Separation of protein mixtures using pH-gradient cation-exchange chromatography.
Historically, separation of a protein mixture after adsorption to a cation-exchange column is effected by alteration in ionic strength. An alternative separation method using pH induced gradient in the range of 4-7.5 was studied. A cation-exchange column with large particle beads containing excessive carboxyls was employed. A pH gradient across the column was generated by a step change at the column entrance using a non-retained buffer system. Consistency and accuracy of pH values in timed intervals were demonstrated in three different batches. In development of the application, we found a correlation coefficient of >0.9 between the elution pH values of six acidic proteins and their isoelectric points. One case study showed the resolution between a monoclonal antibody and non-retained protein species from a protein A column. Another case study showed the feasibility of separating polyethylene glycol conjugated protein from native protein.
Ng
Paul K
PK
Process R&D Applications, Process Chromatography Division, Bio-Rad Laboratories, Hercules, CA 94547,USA. paul_ng@bio-rad.com
He
Jie
J
Snyder
Mark A
MA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000302:isolation & purification / Q000378:metabolism; D007526:Isoelectric Point; D011092:Polyethylene Glycols / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*; D013205:Staphylococcal Protein A / Q000378:metabolism; D013482:Superoxide Dismutase / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011506:Proteins; D013205:Staphylococcal Protein A; D011092:Polyethylene Glycols; D013482:Superoxide Dismutase