Antibody fragment separations by capillary zone electrophoresis.
This study investigated methods of improving the separation and identification of an IgA antibody, McPC603, and its pepsin fragments. The problem presented by purification of antibody fragments (Fabs) and the antibody light chain required accurate and informative analysis of highly hydrophobic proteins, which can polymerize and fold to form secondary structures. Capillary zone electrophoresis (CZE) permits the separation of peptides and small proteins by a method which is orthogonal to the traditional method of reversed-phase HPLC. To facilitate planned studies of the antibody's biological activity, our buffer composition was kept as simple as possible. During CZE analysis, if the buffer pH is below the isoelectric point of the protein, or the protein is large (with a heterogeneous distribution of surface charges), it can irreversibly blind to the capillary wall unless the capillary is coated. We found that C1-coatings in RP-capillaries at pH 9.5 adequately prevented the antibody fragments from binding to the wall. However, the coating did not remain stable at such high pH, so different conditions were sought. We achieved adequate separations in several buffers at nearly physiological pH, in a bare silica capillary which had been coated once with a soluble cationic polymer coating (Micro-Coat applied during column conditioning). Antibody electropherograms changed depending on the type of inorganic buffer salt used in a separation. Phosphate binds to the antigen-binding site of the IgA with low affinity, and interesting effects were observed in separations using phosphate buffer. These effects will de discussed.
Martin
L M
LM
Rockefeller University, New York, NY 10021, USA.
D000912:Antibodies, Neoplasm / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000302:isolation & purification*; D009994:Osmolar Concentration; D010434:Pepsin A / Q000737:chemistry
D000912:Antibodies, Neoplasm; D007128:Immunoglobulin Fragments; D010434:Pepsin A