Method Article

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

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DOI:

10.3791/51023

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January 8th, 2014

 ,  ,  ,  ,  ,  ,  ,  , 
1Department of Chemical and Biomolecular Engineering, Ohio University, 2Biomedical Engineering Program, Russ College of Engineering and Technology, Ohio University, 3Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School

In This Article

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 times1-3. It is well-appreciated that affinity chromatography represents the largest expense and processing bottleneck in both lab and industrial settings2,4. As a result, numerous improvements have been developed to decrease cost and processing time without sacrificing overall product recovery from the process train1,2,5-7. Particularly promising for the isolation of antibodies and other Fc-expressing proteins is affinity chromatography using a protein A membrane adsorber2,5,7-10. Whereas Fc capture in column chromatography is diffusion-limited (i.e. the Fc-expressing protein must diffuse into and through internal pores to reach the majority of the protein A) with high pressure drop across the column, mass transport in membrane adsorbers is driven by bulk convection, resulting in avoidance of diffusion limitations8,9,11,12. In addition, pressure drop across the membrane adsorber is low, thereby permitting faster perfusion flow rates compared to bead-packed columns8,9,11,12. Thus, for laboratory scale isolation, membrane chromatography is predicted to reduce isolation time by several hours and increase product capture compared to column chromatography. Furthermore, mathematical models for IgG adsorption in membrane adsorbers have been proposed8,9,11,12, thus allowing end-users to predict performance in the lab.

Another bottleneck in basic science and engineering laboratories is the characterization of the Fc-expressing protein. However, selection of appropriate methods to complete characterization assays, such Western blots, can greatly reduce time spent on product testing. For instance, semidry transfer in a discontinuous buffer system can accomplish electrophoretic transfer of proteins from an SDS-PAGE gel to a polyvinyl difluoridine (PVDF) membrane in a matter of minutes, as opposed to 1-2 hr in tank (wet) transfer in a continuous buffer system13.

A rapid, inexpensive, and effective protocol to isolate and characterize a chimeric fusion protein expressing an Fc fragment of human IgG is described herein. Most equipment is readily available in standard basic biomedical science, biology, chemistry, and (bio)chemical engineering labs, and all reagents are commercially available. Although representative results were generated from the isolation and characterization of a murine galectin-1/human Fc chimeric fusion protein (Gal-1hFc), the streamlined protocol can be applied to the isolation of other molecules that possess an Fc fragment, such as antibodies, Fc fragments themselves, or other Fc fusion proteins. Our improvements dramatically decreased processing time (as few as 4 hr but more typically ~9 hr, compared to 20+ work hours) while achieving similar or improved product recovery compared to standard methods.

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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 A14-17 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 protein A membrane adsorber capacity. Proceed if protein is detected.

2. Prepare Protein A Membrane Adsorber and Cell Culture Supernatant

  1. Follow the manufacturer's instructions for preparing the membrane adsorber. Never let air enter the membrane adsorber. All solutions perfused through the membrane adsorber should be at room temperature and prefiltered using a 0.22 µm filter. Fill a 10 ml syringe with 0.22 µm-filtered Dulbecco's phosphate buffered saline (DPBS) or buffer of choice and discharge air bubbles. Perfuse DPBS to remove the storage solution and to equilibrate the membrane adsorber immediately before use.
  2. Filter the cell culture supernatant immediately before perfusion through the membrane adsorber, using a vacuum-driven 0.22 µm sterile filtration unit.

3. Load the Protein A Membrane Adsorber

  1. Aspirate cell culture supernatant into a Luer Lock syringe (30 ml or larger), and discharge any air bubbles.
  2. Assemble materials and equipment as shown in Figure 1. Connect the syringe to the inlet of the membrane adsorber. Attach flexible tubing to the membrane adsorber outlet. If desired, place a 0.22 µm syringe filter between the syringe and the membrane adsorber. Use a flask or bottle to catch flow through (also known as filtrate), from the adsorber.
  3. Set the syringe pump to the desired volumetric flow rate, but do not exceed the manufacturer's recommended flow rate (e.g. 10 ml/min). Perfuse the cell culture supernatant through the membrane adsorber, collecting flow through in a beaker or other container. Reload the syringe with supernatants needed.
  4. If desired, test the flow through for the presence of protein using flow cytometry, Western blotting, ELISA, etc. based on lab preference. It is typically unnecessary to reperfuse the flow through.

4. Elute Protein from the Membrane Adsorber

  1. Wash the membrane adsorber with 10 ml DPBS to remove any nonbound protein.
  2. Elute protein from the membrane adsorber at the desired flow rate (e.g. 1 ml/min) using 10-15 ml of elution buffer (e.g. amine-based elution buffer (pH 2.8)). Catch eluate in a tube containing neutralizing buffer (e.g. 1 M Tris (pH 9.4)) at 10% of elution volume (1.0-1.5 ml).
  3. Alternatively, elute protein in one ml increments into tubes containing 100 µl of neutralization buffer, using a total volume of 10-15 ml elution buffer. Use the preferred characterization method to test each fraction for the presence of protein.
  4. Regenerate the membrane adsorber according to the manufacturer's instructions. Perfuse 10 ml of 0.22 µm-filtered DPBS or buffer of choice, then 10 ml of 0.22 µm-filtered 50 mM NaOH in 1 N NaCl, and finally 10 ml of 0.22 µm-filtered DPBS. Fill the membrane adsorber with 20% ethanol in DPBS for long-term storage at 4 ºC.

5. Concentrate and Dialyze the Protein

  1. Deposit all eluate (or elution fractions containing protein as determined in step 4.3) in a 10 kDa molecular weight cut-off centrifugal filter unit. Follow the manufacturer's instructions for centrifugation.
  2. Dialyze the retentate in a small volume dialysis unit (10 kDa molecular weight cut-off) against the buffer of choice, following the manufacturer's instructions. Buffer may need to be added to dialyzed material if protein precipitation is a concern. If desired, the dialyzed material can be refrigerated until step 6 can be performed, but long term storage without preservatives is not recommended.

6. Quantify and Characterize Purified Product in an Expedited Western Blotting Procedure

  1. Resolve purified protein and Fc standards on the gel of choice by SDS-PAGE, under reducing or nonreducing conditions as desired18-20. Use a mini gel rather than midi gel to minimize run time.
    1. If not already known, determine the Fc standards range over which band signal varies linearly with respect to quantity of loaded (e.g. 0.1-1 mg). Use the same gel, running conditions, transfer conditions, and reagents that will be used to quantify and characterize purified protein.
    2. Load multiple sample amounts of purified protein, including samples diluted with DPBS, to ensure that the purified protein band signals are within the linear signal range of Fc standards in image analysis.
  2. Transfer proteins from the gel to a polyvinylidene fluoride (PVDF) membrane in a discontinuous buffer system using a semidry blotter13, following the blotter manufacturer's instructions. Transfer time is typically 5-10 min. If desired, Coomassie stain the gel after transfer to verify transfer efficiency21.
  3. Perform immunoblotting with anti-Fc or anti-IgG conjugated to alkaline phosphatase (AP) or horse radish peroxidase (HRP), based on lab preference, using a vacuum-assisted protein detection system. Immunoblotting time is typically less than 1 hr.
  4. Develop immunoblot using the substrate and method of choice (e.g. AP substrate and enhanced chemiluminescence).
  5. Quantify purified protein by image analysis. Perform a linear regression on the band signals from Fc standards. If R2>0.90, use the equation for the line to calculate protein quantity from its band signal(s). Account for sample dilution if necessary. Since quantification of the protein is performed on the basis of Fc, adjust the value for molecular weight differences between the protein and Fc. If R2<0.90, repeat step 6 in its entirety.
  6. Validate protein using functional assays or methods to detect Fc via flow cytometry, Western blotting, ELISA, etc. based on lab preference.
  7. Dilute protein to desired concentration and/or add any desired preservatives or stabilizers to the purified protein solution before aliquoting for long-term storage, frozen or otherwise.

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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...

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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...

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Disclosures

There is nothing to disclose.

Acknowledgements

The authors wish to thank Mr. Luis F. Delgadillo (Department of Chemical and Biomolecular Engineering, Ohio University), Mr. Matthew H. Williams (Department of Chemical and Biomolecular Engineering, Ohio University), and Dr. Filiberto Cedeno-Laurent (Brigham and Women’s Hospital and Harvard Medical School) for expert technical assistance. The authors also wish to thank the Winter 2011 CHE 404/BME 504 and Fall 2012 CHE 4830/BME 5830 students (Department of Chemical and Biomolecular Engineering and Biomedical Engineering Program, Ohio University) for technical advice and discussion. This work was supported by National Science Foundation (NSF) Major Research Instrumentation grant CBET-1039869 (MMB), NSF CBET-1106118 (MMB), Dermatology Foundation Research Grant A050422 (SRB), National Institutes of Health (NIH) NCI grant 1R15CA161830-01 (MMB), NIH Kirschstein-NRSA Postdoctoral Fellowship F32CA144219-01A1 (SRB), NIH/NCI R01CA173610 (CJD), and NIH NCCAM grant R01AT004628 (CJD).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Protein A membrane adsorber (Sartobind Protein A 2 ml) Sartorius-Stedim93PRAP06HB-12--A
Amicon Ultra centrifugal filter, 10 kDa, 4 mlMilliporeUFC801008Use 15 ml volume if needed
Sterile vacuum filter unit, 0.22 µm, 500 mlMilliporeSCGPU05RE
10 ml Syringes with Luer Lock tipsBD301604
30 ml Syringes with Luer Lock tipsBD309650
Tygon lab tubing (1/16 in x 1/8 in x 50 ft)Cole ParmerWU-95903-16
Slide-A-Lyzer MINI Dialysis Devices (10K MWCO)Thermo Scientific69576
Snap i.d. antibody collection trayEMD MilliporeWBAVDABTR
Snap i.d. single well blot holderEMD MilliporeWBAVDBH01
Elution bufferThermo Scientific21004
Tris, ultra pureFisher Scientific819623
Sodium chlorideFisher Scientific7647-14-5
Tween 20Fisher ScientificBP337-100
DPBSThermo ScientificSH30028.02
DPBS with Ca2+/Mg2+Life Technologies14080-055
BSASigmaA9647
Human FcBethyl Research LaboratoriesP80-104
Anti-human Fc-APCJackson Immunoresearch109136170
Anti-human Fc-APBio-Rad170-5018
Alkaline phosphatase substratePromegaS3841
Flow cytometry tubes (5 ml polystyrene or polypropylene)BD352054
Syringe pumpHarvard Apparatus55-2226A peristaltic pump is also acceptable
Protein gel electrophoresis systemBio-Rad Laboratories552BR094876Any gel electrophoresis system is acceptable, but mini gel systems run fastest
Semidry blotterBio-Rad Laboratories690BR006163Any transfer system is acceptable, but discontinuos transfer systems perform electrophoretic transfer fastest
SNAP i.d. vacuum-assisted protein detection systemEMD MilliporeWBAVDBASEAn upgraded model has replaced this specific instrument, but either should work just as well

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Cell Culture SupernatantFlow CytometryWestern BlottingUltra FiltrationDialysisProtein QuantificationProtein Characterization

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