APPLICATION NOTE POROS CaptureSelect affinity chromatography columns POROS CaptureSelect affinity columns for highspeed quantification of IgG Fc fusion proteins Introduction POROS columns, containing highperformance 2 μm analytical beads functionalized with recombinant CaptureSelect affinity ligands, enable the rapid and precise quantification of target biomolecules in process samples including clarified cell culture harvest and downstream product pools. The family of unique POROS CaptureSelect affinity columns includes ligands for the quantification of a wide range of target molecules such as immunoglobulins, Fc fusion proteins, Fab fragments, bispecific antibodies, and more [1]. This application note describes a model system in which Enbrel protein, an Fc anti-tnf fusion protein, is quantified during Chinese hamster ovary (CHO) cell based production and subsequent purification. This data set serves as a model for the capability of the entire POROS CaptureSelect affinity column product line for the rapid and precise quantification of biomolecules. The data presented show the construction of a standard curve and the quantitative recovery of Enbrel protein from a complex analytical sample using a POROS CaptureSelect IgG Fc, PEEK 2 µm column (dimensions: 2.1 mmd x 3 mml; volume: 1 µl), which is the most common analytical column size. This application note provides recommended operating conditions that will help maximize assay performance and column lifetime. However, since target molecules and sample solutions differ widely between various applications, operating conditions should be optimized to accommodate the specific needs of each assay. Materials and methods Buffer systems and method conditions A two-buffer system is recommended for column operation. A typical equilibration/wash buffer (Buffer A) is 25 1 mm phosphate, ph 6.6 7.5, with or without sodium chloride up to 15 mm. The elution buffer ph should be <3.5 for efficient elution. In this study, Buffer A consisted of 5 mm sodium phosphate, ph 7., 15 mm sodium chloride; and Buffer B consisted of 12 mm hydrochloric acid, ph 1.9, 15 mm sodium chloride. Alternative elution buffer systems include sodium phosphate, glycine, citrate, acetate or other components that buffer well at low ph. It is important to prepare the elution buffer with a buffer concentration equal to or greater than that of the equilibration buffer to ensure a good ph transition during elution. Preparing the equilibration buffer and elution buffer with a matched salt concentration will minimize
refractive index shifts in the UV baseline during elution that are particularly evident when monitoring at 215 nm and when analyzing dilute samples. All solutions should be.2 µm filtered prior to use. The characteristics of the chromatography baselines achieved with equilibration and elution buffers can be assessed by running blank samples. A buffer blank run should be performed when a column is new. NOTE: It is important that complete elution be attained. If only partial elution is attained, then the retained target protein will remain bound to the column between injections, column fouling will begin, and product carryover may affect results. The completeness of elution is assessed by recovery of standards. The detailed HPLC method used in this study is summarized in Table 1. Operating flow rates can run up to 5,2 cm/hr or a maximum operating pressure of 18 bar. In order to ensure effective ph transition, elution should consist of a step to 1% Buffer B. Elution should not consist of a gradient or a blend of Buffer A and Buffer B that results in less than 1% Buffer B being used. The duration of wash, elution, and equilibration stages should provide sufficient buffer volume flow to allow for complete ph transition and establishment of UV baselines. For optimized assay performance, system hold-up volumes and delay volumes should be understood. Samples can be assayed at room temperature. If numerous samples are being assayed using an autosampler, the loading tray can be set at 2 8 C to help ensure product stability. Sample preparation Prior to analysis, all samples should be clarified with a.2 µm, low protein binding filter. Centrifugation of samples may not be sufficient to achieve required sample clarity. A recommended injection volume range for standards and samples is 2 1 μl for a 2.1 mmd x 3 mml column. The injection volume should be equivalent for all standards and samples, and should not exceed the column bed volume. A volume of 2 µl was used for all standards and samples in this study. Highly concentrated samples should be diluted with equilibration buffer to ensure samples are within the linear range of the standard curve that has been established. For dilute samples in the range.2.1 mg/ml, injection volumes of up to 1 µl can be used to achieve sensitive detection. Chinese hamster ovary (CHO)-conditioned supernatant cgmp-banked CHO-S cells (Life Technologies) were thawed in CD FortiCHO Medium (Life Technologies) supplemented with 8 mm L-glutamine (FC + gln) and placed into culture at 37 C. On day 7 of culture, conditioned medium was clarified by centrifugation (1 x g for 5 min) and.2 µm sterile filtration. Standard curve construction Standard curves for this study were generated from high-purity material by serial dilution with Buffer A or CHO-conditioned supernatant to the desired concentration ranges. Purified Enbrel protein (5 mg/ml solution in phosphate-buffered saline, ph 7.4) was added to the CHO-conditioned supernatant to a final concentration of.2 1 mg/ml for construction of standard curves. Samples (2 µl) were run in quadruplicate with the HPLC method described in Table 1. Standard curves generated with purified Enbrel protein diluted in Buffer A are shown in Figure 1. Figure 1A shows a standard curve (.5 1. mg/ml) using a less sensitive wavelength of Table 1. HPLC method conditions. Component Description Column POROS CaptureSelect IgG Fc, PEEK 2 µm 2.1 mmd x 3 mml, 1 µl volume (Cat. No. 4469148) System Agilent 12 HPLC Flow 1.5 ml/min (2,6 cm/hr) Injection 2 µl of Buffer A or CHO-conditioned supernatant spiked with purified Enbrel protein, in the final concentration range.1 1 mg/ml (.2 µm filtered) UV detection 28 or 215 nm Equilibration/wash A 5 mm sodium phosphate, ph 7., 15 mm sodium chloride Elution B 12 mm HCl, ph 1.9, 15 mm NaCl Method timetable: Time (min) % B Description. Sample inject.5 Wash with 7 column volumes of Buffer A.51 1 Step to Buffer B 1.5 1 Elution with 14 column volumes of Buffer B 1.51 Step to buffer A 4. Re-equilibration with 35 column volumes of Buffer A END
A Peak area at 28 nm (mau*s) B Peak area at 215 nm (mau*s) 8, 7, 6, 5, 4, 3, 2, 1, 12, 11, 1, y = 724.43x R =.9983 (regression forced through zero). 2. 4. 6. 8. 1. 9, 8, 7, 6, 5, 4, 3, 2, 1, Enbrel protein concentration (mg/ml) y = 11292x R =.9992 (regression forced through zero)..2.4.6.8 1. Enbrel protein concentration (mg/ml) Figure 1. Enbrel protein standard curves at different UV monitoring wavelengths. A POROS CaptureSelect IgG Fc affinity column was operated on an Agilent 12 HPLC system (Table 1). 2 µl injections of Enbrel protein samples diluted in Buffer A, in the concentration ranges of (A).5 1 mg/ml and (B).2 1. mg/ ml, were analyzed at 28 nm and 215 nm, respectively, and areas of the elution peak at 2.7 min retention time were used to construct the standard curves. 28 nm. The standard curve in Figure 1B was generated using a more sensitive detection wavelength of 215 nm and a protein concentration range of.2 1. mg/ml. The resulting integrated peak areas (mau*s) showed a measurement precision of <5% CV at 28 nm and <2% CV at 215 nm across the entire, specified linear concentration range. Curve data fit by regression analysis (forced through zero) showed linear correlation coefficients of R 2 >.99 for both standard curves. Quantitative capture by analytical column To assess quantitative capture, flow-through (FT) fractions were collected from a 1 mg/ml Enbrel protein spiked CHO-conditioned supernatant sample. These dilute samples were reconcentrated using a centrifugal ultrafiltration device with a 5, MW cutoff membrane (Microcon YM-5 P/N, EMD Millipore) and analyzed by SDS-PAGE. For SDS-PAGE analysis, samples of CHO-conditioned supernatant and FT fractions were precipitated with 3 volumes of cold (prechilled at 2 C) acetone for 3 min at 2 C and the precipitate collected at 14, x g for 1 min. After removal of the supernatant and air-drying, the samples were resuspended in SDS sample buffer + 5 mm DTT and heated to 95 C for 3 min. Samples (1 µl) were then analyzed on a 12% precast SDS-PAGE gel. The gel was stained with colloidal Coomassie blue and destained in water. Column reuse and cleaning Extended column reuse performance should be assessed by monitoring the column backpressure and assessing the performance of an assay control sample over time. In order to prolong column lifetime, column cleaning should be done periodically throughout use (1. M NaCl for 2 column volumes, and return to Buffer A) to remove residual material from the column frits and to clean the resin before column backpressure increases. In between analysis sessions, the column was flushed with 2 column volumes of 2% (v/v) ethanol removed from the HPLC, the ends plugged and then stored at 4 C. When column backpressure increases or performance changes, the column can be cleaned. Typical cleaning solutions such as 2 6 M guanidine hydrochloride, 1 M acetic acid, 2% ethanol, 1 M acetic acid plus 2% ethanol, 2% isopropanol, elution buffer titrated to ph 1.5 2., and elution buffer plus 1 2 M sodium chloride can be used to restore full functional performance. Immobilized CaptureSelect affinity ligands are stable at ph 1 1 and ionic strength 5 M in all common salts. All common agents such as 4 M urea, 3 M guanidine hydrochloride, ethylene glycol, and detergents are compatible. A cleaning cycle should involve 2 or 3 injections of cleaning solution at a volume equal to the column bed volume, followed by 2 or 3 injections of equilibration buffer for example, 2 x 1 µl cleaning solution, 2 x 1 µl equilibration buffer on the 2.1 mmd x 3 mml, 1 µl column. Alternatively, cleaning solutions can be put in-line, and multiple column volumes of a desired solution can be run over the column.
If desired, the cleaning can be run in reverse flow to help clean the top frit, with flow direction returned to normal after cleaning. If the system does not allow for flow reversal, the column can be plumbed in reverse, cleaned, and returned to normal position after cleaning. The column cleaning sequence should be monitored by absorbance at different wavelengths, if possible, for impurity clearance to determine the effectiveness of each solution. See the operating instructions that accompany the product [2]. A Absorbance at 28 nm B Absorbance at 28 nm 45 4 35 3 25 2 15 1 5 mau 4 3 2 1.47.131 Load 1.5 Wash Time (min) Elute Elution time (min) Figure 2. Typical POROS CaptureSelect affinity column chromatogram showing elution of Enbrel protein recovered from CHO-conditioned supernatant spiked to a final concentration of 1 mg/ml. (A) 2 µl injection of the above Enbrel protein-spiked CHO supernatant analyzed as described in Table 1. The load, wash, and elute segments of the program and corresponding absorbance are shown. (B) Sample of CHO-conditioned supernatant with no added Enbrel protein, run in parallel. 2.69 2.72 3.25 3.28 Results and discussion A typical chromatogram for a 2 μl injection of CHOconditioned supernatant spiked with Enbrel protein to a final concentration of 1. mg/ml is shown in Figure 2A. The Enbrel protein specific peak eluted at 2.7 minutes. A blank CHO cell sample with no spiked protein is shown in Figure 2B and demonstrates the absence of a specific elution peak. In quadruplicate samples, the average elution peak areas were used to estimate concentration of Enbrel protein using the standard curve from Figure 1A. Protein recovery data are summarized in Table 2 for a single spiked Enbrel protein concentration of 1. mg/ml in a Buffer A or CHO-conditioned supernatant. The data showed an assay %CV of <4% (n = 4 samples), demonstrating strong precision and that Enbrel protein spiked into a complex sample can be quantitated with a high degree of accuracy. Table 2. Recovery and precision of Enbrel protein peak area from CHO-conditioned supernatant. Enbrel protein was added to CHOconditioned supernatant to a final concentration of 1. mg/ml; 2 µl of the spiked complex sample was then injected onto the POROS CaptureSelect IgG-Fc affinity column, and run according to the HPLC method described for Table 1. The elution peak (at 2.7 min) was integrated and the area used to estimate the Enbrel protein concentration from the 28 nm standard curve shown in Figure 1A. The values represent four replicate injections of the same sample. Sample CHO + 1 mg/ml Enbrel protein Buffer A + 1 mg/ml Enbrel protein Calculated Enbrel protein concentration (mg/ml) Precision (%CV) (n = 4) 1.5 1.4 1.4 3.8 Affinity capture of Enbrel protein was shown to be quantitative by SDS-PAGE analysis of flow-through (FT) fractions. The collected samples were adjusted to their original volume as described in Materials and Methods and then screened for the presence of Enbrel protein by SDS-PAGE gel analysis. The resulting gel image (Figure 3) showed that there was no detectable Enbrel protein in the FT fractions tested, confirming quantitative capture and recovery of Enbrel protein from a complex sample.
26 kda 16 kda 11 kda 8 kda 6 kda 5 kda 4 kda 3 kda 2 kda 15 kda M 1 2 3 Conclusions The flexibility and robustness of POROS CaptureSelect affinity columns make the selection of suitable operating conditions for the rapid quantification of biomolecules a straightforward and easily optimized process. Finetuning of assay parameters, within the broad guidelines outlined above, is recommended. When properly used, POROS CaptureSelect affinity columns enable a long lifetime of rapid, reproducible, accurate, and precise quantitation of biomolecules from harvest materials or purification intermediates. The resulting assay developed for the therapeutic Enbrel protein, as a model for Fccontaining fusion proteins, showed specific binding of the target analyte to the POROS CaptureSelect IgG Fc column, effective elution as shown by good recovery, and a wide dynamic range with excellent linearity and robust precision. These performance features enable simple implementation of a routine high-speed, automated quantitative assay in an analytical development or quality lab supporting cell culture and purification process development or manufacturing. Scientific contributors Malcolm G Pluskal, PhD, Shelly Cote-Parra, MS, Christine Gebski, MS Figure 3. Analysis of flow-through (FT) samples by SDS-PAGE. Samples of CHO-conditioned supernatant and FT fractions were analyzed on a 12% precast SDS-PAGE gel, stained with colloidal Coomassie blue and destained in water. M: Novex Sharp unstained protein standard (Life Technologies); lane 1, CHO-conditioned supernatant + 1 mg/ml Enbrel protein; lane 2, FT from CHO-conditioned supernatant + 1 mg/ml Enbrel protein; lane 3, FT from Buffer A + 1 mg/ml Enbrel protein.
References 1. Product Bulletin CO24355: POROS Affinity Chromatography Columns: High-speed quantitation or purification (lifetechnologies.com/captureselect) 2. Operating Instructions: 8-3-4-193 POROS A and G Affinity Columns, POROS CaptureSelect Affinity Columns Ordering information Product Column dimensions (diameter x length) Column volume (ml) Cat. No. POROS CaptureSelect HSA 2.1 mm x 3 mm.1 4469151 POROS CaptureSelect IgG Fc 2 µm (PEEK polymer column) 2.1 mm x 3 mm.1 4469148 POROS CaptureSelect IgM 2 µm (PEEK polymer column) 2.1 mm x 3 mm.1 4469152 POROS CaptureSelect LC Kappa 2 µm (PEEK polymer column) 2.1 mm x 3 mm.1 4469149 POROS CaptureSelect LC Lambda 2 µm (PEEK polymer column) 2.1 mm x 3 mm.1 446915 POROS CaptureSelect CH1 2 µm (PEEK polymer column) 2.1 mm x 3 mm.1 4481823 POROS CaptureSelect FSH 2 µm (PEEK polymer column) 2.1 mm x 3 mm.1 4481822 Note: All POROS CaptureSelect affinity columns are developed on a 2 µm cross-linked poly(styrene-divinyl benzene) resin. Speak to a technical specialist about how these columns can improve your current process at bp@lifetech.com, or go to lifetechnologies.com/captureselect For Research Use Only. Not for use in diagnostic procedures. 213 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. Enbrel is a registered trademark of Immunex Corporation. Microcon is a registered trademark of Merck KGAA. PEEK is a trademark of Victrex PLC. CO27158 313