Poros a 20 m column

Manufactured by Thermo Fisher Scientific

The POROS A 20 μm column is a chromatographic resin designed for the purification of antibodies and other Fc-containing proteins. It features a rigid, macroporous polystyrene-divinylbenzene matrix with Protein A ligands immobilized on the surface. This column is intended for use in preparative and process-scale chromatographic separations.

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Lab products found in correlation

Chromeleon 7, by Thermo Fisher Scientific (2 mentions) 0.22 m filter, by Merck Group (1 mentions) Dionex ultimate 3000, by Thermo Fisher Scientific (1 mentions) Dionex ultimate 3000 hplc, by Thermo Fisher Scientific (1 mentions) Ghp acroprep 96 filter plate, by Pall Corporation (1 mentions) Gswp04700, by Merck Group (1 mentions)

3 protocols using poros a 20 m column

Detailed Protocols for Bispecific Antibody Analysis

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Soluble aggregate quantification was achieved by two ACQUITY UPLC PrST SEC Columns, 200 Å, 1.7 µm, 4.6 × 150 mm² cat#186005225 in series in a 10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 mobile phase. Bispecific purity was measured using three prepacked POROS A 20 µm columns (2.1 × 30 mm², 0.1 mL) cat#2‐1001‐00 in series and an isocratic elution buffer system. Bispecific and FcFc titers were measured using a POROS A 20 µm column (2.1 × 30 mm², 0.1 mL) cat#2–1001‐00, and Fc*Fc* titers were measured by loading the Protein A flowthrough over a POROS G 20 µm column (2.1 × 30 mm², 0.1 mL) cat#2‐1002‐00 (Thermo Scientific). HCP quantification was performed using a commercially available ELISA kit cat#F550 (Cygnus Technologies). Leached affinity ligand was quantified using the commercially available ELISA kit cat#F400 (Cygnus Technologies).

Tustian A.D., Laurin L., Ihre H., Tran T., Stairs R, & Bak H. (2018). Development of a novel affinity chromatography resin for platform purification of bispecific antibodies with modified protein a binding avidity. Biotechnology Progress, 34(3), 650-658.

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Antibody Concentration Determination by HPLC

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HPLC Protein A affinity chromatography was used to determine the antibody concentration. We used a Dionex UltiMate 3000 HPLC system equipped with a diode array detector (Thermo Fisher Scientific). Mobile phase A was 50 mM phosphate buffer, pH 7.0. Mobile phase B was a 100 mM glycine buffer, pH 2.5. Before usage, all buffers were filtered through 0.22 µm filters (Merck KGaA) and degassed. The system was run at a flow rate of 2.5 mL/min. We loaded 20 µL of the sample, filtered, on a POROS A 20 µm Column (2.1 × 30 mm, 0.1 mL; Thermo Scientific). The column was equilibrated with 10 column volumes of mobile phase A, eluted with a step gradient with 20 column volumes of 100% mobile phase B, and re‐equilibrated with 30 column volumes of mobile phase A. The absorbance at 280 nm was measured. We used a similar protein A purified IgG₁ as the calibration standard. The calibration range was 0.1–8 mg/mL. We evaluated and quantified the results with the Chromeleon™ 7 software (Thermo Fisher Scientific).

Dutra G., Komuczki D., Jungbauer A, & Satzer P. (2020). Continuous capture of recombinant antibodies by ZnCl2 precipitation without polyethylene glycol. Engineering in Life Sciences, 20(7), 265-274.

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Quantification of Antibody Concentration

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Protein A affinity chromatography was used to determine the antibody concentration. For HPLC, we used a Dionex UltiMate 3000 HPLC system equipped with a diode array detector (Thermo Fisher Scientific). Mobile phase A was a 50 mM phosphate buffer with 150 mM NaCl, pH 7.0. Mobile phase B was a 100 mM glycine buffer, pH 2.5. All buffers were filtered through 0.22‐µm filters (GSWP04700; Merck KGaA) and degassed. The system was run at a flow rate of 2.5 ml min⁻¹. We loaded 20 µl of the sample, filtered through 0.2‐µm filters (0.2‐µm GHP AcroPrepTM 96 filter plate; Pall Life Sciences, Ann Arbor, MI), on a POROS A 20 µm Column (2.1 × 30 mm, 0.1 ml; Thermo Scientific). The column was washed with 10 column volumes of mobile phase A, eluted with 20 column volumes of 100% mobile phase B, and re‐equilibrated with 30 column volumes of mobile phase A. The absorbance at 280 nm was measured. We used a similar protein A‐purified IgG1 as the calibration standard. The calibration range was 0.1 to 3 mg mL⁻¹. We evaluated and quantified the results with the Chromeleon^TM 7 software (Thermo Fisher Scientific).

Burgstaller D., Jungbauer A, & Satzer P. (2019). Continuous integrated antibody precipitation with two‐stage tangential flow microfiltration enables constant mass flow. Biotechnology and Bioengineering, 116(5), 1053-1065.

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