It is known that lower flow rates produce smaller initial droplet sizes.6 However, it is not known precisely how droplet size influences ionization efficiency. The widely accepted ion evaporation10 and charge residual models11 focus on how the majority of the charges are transferred from the droplet to the analyte, but the molecular dynamics of ESI are still not completely understood.12 Our experiments attempted to shed light on the nature of ESI phenomena from a physical chemistry point of view.
As a first approximation, we considered that specific surface-tovolume ratios play an important role during the ion suppression phenomena. For a binary system, where aqueous droplets are dispersed in the air, the liquid phase (εrl, relative permittivity ~80) acts as a conductor, while air acts as an insulator (εr, relative permittivity ~ 1). Thus charge carriers are situated at the interface surface, i.e., an electrospray droplet carries a net positive charge at the droplet surface, so molecules with inherent positive charges, such as peptides (in our case, neurotensin), accumulate in the bulk phase of the droplet due to repel forces (Coulomb’s law). Consequently, assuming equal initial concentration of both analytes, neutral molecules will have a partially higher concentration at the surface since they do not repel each other. This can be considered a valuable competitive advantage for charges during the droplet fission, as a higher surface-to-volume ratio ensures a higher concentration of neutral compounds in the surface layer. We also considered that the inherent charge of neurotensin had no significant effect on the individual ionization efficiency of the maltotetraose molecules.
The intact mAb analysis showed that robust, comprehensive and reproducible characterization of intact protein therapeutics is very important for the biopharmaceutical industry. As more complex therapeutic molecules are created, the demand for high-performance and sensitive bioanalytical methods is pushed to its limits. In addition to molecular complexity, other challenges may arise when sample availability is limited,13 such as during the discovery phase or in pharmacokinetics/ pharmacodynamics studies. mAbs are subject to co- and posttranslational modifications, such as glycosylation site occupancy and micro-heterogeneity as well as degradative PTMs, resulting in changes that may affect effector function, antigenicity and immunogenicity.14 One type of mAb analysis is performed at the peptide level after tryptic digestion (level 3 analysis), but information about the intact structure of the molecules, such as modification stoichiometry and heterogeneity, is lost.15 CESI provides mAb analysis at the intact protein level (level 1) even from very small amounts of samples. The sensitivity of the CESI-MS setup for intact protein analysis was evaluated by comparing the MS spectra measured at different flow rates. Figure 4 compares the MS spectra from the analysis of an intact therapeutic monoclonal antibody (Humira) by simple infusion using the CESI sprayer at ultra-low (A: 20 nL/min) and low (B: 250 nL/min) flow rates. Please note that the MS conditions were not optimized, as our interest in this work was to demonstrate the capabilities and advantages of CESI-MS analysis with ultra-low flow rates. The spectra in Figure 4A were obtained by infusing a non-desalted Humira sample (3 μM in 3% formic acid) with a 20 nL/min flow rate. Figure 4B shows the same experiment with a higher flow rate (250 nL/min). Although qualitative comparison of the spectra in Figure 4 revealed no significant differences between 20 and 250 nL/min flow rates, upon further consideration, a notable advantage in sensitivity was observed that was not previously shown with other protein molecules [2, 8]. By integrating both spectra over the same infusion times, a lower flow rate required significantly less sample to obtain the same information. In this case, the signal intensity (in counts per mole) was 12.5 times greater at 20 nL/min than at 250 nL/min. This ratio was proportional to the flow rate and clearly illustrated the benefits of performing nano-ESI mAb analysis using CESI-MS, in terms of sensitivity at ultra-low flow rates. While this is not only an interesting attribute of CESI-MS, it is also most applicable when sample availability is limited. For instance, over the 20 min course of the 2 different flow rate analyses, only 173 ng of Humira was used at 20 nL/min, while 2.16 μg was used at 250 nL/min.