Affinity-based precipitation via a bivalent peptidic hapten for the purification of monoclonal antibodies

Analyst. 2014 Sep 7;139(17):4247-55. doi: 10.1039/c4an00780h.


In a previous study, we demonstrated a non-chromatographic affinity-based precipitation method, using trivalent haptens, for the purification of mAbs. In this study, we significantly improved this process by using a simplified bivalent peptidic hapten (BPH) design, which enables facile and rapid purification of mAbs while overcoming the limitations of the previous trivalent design. The improved affinity-based precipitation method (ABP(BPH)) combines the simplicity of salt-induced precipitation with the selectivity of affinity chromatography for the purification of mAbs. The ABP(BPH) method involves 3 steps: (i) precipitation and separation of protein contaminants larger than immunoglobulins with ammonium sulfate; (ii) selective precipitation of the target-antibody via BPH by inducing antibody-complex formation; (iii) solubilization of the antibody pellet and removal of BPH with membrane filtration resulting in the pure antibody. The ABP(BPH) method was evaluated by purifying the pharmaceutical antibody trastuzumab from common contaminants including CHO cell conditioned media, DNA, ascites fluid, other antibodies, and denatured antibody with >85% yield and >97% purity. Importantly, the purified antibody demonstrated native binding activity to cell lines expressing the target protein, HER2. Combined, the ABP(BPH) method is a rapid and scalable process for the purification of antibodies with the potential to improve product quality while decreasing purification costs.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Ammonium Sulfate / chemistry
  • Animals
  • Antibodies, Monoclonal / chemistry
  • Antibodies, Monoclonal / immunology
  • Antibodies, Monoclonal / isolation & purification*
  • Antibodies, Monoclonal, Humanized / chemistry
  • Antibodies, Monoclonal, Humanized / immunology
  • Antibodies, Monoclonal, Humanized / isolation & purification
  • CHO Cells
  • Cell Line
  • Chemical Precipitation
  • Chromatography, Affinity / methods
  • Chromatography, Reverse-Phase / methods
  • Cricetinae
  • Cricetulus
  • Filtration / methods
  • Haptens / chemistry
  • Haptens / immunology*
  • Humans
  • Peptides / chemistry
  • Peptides / immunology
  • Protein Denaturation
  • Trastuzumab


  • Antibodies, Monoclonal
  • Antibodies, Monoclonal, Humanized
  • Haptens
  • Peptides
  • Trastuzumab
  • Ammonium Sulfate