High-throughput mAb expression and purification platform based on transient CHO

Biotechnol Prog. Jan-Feb 2015;31(1):239-47. doi: 10.1002/btpr.2012. Epub 2014 Nov 28.

Abstract

A high-cell-density transient transfection system was recently developed in our laboratory based on a CHO-GS-KO cell line. This method yields monoclonal antibody titers up to 350 mg/L from a simple 7-day process, in volumes ranging from 2 mL to 2 L. By performing transfections in 24-deep-well plates, a large number of mAbs can be expressed simultaneously. We coupled this new high-throughput transfection process to a semiautomated protein A purification process. Using a Biomek FX(p) liquid handling robot, up to 72 unique mAbs can be simultaneously purified. Our primary goal was to obtain >0.25 mg of purified mAb at a concentration of >0.5 mg/mL, without any concentration or buffer-exchange steps. We optimized both the batch-binding and the batch elution steps. The length of the batch-binding step was important to minimize mAb losses in the flowthrough fraction. The elution step proved to be challenging to simultaneously maximize protein recovery and protein concentration. We designed a variable volume elution strategy based on the average supernatant titer. Finally, we present two case studies. In the first study, we produced 56 affinity maturation mAb variants at an average yield of 0.33 ± 0.05 mg (average concentration of 0.65 ± 0.10 mg/mL). In a second study, we produced 42 unique mAbs, from an early-stage discovery effort, at an average yield of 0.79 ± 0.31 mg (average concentration of 1.59 ± 0.63 mg/mL). The combination of parallel high-yielding transient transfection and semiautomated high-throughput protein A purification represents a valuable mAb drug discovery tool.

Keywords: high-throughput purification; protein A; transient CHO.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / analysis
  • Antibodies, Monoclonal / isolation & purification*
  • Antibodies, Monoclonal / metabolism*
  • CHO Cells
  • Cell Culture Techniques / methods*
  • Chromatography, High Pressure Liquid
  • Cricetinae
  • Cricetulus
  • Electrophoresis, Polyacrylamide Gel
  • High-Throughput Screening Assays / methods*
  • Staphylococcal Protein A / chemistry
  • Staphylococcal Protein A / metabolism

Substances

  • Antibodies, Monoclonal
  • Staphylococcal Protein A