plastic components only and therefore no MSDS is needed. An MSDS for the chromatography resin packed into an OPUS Column may be obtained from the resin manufacturer. 26

27 3. Manufacturing Information 3.1 Introduction All OPUS 5, 10 60/R cm Columns are manufactured at the Repligen corporate headquarters, located at 41 Seyon Street, Waltham, Massachusetts 02453, USA. The QA and QC operations are also based in the same location. Neither the facility nor products manufactured require registration or market approval. Therefore, Repligen s column packing facility and products manufactured herein are not subject to regulatory review or audit by organizations such as the US Food and Drug Administration or European Medicines Agency. 3.2 Manufacturing Quality Assurance Standards and Policy Repligen recognizes the need for high quality standards, and has therefore established an ISO 9001 Quality Management System suitable for the needs of our clients and our industry. Refer to Section 1 for Repligen s Quality Policy. 3.3 Manufacturing Facilities The OPUS Column packing suite consists of multiple ISO Class 7 column packing rooms and a central ISO 8 prep area. Two airlock doorways are maintained, one for people and one for finished goods and equipment. In general, column parts enter the suite through a validated parts washer or are cleaned with NaOH followed by a rinse with RODI water. The OPUS Column suite is completely segregated from all other product manufacturing at Repligen and is a restricted-access area. The clean-room environment is controlled and monitored as follows: Air quality is maintained by 100% HEPA filtered air Preparation room air quality is tested to ISO Class 8 standards for non-viable particulates Column packing room air quality is tested to ISO Class 7 standards for non-viable particulates Room pressure differentials are maintained and monitored according to SOPs All rooms are on a routine cleaning and disinfection schedule Access is restricted to authorized personnel only Gowning is required for entry into controlled areas including a secondary gowning procedure for entry into the ISO Class 7 packing rooms Environmental monitoring is performed to check for viable and non-viable contamination 3.4 Manufacturing Controls and SOPs Training [SOP-1498]: Manufacturing is performed by qualified and trained operators. Training documentation is maintained by Quality Assurance. Process Documentation [SOP-1302, SOP-1098]: Repligen manufacturing processes are governed by an ISO 9001 compliant Quality Management System. All manufacturing work instructions are contained in controlled documents, which are issued in advance of each manufacturing batch. Batches and process intermediates are 100% traceable through an internal lot numbering system. All manufacturing data are recorded by operators at the time of manufacturing. Batch records are archived for 3 years on site, and then stored off site for a minimum of 10 years. 27

28 Raw Materials [SOP-1094]: All raw materials are controlled, and each raw material has a preapproved specification. Receipt of material is verified and released by QA prior to use in manufacturing. Customer Property [SOP-10035]: All customer property including customer supplied resin is documented, tracked, and released prior to the start of packing. Customer property is stored in segregated areas within Repligen s warehouse. Supplier Management [SOP-1496]: Repligen identifies critical suppliers of raw materials and components based on the impact to the quality of the product they are used to manufacture. Critical suppliers are subject to a qualification process and are monitored and routinely audited according to a pre-determined schedule. The supplier audit schedule is established based on critical supplier audit cycle, supplier performance, past audit results, and business requirements. Components [SOP-10042]: All components are controlled and each component has a pre-approved specification. Every receipt of material is verified and released by QA prior to use in manufacturing. In addition, each OPUS Column component has a pre-approved engineering drawing, and is subject to incoming QC inspection, which includes review of materials certificates, and verification of critical dimensions. Process Change Management [SOP-1447]: Manufacturing process changes are governed by change management procedures that include provisions for customer notification of major changes. Product Storage Control [SOP-MFG-1212; SOP-1263]: Product is stored either at ambient temperature, or in temperature controlled units as dictated by the packed resin. All temperature controlled storage units have backup power, 24/7 electronic monitoring, and alarms that are monitored. Preventive Maintenance [SOP-1501] and Calibration [SOP-1181]: Equipment and monitoring devices are controlled through the Repligen Equipment Control process [SOP1171]. Each piece of equipment is uniquely identified and has a preventive maintenance and/or calibration schedule as necessary. High Purity Water [SOP-MFG-1209]: Purified water is supplied to all manufacturing areas from a Reverse Osmosis/Deionization (RODI) system. The RODI system is fully automated, and provides high quality water in a continuously circulating loop. The water system design performance specifications are ASTM Type I Reagent Grade Water, with the addition of low endotoxin and bioburden specifications (Table 4.1). Water quality is monitored continuously using an on-line TOC device, and is routinely sampled and tested by Repligen Quality Control. 28

29 Table 3.1 Repligen Water Specifications Compared with ASTM, USP Purified Water and Water for Injection Parameter ASTM Type I USP Purified water WFI Repligen Specification Conductivity 0.05 µs/cm 1.3 µs/cm 1.3 µs/cm < 0.01 ms/cm Endotoxin N/A N/A < 0.25 EU/mL < 0.5 EU/mL Bioburden N/A 100 CFU/mL 0.1 CFU/mL 10 CFU/mL ph N/A N/A TOC 0.1 ppm 0.5 ppm 0.5 ppm 0.1 ppm High purity water is one of the key raw materials for any bioprocessing product. Repligen believes that these specifications, in conjunction with regular maintenance, ensure that the water system performs at a suitably high quality standard. 3.5 OPUS Column Manufacturing Packing Environment and Environmental Controls All OPUS columns are packed in ISO Class 7 classified clean rooms and buffers prepared in an ISO 8 classified clean room. The rooms are monitored on a weekly basis for viable contaminants using TSA settle plates. Microbial contaminants (bacteria) are counted and reported. Viable contamination limits have been set for the ISO 8 rooms as follows: Alert Limit: > 20 CFU/plate Action Limit: > 35 CFU/plate Viable contamination limits have been set for the ISO 7 rooms as follows: Alert Limit: > 10 CFU/plate Action Limit: > 25 CFU/plate Counts which exceed an alert or action limit are investigated according to SOP-MFG The OPUS manufacturing suite is monitored on a weekly basis for Non-Viable Particles ( 0.5 µm). Each room is monitored in predetermined locations, measuring 1 cubic foot of air over the course of 1 minute. NVP limits have been set for the ISO Class 8 clean room spaces as follows: Alert Limit: 25,000 particles per ft 3 (880,000 particles per m 3 ) Action Limit: 50,000 particles per ft 3 (1,760,000 particles per m 3 ) ISO 8 Specification: 100,000 particles per ft 3 (3,520,000 particles per m 3 ) NVP limits have been set for the ISO Class 7 clean room spaces as follows: Alert Limit: 2,500 particles per ft 3 (88,000 particles per m 3 ) Action Limit: 5,000 particles per ft 3 (176,000 particles per m 3 ) ISO 7 Specification: 10,000 particles per ft 3 (352,000 particles per m 3 ) 29

30 OPUS Work Order and User Specified Inputs Once a purchase order for a column has been received, the OPUS Work Order (QA-FM-10015) is created. The OPUS Work Order compiles information from the purchase order as well as the user specified inputs which were recorded from the OPUS quote request form ( Note: All OPUS columns are built to order and each column ordered must have a quote request form and unique Work Order. These forms document critical user specified inputs and therefore cannot be eliminated from the ordering process. In addition, each OPUS Work Order is unique to a PO number and column part number. The OPUS Work Order captures a variety of information including the following, Quote number, purchase order number and column part number Column size, type, and resin Column packing method and solutions used Column performance release method and specifications (ex: Asymmetry and plate count) Revision history Once all required fields have been populated, Repligen sends the OPUS Work Order to the appropriate customer contact for review. If all the details in the document are satisfactory, the customer signs and dates the form, and then sends the signed form back to Repligen. Once the signed form is received, Repligen can begin column packing. Note: Column packing cannot begin without a customer approved work order. See below for an example of the OPUS Work Order. Figure 3.1 OPUS Work Order Example (blank) 30

31 Chromatography Resin Control All chromatography resins are tested for identity using FTIR prior to being released for production. A copy of the FTIR spectrum is maintained with the completed batch record. The SOPs governing chromatography resin control procedures are SOP-1094 (Control of Incoming Materials), SOP (Control of Incoming Components) and SOP (Customer Property). Repligen Supplied Resins Resins purchased and supplied by Repligen for column packing are controlled as a raw material according to SOP-1094 (Control of Incoming Materials). Upon arrival, each resin is placed into quarantine pending disposition by Repligen Quality assurance. Upon release, each batch of resin is given a unique internal lot number and expiration date to ensure full traceability throughout the manufacturing process. Customer Supplied Resins Repligen s customer property procedures (SOP-10035) are designed to protect our customer s financial and intellectual property interests to prevent unauthorized use, disclosure, or disposal. All resins shipped to Repligen for column packing must be unused. Each customer supplied resin is assigned a unique customer property number which is typically linked to a specific purchase order and part number. The customer property number is noted on the form entitled, Health and Safety Declaration for Accepting Customer Supplied Resin, which must be filled out by an end user prior to shipping resin to Repligen. Upon arrival, the resin is managed according to SOP-10035, which dictates the resin must be labeled, segregated, and then stored according to the resin vendor s recommendation. All customer supplied resin is also tested for identity using FTIR prior to being released for production. Unused media is either returned or discarded according to the customer s instructions. Repligen understands in some instances customers may have a requirement to sample and test a small aliquot of the resin for identity (ex. FTIR). Unused resin in containers, which have been sampled for QC testing, are compatible with Repligen s quality requirements and therefore can be accepted for column packing. Resin Preparation Once the appropriate resin has been released, parts cleaned, and column packing batch record documentation have been issued, the follow steps occur: 1. Manufacturing operators move sealed resin containers into one of the OPUS ISO 7 clean rooms. Note: only the released resin is allowed in the packing room, and only one resin type at a time can be present. 2. Manufacturing operators verify the appropriate resin by checking against the OPUS Work Order. 3. After verifying the resin, containers are opened for resin preparation (decantation of storage solution, fines removal, etc.). 4. For OPUS GMP Run Ready columns only, prior to beginning resin prep an operator removes ml of resin per resin lot from the original container. This operation is performed using a sterile pipette and the operator dispenses the resin aliquot into a 60 ml gamma irradiated, tamper evident PETG (polyethylene terephthalate) bottle. The bottle is then sealed and labeled with resin name, lot number, part number, date, and Repligen operator initials. In the event multiple resin lots are packed into a single column, a single side sample of each resin lot will be taken. Therefore, multiple side samples will be delivered with a GMP Run Ready column if multiple resin lots are packed. 31

32 5. After column packing, the resin QC sample/s or resin side sample/s are stored with the finished the OPUS GMP Run Ready column for shipment. In general, Repligen uses the following approach for slurry preparation and percent solids determination: 1. Decant off storage solution 2. Perform defining of resin as specified by resin MFG. 3. Suspended resin in packing buffer to target the desired % solids 4. Combine resin bottles in a holding tank if required 5. Pull resin samples 6. Record mass and volume added for density calculations 7. Spin samples in a centrifuge 8. Read spin down solids volume 9. Determine average % solids (solids vol. / total vol.) 10. Use target compression factor (CF = target column vol./uncompressed resin vol.) as derived by a. Past empirical data b. Resin manufacturer recommendations c. Repligen R&D recommendations 11. Calculate target solids volume to add (target bed volume / CF) 12. Apply % solids (Step 9) and density (Step 6) to target solids calculations (Step 11) to determine mass of slurry required 13. (Target solids vol. / % solids) * density = mass slurry to dose Assembly, Packing, and Performance Testing OPUS columns are assembled from qualified and controlled components. Inspected and released component parts are taken from inventory and brought into the ISO 8 classified cleanroom through an automated parts washer. Parts which do not enter through the parts washer are chemically cleaned with NaOH, followed by rinse with RODI water to depyrogenate and reduce bioburden. Both processes have been qualified and validated to the following specifications: Table 3.2 Parts Cleaning Specifications Method Parts Cleaning Specifications Bioburden Endotoxin TOC Conductivity ph Parts Washer < 10 cfu/ml < 0.5 EU/ml < 10 PPM < 10 us/cm NaOH Soak < 10 cfu/ml < 0.25 EU/ml N/A < 50 us/cm The following points summarize the major steps which occur during OPUS Column packing: 1. Solutions and buffers are prepared in the ISO 8 area of the OPUS clean rooms. All buffers and solutions are filtered with a 0.2um filter. 2. Packing solutions/buffers and allocated parts, which have been cleaned, are brought into the designated ISO 7 packing room. 3. All subsequent steps are performed in the designated ISO 7 clean room. 4. The bottom flow distributor with the ultrasonically welded mesh is inserted into the bottom end of the tube to form a water-tight seal. The flow distributor assembly is then secured in place with a polypropylene weld or adhesive bond. 32

33 5. The chromatography resin is added to the open ended column by dosing directly into the column or using a peristaltic pump. 6. The top flow distributor is inserted into the column tube and packing is initiated to establish a uniform bed. OPUS columns utilize flow packing, axial compression, and/or a combination of both methods to achieve a well packed bed. In general, packing protocols are developed in Repligen s R&D department and subsequently transferred to the production team. 7. Following the prescribed packing procedure, the top flow distributor is precisely positioned to create the correct resin compression. Note: Rigid particle resins such as ceramic hydroxyapatite are not compressed, rather the top flow distributor is positioned so there is no significant gap between the consolidated resin bed and the flow distributor assembly. 8. Bed performance is tested according to the procedure and specifications as documented on the OPUS Work Order. Note: A column may be tested and flow distributor adjusted during the packing process to ensure optimal performance. 9. Once the final flow distributor position is set and the column has met the performance specifications (theoretical plates/meter and Asymmetry) as documented on the OPUS Work Order, the packing process can conclude. 10. The column is then sanitized using a procedure recommended by the resin manufacturer and/or as documented on the OPUS Work Order. 11. Post sanitization, the column is then neutralized (typically with RODI water), and Bioburden or Endotoxin samples taken (OPUS GMP Run Ready Columns only). 12. After neutralization, each pre-packed column is put in its final storage solution as documented on the OPUS Work Order. 13. The column assembly is then finished by securing the flow distributors in place using a polypropylene weld or adhesive bond, and assembling the top cap, bottom cap, and side-guard. Note: The top cap, bottom cap, and side guard serve cosmetic and ergonomic functions, and therefore do not impact the performance of the column. 14. Inlet and outlet ports are capped and sealed using a tamper evident SaniSure clamp. 15. Each OPUS 5, cm Column is sealed in a polyethylene bag before being removed from the OPUS cleanroom suite. Note: OPUS 45/R and 60/R cm Columns are not bagged since they are on wheels. 16. Columns are then stored according to storage conditions as noted on the OPUS Work Order. Client resin verification upon arrival: Included with each column lot is a copy of the resin vendor s Certificate of Analysis 1 and a small sample/s (10-30 ml) of resin (GMP Run Ready columns only) intended for incoming quality control analysis. As described above, in the event multiple resin lots are packed into a column, a resin aliquot is taken for each resin lot and delivered with the column. Finally, the column label contains the resin description, and the certificate of analysis for each column indicates the part number and lot number of the pre-packed resin. Equipment Used in the Manufacturing of OPUS Columns: Wherever possible, all equipment used to pack OPUS columns is dedicated single use equipment such as: Slurry tanks and carboys Single-use liners for buffer tanks Single-use platinum cured tubing for pipe transfers 1 In rare cases a resin vendor s certificate of analysis may not be included with the OPUS CoA package. For example, when a customer ships custom resin without a CoA to Repligen for packing, Repligen may not be able to include a resin CoA in the OPUS CoA package if the custom resin CoA is not readily available from the manufacturer. 33

34 Where reusable components are used in the manufacturing process which might come into contact with the resin or the inside of the column, Repligen employs validated cleaning protocols to ensure the risk of cross resin contamination is mitigated. 3.6 Bioburden and Endotoxin Release Testing Columns are tested according to the specifications detailed in the OPUS Work Order. Bioburden and endotoxin testing is performed on OPUS GMP Run Ready columns and samples are taken in RODI water post sanitization and just prior to introducing the storage solution. The release specifications are as follows: Bioburden: <10 CFU/mL Endotoxin: <0.25 EU/mL Repligen performs endotoxin and bioburden testing in-house using the following two methods. Bioburden assay method Bioburden is analyzed by a vacuum filtration method. The sample is applied to a sterile filter under vacuum and then rinsed with peptone water. The filter is collected aseptically and placed onto TSA media. The plate is placed into an incubator at 32C +/- 2C for 5 days. An analyst reads the plates at day 3 and day 5 and documents the results. A negative control of the peptone water is also plated and incubated to demonstrate the reagents used during the analysis were not contaminated. Endotoxin assay method Endotoxin is analyzed by gel clot method. Repligen uses a commercially available kit and performs the test according to the manufacturer s instructions which have been transcribed into an internal test procedure. A column rinse sample with a volume of 0.25 ml is introduced to the gel clot tube and incubated in a 37C water bath. The tubes are then read by an analyst and the data is recorded. A positive control is run alongside the sample for this assay. Bioburden and Endotoxin test results are documented on the Certificate of Analysis as a pass/fail result for each GMP Run Ready column. See Section 2.0 for sample CoA documents. Note: OPUS Pre-GMP columns are not tested for bioburden or endotoxin. 34

35 4. Shipping Qualification 4.1 Summary To certify the packaging, column components, and column performance remains intact during and after shipping, International Safe Transit Association (ISTA) tests were conducted on a variety of OPUS column sizes. A third party certified laboratory performed the shipping tests, and Repligen tested chromatographic performance pre and post shipping. Results from the studies show the packaging withstands the stressors of commercial shipping, and chromatographic performance is maintained. 4.2 Introduction Objective The objective of the shipping studies is to demonstrate the custom made packaging (Figures 5.1 and 5.2) and pre-packed columns of multiple diameters arrive intact after worst case shipping simulations. See Section 6 for engineering drawings of packaging used for each internal diameter column. OPUS columns employ different packaging based on size and weight. To cover the range of different column dimensions and packaging methods/materials the following column sizes were evaluated. OPUS 5 cm Column OPUS 10 cm Column (uses identical packaging as the OPUS 14 cm Column) OPUS 20 cm Column (uses identical packaging as the OPUS 25, and 30 cm Columns) OPUS 45 (uses identical packaging as the OPUS 45R Column) OPUS 60 (uses identical packaging as the OPUS 60R Column) OPUS 60R (representative of the R design) Figure 4.1 OPUS 5 cm Column Shipping Container Design 35

36 Figure 4.2 OPUS cm Column Shipping Container Design Figure 4.3 OPUS 45/R - 60/R Column Shipping Container Design Acceptance Criteria The shipping tests documented in this regulatory support file have two different acceptance criteria categories as detailed below. Visual inspection of packaging Per ISTA guidelines, procedures 2A and 2B call for a simple pass/fail assessment based on a visual test of packaging materials. This assessment is made by the certified ISTA test facility and documented in a summary report. Chromatographic performance Repligen assessed chromatographic performance by measuring plate count and asymmetry to compare pre-shipping performance with post-shipping performance. In order to pass the chromatographic portion of the test, the minimum post-shipping theoretical plate count and asymmetry measurements had to be within +/-25% of pre-shipping results. 36

37 4.3 Method: ISTA 2A and 2B Tests ISTA Test Methods ISTA Procedure 2A was used for the OPUS 5 cm ID and 2B 2 was used for OPUS 10 60/R cm IDs. The selection of the two tests has to do with weight and shipping methods (see below). ISTA Procedure 2A is a worst case simulation test for individual packaged-products shipped in boxes ( parcel shipments). ISTA Procedure 2B is a worst case simulation test for individual packaged-products shipped on pallets Both tests are used to: Evaluate the performance of a packaged product Compare relative performance of the package and product design During the test, the package and product are considered together and not separately. Tests and Conditions All studies were performed by Atlas Box and Crate in Sutton, MA per ISTA guidelines. The specific test conditions are described further in Table 5.1 below. The 5, 10, 20, 45, 60, and 60R columns were manufactured by Repligen, packaged into their respective shipping containers, picked up by Atlas, shipped to their certified testing facility, and tested according the applicable standards. After the tests were complete, Atlas returned the shipping containers to Repligen for post testing analysis. Table 4.1 ISTA 2A and 2B Testing Conditions (OPUS 5, cm Columns) Test Name Test Details Atmospheric Conditioning Controlled Temperature and Humidity Conditioning Compression Testing Random Vibration Testing Incline Impact (2B only) Ambient Conditions for 36 hours 38 ±2 C; Relative Humidity 85% ±5% for 36 hours 4 C±2 C; RH uncontrolled for 36 hours 248 lbs. (OPUS 5), 370 lbs. (OPUS 10), 744 lbs (OPUS 20), 1728 lbs (OPUS 45), 2,392 lbs (OPUS 60), and 2,836 lbs (OPUS 60 R) on top of crate/box 60 minutes of random vibration Frequency (Hz) PSD Level, g 2 /Hz Slide crate down ramp and into a wall Drop Testing Crate dropped 8 to ground (2B) 2 See, Accessed on 02/04/

38 Random Vibration Testing Crate bottom edges dropped 8 to ground (2B) Box dropped 38 inches to the ground (2A) 60 minutes of random vibration Frequency (Hz) PSD Level, g 2 /Hz Figure 4.4 OPUS 5 cm Column Compression and Drop Testing Figure 4.5 OPUS 20 cm Column Vibrational and Compression Testing 38

39 Figure 4.6 OPUS 60/R Column Vibrational, Incline Impact and Drop Testing Visual Inspection Per ISTA Procedures 2A and 2B, the certified test facility performed a visual pass/fail inspection of the packaging materials Column Efficiency Test Methods Overview: Column efficiency testing was designed to assess and uncover any discernible differences in chromatographic performance pre-shipping and post-shipping. Repligen conducted all chromatographic performance tests. 5 cm Study: 5 cm x 10 cm OPUS Column packed with Sepharose 6 Fast Flow cm Study: 10 cm x 15.3 cm OPUS Column packed with Q Sepharose Fast Flow resin cm Study: 20 cm x 19 cm OPUS Column packed with a silica based protein A resin 45 cm Study: 45 cm x 21 cm OPUS Column packed with Q Sepharose Fast Flow resin 60 cm Study: 60 cm x 20 cm OPUS Column packed with Sepharose 6 Fast Flow resin 60R Study: 60 x 20.5 cm OPUS Column packed with 90% Phenyl Sepharose 6 Fast Flow with 10% SP Sepharose XL Each column was tested at 100 cm/hr with pulse injection of 1% CV acetone solution, except for the OPUS 5 cm Column which was tested at 100 cm/hr using a 0.2M NaCl mobile phase and 2M NaCl spike Results: ISTA and Column Efficiency Tests Results: Visual Inspection ISTA test facility: After the worst case shipping simulation tests were completed, Atlas visually inspected the packaging for signs of damage. For all tests, the custom designed OPUS packaging passed the visual inspection without any significant signs of damage. Note: Visual inspection results are documented as part of the ISTA summary reports issued by the test facility. Repligen: Upon receipt from Atlas Box and Crate, all shipping containers and columns were inspected and found to be undamaged and intact. 39

40 Note: visual inspection results are documented in Repligen s summary report for each shipping test Chromatographic Performance Test Results The data summarized in Table 5.2 shows the packing efficiency of 10, 20, 45, 60, and 60R OPUS columns remain stable following the applicable shipping simulation. Stability is exhibited by a negligible change in asymmetry and plate count, while meeting the acceptance criteria. Table 4.2 OPUS 10 cm, 20 cm and 45 cm Column Performance Data Column Date N/m As Pass / Fail 5 cm ID Pre-shipping PASS Post-shipping PASS 10 cm ID Pre-shipping PASS Post-shipping PASS 20 cm ID Pre-shipping PASS Post-shipping PASS 45 cm ID Pre-shipping PASS Post-shipping PASS 60 cm ID Pre-shipping PASS Post-shipping PASS 60R Pre-shipping PASS Post-shipping PASS *Pre-ship and post-ship test flow rates vary slightly due to manual flow control of testing set-up Conclusion In summary, the following was observed post worst case shipping simulation: No significant damage to the shipping containers No damage to the column structures No discernable damage to the packed beds No significant differences in chromatographic performance Therefore, OPUS columns designed and tested by Repligen for chromatographic performance, and the shipping containers designed and tested in conjunction with Atlas Box and Crate, demonstrate suitable robustness for surviving the harsh environments of commercial shipping. 40

41 5. Extractables and Leachables 5.1 Introduction and Background Plastic materials have been used in the manufacturing of therapeutics for many decades. Over the last 15 years, focused product development by many vendors and biotechnology companies has resulted in a plethora of single-use technologies. During this time, the industry has witnessed the development and adoption of critical disposable and single-use technologies like mixers, bioreactors, filters, and connectors. As a result, standards and best practices for evaluating component safety have been set for the selection and qualification of plastics. In general, plastics used in biopharmaceutical manufacturing have low defined extractables and have been determined to be non-toxic at equivalent therapeutic doses. Many base standards used for guidance have been set by regulatory publications including USP, CFR 21, and EMEA. These basic standards have been elaborated on by industry organizations like the Bio-Process Systems Alliance (BPSA) and Parenteral Drug Association (PDA) as well as product manufacturers through the publication of best practices of testing and assessment of data. In addition, end user therapeutic manufacturers have become more demanding in their analytical requirements, assessment of data, determination of risk, and minimum threshold for meeting internal standards. Repligen is sensitive to the demands of the industry and will therefore supply relevant and applicable information about the plastics used in the product contact components of OPUS columns. In accordance with industry standards, Repligen uses the definitions for extractables and leachables as stated in the 2007 BPSA document, "Recommendations for Extractables and Leachables Testing: Part 1. Extractables: Chemical compounds that migrate from any product-contact material when exposed to an appropriate solvent under exaggerated conditions of time and temperature. Leachables: Chemical compounds, typically a subset of extractables that migrate into a drug formulation from any product contact material as a result of direct contact under normal process conditions. There is a general consensus that it is the responsibility of the product technology vendor to provide an extractable data package. In recent years there have been efforts to standardize the testing procedures for extractables, and wherever possible it is Repligen s intent to comply with the standardization efforts. In addition, in compliance with CFR 21, Part , OPUS columns are designed such that all product contact materials are not reactive, additive, or absorptive so as to alter the safety, identity, strength, quality, or purity of the drug product beyond established requirements. Therefore, using the BPSA guidance as a backdrop, Repligen has designed an extractables program to produce a robust data package by conducting extractables testing using three solvents as well as exaggerated time and temperature conditions. Solvents were chosen for their common usage in chromatography columns. Leachables, however, are considered to be process specific and the responsibility of the end user to define within specific process parameters. 41

42 5.2 OPUS Extractables Strategy Based on the above discussion, Repligen has applied the following philosophy in its approach to developing a meaningful extractables and leachables package for OPUS columns: 1. All plastics used will be certified to meet four criteria (see Sections for more detail): a) Certified to meet USP <88> Biological Reactivity Tests, In Vivo (USP Class VI) b) Certified to meet CFR 21 Part 177 Indirect food Additives: Polymers i. Sec Olefin polymers (Polypropylene) ii. Sec Rubber articles intended for repeated use (Silicone) c) USP General Chapters 661: Polypropylene Containers d) Certified Animal Free or compliant with EMEA 410/01 2. Extractables from the OPUS 5, and 10 60/R cm Column platform were determined according to an approved written experimental rationale for exaggerated time and temperature conditions and tested against an approved protocol. 3. Leachables from the OPUS 5, and 10 60/R cm Column platform: Repligen will not conduct, nor present data for leachables in this for any process specific solution beyond what is determined in the extractables testing. Repligen will on request, support client based leachable testing by providing component test materials. 5.3 USP<88> Biological Reactivity Tests, In Vivo Six plastic classes are defined in Table 3.1. This classification is based on responses to a series of in vivo tests for which extracts, materials, and routes of administration are specified. These tests are directly related to the intended end-use of the plastic articles. Table 5.1 Summary of USP <88> Plastics Classes Plastic Classes Tests to be Conducted I II III IV V VI Extract Species Procedure X X X X X X USP 0.9% NaCl Mouse Systemic-Intravenous X X X X X X Rabbit Intracutaneous Irritation X X X X X 1:20 EtOH/NaCl Mouse Systemic-Intravenous X X X X X Rabbit Intracutaneous Irritation X X X PEG 400 Mouse Systemic-Intravenous X X Rabbit Intracutaneous Irritation X X X X Cottonseed Oil Mouse Systemic-Intravenous X X X Rabbit Intracutaneous Irritation X X Muscle Implant Rabbit Intramuscular (7 day, no Histopathology) 42

43 Acute Systemic Toxicity Test Purpose: In-vivo systemic tests evaluate the impairment or activation of a system rather than the impairment of individual cells or organs. In acute systemic toxicity tests, the test material (extract) is tested for systemic toxic effects as a result of a single, acute exposure. This test is designed to evaluate systemic responses to the extracts of materials following injection into mice. Irritation Test: Intracutaneous Irritation Test Purpose: The irritation tests are in-vivo screening tests to evaluate the potential of test materials or their extracts to cause irritation on the exposed part of the body. This test is designed to evaluate local responses to the extracts of materials following intracutaneous injection into rabbits. Implantation Test: Intramuscular Purpose: Implant studies evaluate the local pathological effects on living tissue at both the gross and microscopic level of a test article surgically implanted into an appropriate implant site. The implantation test is designed for the evaluation of plastic materials and other polymeric materials in direct contact with living tissue CFR 21 Part 177 Indirect food Additives: Polymers Subpart B: Substances for Use as Basic Components of Single and Repeated Use Food Contact Surfaces. Section Olefin Polymers (Applied to Polypropylene). The olefin polymers from polypropylene referenced in CFR 21 Part 177 Sec may be safely used as articles or components of articles intended for use in contact with food, subject to the provisions of this section. Subpart C: Substances for use only as components of articles intended for repeated use Sec , and rubber articles intended for repeated use (applied to silicone). Rubber articles intended for repeated use may be safely used in producing, manufacturing, packing, processing, preparing, treating, packaging, transporting, or holding food, subject to the provisions of this section USP General Chapters 661: Polypropylene Containers The standards and test provided in this section characterize polypropylene containers produced from homopolymers or co-polymers that are suitable for packaging dry solid or liquid oral dosage forms Animal Free and EMEA 410/01 Note: This is for guidance on minimizing the risk of transmitting animal spongiform encephalopathy agents via human and veterinary medicinal products. Scientific principles for minimizing risk (EMEA 410/01): Use of materials from non TSE-relevant animal species or non-animal origin is preferred. If materials from TSE-relevant animal species have to be used, consideration should be given to all the necessary measures to minimize the risk of transmission of TSE. 43

44 5.4 Extractable Test Protocol and Results By Michael Ruberto, Ph.D. Background Repligen has performed polymer deformulation and extractables testing on the product contact components of their OPUS columns. This data has been consolidated into a format that will be suitable for use in the OPUS product literature. Extractables and Leachables Materials The materials used to fabricate single-use processing equipment for biopharmaceutical manufacturing are often polymers, such as plastic or elastomers (rubber), as opposed to the traditional metal or glass. Polymers offer more versatility, since they are light-weight, flexible and much more durable than their traditional counterparts. Plastic and rubber are also disposable, so issues associated with cleaning and its validation are often avoidable. Whenever there is contact between a material of construction for a manufacturing component and therapeutic, there is always the possibility for chemicals from the material to migrate or leach into the drug. All materials can produce leachables. Leachables are the chemicals that migrate from single-use processing equipment into the various components of the drug product during manufacturing. Extractables are chemicals entities, organic and inorganic, that can be extracted from single-use processing equipment using common laboratory solvents in controlled experiments. They represent the worst case scenario and are used as a tool to predict the types of leachables that can be encountered during pharmaceutical production. So, extractables are the potentials and leachables are the actuals. An effective way to minimize and control leachables is to use well characterized materials to fabricate the manufacturing equipment. The OPUS column product contact materials are mainly constructed from polypropylene (PP) homopolymers and platinum cured silicone. PP homopolymer is much less prone to leachables than other types of polyolefins due to its mechanical and physical properties (such as melting point, glass transition temperature, molecular weight distribution, and percent crystallinity.) All of these important polymer characteristics have been measured and benchmarked for each PP product contact component and can be used as a quality control measure to ensure that all incoming raw materials have similar properties. This can help to ensure consistent, batch-to-batch leachables profiles for the OPUS product contact components. The additives package has also been determined for each polymer in an effort to predict and control leachables. The platinum cured silicone components are very durable materials that are often used in medical applications. These silicone elastomers are amorphous polymers that usually do not require formulation with additives such as phthalates, antioxidants, or heat and light stabilizers. This less complex formulation greatly reduces the risk of leachables. The silicone polymers have also been fortified with silica fillers to provide the essential mechanical properties required for the OPUS chromatography applications. As in the case of the PP components, the mechanical and physical properties of the platinum cured silicone gaskets and tubing have all been measured and benchmarked. All polymers used to construct the product contact components have met the requirements of a USP Class VI polymer and are regulated for food contact application according to 21CFR Part 177 Indirect Food Additives. The specific polymer and regulatory information is summarized in the table below. 44