Enhanced pepsin digestion: a novel process for purifying antibody F(ab')(2) fragments in high yield from serum.
Enzyme-cleaved antibodies are used widely for the treatment of envenoming. Such products should comprise only 'highly pure' immunoglobulin fragments since Fc or other contaminating protein fragments or their aggregates may lead to side effects. The digestion of ovine antiserum and its purified IgG were investigated using pepsin and trypsin. Trypsin was effective at digesting purified IgG but unsuitable for the direct digestion of serum. In contrast, pepsin was highly effective at digesting all unwanted serum components to low molecular weight (< or =13 kDa) fragments while leaving the approximately 100-kDa F(ab')(2) intact. The optimum pH for pepsin digestion was between 3.25 and 3.50. The effects of salt concentration and pH on the digestion products were investigated by size exclusion chromatography under various conditions, which revealed a pH-dependent aggregation of some of the low molecular weight Fc and non-IgG fragments. These high molecular weight aggregates were not shown by SDS-PAGE. Unwanted low molecular weight fragments could be removed simply by diafiltration with a 30-kDa nominal molecular weight cutoff membrane and piperazine buffer (containing 150 mM NaCl, pH 6), leaving an F(ab')(2) solution contaminated only with some pepsin and a small amount of the aggregated low molecular weight fragments. These highly acidic contaminants were then removed easily using an anion exchange column and the F(ab')(2) produced following a subsequent concentration step was essentially free from pepsin and aggregates with a purity of over 96% and a yield of 19.3 g F(ab')(2)/l serum. This novel, high yield method for processing serum to highly pure F(ab')(2) avoids salt precipitation and centrifugation and should be suitable for large-scale production.
Jones
R G A
RG
Department of Clinical Biochemistry, Barts and The London, Queen Mary's School of Medicine and Dentistry, UK. r.g.jones@qmul.ac.uk
Landon
J
J
D000818:Animals; D001514:Bee Venoms / Q000276:immunology*; D001516:Bees; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange / Q000379:methods; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000097:blood*; D007074:Immunoglobulin G / Q000097:blood; D008970:Molecular Weight; D010434:Pepsin A / Q000378:metabolism*; D012756:Sheep; D012965:Sodium Chloride; D012995:Solubility; D014357:Trypsin
D001514:Bee Venoms; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D012965:Sodium Chloride; D014357:Trypsin; D010434:Pepsin A