Elution conditions and degradation mechanisms in long-term immunoadsorbent use.
The limited life of immunoadsorbents used for the large-scale purification of biological macromolecules poses a significant limitation to the more widespread application of this technology. In this study, the binding activity of a monoclonal antibody (MAb) to bovine serum albumin (BSA) was measured as a function of pH, ionic strength, and varying concentrations of KSCN, ethylene glycol, or DMSO. Low pH (2.5) and 3 M KSCN each reduced the antibody binding constant below 6 x 10(5) L/mol, meeting criteria derived from a simple chromatographic model for identifying effective eluents. A panel of six MAb to BSA was exposed repeatedly to adsorption conditions and the two eluents. Four MAb lost less than 50% of their initial binding capacity over 100 cycles. The other two lost 75% of their initial capacity. One MAb was stable when exposed to low pH but lost binding capacity with KSCN. In all cases, the equilibrium constant was unchanged. The loss of capacity was also shown to be a strong function of antibody loading: at 14.5 mg/mL, 98% of the initial binding capacity of one MAb was lost within 40 cycles, versus 75% loss at 1 mg/mL. Antibody leakage and nonspecific adsorption of contaminants were not responsible for significant loss of antibody activity over time.
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge 02139.
K P
KP
Antonsen
C K
CK
Colton
M L
ML
Yarmush
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity; D002417:Cattle; D002846:Chromatography, Affinity; D004121:Dimethyl Sulfoxide; D019855:Ethylene Glycol; D005026:Ethylene Glycols; D006863:Hydrogen-Ion Concentration; D007164:Immunosorbents / Q000737:chemistry*; D007700:Kinetics; D009994:Osmolar Concentration; D011863:Radioimmunoassay; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000276:immunology*; D013816:Thermodynamics; D013861:Thiocyanates; D013997:Time Factors
D000911:Antibodies, Monoclonal; D005026:Ethylene Glycols; D007164:Immunosorbents; D013861:Thiocyanates; D012710:Serum Albumin, Bovine; D019855:Ethylene Glycol; C009941:potassium thiocyanate; D004121:Dimethyl Sulfoxide