Liquid Chromatographic Separation of Therapeutic Monoclonal Antibodies with Submicrometer Particles
Abstract
The development of monoclonal antibodies (mAbs) has been one of the hottest areas in the pharmaceutical industry. In this work, submicrometer nonporous particles were used to prepare stationary phases for reversed-phase liquid chromatography (RPLC), ion exchange chromatography (IEX) and hydrophilic interaction liquid chromatography (HILIC), targeting at different problems in mAbs characterization. IgG2 suffers from disulfide scrambling and RPLC is the standard method for disulfide isoforms characterization. Four overlapping peaks are resolved by a standard commercial column, and the chromatograms change with sample concentration. A shallower gradient improves resolution, but the chromatograms are still concentration dependent. A new method is introduced to further improve resolution. The sample is injected at isocratic condition followed by a shallow gradient. Since pre-concentrating sample at the head of the column is avoided, the new method not only greatly improves resolution, but also alleviates nonlinearity of the chromatography, which means the chromatograms at different concentrations are more consistent. The resolution can be further improved by using submicrometer nonporous particles. Compared to the standard commercial column, the 0.69 μm particles based column exhibits improved resolution and sensitivity. Using the isocratic-gradient elution method, our lab-made RPLC column can resolve different subvariants of IgG2 disulfide isoforms, which is the first time that these subvariants are separated in chromatography. Charge heterogeneity is very common in mAbs and IEX is most widely used for charge variants separations. A polymer based stationary phase has been developed for anion exchange chromatography. Monomers carrying tertiary amines, together with acrylamide, are grafted to silica surface by polymerization. Compared to a leading commercial ion exchange column, the polymeric stationary phase gives sharper peaks and better resolution. Following a similar procedure, A HILIC stationary phase has been prepared for mAbs glycosylation analysis. In the HILIC stationary, only acrylamide is used as the monomer for polymerization, and the chain length is much longer in order to form a thick polymer layer. Since the N-glycosylation site is on the Fc fragment, mAbs are digested with enzyme to generate fragments. The highly efficient HILIC stationary phase not only separates out non-glycosylated Fc fragments, but also resolves the different glycoforms of Fc fragments.
Degree
Ph.D.
Advisors
Wirth, Purdue University.
Subject Area
Chemistry
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