Rapid, Ultrafiltration-based Antibody Purification

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.