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

Overview

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 min. Stop the manual run after equilibration.
   2. In the fraction collector, place 15 mL conical tubes to collect purified antibody eluate and 50 mL conical tubes to collect flow-through during high salt wash. Ensure that the fraction collector is reset to the start position by opening and closing the fraction collector before the beginning the run. The fraction collector is maintained at 7 °C.
      NOTE: The fraction collector can be reset manually under the Fraction Collector tab in Settings, for both 15 mL and 50 mL tubes.
2. Sample injection
   NOTE: The harvested cell culture fluid used in the following procedures has been obtained from chinese hamster ovary cells cultured in automated micro-bioreactors.
   1. Add the 0.22 µm filtered harvest cell culture fluid to an empty 12 mL syringe whose nozzle end is capped.
   2. Holding the syringe with the nozzle facing down, insert the syringe plunger till a small portion of the plunger is in. Making sure that the fluid is not leaking, turn the syringe with the nozzle facing up and remove the cap.
   3. Still holding the syringe with the nozzle facing up, push the cylinder to dispel any air until the cell culture fluid is at the tip of the nozzle. Insert the syringe nozzle into the manual injection port on the purification system and twist to tighten.
   4. Push down on the plunger until all the sample is injected and is visible in the attached 10 mL large volume sample loop.
   5. Open the saved method file. Save the result file in the required location and specify file name when prompted. Hit run after the sample has been injected into the large volume sample loop.
3. Running the purification method
   1. Select the saved method and click run when prompted by instrument software (step 1.2.5).
      NOTE: The system is set up to run the following steps. The user needs not do anything while the instrument is running.
   2. Equilibrate the column with three column volumes (CVs) of equilibration buffer at a flow rate of 2 mL/min. Once the column is equilibrated, the system, using the large volume sample loop, will inject the sample onto the column at a flow rate of 1 mL/min.
   3. A drop in UV signal at 280 nm indicates that the sample is finished loading. Wash the column with equilibration buffer at a flow rate of 2 mL/min until the UV signal drops below 25 mAU.
   4. Use four CVs of 25 mM Tris with 1 M NaCl at pH 7.5 to perform a secondary high salt wash at a flow rate of 2 mL/min. The system fraction collector will collect any protein/DNA that comes off the column during the salt wash in 50 mL tubes.
   5. Apply five CVs of elution buffer at a flow rate of 1 mL/min to elute the antibody off the column. Collect the eluate in 15 mL tubes based on UV signal; when the UV 280 signal is above 35 mAU, collection starts; collection ends when the signal drops below 50 mAU; this is called peak cutting.
      NOTE: Peak cutting ensures normalization of elution profiles and to avoid elution peak tailing which may contain protein aggregates.
   6. Wash the column using three CVs of equilibration buffer. The run ends after the wash step.
   7. After elution immediately neutralize the purified protein using 1 M Tris base to a pH of ~5.5. Measure the protein concentration using a microvolume UV-Vis spectrophotometer at 280 nm and 260 nm and store at 4 °C.

Materials

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