by Darshan Koticha, Christopher Barbagallo and Peter Rapiejko
 Figure 1. Concentration and purification of antibody from clarified supernatant. Figure 1A, SDS-PAGE gel shows equal amounts of clarified supernatant (lane 1), supernatant concentrated using ammonium sulfate and dialysis (lane 2), or ultrafiltration (lane 3). Figure 1B, SDS-PAGE gel shows equal amounts (2.0 mg) of antibodies purified using the ultrafiltration method (lane 3), ammonium sulfate precipitated antibody (lane 4), control antibody (Invitrogen) (lane 1), and molecular weight markers (Invitrogen) (lane 2). Heavy (H) and light (L) chains of the antibodies are indicated. Samples were run on 4–12% NuPAGE gels (Invitrogen), under reducing conditions, stained with Sypro Orange dye (Invitrogen) and imaged on the Kodak 4000 MM gel scanner (Kodak, Rochester, NY). |
Monoclonal antibodies are important research tools and potent therapeutic agents for many different cancers.
(1,2) Purification of these proteins, on the laboratory scale, is a time-consuming process. Traditionally, antibodies from hybridomas have been purified using a complex method that involves an ammonium sulfate precipitation, dialysis and protein-A/G affinity chromatography.
(3,4) The entire process typically requires several days to complete and can be particularly challenging if multiple antibodies are to be evaluated in parallel.
This report describes a simple, filtration-based method for the rapid purification of monoclonal antibodies. The entire process is completed in less than two hours. The feasibility of this approach was demonstrated by purifying an anti c-myc antibody secreted by the hybridoma clone 9E10.
(5) This clone has been used extensively to detect myc-tagged proteins.
Clarification of hybridoma cells to obtain supernatants
The first step in the purification of antibodies requires the removal of cells, generally by centrifugation. However, centrifugation involves multiple steps and takes 15-30 minutes for completion. An easier alternative is removing the cells by filtration (Stericup filter cup, Millipore, Billerica, MA). To demonstrate this, hybridoma cells, clone 9E10 (ATCC, Manassas, VA), were grown in RPMI medium containing 10% Low Ig FBS (Invitrogen, Carlsbad, CA) to a final density of 1.5 × 10
6 cells/ml. A glass fiber prefilter, AP40 (Millipore), was inserted into the notch of the filter cup. The prefilter is required to prevent membrane fouling and increase the speed of filtration. The hybridoma cells were added and vacuum was applied. Less than two minutes were required to filter 200 ml of culture compared to more than 15 minutes using centrifugation (Table 1). The clarified supernatant was collected directly into a sterile container and ready to use.
Concentration of the clarified supernatant
Hybridoma supernatants (Fig. 1A, lane 1) typically contain low concentrations of mABs in the range of 5-25 μg/m. These low levels necessitate the concentration of the supernatant, since loading 200-500 ml on a Protein A or G affinity column at the standard flow rate of 1-2 ml per minute would require more than three hours without the use of complex equipment. Equal volumes (200 ml) of clarified hybridoma supernatant were concentrated using ammonium sulfate precipitation (Fig. 1A, lane 2) or using ultrafiltration (Fig. 1A, lane 3).
(6)For the sample concentrated by precipitation, ammonium sulfate (Sigma, St. Louis, MO) was added to the supernatant to a final volume of 50% w/v. The salt precipitate was collected by centrifugation and re-solubilized in 30 ml of PBS. As the hybridoma cells were grown in 10% serum, the volume of the salt precipitate was higher due to the excess serum proteins. The re-solubilized precipitate was desalted using dialysis with a 10K molecular weight cutoff (MWCO) membrane (Spectrum Labs, Rancho Dominguez, CA) and three rounds of buffer exchange in PBS. This was followed by more centrifugation to remove precipitated material. Desalting was followed by Protein G chromatography for antibody purification. This entire process required two days for completion.
 Figure 2. Specific activity of antibodies purified after concentration with ultrafiltration (green) is comparable to antibody purified using an ammonium sulfate precipitation method (blue). Activities were compared using an ELISA-based assay as described in the text. Activity of antibody obtained from commercial sources was used as a control (black line).
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For the sample concentrated by ultrafiltration, aliquots of 70 ml were added to the Centricon Plus-70 devices, 100K mwco (Millipore). The devices were centrifuged at 3500 ×g for 30 minutes. The concentrated supernatant, final volume 6 ml, was recovered into the collection tube using an invert spin. The entire process required 40 minutes for completion.
The results show that ultrafiltration is significantly faster than precipitation when concentrating hybridoma supernatants (40 minutes compared to 2 days). In addition, ultrafiltration requires only three steps while precipitation requires 10 steps to concentrate proteins .
Affinity chromatography to purify mAB
Affinity chromatography with protein A or G media is the most popular method to purify antibodies. A Montage Antibody Purification Spin Column with Prosep G media (Millipore) was used for the final purification of antibody from the concentrated supernatant. The concentrated supernatants from the previous section were equilibrated in the binding buffer (1.5 M Glycine/NaOH, 3 M NaCl, pH 9.0) and added to the centrifugal column. The column was centrifuged at ~150 ×g for 20 minutes to allow the binding of antibody to protein G. The column was washed three times by adding binding buffer and spinning for 2 minutes at 500 ×g. The bound antibody was eluted using a glycine buffer (0.2M, pH 2.5) into the collection tube containing Tris-Cl (1.0 M, pH 9.0) to neutralize the antibody solution. The entire purification process was completed in less than 1 hour.
Recovery, purity and activity of the mAB
Antibody recovery for both samples was measured using the Commassie assay (Pierce, Rockford, IL). As shown, the yields were comparable between the two methods (Table 1). Purity was measured using SDS gel electrophoresis (Fig. 1B). The antibody purified using the ultrafiltration-based method (Fig. 1B, lane 3) was equivalent to the antibody purified by the traditional method (Fig. 1B, lane 4) by this method. The monoclonal antibody purchased from Invitrogen was used as a control (Fig. 1B, lane 1).
An ELISA-based assay was performed to show that ultrafiltration did not affect the binding activity of the antibodies compared to ammonium sulfate precipitation (Fig. 2). The specific activity of the purified antibodies was compared to the commercially purified antibody. HEK 293 cells (Invitrogen) were grown on 96-well plates (BD Biosciences, Franklin Lakes, NJ). After fixation and epitope-retrieval (10 minutes in a microwave), cells were permeabilized with 1% saponin (Sigma) in PBS +2% normal donkey serum (Jackson Immunoresearch, West Grove, PA) and treated with serial dilutions of the indicated purified mABs. The cells were then washed and treated with goat anti-mouse HRP-conjugate antibody (Sigma). The reactions were developed using a SureBlue TMB HRP substrate (KPL, Gaithersburg, MD). The readings were measured on a Spectromax plate reader (Molecular Devices, Sunnyvale, CA). The results indicate that ultrafiltration did not result in the loss of activity since the specific activity of the antibodies purified by the two methods was similar.
Conclusion
Monoclonal antibodies represent one of the fastest growing segments of therapeutic agents, in addition to being invaluable diagnostic reagents. There is a need for accelerating the purification of antibodies to characterize potential candidates. In this report we demonstrate a convenient method for the rapid purification of antibodies. Identification of a candidate antibody requires screening a large number of hybridoma supernatants. We show that filtration is a fast and easy method for clarifying and concentrating hybridoma supernatants. Further evaluation and validation requires purifying antibodies on the bench scale. Spin columns using pre-packed protein A or G media enable convenient affinity purification of antibodies.
About the authors
Darshan Koticha, Christopher Barbagallo and Peter Rapiejko are (need short author information). More information about the techniques discussed in this report are available from:
Millipore Corp., Bioscience Div. 800-645-5476
www.millipore.com Acknowledgements
The authors would like to thank Drs. Ajay Sharma and John Lynch for critically reviewing the manuscript.
References
1. Adams, G.P. and Weiner, L.M. Monoclonal antibody therapy of cancer.
Nat. Biotech. 23:1147-1157 (2005).
2. Woof, J.M. Immunology. Tipping the scales toward more effective antibodies.
Science 310:1442-1443 (2005>.
3. Harlow, E. and Lane, D.
Antibodies. A
Laboratory Manual. CSH Laboratory Press, Cold Spring Harbor Laboratory NY (1988).
4. Page, M. and Thorpe, R. Purification of monoclonal antibodies.
Methods Mol. Biol. 80:113-119 (1998).
5. Evan, G.I., Lewis, G.K., Ramsay, G. and Bishop, J.M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.
Mol. Cell. Biol. 5:3610-3616 (1985).
6. Saha, K., Case, R. and Wong, P.K. A simple method of concentrating monoclonal antibodies from culture supernatant by ultrafiltration.
J. Immunol. Methods 151:307-308 (1992)