Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Summary

Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins. Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours.

Abstract

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography. While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process. Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.). Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment. To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available. To demonstrate this protocol, representative results are presented in which chimeric murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber. Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.

Introduction

Isolation of antibodies and recombinant proteins expressing immunoglobulin G (IgG) Fc fragments from cell culture supernatants and dilute lysed cell solutions can be accomplished using protein A affinity chromatography. In basic science and engineering laboratories and industry, columns packed with porous protein A-coated agarose, glass, or polymeric beads are most commonly used for affinity chromatography, despite high financial costs and long processing times^1-3. It is well-appreciated that affinity chromatography represents the largest expense and processing bottleneck in both lab and industrial settings^2,4. As a result, numerous improvements ....

Protocol

1. Verify the Affinity for Protein A

1. Verify that the Fc-expressing protein (recombinant fusion protein or IgG antibody, heretofore referred to as "protein") has acceptable affinity for protein A^14-17 prior to using the protein A membrane adsorber, and test for the presence of the protein in the stock solution (e.g. cell culture supernatant clarified by centrifugation at 1,000 x g for 5 min to pellet suspended cells). Use flow cytometry, Western blotting, ELISA, etc. to detect protein function and/or presence of Fc, based on lab preference. Ideally, quantify the protein in the cell culture supernatant (step 6) to avoid exceeding....

Results

The streamlined protocol for isolation and characterization of Fc-expressing proteins is routinely used to process chimeric murine galectin-1 fused to human Fc (Gal-1hFc) from dilute cell culture supernatants. The flowchart in Figure 2 illustrates the workflow and time for each step in the protocol. For a typical batch of 300 ml of supernatant, the total processing time is approximately 9 hr when the optional flow cytometry testing of elution fractions is performed. If all flow cytometry analysis is omit.......

Discussion

The protocol described herein was developed to rapidly isolate and characterize a chimeric fusion protein expressing human Fc (Gal-1hFc), without significantly compromising product recovery or inflating cost. The key components of the streamlined workflow are a protein A membrane adsorber for isolation, and semidry transfer and vacuum-assisted immunoblotting for characterization by Western blotting.

The dramatic decrease in processing time for isolation and characterization of Gal-1hFc fr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Protein A membrane adsorber (Sartobind Protein A 2 ml)	Sartorius-Stedim	93PRAP06HB-12--A
Amicon Ultra centrifugal filter, 10 kDa, 4 ml	Millipore	UFC801008	Use 15 ml volume if needed
Sterile vacuum filter unit, 0.22 µm, 500 ml	Millipore	SCGPU05RE
10 ml Syringes with Luer Lock tips	BD	301604
30 ml Syringes with Luer Lock tips	BD	309650
Tygon lab tubing (1/16 in x 1/8 in x 50 ft)	Cole Parmer	WU-95903-16
Slide-A-Lyzer MINI Dialysis Devices (10K MWCO)	Thermo Scientific	69576
Snap i.d. antibody collection tray	EMD Millipore	WBAVDABTR
Snap i.d. single well blot holder	EMD Millipore	WBAVDBH01
Elution buffer	Thermo Scientific	21004
Tris, ultra pure	Fisher Scientific	819623
Sodium chloride	Fisher Scientific	7647-14-5
Tween 20	Fisher Scientific	BP337-100
DPBS	Thermo Scientific	SH30028.02
DPBS with Ca2+/Mg2+	Life Technologies	14080-055
BSA	Sigma	A9647
Human Fc	Bethyl Research Laboratories	P80-104
Anti-human Fc-APC	Jackson Immunoresearch	109136170
Anti-human Fc-AP	Bio-Rad	170-5018
Alkaline phosphatase substrate	Promega	S3841
Flow cytometry tubes (5 ml polystyrene or polypropylene)	BD	352054
Syringe pump	Harvard Apparatus	55-2226	A peristaltic pump is also acceptable
Protein gel electrophoresis system	Bio-Rad Laboratories	552BR094876	Any gel electrophoresis system is acceptable, but mini gel systems run fastest
Semidry blotter	Bio-Rad Laboratories	690BR006163	Any transfer system is acceptable, but discontinuos transfer systems perform electrophoretic transfer fastest
SNAP i.d. vacuum-assisted protein detection system	EMD Millipore	WBAVDBASE	An upgraded model has replaced this specific instrument, but either should work just as well