TechnicalNote. BAKERBOND Multimode Ion Exchange Chromatography Media. Introduction. BAKERBOND PolyPEI

Transcription

1 TechnicalNote BAKERBOND Multimode Ion Exchange Chromatography Media BAKERBOND PolyPEI Introduction BAKERBOND PolyPEI is a multimode ion exchanger functioning as a weak anion exchanger over a wide ph range. The unique selectivity of PolyPEI is the result of proprietary surface chemistry with weak anionic exchange sites due to the presence of primary, secondary and tertiary nitrogen on the Polyethyleneimine (PEI) ligands. The presence of amino groups with different pka offers better selectivity than conventional weak anion exchange media with equivalent capacities, and it is capable of separating proteins and peptides having similar isoelectric points (pi). Unique selectivity of closely related molecules is often achieved with PolyPEI where conventional ion exchangers fail to provide sufficient separation in a process environment. The average particle size of 35 µm with narrow particle size distribution (d 60 /d 10 <1.4) provides high efficiency and will produce narrow elution bands of highly concentrated product. This increases resolution and decreases pool volumes compared to media based on larger particles. High efficiency in combination with high selectivity enables higher loading levels while maintaining separation. PolyPEI is based on highly cross linked rigid spherical polymethacrylate particles with optimal porosity and mechanical strength. The hydrophilic backbone has low non-specific binding and the ability to withstand prolonged contact with all commonly used cleaning and sanitizing solutions. PolyPEI can be easily packed to bed heights of up to 40 cm and operated in the linear velocity range of up to 500 cm/hr using conventional columns with pressure ratings of 2 to 10 bar. PolyPEI is manufactured under the strictest controls and testing and is supported by world class quality systems and application resources. As a result of this unique combination of selectivity, efficiency, and the ability to maintain performance at a high linear velocity, PolyPEI can provide high process throughput, improvements in productivity and enable new processes. Characteristics Functionality Weak Anion Exchanger Functional Group -CH2CH2NH2, (-CH2CH2)2NH, (CH2CH2)3N Anion exchange Capacity Cl-meq/mL Average Particle Size 35 µm Particle Size Distribution (d60/d10) < 1.4 Median Pore Size 500 Å Exclusion Limit Daltons Operating ph Range Cleaning ph Range Chemical Stability All commonly used aqueous buffers, sodium hydroxide, acetic acid, phosphoric acid, guanidine hydrochloride, up to 100% ethanol, methanol, or 2-Propanol. Shipping Solvent Media is shipped as 1:1 slurry in acetate buffer containing benzyl alcohol at ph 4.5 and can be stored for up to 5 years at 4 15 C. The media can also be stored in 20% ethanol or 0.1 M NaOH for up to 5 years. TECHNICAL NOTE

2 Multimode functionality FIG. 1. Ligand structure of PolyPEI The multimode functionality of PolyPEI is obtained by covalently bonding PEI to the surface of highly cross linked polymethacrylate beads. Proprietary surface chemistry results in various weak anionic exchange sites due to the presence of primary, secondary and tertiary nitrogen on the PEI ligands, thereby providing a weak anion exchange behavior. Physical Characteristics FIG. 2. SEM image of polymethacrylate spherical beads at 2,000 magnification FIG.3. Particle size distribution of a typical lot of polymethacrylate beads The median particle size of 35 micron and a particle size distribution of less than 1.4 (d 60 /d 10 ) shows a total lack of fines. The picture details the open pore structure of the bead. As illustrated by the SEM image and particle size distribution graph, PolyPEI is capable of providing high column efficiency leading to narrow elution bands and as a result, concentrated product fractions. The SEM image also shows good porosity with an average pore size of 500 Å and an exclusion limit of Dalton. This open porosity coupled with the bead s high mechanical strength allows the use of PolyPEI at high linear velocities due to good mass transfer. TECHNICAL NOTE 2

3 Separation Performance: Selectivity and Capacity FIG. 4. Separation of Proteins on PolyPEI Column: cm Buffer A: 50 mm TRIS ph 8.0 Buffer B: buffer A plus 1 M NaCl, ph 8.0 Linear gradient from 0 to 100% B in 10 CV Flow rate: 1.2 ml/minute Sample volume: 1.5 ml. Protein pi MW Sample conditions 1. Lysozyme kd 0.4 mg/ml 2. Human IgG kd 4.0 mg/ml 3. BSA kd 2.5 mg/ml 4. B-Lactoglobulin B kd 1.0 mg/ml 5. B-Lactoglobulin A kd 1.0 mg/ml FIG. 5. Dynamic Binding Capacity of BSA on PolyPEI at High Linear Velocity Column: mm sample: 1 mg/ml Bovine Serum Albumin Buffer A: 20 mm Sodium Acetate, ph 6.2 Elution Buffer: 1 M Sodium Acetate, ph 6.2 PolyPEI media provides unique selectivity and can be used to separate closely related proteins due to its unique chemistry and multimodal functionality. As FIG. 4. illustrates, BSA (pi 4.7), B-Lactoglobulin B (pi 5.34) and B-Lactoglobulin A (pi 5.21) having close isoelectric points are well resolved and can be easily separated. As expected, lysozyme flows through without binding while IgG is retained demonstrating the capability to separate lysozyme from IgG as well. This in combination with the media s high efficiency enable high column loading. FIG. 5. illustrates that the breakthrough capacity of BSA does not change significantly over the linear velocity range of 200 to 400 cm/hour. This is due to the particle s open pore structure and rigidity facilitating good mass transfer. High resolution and the ability to maintain capacity at high linear velocities facilitate high throughput for both large and small proteins. TECHNICAL NOTE 3

4 Stability and Consistency FIG. 6. Chemical Stability of PolyPEI Column: mm, sample injected: 5 ml Binding buffer: 20 mm Sodium Acetate, ph 6.2 Elution buffer: binding buffer plus 1 M NaCl, ph 6.2 Sample: lysozyme, rabbit gamma globulin and alpha lactalbumin Red: PolyPEI for 10 months in storage solution at 4 C Blue: PolyPEI for 10 months in storage solution at room temperature Pink: PolyPEI for 10 months in 10 mm phosphoric acid Green: PolyPEI in 0.1 NaOH at room temperature for 10 months Cyan: PolyPEI washed dynamically with 0.5 M NaOH for 48 hours at 1.0 ml/min. The unique selectivity of PolyPEI is not affected by extreme ph conditions over extended periods of time. FIG. 7. Relative Dynamic Binding Capacity (DBC) of BSA under various storage conditions Column: mm Binding buffer: 20 mm Sodium Acetate, ph 6.2 Elution buffer: 1 M Sodium Acetate, ph 6.2 A: Fresh lot in Sterilant buffer at room temperature B: in 10mM H3PO4 at room temperature for 4.5 years C: in 0.1N NaOH at room temperature for 4.5 years D: in Sterilant buffer at low temperature for 4.5 years In FIG. 6, the consistent separation behavior of PolyPEI was evaluated by separating lysozyme, rabbit gamma globulin and alpha lactalbumin from a mixture after the media was subjected to various storage conditions, including extreme ph over a period of 10 months. After each separation, the column was regenerated by washing with 5 CV of elution buffer. There is no measureable change in retention time of the proteins, indicating consistent separation performance under a wide ph range of storage conditions. In FIG. 7, PolyPEI is again shown to be chemically stable since there is no significant change in capacity of initial media compared to media samples stored in different solutions for up to 5 years. Stability of PolyPEI was evaluated by measuring the dynamic binding capacity of BSA. Slight variations are expected within the error of the experiment. TECHNICAL NOTE 4

5 Operational Flexibility FIG. 8 Pressure Flow relation of polymethacrylate FIG. 9. Scale up to a 45 cm ID Column Bed height: 21.5 cm Column diameter: 20 cm PolyPEI can easily be packed in large columns for high throughput applications. The linear behavior of the pressure flow curve indicates that there is no resin compression. FIG. 10. Efficiency of column (20 id x 21.5 cm high) packed with polymethacrylate based media The conductivity peak shows an efficiencyof 11,061 plates/meter at an asymmetry of Repeated column packing of the media yields high efficiencies as well as low asymmetries and is reproducible from a 20 cm to 45 cm column, indicating easy process scalability. The narrow particle size distribution and mechanical stability of PolyPEI provide the ability to operate in conventional low pressure columns at a bed height of up to 20 cm. Modern medium pressure columns, rated at 7 bar, can be used to pack beds up to 40 cm bed height and operate at higher linear velocities. The linear pressure flow curve for polymethacrylate indicates no resin compression at a bed height of 21.5 cm and a column diameter of 20 cm. In addition, the media does not need to be defined prior to the initial packing or subsequent packings, reducing the consumption of buffers and simplifying operations. TECHNICAL NOTE 5

6 Product Ordering Information Product Number Size ml ml ml L L Phillipsburg, NJ 9001: 2008 & 14001: 2004 Paris, KY 9001: 2008 Mexico City, Mexico 9001: 2008 Deventer, the Netherlands 9001: 2008 & 14001: 2004 & 13485: 2003 Selangor, Malaysia 9001: 2008 About Avantor Performance Materials Avantor Performance Materials manufactures and markets high-performance chemistries and materials around the world under several respected brand names, including the J.T.Baker, Macron Fine Chemicals, Rankem, Diagnova, BeneSphera, and POCH brands. Avantor products are used in a wide range of industries. Our biomedical and life science solutions are used in academic, industry and quality control laboratories for research, pharmaceutical production and medical lab testing, while our electronics solutions are used in the manufacturing of semiconductors and flat panel displays. Based in Center Valley, Pennsylvania (USA), Avantor is owned by an affiliate of New Mountain Capital, LLC. For additional information please visit or follow Ordering Information and Assistance Customer Service and Technical Service TOLL FREE: AVANTOR ( ) OUTSIDE OF U.S. TEL: FAX: info@avantormaterials.com AskAvantor Our Web site features ASK Avantor, which includes live chat capabilities with customer service representatives. Lit Number: Avantor Performance Materials, Inc. All rights reserved. Trademarks are owned by Avantor Performance Materials, Inc. or its affiliates unless otherwise noted. Corporate Headquarters Avantor Performance Materials, Inc Corporate Parkway Suite #200 Center Valley, PA USA Worldwide Locations China Malaysia North America India Mexico Taiwan Korea The Netherlands For contact information at these locations, visit Us/Worldwide-Directory.aspx