Rapid, Ultrafiltration-based Method for Purification of Monoclonal Antibodies Using the Centricon^® Plus-70 Device

Introduction

Monoclonal antibodies (MAb) continue to gain importance as therapeutic and diagnostic agents for many cancers. The process of screening hybridoma libraries for candidate MAbs is both time consuming and labor intensive. Once a hybridoma cell line expressing the desired MAb is established, a bench-scale purification methodology (e.g., 50 to 500 mL) must be developed to produce sufficient MAb for further characterization. A traditional method for purifying MAbs involves these steps:

1. Clarifying the hybridoma supernatant by centrifugation
2. Concentrating the Mab by ammonium sulfate precipitation
3. Resolubilizing the MAb and desalting using dialysis
4. Additional purification using protein A/G affinity chromatography
5. Desalting and buffer exchange using dialysis

This process typically requires several days to complete and can be labor-intensive when evaluating multiple MAbs in parallel. Ultrafiltration-based MAb purification, on the other hand, is easier, faster (2–3 hours versus 2 days), and yields higher recoveries.

Method

The method involves clarification of the hybridoma supernatant by microfiltration using a Stericup vacuum filter cup, followed by concentration using ultrafiltration¹ with a Centricon^® Plus-70 device:

1. Add 200 mL supernatant to Centricon^® Plus-70 Device (100K MWCO) as follows:
   1a. Load 65 mL of supernatant to the device
   1b. Centrifuge (20–30 min)
   1c. Discard filtrate
   1d. Repeat steps 1a-1c two more times until all the supernatant has been loaded
2. Invert device and spin to collect sample (2 min)
3. Load on protein-G column
4. Further purify on protein A/G beads
5. Desalt and buffer-exchange using ultrafiltration

Results

Ultrafiltration was successfully used to purify an anti c-myc antibody secreted by the hybridoma clone 9E10² (Figure 1). The purified MAb performed comparably to traditionally/commercially prepared MAbs in Western blotting and ELISA assays (Figure 2). Our data show that the combination of microfiltration and ultrafiltration is a rapid method for Mab purification. Additionally, MAb purified on Montage^® centrifugal columns with Protein G media is comparable to the commercially available MAb in purity, activity and cost.

Figure 1. Purification of anti c-myc antibody using ultrafiltration. C: clarified supernatant; Tr: Supernatant precipitated with ammonium sulfate and dialyzed using 10K MWCO membrane (Spectrapor, Rancho Dominguez, CA, USA); UF: Supernatant concentrated by ultrafiltration on Centricon^®-Plus 70 device.

Figure 2. Upper panel shows activity comparison measured in a Western blot between Mab (derived from HEK 293 cells) purified using ultrafiltration versus traditional methods. Three-fold serial dilutions of HEK 293 cell nuclear extracts were run on 4-12% SDS-PAGE gels and transferred to Immobilon^®-P membrane. Blots were probed with the indicated primary antibodies and secondary anti-mouse alkaline phosphatase conjugate. The antibodies were detected with alkaline phosphatase substrate and imaged on a Kodak imager. Bottom panel shows activity comparison measured in an ELISA. HEK 293 cells were grown on 96-well plates. After fixation and epitope retrieval by heating for 10 minutes in a microwave oven, cells were permeabilized with 1% saponin (MilliporeSigma) in PBS and 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. The reactions were developed using a SureBlue™ TMB HRP substrate (KPL, Gaithersburg, MD, USA). The readings were measured on a SpectraMax^® plate reader (Molecular Devices, Sunnyvale, CA, USA).

References

1. Saha K, Case R, Wong PK. 1992. A simple method of concentrating monoclonal antibodies from culture supernatant by ultrafiltration. Journal of Immunological Methods. 151(1-2):307-308. https://doi.org/10.1016/0022-1759(92)90131-c

2. Evan GI, Lewis GK, Ramsay G, Bishop JM. 1985. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.. Mol. Cell. Biol.. 5(12):3610-3616. https://doi.org/10.1128/mcb.5.12.3610