columns Protein-Pak Hi Res IEX Columns and Standards [ CARE AND USE MANUAL ] I. INTRODUCTION CONTENTS I. INTRODUCTION

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1 CONTENTS I. INTRODUCTION II. CONFIGURING AN ACQUITY SYSTEM FOR USE IN IEX PROTEIN SEPARATIONS III. GETTING STARTED a. ecord Installation b. Column Connectors c. Column Installation d. Column Equilibration e. Useful Functional Tests for Benchmarking a New Column I. INTRODUCTION Thank you for choosing a Protein-Pak ion-exchange column. Waters Protein-Pak Hi Res IEX family of columns were developed to assist in the UPLC characterization of recombinant proteins and monoclonal antibodies found in many of today s novel biopharmaceutical therapeutics. The non-porous, high-ligand density particles overcome some of the high molecular weight limitations present using traditional porous, IEX particles. In addition, use of a multi-layered network of ion-exchange groups (SP, CM, or Q) on non-porous particles assist the effective diffusion and binding of charged proteins or other biomolecules onto the particles to deliver relatively high sample loading capacities and component resolution while minimizing column fouling. IV. COLUMN SPECIFICATIONS AND USE a. Specifications b. Sample Preparation c. Operating ph d. Solvents e. Pressure f. Flow Rate g. Temperature The available ion-exchange chemistries include a strong-anion and weak- and strong-cation exchangers (Table 1), all of which are stable, over a wide ph range (3 10), high salt concentrations and standard pressures (see Column Specifications and Use section) via use of polymer-based particles. Only when the Protein-Pak Hi Res IEX column series is combined with the ACQUITY UPLC system will the full performance benefits of this column be realized. V. TROUBLESHOOTING Table 1 VI. COLUMN CLEANING, REGENERATION, AND STORAGE a. Cleaning and Regeneration b. Storage Abbreviation SP CM Q Ion-Exchange Ligand Sulfopropyl Carboxymethyl Quaternary ammonium VI. INTRODUCING ECORD INTELLIGENT CHIP TECHNOLOGY a. Introduction b. Installation c. Manufacturing Information d. Column Use Information VIII. ORDERING INFORMATION columns

2 II. CONFIGURING AN ACQUITY SYSTEM FOR USE IN IEX PROTEIN SEPARATIONS Although Waters Protein-Pak Hi Res IEX columns do not require the high pressure capability of an ACQUITY UPLC system, the full performance benefits of this column will only be realized with the low system volume and low detector dispersion of the optimized ACQUITY UPLC system. However, ion-exchange chromatography may require modifications to an existing ACQUITY UPLC system. Please refer to Size Exclusion and Ion-Exchange Chromatography of Proteins using the ACQUITY UPLC System, ( A) for specific recommendations that can be obtained at com/chemcu. III. GETTING STARTED An Analysis Report and an Inspection Data Sheet are generated and available for each shipped Protein-Pak Hi Res IEX column. The Analysis Report is specific to each batch of packing material and includes the batch number and chromatographic separation obtained using defined protein standards. This document is available upon request htm?id= &ev= &locale=en_us. The Inspection Data Sheet is included with each shipment and is specific to each individual column and contains the following information: batch number, column serial number, plate count, tailing factor, retention factor, and chromatographic separation conditions. These data should be stored for future reference. b1. The ACQUITY UPLC system utilizes tubing and gold plated compression screws that have been designed to meet stringent tolerance levels and to minimize extra column volumes. The columns should be attached to the ACQUITY UPLC injector with a column stabilizer. b2. Waters Protein-Pak Hi Res columns require end fittings that have a 2 mm depth between the ferrule and the end of the extending stainless steel tubing (Figure A) that must perfectly seat to the end of the Protein-Pak Hi Res column. Connecting a different style connector to a Waters Protein-Pak Hi Res IEX column may leave a gap between the end of the stainless steel tubing and the column that will result in undesired peak broadening (Figure B). To correct this problem, disassemble the finger-tight fitting of the stabilizer and remake the connection (i.e., 2 mm depth) to the Waters Protein-Pak Hi Res IEX column. Figure A Figure B 2 mm a. ecord Installation The ecord button should be attached to the side of the Waters ACQUITY UPLC column heater module. The ecord button is magnetized and does not require specific orientation. b. Column Connectors There are two options for connecting the Protein-Pak Hi Res column to an ACQUITY system. The first option is to use the reusable fitting on the 150 mm and 100 mm column stabilizers (sect b1). This fitting/tubing is supplied with ACQUITY UPLC column heaters. A second alternative is to remove this fitting and replace it with a non removable fitting (i.e. steel). If the second option is employed, specific directions must be followed (sec b2). In a proper tubing/column connection, the tubing touches the bottom of the column end fitting with no void between them. (Figure C). Figure C 2

3 c. Column Installation Note: The flow rates given in the procedure below are for a Protein- Pak Hi Res CM or SP, 7 µm, 4.6 mm i.d. column. 1. Purge the solvent delivery system of any organic or waterimmiscible mobile phases and connect the inlet end of the column to the injector outlet. An arrow on the column identification label indicates the correct direction of solvent flow. 2. Flush column with 100% aqueous buffer (line A1) by setting the pump flow to 0.2 ml/min. 3. When the buffer is flowing freely from the column outlet attach the column outlet to the detector. Monitor the system pressure to ensure the column is within its pressure limitations. 4. Gradually increase the flow rate, by not more than 0.5mL/min at a time (as described in step 2). 5. Once a stable backpressure has been achieved, proceed to the next section. d. Column Equilibration Protein-Pak Hi Res columns are shipped in 20 mm sodium sulfate with 0.05% sodium azide. It is important to ensure mobile-phase compatibility before changing to a different mobile-phase system. Equilibrate the column with a minimum of 10 column volumes of the buffer to be used (refer to Table 2 for column volumes). Table 2. Empty Column Volumes in ml (multiply by 10 for flush solvent volumes). Column Dimension Approximate Volume 4.6 x 100 mm 1.66 ml e. Useful Functional Tests for Benchmarking a New Column It may be useful to benchmark the column performance with a sample that is representative of the intended application. A separation of common proteins with an appropriate method is suggested for your new column. Two standards were specially designed for this purpose. The IEX Cation Test Standard is specifically designed with proteins that resolve well on cationexchange columns. The IEX Anion Test Standard is specifically designed with proteins that resolve well on anion-exchange columns. This test can also be used to monitor the condition of your column after extended use. The following sets of conditions and chromatograms show examples of using the IEX Standards for benchmarking and monitoring column and system health. The exact results observed in any laboratory will depend on the instrument in use. Similar results can only be expected with a Waters ACQUITY UPLC system. These tests are valuable for monitoring the life of the column and for troubleshooting separation difficulties that may arise. Protein-Pak Hi Res Cation-Exchange Columns and Standards Sample Diluent: 20 mm Sodium Phosphate Buffer, ph 6 7 The IEX Cation Test Standard is packaged in a Waters TruView Max Recovery Vial (p/n cv) and is lyophilized for prolonged storage and stability. The standard is shipped at room temperature but it upon arrival, it is recommended to store this standard at -20 C. Protein Sigma P/N Conc. mg/ml Bovine Ribonuclease A R Cytochrome C, bovine heart C Chicken Lysozyme L Column: Protein-Pak Hi Res CM, 4.6 x 100 mm, or Protein-Pak Hi Res SP, 4.6 x 100 mm Injection Volume: 10.0 μl Flow Rate: 1 ml/min Mobile Phase A: 20 mm sodium phosphate buffer, ph 6.6 Mobile Phase B: 20 mm sodium phosphate buffer, 0.5 M NaCl, ph 6.6 Weak Needle Wash: Mobile phase A Strong Needle Wash: Mobile phase B Seal Wash: 90/10 H 2 0/MeOH Temperature: 30 C Detection: 280 nm Gradient: Time %A %B Curve

4 Protein-Pak Hi Res CM Column AU Cytochrome C Ribonuclease A Protein-Pak Hi Res SP Column AU Ribonuclease A Lysozyme min Cytochrome C Lysozyme min Protein-Pak Hi Res Anion-Exchange Column and Standard Sample Diluent: Lyophilized in 20 mm Tris Buffer, ph 8 9, Reconstituted in 1ml 100% Milli-Q Water, vortex and inspect for undisssolved solids. Protein Sigma P/N Conc. mg/ml Horse Myoglobin M Conalbumin from Chicken Egg White C Soybean Trypsin Inhibitor T Column: Inj Vol: 10.0 µl Flow Rate: Mobile Phase A (Acid): Mobile Phase B (Base): Mobile Phase C (Salt): Mobile Phase D (Aqueous): Protein Pak Hi Res Q, 4.6 x 100 mm 0.6 ml/min 100 mm TRIS HCl (aq) 100 mm TRIS (aq) 1000 mm NaCl (aq) Water Sample Manager Wash: 25 mm TRIS, 150 mm NaCl, ph 8.3 Sample Manager Purge: 25 mm TRIS, 150 mm NaCl, ph 8.3 Seal Wash: Column Temp.: 30 C Sample Manager Temp.: 4 C Detection: Auto Blend Plus Gradient: Time Flow (min) (ml/min) 90/10 H 2 O/MeOH 280 nm ph ph Curve Salt (mm) Salt Curve Initial Actual Gradient (with no Auto Blend capability): Flow Time %A %B %C %D (ml/min) Initial Protein-Pak Hi Res Q Column Myoglobin Conalbumin* Trypsin Inhibitor *Contains Isoform A and B which could be resolved under various ph conditions) 4

5 IV. COLUMN SPECIFICATIONS AND USE a. Specifications Description Protein-Pak Hi Res Q Protein-Pak Hi Res CM Protein-Pak Hi Res SP Ion Exchange Strong Anion Weak Cation Strong Cation Functional Group Matrix Quaternary ammonium Hydrophilic polymer Carboxymethyl Sulfopropyl Hydrophilic polymer Hydrophilic polymer Particle Size (µm) Pore Size: i.d. x L (mm) Non porous 4.6 x 100 Non porous 4.6 x 100 Non porous 4.6 x 100 Counter Ion Cl- Na+ Na+ ph Range Small Ion Capacity (µeq/g dry gel) pk a Approximate Protein Binding Capacity in mgs per column (i.e., BSA for Hi Res Q column; Lysozyme for Hi Res CM and Hi Res SP columns) Flow Rates 2. Max Pressure across column ml/min 2175 psi (15MPa) ml/min 1450 psi (10Mpa) ml/min 1450 psi (10Mpa) Salt Concentration No limit No limit No limit Organic Concentration <50%. When switching from aqueous buffers to organic, lower flow rates to <0.25 ml/ min. <50%. When switching from aqueous buffers to organic, lower flow rates to <0.5 ml/min. <50%. When switching from aqueous buffers to organic, lower flow rates to <0.5 ml/min. Temperature ( C) For optimal resolution of complex samples, do not exceed 20% of the column s protein binding capacity. 2. See section e. pressure for details. To ensure the continued high performance of Protein-Pak Hi Res IEX columns, follow these guidelines: b. Sample Preparation 1. It is preferable to prepare the sample in the operating mobile phase or a mobile phase that has a higher ph (anion exchange) or lower ph (cation exchange) than the mobile phase to ensure complete loading of the sample onto the column. The ionic strength of the sample should also be lower or equivalent to that of the starting buffer. 2. If the sample is not dissolved in the mobile phase, ensure that the sample, solvent and mobile phases are miscible in order to avoid sample and/or buffer precipitation. c. Operating ph The recommended operating ph range for Protein-Pak Hi Res IEX columns is A listing of commonly used buffers and additives is given in Table 3. The column lifetime will vary depending upon the operating temperature as well as the type and concentration of buffer used. Table 3. Buffers Commonly Used for Ion Exchange Anion-Exchange Buffers ph Range Additive/Buffer pk a (25 C) Counter-ion Conc. (mm) N-Methylpiperazine 4.75 Cl Piperazine 5.68 Cl-/HCOO bis-tris 6.48 Cl Bis-tris propane 6.80 Cl MOPS 7.28 Cl Triethanolamine 7.76 Cl-/HCH 3 COO Tris 8.06 Cl Diethanolamine 8.88 Cl Ethanolamine 9.50 Cl CAPS Cl- 20 Cation-Exchange Buffers ph Range Additive/Buffer pk a (25 C) Counter-ion Conc. (mm) Lactic acid 3.81 Na Formic acid 3.75 Na+/Li Acetic acid 4.76 Na+/Li Malonic acid 5.68 Na+/Li MES 6.10 Na+/Li Phosphate 7.20 Na HEPES 7.55 Na+ or Li BICINE 8.35 Na+ 20 a Adapted from: 1. P Stanton, Ion-Exchange Chromatography, HPLC of Proteins and Peptide: Methods and Protocols; Aguilar, M.-I. Ed; Method in Molecular Biology, Humana Press, Totowa, NJ, Vol. 251, Ch Buffer Reference Center, Sigma Aldrich, com/life-science/core-bior agents/biological-buffers/learning-center/bufferreference-center.html 5

6 d. Solvents To maintain maximum column performance, use high quality chromatography grade solvents. Filter all aqueous buffers prior to use. Solvents containing suspended particulate materials can damage the fluidic components of the UPLC system and may clog the inlet distribution frit of the column. This will result in higher operating pressure and poor performance. The maximum organic concentration must be <50%. When the solvent is replaced by distilled or ion exchanged water, the flow rate must be less than 0.25 ml/min for Protein-Pak Hi Res Q, 5 µm column or less than 0.5mL/min for Protein-Pak Hi Res CM or SP, 7 µm columns. e. Pressure Protein-Pak Hi Res IEX columns can tolerate pressures across the column of up to 2175 psi for Protein-Pak Hi Res Q, 5 µm column and up to 1450 PSI for Protein-Pak Hi Res SP, 7 µm and Protein- Pak Hi Res CM, 7 µm columns. These suggested limits are for the pressure drop across the column. The actual recorded pressure on an HPLC or UPLC system is a sum of the pressure generated from solvent flow through the LC tubing and detector cell combined to the pressure generated from solvent flow through the packed IEX column. To determine the actual pressure drop across the IEX column, subtract the above value from the system pressure obtained (using the same eluent, flow, and temperature selected for the IEX separation method) when a union is substituted in place of the column on the LC System. Note: Working at the extremes of pressure, ph and/or temperature may result in shorter column lifetimes. f. Flow Rate Standard flow rate: ml/min: Protein-Pak Hi Res Q, 5 µm column ml/min: Protein-Pak Hi Res CM and SP, 7 µm columns Under certain conditions (viscous buffers, low temperatures) the maximum flow rate may have to be reduced so as to not exceed the maximum column pressure limits. g. Temperature Temperatures between 10 C 60 C are recommended for operating Protein-Pak Hi Res IEX columns. V. TROUBLESHOOTING The first step in systematic troubleshooting is comparison of the column, in its current state, to the column when it was functioning properly. The functional tests with the protein mixture may reveal subtle changes in surface chemistry that affect the application. There are several common symptoms of change in the column. 1. An increase in pressure is often associated with lost performance in the application. The first step in diagnosis is to ensure that the elevated pressure resides in the column rather than somewhere else in the system. This is determined by measuring pressure with and without the column attached to the instrument. If the system is occluded, the blockage should be identified and removed. If the pressure increase resides in the column, it is helpful to know whether the problem was associated with a single injection or whether it occurred over a series of injections. If the pressure gradually built up, it is possible that the column can be cleaned as described in Section VI. For future stability, it may be useful to incorporate a stronger regeneration step in the method. If a single sample caused the pressure increase, it likely reflects particulates or insoluble components, such as lipids. Cleaning is still an option, but using the more aggressive options. The sudden pressure increase suggests that the user should consider some sample preparation, such as filtration or high-speed centrifugation. 2. Loss of retention can reflect a change in the column surface chemistry. Before proceeding with diagnostic or corrective measures, check that the mobile phases have been correctly prepared and the correct method has been selected. Then repeat the functional or protein test. If the proteins show loss of retention, the column may require replacement. If the changes are small and reflected only for some proteins, one of the cleaning procedures may be effective. 3. Change in peak shape, resolution, or relative retention of peaks. Follow the same steps as for loss of retention (Symptom 2). 4. Carryover and memory effects are defined as the appearance of the constituents of one sample in the next gradient analysis. First determine whether the column or the system is the source of carryover. Define a gradient method that includes an internal gradient. That is, the analytical gradient is repeated within a single method. If the protein peaks appear in both gradients, at the same time after start, the protein came from the column in what is often described as a memory effect. If the protein peaks only appear when an injection is 6

7 made, they likely originate from adsorption to some system component. In that case follow the instrument manufacturer s recommendations. Memory effects as a source of carryover may be reduced or eliminated in several ways. First, raising the temperature of the separation reduces the possibility of nonspecific protein adsorption. Second, memory effects may be more pronounced with steep gradients. Keep the gradient slope at 10 column volumes or greater. Third, memory effects may be exacerbated by high flow rates. Reduce the flow rate by one half while doubling the gradient time to maintain a constant slope. Finally, apparent memory effects may actually reflect the solubility of the protein in the mobile phase. Reducing the amount injected may eliminate the effect. Note: Useful general information on column troubleshooting problems may be found in HPLC Columns Theory, Technology and Practice, U.D. Neue, (Wiley-VCH, 1997), the Waters HPLC Troubleshooting Guide (Literature code # EN) or visit the Waters Corporation website for information on seminars VI. COLUMN CLEANING, REGENERATION AND STORAGE a. Cleaning and Regeneration Changes in peak shape, peak splitting, shoulders on the peak, shifts in retention, change in resolution, carryover, ghost peaks, or increasing backpressure may indicate contamination of the column. Choose a cleaning option that may be expected to dissolve the suspected contaminant. 3. Flow reversal or back flushing is often suggested as part of a cleaning procedure. This should be reserved as a last resort. It may further damage the column or provide a short-lived improvement in performance. b. Storage For overnight storage, the column can remain in the IEX separation eluent while maintaining a low flow rate (e.g., 0.1 ml/min). For long-term storage, replace the eluent at the suggested flow rate shown below with filtered, high purity water containing 0.05% sodium azide that will inhibit undesired microbial growth. <0.25 ml/min for Protein-Pak Hi Res Q, 5 µm columns <0.5 ml/min for Protein-Pak Hi Res CM and SP, 7 µm columns Completely seal column to avoid evaporation and drying of the bed. VII. INTRODUCING ECORD INTELLIGENT CHIP TECHNOLOGY a. Introduction The ecord intelligent chip is a new technology that will provide the history of a column s maximum backpressure, maximum temperature and the number of injections performed throughout its lifetime. The ecord will be permanently attached to the column to assure that the column s performance history is maintained in the event that the column is moved from one instrument to another. 1. It may be useful to conduct cleaning procedures at one-half the flow rate typically used with that column. In this way the possibility of high pressure events is reduced. 2. Several different cleaning solutions may be injected to strip strongly adsorbed material or particulates from the column. Make the largest injection possible with the system configuration. With such strong cleaning solutions, it is best to disconnect the detector from the column and to direct the flow to waste. a. 0.1mol/L NaOH, or b. 20 ~ 40% acetic acid, or c. Solution containing aqueous organic solvent such as methanol or acetonitrile d. Solution containing a solubilizer such as urea and non-ionic surfactants Figure D. Waters ecord Intelligent Chip 7

8 At the time of manufacture, tracking and quality control information will be downloaded to the ecord. Storing this information on the chip will eliminate the need for a paper Certificate of Analysis. Once the user installs the column, the software will automatically download key parameters into a column history file stored on the chip. In this manual, we explain how the ecord will provide a solution for easily tracking the history of the columns, reduce the frustration of paperwork trails, and give customers the reassurance that a well performing column is installed onto their instruments. d. Column Use Information Figure E. ecord Inserted into Side of Column Heater. b. Installation Install the column into the column heater. Plug the ecord into the side of the column heater. Once the ecord is coupled to the reader, the overall column usage information will be available in the ACQUITY console, allowing the user to access column information on their desktop. The ecord chip provides the customer with column use data. The top of the screen identifies the column including chemistry type, column dimensions and serial number. The overall column usage information includes the total number of samples, total number of injections, total sample sets, date of first injection, date of last injection, maximum pressure, and temperature. The information also details the column history by sample set including date started, sample set name, user name, system name, number of injections in the sample set, number of samples in the sample set, maximum pressure, and temperature in the sample set and if the column met basic system suitability requirements. c. Manufacturing Information The ecord chip provides the user with an overview of the bulk material QC test results. The ecord chip provides the user with QC test conditions and results on the column run by the manufacturer. The information includes mobile phases, running conditions and analytes used to test the columns. In addition, the QC results and acceptance is placed onto the column. 8

9 VIII. ORDERING INFORMATION Product Description Part Number Protein-Pak Hi Res CM, 7 µm, 4.6 x 100 mm Protein-Pak Hi Res SP, 7 µm, 4.6 x 100 mm Protein-Pak Hi Res Q, 5 µm, 4.6 x 100 mm IEX Cation Test Standard IEX Anion Test Standard Note: Only when Protein-Pak Hi Res IEX columns are combined with the ACQUITY UPLC system are the full performance benefits realized. See Waters service notes P/N A for ACQUITY UPLC system configuration guidelines for ion-exchange chromatography. Waters, The Science of What s Possible, ACQUITY UPLC, ACQUITY, and UPLC are registered trademarks of Waters Corporaiton. Protein-Pak, TruView, and ecord are trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Produced in the U.S.A. February 2014 Rev C EN KP-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: