High-speed quantitation or purification

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1 PRODUCT BULLETIN POROS Affinity Chromatography Columns POROS Affinity Chromatography Columns High-speed quantitation or purification Broad utility POROS A, G, and CaptureSelect affinity ligands address most biological drug classes Rapid POROS resins offer fast quantitation or purification of immunoglobulins and fusion proteins Reduced hands-on time HPLC method can be automated for streamlined operation Robust deliver highperformance purification over hundreds of chromatography cycles Analytical groups supporting biological drug development efforts require rapid, sensitive, and precise methods for quantifying proteins from cell cultures, fermentation harvests, or purification intermediates. It is critical that biopharmaceutical manufacturers be able to monitor bioreactor product titers to determine when to harvest or abort a reactor cycle. POROS Affinity Chromatography Columns incorporate the advantages of high-speed, high-performance analytical 2 µm POROS chromatography resins with the unique selectivity of Protein A, Protein G, and CaptureSelect ligands (Figure 1). The combination allows for 3-minute quantitation of immunoglobulins, fusion proteins, Fab fragments, and bispecific antibodies. Alternative assays lack the combined advantages of speed, selectivity, method automation, and high precision. In addition, the affinity-purified protein is suitable for subsequent analysis such as mass spectrometry. V H Protein A or G C H 1 C H 2 C H 3 C L V L Protein L CaptureSelect LC-Kappa or LC-Lambda CaptureSelect IgG Fc Figure 1. POROS A, G, and CaptureSelect ligands target multiple IgG epitopes. This graphic shows the binding sites for affinity ligands Protein A, Protein G, and Protein L. Novel POROS CaptureSelect ligands provide alternative specificities that enable rapid binding for the quantitation or purification of engineered immunoglobulins (POROS Affinity Chromatography Columns represented in orange).

2 The BAC and Life Technologies collaboration creates a range of ready-to-use HPLC columns prepacked with CaptureSelect affinity ligands immobilized on POROS chromatography media. Designed for the high flow rates necessary to provide the real-time results demanded for biopharmaceutical development and quality assurance needs, the unique selectivity of these columns will support Process Analytical Technology (PAT) for most biopharmaceutical manufacturing process. High speed with high specificity The combination of Protein A, Protein G, and CaptureSelect affinity ligands combined with POROS 2 µm resin provides a high flow rate, high-performance chromatography solution for the rapid quantitation and small-scale purification of: IgG all subclasses, specific for human and many other species (Table 1) IgM specific for human, mouse, or rat Kappa light chain specific for human, all subclasses Lambda light chain specific for human, all subclasses Human serum albumin Fusion proteins, Fc, or human serum albumin POROS perfusion chromatography allows the quantitation of product from clarified harvest or partially purified intermediates typically in under 3 minutes. Moreover, only minimal sample preparation (.2 µm filtration) is required, and the eluted product is highly purified and suitable for further analysis. Figure 2 illustrates the chromatographic performance of POROS CaptureSelect LC-Kappa with five unique kappa Fab fragments. The associated standard curves, Figure 3, exhibit excellent linearity. Additionally, Figure 4 displays the specificity of the CaptureSelect HSA ligand Figure 2. POROS CaptureSelect resins exhibit high-speed and efficient chromatography, regardless of the type of kappa light chain. This overlay of 5 kappa light chain runs on a POROS CaptureSelect LC-Kappa column illustrates the speed and efficiency of the analysis. Column: POROS CaptureSelect LC-Kappa column (Cat. No , 2.1 x 3 mm, PEEK polymer). System: Agilent 12, flow rate 2 ml/min. Sample injection: 5 µl purified kappa light chain,.5 mg/ml. Equilibration buffer: phosphate buffer, 15 mm NaCl, ph 7.5. Elution buffer: 12 mm HCl, 15 mm NaCl, ph 1.9. UV detection: 28 nm. Absorbance (mau min) Injection volume (μl) Figure 3. Standard curves for POROS CaptureSelect LC-Kappa exhibit good linear range (8x) and linear correlation coefficients (R 2 range:.9991 to 1). Column: POROS CaptureSelect LC-Kappa column (Cat. No , 2.1 x 3 mm, PEEK polymer). System: Agilent 12, flow rate 2 ml/min. Sample injection: Specified volumes of purified kappa light chain,.5 mg/ml. Equilibration buffer: phosphate buffer, 15 mm NaCl, ph 7.5. Elution buffer: 12 mm HCl, 15 mm NaCl, ph 1.9. UV detection: 28 nm.

3 Table 1. Selection guide. POROS Protein A, G resins POROS CaptureSelect resins Bimolecular type Protein A Protein G IgG-Fc LC-Kappa LC-Lambda HSA IgM IgG1, IgG2, IgG4 IgG3 IgG, C H 2 domain deleted IgM IgA, IgD, IgE Fab, Fab 2 Human serum albumin Albumin fusion protein IgG Fc fusion protein Specifications Product Protein-binding capacity (mg/ml) Species specificity POROS CaptureSelect IgG-FC 8.9 Human POROS CaptureSelect LC-Kappa14.5 Human POROS CaptureSelect LC-Lambda 1. Human POROS CaptureSelect HSA 2.6 Human POROS CaptureSelect IgM 4. Human, mouse, rat POROS Protein A 19. Human, mouse, and 5 other species POROS Protein G 9. Human, mouse, rat, and 8 other species Materials Component Column Frit POROS resin CaptureSelect ligand Protein A, Protein G Material of construction PEEK polymer or stainless steel 2 µm, titanium Crosslinked poly (styrenedivinylbenzene) Single-domain monospecific antibody fragment produced in Saccharomyces cerevisiae Recombinant Protein A and Protein G Automation POROS Affinity Chromatography Columns are suitable for use with HPLC systems, which allow for the automation of chromatography methods, data collection, and data analysis. In addition, the immobilized affinity ligands enable one method and buffer system to be used across a class of molecules, streamlining analytical approaches. Multiple cycles Proper sample preparation, adherence to column-operating instructions, and the use of an optimized columncleaning regime result in strong reuse performance. POROS Affinity Chromatography Columns can be cycled hundreds of times for costeffective and reproducible quantitation or purification.

4 Figure 4. POROS CaptureSelect HSA resin exhibits high specificity for the target molecule. The specificity of a POROS CaptureSelect HSA column was demonstrated by loading (A) 1 mg/ml bovine serum albumin (BSA) and (B) 1 mg/ml monoclonal antibody samples. The chromatograms show both BSA and antibody eluting in the void volume, whereas HSA fusion protein (C) elutes at approximately 2.9 min. A Column: POROS CaptureSelect HSA column (Cat. No , 2.1 x 3 mm, PEEK polymer). System: Agilent 12, flow rate 1 ml/min. Sample injection: 1 µl purified HSA fusion protein,.2 mg/ml. Equilibration buffer: phosphate buffer, ph 7.. Elution buffer:.5 M acetic acid, ph 2.5. UV detection: 28 nm B C

5 Speak to a technical specialist about how these columns can improve your current process, at bp@lifetech.com, or go to Ordering information Product Column dimension (diameter (mm) x length (mm)) Volume (ml) Cat. No. POROS Protein A 2 μm (stainless steel column) 2.1 x μm (PEEK polymer column) 2.1 x μm (PEEK polymer column) 4.6 x μm (PEEK polymer column) 4.6 x μm (PEEK polymer column) 1 x POROS Protein G 2 μm (PEEK polymer column) 2.1 x μm (PEEK polymer column) 4.6 x μm (PEEK polymer column) 4.6 x μm (PEEK polymer column) 1 x POROS CaptureSelect HSA 2 μm (PEEK polymer column) 2.1 x μm (PEEK polymer column) 4.6 x μm (PEEK polymer column) 4.6 x μm (PEEK polymer column) 1 x POROS CaptureSelect IgG-Fc 2 μm (PEEK polymer column) 2.1 x μm (PEEK polymer column) 4.6 x μm (PEEK polymer column) 4.6 x μm (PEEK polymer column) 1 x POROS CaptureSelect IgM 2 μm (PEEK polymer column) 2.1 x μm (PEEK polymer column) 4.6 x μm (PEEK polymer column) 4.6 x μm (PEEK polymer column) 1 x POROS CaptureSelect LC-Kappa 2 μm (PEEK polymer column) 2.1 x μm (PEEK polymer column) 4.6 x μm (PEEK polymer column) 4.6 x μm (PEEK polymer column) 1 x POROS CaptureSelect LC-Lambda 2 μm (PEEK polymer column) 2.1 x μm (PEEK polymer column) 4.6 x μm (PEEK polymer column) 4.6 x μm (PEEK polymer column) 1 x

6 For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. 212 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. CaptureSelect is a registered trademark of BAC BV. PEEK is a trademark of Victrex PLC. Printed in the USA. CO lifetechnologies.com