A novel method for controlling the pepsin digestion of antibodies

J Immunol Methods. 1993 Jan 4;157(1-2):165-73. doi: 10.1016/0022-1759(93)90083-j.


We have discovered that 0.5-0.8 M ammonium sulfate has a beneficial effect on the peptic digestion of several antibodies. Initially, the project was begun to address the troublesome precipitation observed when pepsin was used to digest monoclonal antibody CYT-368 (C46), an IgG2a that binds carcinoembryonic antigen. High concentrations of various salts effectively blocked this precipitation, and also accelerated the rate of digestion. Of these, ammonium sulfate had the greatest effect. Therefore, it was tested on the peptic digestion of ten other murine IgG antibodies as well as rabbit, sheep and goat immunoglobulins. Besides preventing precipitation, ammonium sulfate usefully modulated the digestion rate, accelerating the digestion of some antibodies and reducing it for others. Finally, ammonium sulfate altered the specificity of pepsin during the digestion of antibody CYT-099 (B72.3) preventing the enzyme from cleaving in the F(ab')2 region, and thereby preserving the activity of the fragment. With ammonium sulfate we were able for the first time to use pepsin to obtain active F(ab')2 in good yield from this widely used antibody.

MeSH terms

  • Ammonium Sulfate / pharmacology*
  • Animals
  • Antibodies, Monoclonal / metabolism
  • Goats
  • Immunoglobulin G / metabolism*
  • Isoelectric Point
  • Mice
  • Pepsin A / pharmacology*
  • Peptide Fragments / immunology
  • Rabbits
  • Sheep


  • Antibodies, Monoclonal
  • Immunoglobulin G
  • Peptide Fragments
  • Pepsin A
  • Ammonium Sulfate