A Technique to Purify Monoclonal Antibodies Produced by Chinese Hamster Ovary Cells

Published: March 29, 2024

Abstract

Source: Velugula-Yellela, S. R., et al. Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System. J. Vis. Exp. (2019).

This video demonstrates an assay for purifying monoclonal antibodies from cell culture fluid harvested from Chinese Hamster Ovarian cells. It employs a porous glass resin with immobilized protein A, which binds to the Fc region of the antibodies in the sample, effectively separating them from contaminants. The use of a low-pH elution buffer weakens the interaction, facilitating the retrieval of purified antibodies.

Protocol

1. Purification of antibody NOTE: The equilibration buffer for the in-house antibody is 25 mM Tris, 100 mM NaCl, pH 7.5. The elution buffer used is 0.1 M acetic acid. The buffers and resin (Protein A) are dependent on the specific antibody purified. Column volume is equivalent to the bed height of the resin. The amount of mobile phase used is determined in terms of column volume. Initializing the purification system Open the software at…

Offenlegungen

The authors have nothing to disclose.

Materials

CHO DG44 Cell Line Invitrogen A1100001
Akta Avant 25 General Electric Life Sciences 28930842
Pro Sep vA Ultra Chromatography Resin Millipore Sigma 115115830 Purification Stationary Phase
Omnifit 10cm Column Diba Fluid Intelligence 006EZ-06-10-AA Housing for Stationary Phase
Tris Base Fisher Scientific BP154-1
Superloop 10 mL GE Healthcare 18-1113-81
µDawn Multi Angle Light Scattering Detector Wyatt WUDAWN-01
0.22 µm Millex GV Filter Unit PVDF Membrane Merck Millipore SLGV033RB
10X Phosphate Buffered Saline Corning 46-013-CM
12 mL Syringe Covidien 8881512878
50 mL Falcon tube Corning Inc. 352070
96-Well Plate Bio-Rad 127737
Acetic Acid Sigma-Aldrich 695072
Amicon Ultra-4 100 kDa centrifugal filters Merck Millipore UFC810096
Amino Acid Standard, 1 nmol/µL Agilent Technologies 5061-3330
Amino Acid Supplement Agilent Technologies 5062-2478
Ammonium Formate Solution – Glycan Analysis Waters Corporation 186007081
Blue Screw Caps with Septa Agilent Technologies 5182-0717
Chromatography Water (MS Grade) Fisher Chemical W6-4
Hydrochloric Acid Fisher Scientific A144-500
Intact mAb Mass Check Standard Waters Corporation 186006552
Intrada Amino Acid Column 150 x 2 mm Imtakt WAA25
NanoDrop One Microvolume UV-Vis Spectrophotometer Thermo Fisher Scientific 840274100

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A Technique to Purify Monoclonal Antibodies Produced by Chinese Hamster Ovary Cells. J. Vis. Exp. (Pending Publication), e22095, doi: (2024).

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