Poly(alkyl methacrylate) Bonded Phases for RPLC Separation of Monoclonal Antibodies
Monoclonal antibodies (mAbs) and antibody-drug complexes (ADCs) are promising therapeutics that have potential to significantly impact how diseases, such as cancer, are treated. One of the issues plaguing monoclonal antibodies is their high degree of heterogeneity. Current commercial columns are unable to fully resolve monoclonal antibody samples. The bonded phase is typically the limiting factor when it comes to chromatographic separations, thus an improved bonded phase has potential to significantly improve mAb separations. Alkylmethacrylates of various hydrophobicities were polymerized onto the surface of silica particles. These particles were packed into stainless steel columns. In addition to these columns, additional columns were made which had the polymer grown in the column following particle packing. The polymer was expected to provide a thicker bonded phases surface than traditional silane bonded phases. This extra thickness was expected to reduce the number of strong adsorption sites found on the surface. The columns were tested with a fragmented IgG4 sample and an IgG2 sample via RPLC. The results from the IgG4 sample indicated that a poly(methyl methacrylate) bonded phase provided the best resolution. The resolution of the fragment region of the IgG4 was unprecedented and performed better than leading commercial columns. A general trend observed when comparing the different polymers was that lower hydrophobicity provided enhanced resolution. IgG2 is susceptible to disulfide scrambling. This results in four peaks that heavily overlap. Again, the poly(methyl methacrylate) bonded phase provided the best resolution. This column was able to resolve all four IgG2 isomers and began to resolve additional peaks that had not been seen before. Again, a less hydrophobic polymer improved resolution. Polymer bonded phases offer advantages over traditional bonded phases and have proven to improve monoclonal antibody separations in RPLC. Pursuing these types of bonded phases for other forms of chromatography could yield positive results.
Wirth, Purdue University.
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