Reversed phase liquid chromatography of intact monoclonal antibody, fragments and aggregates using nonporous silica particles
Conventional reversed phase materials such as fully porous and superficially-porous packings currently separate intact mAb fragments, but are still limited in the separation of intact mAb and its aggregates. This is because these particles suffer from insufficient pore size which causes obstructed diffusion for intact mAb and aggregates and translates to poor sample recovery. Studies have shown that the conventional silica with an average pore size of 300 Å excludes about 45 % of the pore volume of IgG4 mAb monomer. Nonporous particles were used to eliminate the porous medium heterogeneity present in porous media. Due to its compactness and high packing order marked by narrow pore distribution, a 4200 Å pore size which is more than 10-fold in magnitude to the pore size of the commercial RPLC packings, was used to form a large enough pore size provides maximum accessible surface area for the diffusion of mAb aggregates with minimal exclusion. By harnessing these advantages, a reversed phase column was developed with nominal 1.5 µm nonporous particles in the stainless steel format (50 mm X 2.1mm i.d). Results showed that this column offered complete recovery and improved separation to the RPLC of IgG4 mAb fragments and aggregates from the intact monomer. From the particle size dependency results of these particles with the resolution of intact mAb, its fragments and aggregates, the 1500 nm particle showed better resolution and close-enough efficiency with the 750 nm particle. These combined results enables the simultaneous separation of mAb fragments and aggregates in RPLC with complete recovery and improved resolution. It has the potential to serve as an orthogonal technique to size-exclusion chromatography for the analysis of mAb fragments and aggregates.
Wirth, Purdue University.
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