<p>LC-MS of protein drugs requires new ideas in bonded phase
design rather than adapting bonded phases from the realm of small-molecule
drugs. The polymer-shell bonded phase is designed to interact with larger
molecules and to shield proteins from the silica substrate. The particles
consist of a core of solid silica and a shell of dense polymer brush. The
polymer layer is thick enough to protect the protein from interactions with
silanols to reduce peak tailing. The polymer contains multiple functional
groups that introduce more selectivity. This design gives unprecedented LC
resolution and MS sensitivity. Our group has developed polymer shell bonded
phases for hydrophobic interaction chromatography (HIC-MS) of antibody-drug
conjugates (ADCs), hydrophilic interaction liquid chromatography (HILIC-MS) of
glycoproteins, and reversed-phase liquid chromatography (RPLC-MS) of monoclonal
antibodies. Since HIC is not in-line compatible with MS due to the high salt
levels, it is laborious to identify the constituents of HIC peaks. An
MS-compatible alternative to HIC is reported here: native reversed phase liquid
chromatography (nRPLC). This employs a mobile phase 50 mM ammonium acetate for
high sensitivity in MS, and elution with a gradient of water/isopropanol. The
nRPLC-MS data show that all ADC species, ranging from drug-to-antibody ratios
of 1 to 8, remained intact and native on the column. As we adapt this concept
to intact proteins, we find that lysozyme and α-chymotrypsinogen A are both
eluted in their native conformations. We also use the polymer-shell concept to
resolve IgG1 free thiol variants by RPLC-MS with 0.5% formic acid. Since there
are always other variants besides the intended ones, the need for high MS
sensitivity is desired to distinguish subtle mass change between disulfide bond
and free thiols. Overall, MS sensitivity increases 10X relative while all of
the thiol variants are well resolved by the polymethylmethacrylate bonded phase.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/8970680 |
| Date | 13 August 2019 |
| Creators | Tse-Hong Chen (7013258) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/Polymer-Shell_Bonded_Phase_for_Improving_Online_LC-MS_Analysis_of_Intact_Proteins_mAbs_and_ADCs/8970680 |
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