POROS CaptureSelect affinity columns for rapid, small-scale purification and sample preparation of recombinant proteins

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1 APPLICATION NOTE POROS CaptureSelect affinity chromatography columns POROS CaptureSelect affinity columns for rapid, small-scale purification and sample preparation of recombinant proteins Introduction Thermo Scientific POROS columns, containing highperformance 20 μm analytical beads functionalized with recombinant Thermo Scientific POROS CaptureSelect affinity ligands, can be used for preanalytical sample preparation of biomolecules from complex samples such as clarified cell culture harvests. The family of unique Thermo Scientific POROS CaptureSelect affinity columns includes ligands for the sample preparation of a wide range of target molecules, such as immunoglobulins, Fc fusion proteins, human serum albumin (HSA) fusion proteins, Fab fragments, bispecific antibodies, and other recombinant proteins [1]. The resulting preparative samples can be used in common analytical assays for assessment of posttranslational modifications, including glycosylation and deamidation. In addition, samples can be used for molecular aggregation, protein fragmentation, amino acid sequencing, and bioassay studies. This application note describes a model system in which an IgG1-Fc fusion protein is purified from a dilute sample matrix for further analytical characterization. The resulting purified protein was subjected to structural analysis of N-linked glycans by mass spectrometry (MS), and its aggregation state was assessed by analytical ultracentrifugation (AUC). This data set serves as a model to demonstrate the capability of the POROS CaptureSelect product line for the affinity purification of biomolecules to enable further analytical characterization. This note provides recommended operating conditions that will help maximize purification performance and column lifetime. However, since target molecules and sample solutions differ widely, operating conditions should be optimized to accommodate the specific needs of each application. 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 mm phosphate, ph , with or without sodium chloride up to 150 mm. The ph of the elution buffer (buffer B) should be <3.5 for efficient elution. In this study, buffer A consisted of 50 mm sodium phosphate, ph 7.4, with 150 mm sodium chloride; and buffer B consisted of 100 mm glycine, ph 2.9. Alternative elution buffer systems include sodium phosphate, glycine, citrate, acetate, or other components that buffer well at low ph. If your protein of interest is acid-labile, a higher-ph elution buffer with excipients has proven useful in some applications.

2 An elution buffer of 100 mm sodium acetate, ph 5.0, 1 M magnesium chloride, is an example. If elution at a higher ph still proves challenging, up to 40% (v/v) propylene glycol may also be added. It is important to prepare the elution buffer with a buffering capacity 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, which are particularly evident when monitoring at 214 nm and when analyzing dilute samples. All solutions should be 0.2 µm filtered prior to use. The chromatography baselines of the equilibration and elution buffers can be assessed by running blank samples. A buffer blank run should be performed when a column is new. The detailed HPLC method used in this study is summarized in Table 1. Operating flow can run up to 5,000 cm/hr or a maximum operating pressure of 180 bar. To ensure effective ph transition, elution should consist of a step to 100% buffer B. Elution should not consist of a gradient or a blend of buffer A and buffer B that results in buffer B being used at less than 100% strength. The durations of wash, elution, and equilibration steps should provide sufficient column volumes of flow to allow for complete ph transition and establishment of UV baselines (Figure 1A). For optimized performance, system hold-up and delay volumes should be understood. Samples can be run at room temperature. If numerous samples are being purified using an autosampler, the loading tray can be set at 2 8 C to help ensure product stability. Note: It is important to attain complete elution. If only partial elution is attained, the target protein will remain bound to the column between purification runs, column fouling will begin, and product carryover may affect results. The completeness of elution is assessed by recovery of standards. Table 1. HPLC method conditions. Component Column System Flow Injection Description POROS CaptureSelect IgG Fc, 20 µm PEEK polymer column, 2.1 mm x 30 mm, 100 µl volume (Cat. No ) HPLC system with a quaternary pump 0.5 ml/min (866 cm/hr) 7 ml of a CHO-conditioned medium spiked with purified IgG1-Fc fusion protein to 0.1 mg/ml final concentration (0.2 μm filtered) 280 nm 50 mm sodium phosphate, ph 7.4, 150 mm sodium chloride UV detection Equilibration/wash (buffer A) Elution (buffer B) 100 mm glycine, ph 2.9 Method timetable Time (min) % buffer B Description * Fractions were collected at the outlet of the HPLC UV detector and pooled Pump 7 ml (70 column volumes) of sample onto the column using the system pump Wash with 50 column volumes of buffer A Step to buffer B Elute with 14 column volumes of buffer B* Step to buffer A Re-equilibrate with 37 column volumes of buffer A End

3 Load preparation, column loading, and 25 sample collection 16.6 All load material should be clarified with a µm, low 20 protein-binding filter. Centrifugation may not be sufficient to ensure load clarity. As a starting 15point, target analytes can 10.3 be loaded to 80% of the reported dynamic binding capacity 10 of the column. A Thermo Scientific POROS 6.0 CaptureSelect IgG Fc, 2.1 mm x 30 mm, 100 µl column was used for this 5 study. Depending on the HPLC system capabilities, larger load volumes can be introduced to the column. Fluorescence intensity (FU) A Absorbance at 280 nm (mau) Load Wash Elute Retention time (min) Table 2 summarizes the POROS CaptureSelect IgG Fc affinity ligand family of columns and their potential preparative yield available in this format. The dynamic binding capacities of other members of the POROS CaptureSelect affinity ligand column family are summarized in the operating instructions [2]. Fractions were collected directly at the outlet of the UV detector on the HPLC system and pooled based on the UV trace. Time (min) B 1 2 CHO-conditioned supernatant with added IgG1-Fc fusion protein cgmp-banked Gibco CHO-S Chinese hamster ovary cells were thawed in Gibco CD FortiCHO Medium supplemented with 8 mm L-glutamine and placed into culture at 37 C. On day 7 of culture, the conditioned medium was clarified by centrifugation (100 x g for 5 min) and 0.2 µm sterile filtration. IgG1-Fc fusion protein (50 mg/ml in phosphate- buffered saline, ph 7.4) was added to the conditioned supernatant to a final concentration of 0.1 mg/ml for preparative loading. Figure 1. Small-scale purification from dilute feed. The POROS CaptureSelect IgG Fc column was operated on a HPLC system. A 7 ml sample of clarified CHO-conditioned supernatant spiked with IgG1-Fc fusion protein to a final concentration of 0.1 mg/ml was loaded following the method described in Table 1. Elution was performed at ph 2.9 at a flow rate of 0.5 ml/min. (A) Representative chromatogram of the protein purification monitored at 280 nm. (B) SDS-PAGE analysis of samples before and after purification. The samples were run on an SDSpolyacrylamide (12%) gel under reducing conditions, stained with colloidal Coomassie blue, and destained in water. Lane 1: column load; lane 2: elution pool. Table 2. Potential preparative yield of POROS CaptureSelect IgG Fc affinity ligand columns. Column type POROS CaptureSelect IgG Fc column Column dimensions (diameter x length) Column volume (ml) Dynamic binding capacity (mg per ml of resin volume) Potential preparative yield* (mg) 2.1 mm x 30 mm mm x 50 mm mm x 100 mm mm x 100 mm * Up to 80% of the stated dynamic binding capacity can be loaded without product breakthrough, but should be optimized for each ligand and target analyte. The dynamic binding capacities of other members of the POROS CaptureSelect affinity ligand column family are summarized in the operating instructions [2].

4 Analysis of PNGase F released N-linked glycans by HILIC ion-trap MS/MS Approximately 100 µg of the elution pool described in Figure 1 was treated with PNGase F (Prozyme) overnight at 37 C, per the supplier s recommendation. The released glycans were harvested by ethanol extraction. The precipitated protein from the ethanol extraction procedure was saved for further analysis by ESI-ToF MS (data not shown), while the extracted glycans were brought to dryness and then derivatized with 2-aminobenzamide (2- AB) in the presence of sodium cyanoborohydride. After cleanup by gel filtration for the removal of excess dye, the derivatized glycans were analyzed by hydrophilic interaction chromatography (HILIC) using an NH2P-50 2D column (Shodex) at a flow rate of 0.5 ml/min and a 10 column volume (CV) linear gradient. Mobile phase A contained 2% (v/v) acetic acid and 1% (v/v) tetrahydrofuran (THF) in acetonitrile, and mobile phase B contained 5% (v/v) acetic acid, 3% (v/v) triethylamine, and 1% (v/v) THF in water. Detection was by fluorescence with excitation at 320 nm and emission at 430 nm. Peaks of interest were analyzed using an ion-trap mass spectrometer to identify glycan structures. Sialic acid content of proteins purified using POROS CaptureSelect IgG Fc affinity column Approximately 43 µg of the purified protein and 140 µg of bovine fetuin control were subjected to mild acid hydrolysis in a solution of 2.5 M acetic acid for 45 min at 80 C. (Fetuin is a well-characterized glycoprotein and was added as an internal control for sample processing.) The released sialic acids were derivatized with o-phenyldiamine and then chromatographed using a 4.6 mm (D) x 150 mm (L) reverse phase C18 column (Prodigy 3 µm ODS, Phenomenex). Elution was performed using a 10 CV linear gradient at a flow rate of 0.6 ml/min. Mobile phase A consisted of 0.5% (v/v) phosphoric acid, 0.3% (v/v) butylamine, 1% (v/v) THF in water. Mobile phase B consisted of 50% (v/v) acetonitrile in mobile phase A. Analytical ultracentrifuge (AUC) analysis of proteins purified using POROS CaptureSelect IgG Fc affinity column Prior to analysis by AUC, the affinity-purified IgG1-Fc fusion protein was adjusted from ph 4.0 to ph 7.4 with 1 N NaOH. The 50 mg/ml stock IgG1-Fc fusion protein sample was diluted to 0.5 mg/ml with 1X PBS, ph 7.4. The protein samples were loaded into the sample channels of AUC cells having quartz windows and 12 mm doublesector centerpiece. A 1X PBS buffer blank was loaded into the corresponding reference channel of each cell. The centrifugation was carried out at 20 C, 45,000 rpm, for hours. Radial scans of the concentration profile were collected sequentially by absorbance at 280 nm until full sedimentation was reached. The resulting data sets were analyzed using the program SEDFIT with a continuous c(s) distribution model, yielding best-fit distributions for the number of sedimenting species and the effective molecular weights. 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 (1.0 M NaCl for 20 CV 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 20 CV of 20% (v/v) ethanol, removed from the HPLC, plugged at the ends, and then stored at 4 C. When column backpressure increases or performance changes, the column should be cleaned. Typical cleaning solutions include 2 6 M guanidine hydrochloride, 1 M acetic acid, 20% ethanol, 1 M acetic acid plus 20% ethanol, 20% isopropanol, elution buffer titrated to ph , and elution buffer plus 1 2 M sodium chloride. These cleaning solutions can be used to restore functional performance. Immobilized POROS CaptureSelect affinity ligands are stable at ph 1 10 and ionic strengths of 0 5 M of all common salts. All common agents such as 4 M urea, 3 M guanidine hydrochloride, propylene glycol, and detergents are compatible with the columns. A cleaning cycle should involve 2 or 3 injections of cleaning solution (1 CV per injection), followed by 2 or 3 injections of equilibration buffer. For example, use 2 x 100 µl cleaning solution, then 2 x 100 µl equilibration buffer on the 2.1 mm x 30 mm, 100 µl column. Alternatively, multiple column volumes of a desired solution can be run over the column. If desired, the solution can be run in reverse flow to help clean the top frit, with the 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 the 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].

5 Results and discussion A POROS CaptureSelect IgG Fc 2.1 mm x 30 mm affinity column was used for small-scale purification of a dilute target biomolecule from a complex biological sample. In this case, we used CHO-conditioned supernatant spiked with IgG1-Fc fusion protein. Subsequent elution was carried out at low ph. The target protein was purified from CHO-conditioned supernatant at a dilute concentration of 0.1 mg/ml, and the resulting chromatogram is shown in Figure 1A. The eluate showed a high degree of purity, as assessed by SDS-PAGE (Figure 1B). This elution pool of purified protein was used in the following methodologies for subsequent analytical characterization: Analysis of N-linked glycans, with the released N-linked glycan structures from the IgG1-Fc fusion protein resolved by HILIC (Figure 2) coupled to MS/MS detection. From this analysis, the putative structure of the released glycans can be derived. Fluorescence intensity (FU) Retention time (min) Figure 2. Analysis of PNGase F released N-linked glycans by HILIC ion-trap MS/MS. Approximately 100 µg of the elution pool from Figure 1 was treated with PNGase F enzyme (Prozyme) overnight at 37 C, per the supplier s recommendation. The released glycans were harvested by ethanol extraction and subjected to analysis as described in the methods section Absorbance at 280 nm (mau) Sialic acid content of released N-linked glycans was determined as o-phenyldiamine derivatives and resolved by reverse phase HPLC analysis, employing bovine fetuin as an assay control. A summary of the results is shown in Table 3. Table 3. Sialic acid (SA) content of protein purified using POROS CaptureSelect IgG Fc affinity column. SA content Eluted peak SA content (mol SA/ mol protein) Wash Fetuin control (mol SA/mol protein) Glycolyl Acetyl Total Analytical ultracentrifugation (AUC) to characterize the molecular size, aggregation state, and shape of the protein. Material prepared using the analytical POROS column was high enough in purity to be subjected directly to AUC analysis. The resulting SEDFIT analysis of the AUC sedimentation data is summarized in Table 4. The data show that the purified IgG1-Fc fusion protein had an aggregate profile similar to that of a control Load sample of the target protein that was not processed with a POROS CaptureSelect column. The analytical methods above are just a few examples of Elute the processes that can be used to further characterize a target protein purified from small amounts of starting sample. The pre-analysis workflow employing a POROS CaptureSelect affinity column can quickly yield pure protein in sufficient amounts Time (min) (up to 56 mg in this application) to facilitate more detailed protein characterization by a wide range of analytical methodologies.

6 Table 4. Analytical ultracentrifugation (AUC) analysis of protein purified using POROS CaptureSelect IgG Fc affinity column, and protein control. SEDFIT data Purified protein Protein control sample Time (sec x ) Approx. MW % Total Approx. MW % Total < Conclusions The use of the POROS CaptureSelect IgG Fc Affinity Column for small-scale, rapid purification of a recombinant Fc protein has been demonstrated. The resulting affinitypurified protein was shown to be suitable for use in subsequent analytical characterization. References 1. Product Bulletin CO24355, POROS Affinity Chromatography Columns: High-speed quantitation or purification. 2. Operating Instructions, POROS CaptureSelect Affinity Columns. P/N The POROS CaptureSelect IgG Fc affinity column format can be used to purify 0.7 to 56 mg of material per cycle for further protein characterization (Table 2), depending on the size of the column used. The range of column sizes and the various ligands available enable the preparation of tens of milligrams of high-purity protein for a range of target molecules. This material can then be used in a number of different protein analytical methods for further characterization that targets glycosylation, molecular aggregation, protein fragmentation, oxidation, or deamidation. POROS CaptureSelect affinity columns offer robust performance and allow for efficient and easily optimized sample preparation and purification processes. The columns can provide highly pure protein that is suitable for analytical characterization directly from complex samples. The coupling of highly specific, stable POROS CaptureSelect affinity ligands to the highly efficient 20 µm POROS chromatography resin creates a unique, highspeed, reusable affinity column perfectly suited for sample preparation of recombinant proteins.

7 Ordering information Product POROS CaptureSelect HSA POROS CaptureSelect IgG Fc POROS CaptureSelect IgM POROS CaptureSelect LC Kappa POROS CaptureSelect LC Lambda POROS CaptureSelect FSH Column dimensions (diameter x length) Column volume (ml) Cat. No. 2.1 mm x 30 mm mm x 50 mm mm x 100 mm mm x 100 mm mm x 30 mm mm x 50 mm mm x 100 mm mm x 100 mm mm x 30 mm mm x 50 mm mm x 100 mm mm x 100 mm mm x 30 mm mm x 50 mm mm x 100 mm mm x 100 mm mm x 30 mm mm x 50 mm mm x 100 mm mm x 100 mm mm x 30 mm mm x 50 mm mm x 100 mm mm x 100 mm New columns are added periodically to expand the product catalog. Please visit our website for the most current selection at thermofisher.com/captureselect

8 Ordering information Product Column dimensions (diameter x length) Column volume (ml) Cat. No. POROS CaptureSelect GCSF Lab Scale Columns POROS CaptureSelect GCSF POROS CaptureSelect hgh Lab Scale Columns POROS CaptureSelect hgh POROS CaptureSelect IgA Lab Scale Columns POROS CaptureSelect IgA 2.1 mm x 30 mm mm x 50 mm mm x 100 mm mm x 100 mm mm x 30 mm mm x 50 mm mm x 100 mm mm x 100 mm mm x 30 mm mm x 50 mm mm x 100 mm mm x 100 mm Contact a technical specialist about how these columns can improve your current process at or go to thermofisher.com/captureselect For Research Use Only. Not for use in diagnostic procedures Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. PEEK is a trademark of Victrex PLC. COL

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