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

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.

1. Initializing the purification system
   1. Open the software attached to the purification system. Using manual instructions, equilibrate the column with the equilibration buffer at a flow rate of 2 mL/min for 40 m....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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