The information contained in this document is subject to change without notice.

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2 The information contained in this document is subject to change without notice. Copyright 2015 Repligen Corporation. 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

3 Safety Notices: Please review the Regulatory Support File for this product prior to use Unless otherwise specified by the end-user, the column is generally shipped with the resin in 18.5% ±1% ethanol solution, a recognized bacteriostatic agent. It is flushed from the resin during equilibration and preparation for use. Follow all local regulations for safe disposal For laboratory and manufacturing production use only Not for administration to humans 2

4 Contents Introduction... 4 What is Open Platform User Specified?... 4 Column Design... 4 Materials of Construction... 5 Product Contact Materials... 5 Non-Product Contact Materials... 6 OPUS Column Physical Specifications... 8 Solvent Compatibility Connecting and Operating Your OPUS Column General Usage Instructions Cleaning and Sanitization Recommendations Troubleshooting Air in the column Pressure increase during run Pressure drop during run Appendices: Appendix 1: Use of a 3-Way Valve for Inlet Connection and Air Purging Appendix 2: Connecting an OPUS column to an AKTA Ready tubing set Appendix 3: A Note on Bubble Traps Appendix 4: Column Performance Testing

5 Introduction What is Open Platform User Specified? OPUS Open Platform User Specified Columns are designed to perform chromatography purification of biological molecules in either GMP or non GMP applications. The OPUS pre-packed disposable column platform offers an alternative to conventional pack in place glass or stainless steel columns and can be reliably packed with virtually any resin from any source. To accommodate a wide range of biopharmaceutical applications, Column Design The OPUS cm ID column platform has been designed to meet the requirements of GMP manufacturing in the pharmaceutical and biopharmaceutical industries for campaign-use and single-use applications. Platformable OPUS columns are designed to be broadly configurable to accommodate a wide range of purification and polishing applications for vaccines, monoclonal antibodies, and recombinant proteins. For example, OPUS columns: Accept virtually all commercially available bioprocessing resins Are available in a wide range of bed heights and industry standard column diameters Are configurable for specific packing procedures, release tests, and storage solutions Quality Standards In order to meet the needs of GMP manufacturing, OPUS cm ID columns are manufactured in the U.S.A. under the following quality standards: ISO 9001 certified Quality Management System 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 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 Disposable Columns can be customized according to two levels of GMP readiness (see Table 1 for more information): Pre-GMP GMP Run Ready 4

6 Table 1. Certificate Component Manufacturing Pre-GMP cm IDs Manufacturing GMP Run Ready cm IDs Certificate of Analysis X X Catalog Number X X Lot Number X X Serial Number X X Actual Column Diameter X X Actual Bed Height X X Resin Type X X Resin Lot Number X X Shipping/Storage Buffer X X Packing Date X X Asymmetry Result X X N/m Result X X ISO 9001 Compliance Statement X X Product Contact Materials Compliance Statement (animal free, EMEA 410/01, USP Class VI) Copy of Resin Certificate of Analysis X X Packing Environment X X QC Chromatogram X X Chromatography Resin Control Statement Resin sample for QC analysis Individual packed column Endotoxin result Individual packed column Bioburden result Materials of Construction OPUS cm ID columns are designed using polymers which are best suited to downstream processing applications (Figure 1, Figure 2 and Table 2). Product Contact Materials For cm ID columns, polypropylene is used for the column shell, flow distributors, inlet, outlet, and bed support screens The column shell, flow distributors, inlet and outlet are made from the same medical grade polypropylene homo-polymer formulation The polypropylene bed support screens are made using a different polypropylene formulation Note: the polypropylene mesh is ultrasonically welded to the polypropylene flow distributor X X X X X X 5

7 The cm column shells are made from a 70% w/w E-glass/Polypropylene engineered composite structure. This material exhibits a high strength-to-weight ratio, excellent toughness and chemical resistance. Platinum cured silicone tubing is used for the return line and O-rings Non-Product Contact Materials Top & 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 Figure 1. Materials of Construction Summary: cm ID Columns 6

8 Figure 2. Materials of Construction Summary: cm ID Columns Table 2. Product Contact Materials Component Column Tube (10-30 cm ID) Column Tube (45 cm ID) Flow Distributors Material USP <88> Class CFR Polypropylene Class VI % w/w E-Glass / PP engineered composite structure Class VI Polypropylene Class VI Inlet & Outlet Polypropylene Class VI Bed Support Screens Polypropylene Class VI O-Rings Platinum-cured medical grade silicone Class VI Return Line Platinum-cured medical grade silicone (braided polyester fiber) Class VI Animal Origin Animal Free Animal Free Animal Free Animal Free EMEA 410/01 Animal Free Animal Free 7

9 OPUS Column Physical Specifications Table 3.1. Physical Specifications Summary (10 30 cm ID) Column Diameter Physical Attributes 10 cm 14 cm 20 cm 25 cm 30 cm Internal cross section Column Body Pressure Rating 78.5 cm cm cm cm cm 2 4 Bar 4 Bar 4 Bar 4 Bar 4 Bar Bed height range Configurable Configurable Configurable Configurable Configurable Column Volumes 10 cm bed height 0.8 L 1.5 L 3.1 L 4.9 L 7.1 L 20 cm bed height 1.6 L 3.1 L 6.3 L 9.8 L 14.1 L 30 cm bed height 2.4 L 4.6 L 9.4 L 14.7 L 21.2 L Assembled Column Height (cm) ~20 + bed height ~30 + bed height ~30 + bed height ~33 + bed height ~35 + bed height Outer Diameter (including caps) 16 cm 21 cm 27 cm 33 cm 38 cm Inlet/Outlet Flow Path Internal Diameter 6.35 mm mm mm mm mm Inlet and Outlet Port Connectors ¾ inch mini Tri-Clamp ¾ inch mini Tri-Clamp ¾ inch mini Tri-Clamp ¾ inch mini Tri-Clamp ¾ inch mini Tri-Clamp 8

10 Table 3.2. Physical Specifications Summary (45 cm ID) Column Diameter Column Diameter Physical Attributes 45.7 cm 59.9 cm 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 Column Volumes 10 cm bed height 20 cm bed height 30 cm bed height 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 Inlet and Outlet Port Connectors Per ASME BPE Standards, Current Edition mm 0.43 inches ¾ inch mini tri-clamp mm 0.75 inches 1 inch tri-clamp Column Mass Table Table 4. Approximate Column Weight Table Column Diameter Bed Height 10 cm 14 cm 20 cm 25 cm 30 cm 45 cm 60 cm 5 cm 2 kg 4 kg 6 kg 10 kg 14 kg 69 kg 117 kg 10 cm 2.5 kg 5 kg 8 kg 13 kg 18 kg 77 kg 131 kg 15 cm 3 kg 6 kg 10 kg 16 kg 22 kg 86 kg 145 kg 20 cm 3.5 kg 7 kg 12 kg 19 kg 26 kg 94 kg 159 kg 30 cm 4 kg 9 kg 16 kg 24 kg 34 kg 110 kg 187 kg OPUS columns with cm IDs are designed with handles. The 10 cm, 45 cm, and 60 cm ID columns have no lifting handles. Columns with internal diameters of 10 cm through 30 cm may be lifted safely by one or two operators cm ID columns are equipped with castors for better transportability. 9

11 Solvent Compatibility Table 5. Solvent Compatibility Summary Solvent Compatibility Water 20% Ethanol A 2% Acetone A 2% (w/v) Detergents A 8M Urea or 6M Guanidinium HCl 1M HCl 2M NaOH A = Excellent - No effect. B = Good - Minor Effect, slight corrosion or discoloration. C = Fair - Moderate Effect, not recommended for continuous use. Softening, loss of strength, swelling may occur. D = Severe Effect - Not recommended for ANY use. N/A = Information not available. Connecting and Operating Your OPUS Column General Usage Instructions 1. Prior to removing your OPUS column from the box or crate, please review the Uncrating Guide which provides step-by-step instructions to help you uncrate/unpack your OPUS column safely. 2. When you are ready to use your OPUS column, remove the clear plastic bag containing the (10 30 cm) column. There is no bag for 45 cm & 60 IDs, so the column should be wiped down prior to use if necessary. Using a wire cutter or sharp scissors, remove the white cable-tie on the SaniSure CLAMPs* (see for more information) on both the inlet and the outlet. 3. With the inlet and the outlet open, hook up the column inlet under low flow with a fractional triclamp (check tables 3.1 & 3.2 the inlet & outlet size) so that no air is introduced into the column (a 3-way valve can also be used for this connection). Once the inlet has been connected under low flow, the outlet can then be connected. Instructions for using a 3-way valve to connect the column inlet as well as to purge air from the inlet line can be found in Appendix 1. Instructions on how to connect an OPUS column to an AKTA Ready tubing set can be found in Appendix 2. Information on recommended bubble traps can be found in Appendix Flush the storage solution (example: 18-20% EtOH) from the column with 6-10 column volumes of RO/DI water or mild buffer (e.g. PBS) at 100 cm/h. Note: all buffers and cleaning solutions should be filtered through a 0.22 µm filter membrane. 5. To test for chromatographic performance and compare results to the certificate of analysis, a short instruction guide can be found in Appendix 4. A A B A 10

12 6. Equilibrate your column using 6-10 CVs of your beginning process buffer or mobile phase. 7. Reference the technical specifications below when using your OPUS column during processing: Cleaning and Storage: OPUS columns do not require special in-process cleaning or storage protocols. Please consult the chromatography resin manufacturer for cleaning and storage information. Mechanical Strength: Recommended maximum 4 bar working pressure for cm ID columns, and 3 bar working pressure for 45 cm & 60 cm ID columns. Temperature: Column construction is designed to support a working temperature range of 2 C to 40 C. 8. Once your chromatography process is completed, the column should be prepped for disposal or storage. Disposal: Clean and sanitize the column prior to disposal according to local government regulations. Storage: Clean, flush, and prepare the column for storage per the recommendations of the resin manufacturer or other validated procedure. 9. Reuse post storage: Start with the general usage instructions in Step 5. Cleaning and Sanitization Recommendations Please consult the resin manufacturer for recommended cleaning and storage protocols. OPUS columns can be cleaned with any sanitization agent that is compatible with the materials of construction (see Table 5 for Solvent Compatibility Summary). Prior to sanitizing your column, check for solvent compatibility with the chromatography resin manufacturer. Troubleshooting Air in the Column Potential Fixes If air entered the inlet port and did not reach the column (to the best assessment of the operator), follow the air purge procedure described in Appendix 1. If air entered the packed chromatography bed, recondition the column by running a solution with low surface tension in reverse flow at 20% of operating flow rate for a minimum of 5 hours. Prior to reconditioning your column, check for solvent compatibility with the chromatography resin manufacturer. Examples of low surface tension solutions include 1% surfactant, 20% ethanol for normal phase resins, and 60% ethanol for reverse phase resins. Retest column performance (HETP, asymmetry) according to instructions in Appendix 4. Pressure Increase during Operation Causes Obscuration of the polypropylene mesh Incorrect column valve position Operation under higher flow rate than recommended for the packed resin Residue build up at the head of the column Compromised chromatography resin 11

13 Potential Fixes Run the column in reverse at normal operating flow rate for the specific resin for at least 10 CV. Recheck pressure and column performance (HETP, asymmetry, flow delta P) under normal operating conditions Clean the column with the appropriate cleaning method for the residue that clogged the mesh and/or resin. Running in reverse flow, or up-flow mode is recommended. Check valve position Pressure Drop during Operation Cause Line or fitting leaks Potential Fixes Check lines and connections Appendices: Appendix 1: Use of a 3-Way Valve for Inlet Connection and Air Purging To purge air when the column is first connected to the chromatography system: 1. Connect one end of the 3-way valve to the column inlet and the other end to the chromatography system pump. Leave the column outlet closed. 2. Configure the 3-way valve flow path as shown in Figure 3 below. Figure3. 3-way valve position for purging air from the column inlet From Pump To Purge To Column 3. For 10 and 14 cm ID columns, attach a syringe to the purge line while pumping the mobile phase at low flow rate (half the normal operating flow rate), and draw the plunger to create negative pressure. Air bubbles will be drawn into the syringe, and mobile phase will immediately fill the space created. 4. For 20, 25, 30, 45 & 60 cm ID columns, begin pumping mobile phase at a slow flow rate (about half of the normal operating flow rate). The mobile phase will travel into the column inlet and out of the purge line. This path will engage and dislodge any air bubbles trapped in the column inlet line. 12

14 5. To ensure all the air has been purged, tap the inlet line with your finger, or grab the column with two hands and shake it (if possible). This process will dislodge any trapped air remaining in the line. 6. After all the air has been purged from the inlet line, engage the 3-way valve as shown in Figure 4 below. Figure 4. 3-way valve From Pump To Column 7. With the flow off and the 3-way valve configured as shown in Figure 4, open the column outlet and connect it to the chromatography system. 8. Introduce flow to the column at a low flow rate (ex. half the operating flow rate) to flush trapped air from the column outlet. In the absence of a 3-way valve: 1. A T line can be connected between the column and chromatography system. 2. The T line can be used as described above for purging air. 3. After air has been purged, the purge line can be clamped or closed with a stopper. 4. The column outlet can then be connected to the chromatography system for normal use. 13

15 Appendix 2: Connecting an OPUS column to an AKTA Ready tubing set 1 1. Inlet manifold 2. Pump tubing 3. Flow cell for pressure sensor 4. Air trap 5. Air vent tubing 6. Column inlet connection 7. Column outlet connection 8. Flow cell for pressure sensor 9. ph electrode (optional) and has to be ordered separately 10. Flow meter cell 11. Temperature cell 12. Conductivity cell 13. UV flow cell 14. Flow cell for pressure sensor 15. Outlet manifold 1 GEHC website: Accessed on April, 22 nd

16 Instructions: Close-up of flow path 1. Connect the inlet of the column to the AKTA Ready system tubing set per AKTA Ready instructions (see AKTA Ready wizard). At this point, the outlet of the column should be closed and disconnected. Alternatively, connect the outlet to the AKTA Ready tubing set with the outlet pinch valve closed. Then open the proper pinch valve to allow flow to be introduced to the column. 2. Open the pinch valve for the column bypass. Make sure the column outlet is either disconnected or the pinch valve for the column outlet is closed. 3. Start flowing the mobile phase through the system at less than half of the recommended operating flow rate for your chromatography resin/process. During this operation, the flow will be split. One portion of the mobile phase will enter the bypass line and other portion will enter the inlet line. Note: The mobile phase will not enter the column because the column outlet is closed (with the pinch valve, or outlet cap) thus creating a stop barrier for the flow. The fraction of the flow that enters the inlet line will dislodge the trapped air in the tube and connector. 4. With the flow split to the bypass and inlet lines, air bubbles will begin to travel upward in the inlet tube, and will be evacuated through the column bypass line into the system outlet. To ensure all the air is evacuated, tap and/or shake the inlet tube and inlet connector (alternatively the column can be tapped or shaken). 15

17 5. After all the air has been removed from the column inlet and connector, close the bypass line pinch valve and connect the outlet line to the column with the outlet pinch valve in the open position. 6. Run mobile phase through the column to purge the outlet of air. Appendix 3: A Note on Bubble Traps A bubble trap connected to the inlet of the column can prevent air from entering the column. If your chromatography system comes with a bubble trap, then an additional bubble trap at the inlet is not recommended. For disposable bubble traps, we recommend disposable filter cartridges from Sartorius or Millipore. Note the membrane in the filter capsule should be compatible with your chromatography process. Sartorius Filter Cartridges: Millipore Filter Cartridges: 0:0 Cartridge or Bubble Trap Size Recommendations: To ensure air does not enter the column, the volume of the bubble trap should be just large enough to allow the trap to drain completely in about 10 seconds at the maximum process flow rate. A bubble trap with a volume too large will create unnecessary band broadening and result in poor chromatographic performance. Appendix 4: Column Performance Testing Follow the steps below to measure the plate count and asymmetry of your OPUS column. Please note, minor differences (plus or minus 10-20%) in the measured plate count and asymmetry noted on the column COA are to be expected. Sources of variation include, the use of different chromatography instruments for measurement, operator variability, normal variability within the test methods (i.e. flow rate and sample volumes), and transit conditions. If the plate count and asymmetry measurements are within defined acceptance limits (reference the column s COA QC Release Data ) then the column should be considered fit for purpose. Please consult Repligen technical support (option 3) or bioprocessing@repligen.com for further questions. 1. Remove column storage solution: a. If column storage solution is 18-20% Ethanol, run RO/DI water at low flow rate (ex. 100 cm/ h) for 3-4 column volumes. Because ethanol solution is more viscous than water, the flow rate of this step should be chosen such that the pressure drop on the column does not exceed the maximum operating pressure for the chromatography resin and the column (4 bar). b. If column storage solution is 0.1M NaOH, run a higher ionic strength solution, such as 0.2M NaCl or PBS, at normal operating flow rate for 3-4 column volumes. 2. After the storage solution has been removed, condition the column with the packing buffer for 10 column volumes at packing flow rate (typically 1.5 2x the normal operating flow rate or consult packing instructions for the resin). 16

18 a. Note: refer to your column s OPUS column Work Order Manufacturing Details for the packing buffer used. 3. Proceed to testing the column: a. Equilibrate with 10 column volumes of test mobile phase at the test flow rate (linear velocity). i. Note: refer to your column s OPUS column Work Order Quality Control Specifications for the test mobile phase and test flow rate used by Repligen. b. Conduct a pulse injection of 1-2 % CV of the test injection solution. i. Note: refer to your column s OPUS column Work Order Quality Control Specifications for the test injection solution used by Repligen. c. Elute with mobile phase for 3-4 CV at the same test flow rate while monitoring UV or conductivity. 4. Calculate number of theoretical plates and asymmetry of the eluted peak: V R Theoretical Plate Count: N= 5.54 x (V R /W 1/2 ) 2, assuming a Gaussian peak Where: N = number of theoretical plates V R = peak retention (elution) volume W 1/2 = peak width at half height Asymmetry: As = b/a Where: a = partial peak width at 10% of the peak height for the leading part of the peak b = partial peak width at 10% of the peak height for the tailing part of the peak 5. If the plate count and asymmetry measurements are within defined acceptance limits (reference the column s COA QC Release Data ) then the column should be considered fit for purpose. 17

19 For more information or questions, please contact Repligen: Repligen Corporation 41 Seyon Street Building #1, Suite 100 Waltham, MA