High Sensitivity Intact Antibody Drug Conjugate Analysis Using an Integrated Microfluidic Device Coupled to HRMS

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Gregory T. Roman, James P. Murphy
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This poster aims to characterize the analytical capabilities of the ionKey/MS system for the mass determination of antibodies and antibody drug conjugates.

The iKey separation device is packed and assembled using reversed-phase bridged ethyl hybrid (BEH) 1.7μm C4 particles. The iKeys are integrated with an ESI emitter shown in Figure 1 below; iKey dimensions were 150 μm id x 10 cm. In addition to the iKey, trapping columns, 300 m x 5 cm, were packed with either BEH 5.0 m C4 particles or MassPrep desalting column technology. These columns were plumbed to the trapping valve manager (TVM), which was consequentially plumbed to the iKey.

These dimensions provide improved sensitivity compared to identical injection volumes on an ACQUITY UPLC 2.1 mm column. Much of this sensitivity improvement is based on improvements in a) ionization efficiency, b) sampling efficiency, and c) ion suppression.

Electrospray ionization efficiency is inversely related to flow rate. Generally, as flow rates are reduced, electrospray efficiency increases non-linearly. The major difference between microflow ESI and high flow ESI is the diameter of droplets that are generated between the two flow regimes. In the microflow regime, it is possible to generate smaller droplets as compared to the high flow. This assists in electrospray ionization by enabling a relatively high charge density within a droplet, and also increasing the effective electric field at the surface of the droplet. Since there is a proportionally higher analyte to solvent ratio in the spray plume for microflow ESI, the sampling of a greater fraction of the analyte will occur. Finally, ion suppression is reduced in smaller droplets because the ions have an “easier” path to the surface of the droplet.

As is the case with monoclonal antibodies, improvements in electrospray efficiency will further assist in the reduction of the barrier of entry into the gaseous phase for large macromolecules.

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