Capture of a monoclonal antibody and prediction of separation conditions using a synthetic multimodal ligand attached on chips and beads.
A synthetic ligand called 2-mercapto-5-benzimidazolesulfonic acid has been successfully used for the specific chromatographic capture of antibodies from a cell culture supernatant. Adsorption occurred at physiological ionic strength and pH range between 5.0 and 6.0, with some binding capacity variations within this pH range: antibody uptake increased when the pH decreased. With very dilute feedstocks, as was the case with the cell culture supernatant under investigation, it was found that the pH had to be slightly lowered to get a good antibody sorption capacity. To optimize separation conditions, a preliminary study was made using ProteinChip Arrays that displayed the same chemical functionalities as the resin. Arrays were analyzed using SELDI-MS. By this mean, it was possible to cross-over simultaneously different pH conditions at the adsorption and the desorption steps. Best conditions were implemented for preparative separation using regular lab-scale columns. At pH 5.2, antibody adsorption was not complete, while at pH 5.0 the antibody was entirely captured. pH 9 was selected at elution, rather than pH 8.0 or 10.0, and resulted in a complete desorption of antibodies from the column. Benefits of the prediction of separation conditions of antibodies on MBI beads using SELDI-MS were a significant reduction in analysis time and in sample volume. This was possible because the separation of IgG on the chip surface did mimic very well the separation on beads.
Brenac
Virginie
V
BioSepra, Process Proteomics, Division of Ciphergen Biosystems Inc., 48 Avenue des Genottes, BP 38347 Cergy Saint Christophe, 95804 Cergy Pontoise Cedex, France. vbrenac@ciphergen.com
Ravault
Vincent
V
Santambien
Patrick
P
Boschetti
Egisto
E
D000327:Adsorption; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001562:Benzimidazoles / Q000737:chemistry*; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D003470:Culture Media / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000302:isolation & purification; D007834:Lasers; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D040081:Protein Array Analysis / Q000379:methods*; D013451:Sulfonic Acids / Q000737:chemistry*
C497089:2-mercapto-5-benzimidazolesulfonic acid; D000911:Antibodies, Monoclonal; D001562:Benzimidazoles; D003470:Culture Media; D007074:Immunoglobulin G; D008024:Ligands; D013451:Sulfonic Acids