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A Robust High-Throughput Method for Antibody Purification using Magnetic Beads on the KingFisher® Flex Platform

Mark Bratz, Becky Godat, Doug Wieczorek, Nidhi Nath

Promega Corporation
Publication Date: March 2016, tpub_170

Abstract

In this report we demonstrate the KingFisher® Flex platform for automated 96-well processing of magnetic particles for parallel purification of eight different mouse and human antibodies using Magne® Protein A and Magne® Protein G Beads.

Introduction

As the antibody therapeutics industry expands, new methods are being sought to improve the efficiency of lead antibody identification in the early discovery phase. A key step in this direction is the ability to assay large numbers of test antibodies in multiple biologically relevant assays. Some antibody assays, such as enzyme-linked immunosorbent assays (ELISAs) for binding to antigen, can be performed on antibodies in cell media. However, many functional assays, especially cell-based assays, require highly pure antibodies at high concentrations. 

We have developed high-capacity, magnetic Protein A and Protein G beads for antibody purification (Magne® Protein A Beads, Cat.# G8781;  Magne® Protein G Beads, Cat.# G7471) that are compatible with automated platforms. In this report we demonstrate the KingFisher® Flex platform for automated 96-well processing of magnetic particles for parallel purification of eight different mouse and human antibodies using Magne® Protein A and Magne® Protein G Beads. To demonstrate the reproducibility of the purification process, six replicates of each antibody were purified.

Methods

Materials to be Supplied by the User

  • KingFisher® Flex with 96 Deep-well Head
  • Deepwell 96 Plates (Thermo Scientific Cat.# 95040460)
  • KingFisher® 96 Tip Comb for DW Magnets (Thermo Scientific Cat.# 97002534)
  • Magne® Protein A Beads (Cat.# G8781) and Magne® Protein G Beads (Cat.# G7471) 
  • 1X PBS (pH 6.8)
  • 100mM glycine (pH 2.7)
  • 2M Tris (pH 7.5)

Plate Setup

  • (Total Run Time = 1 hour, 20 minutes)
  • Plate 1 = Sample. Add up to 1,000μl of sample per well, adjusting the remaining volume using 1X PBS. 
  • Plate 2 = Resin. Add 450μl of 1X PBS and 50μl of bead slurry per well.
  • Plate 3 = Wash 1. Add 1,000μl of 1X PBS per well.
  • Plate 4 = Wash 2. Add 500μl of 1X PBS per well.
  • Plate 5 = Wash 3. Add 500μl of 1X PBS per well.
  • Plate 6 = Elution Plate. Add 100μl of 100mM glycine (pH 2.7) per well.  
  • Plate 7 = Tip Plate. Add KingFisher® 96 Tip Comb for DW Magnets.

Protocol

The manufacturer’s protocol was followed (Magne® Protein A Beads and Magne® Protein G Beads for Antibody Purification Technical Manual, #TM371) as described briefly here:

  1. Beads were incubated with the samples for 60 minutes.
  2. Beads were washed three times with PBS.
  3. Captured antibodies were eluted by 5-minute incubation with elution buffer.
  4. Eluted samples were immediately neutralized by adding 20µl of 2M Tris (pH 7.5) to each well of the elution plate.
  5. An automated method for the KingFisher® Flex platform was developed. For more information about this method please contact Promega Technical Services (techserv@promega.com).

Results and Discussion

Typical antibody concentrations during early-stage monoclonal antibody development are approximately 50µg/ml. We selected eight purified antibodies (Table 1) and spiked them into the spent cell media for a final concentration of approximately 50µg/ml. The use of spiked samples allowed us to calculate the efficiency of antibody recovery as shown in Table 1. Six replicate samples of each antibody were purified with Magne® Protein G and Magne® Protein A Beads, and high recoveries (60% to >90%) were obtained for all antibodies (Table 1). Efficient recovery of antibody is dependent on the length of incubation of samples with the magnetic beads. A typical incubation time of 1 hour is sufficient, but for dilute samples longer incubation times (up to 2 hours) may be needed. Well-to-well reproducibility was high, with %CVs of the six replicate samples less than 10% for most samples. The high reproducibility can be attributed to excellent magnetic properties of the beads. Consistent and high recovery of antibody is important at early stages of the monoclonal antibody development phase to allow for evaluation of antibodies in relevant functional assays and selection of good lead candidates for downstream applications.

13578LA

Table 1. Antibody Purification from Deep-Well 96-well Plate using the KingFisher® Flex Instrument. Sample volume = 1ml.

Testing the purified antibodies in functional assays often requires further modifications, such as labeling with fluorescent dyes or immobilization on biosensor chips, which may require dialysis into appropriate buffer or have certain concentration requirements. High-capacity magnetic beads have an advantage in this regard because small volumes of beads can capture and concentrate antibody from dilute cell media and allow elution of antibody in a small volume at a relatively high concentration as shown in Table 2. Availability of high-concentration antibodies will allow functional assays such as ELISA, affinity measurements and cell-based imaging to be performed without any additional buffer exchange.

13579LA

Table 2. Antibody Concentration of Starting Material and Purified Antibody.

Conclusion

The ability to purify hundreds of antibodies with good recovery, high concentration and in a time-effective manner will enable screening of antibodies in multiple biologically relevant formats, providing more information for selecting good lead candidates.

Magne® Protein G and Magne® Protein A Beads

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