Ionization Efficiency, Ion Suppression and Detection
Sensitivity of CESI-MS
Gabor Jarvas,1,2 Bryan Fonslow,1,3 and Andras Guttman2,3
1 The Scripps Research Institute, La Jolla, CA, US
2 Horvath Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Hungary
3 SCIEX, Brea, CA, US

Introduction
Regulatory approval requires full structural and functional
characterization, but the heterogeneity and complexity ofbiologics in most cases are not easily addressable with theanalytical techniques available today.1 Full characterization canreveal changes in primary amino acid sequences, quality attributemodifications and post-translational modifications (PTMs),
including glycosylation, that may affect therapeutic efficacy,bioavailability and biosafety. Monoclonal antibodies (mAbs) are
widely used in therapies for autoimmune and infectious diseases
as well as to combat cancer. With a number of innovator

Figure 1. The OptiMS cartridge from SCIEX provides an ultra-low flow
ESI interface.

patents expiring, a growing number of biosimilar alternatives

is a valuable advantage when the sample availability is limited,are expected to enter the market. Liquid phase separationas it is during clone selection.

methods (liquid chromatography and capillary electrophoresis)
followed by mass spectrometry will play a crucial role in thefield of biotherapeutics analysis.2 Capillary electrophoresis (CE)
coupled with mass spectrometry (MS) is a powerful combinationof a high-performance liquid phase separation technique and a
versatile detection method, providing excellent selectivity, highsensitivity and structural information.3 Due to the electrophoretic
mobility-based separation of CE, results are orthogonal toLC-MS approaches, providing new or confirmatory results. Theunique design of the CE separation capillary with an integrated
etched tip4 for electrospray ionization (CESI) provides muchpromise to fulfill some of the new requirements in biotherapeutics
characterization at all levels, including intact (level 1), reduced(level 2), bottom up (level 3) and glycosylation (level 4) analyses.
In particular, CESI-MS benefits from the inherent ultra-low flowrate of the method (a few nL/min), generally termed as nanoelectrospray ionization (nano-ESI), featuring high ionization
efficiency and excellent sensitivity. The use of very low flow rates
enables the application of highly aqueous solutions for ESI-MS,5
ideally suited for high-performance CE separations. Notably,aqueous buffers are suitable electrolytes for the analysis ofhydrophilic molecules such as glycopeptides and glycoproteins.
Furthermore, nano-ESI has reduced sample requirements, which

In the pioneering paper of Schmidt et al.,6 some of theadvantageous characteristics of nano-ESI were demonstrated,
including the suggestion that ion suppression is apparently
eliminated at flow rates below 20 nL/min. Subsequently, alow flow rate ESI-MS interface was introduced for capillaryelectrophoresis with ESI-MS.4 This design included an etched
tip at the capillary outlet providing adequate connection to close
the high-voltage separation circuit and simultaneously providing
the high voltage necessary for the electrospray process. The
approach is termed CESI, referring to the integration of capillaryelectrophoresis and electrospray ionization into a single dynamic
process.7 A handful of CESI infusion studies have already shown
the ionization efficiency benefits at ultra-low flow rates. The
first of these experiments illustrated an ~18-fold improvementin peptide sensitivity (counts/mole) when comparing flow
rates at tens to hundreds of nanoliters per minute.8 Infusionof a phosphopeptide mixture at 10 and 100 nL/min showed
dramatically reduced ion suppression for highly phosphorylated
peptides,9 an otherwise common problem in shotgun (phospho)
proteomics. Most recently, a set of intact protein and native
MS infusion experiments using CESI illustrated sensitivity
improvements when operating in the ultra-low flow range.2
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This technical note reports the effect of CESI-MS flow rate on
ionization efficiency, ion suppression and detection sensitivity for
analytes of biotherapeutic interest. All experiments were carriedout in infusion mode, thus no electric field mediated variations(electrophoretic and/or electroosmotic mobility) was considered.
The ion suppression phenomenon was systematically studied
using a well-defined oligosaccharide and peptide mixture

Important:• A separation current above 5 μA might cause permanent
damage to the separation capillary.
• Generally, please do not apply >2000V to generate
electrospray as it may result in capillary damage.

representing molecules of contrasting ionization capability.

TheoryThe sensitivity of the approach at the intact protein level wasPreviously, we calculated flow rates based on the time for

examined by infusing Humira, a commercially available mAb.
While most previous studies focused on the advantageous
characteristics of nano-ESI with sprayers pulled from glass
capillaries, we report on continued studies to investigate theeffect of ultra-low flow rates with a commercial CESI capillary.

the sample to fill an entire capillary, and we concluded thatneither electrospray nor the MS vacuum had an effect on theflow inside the capillary.2 Based on these results, flow rates
were controlled with the built-in pressure system in the CESI
8000 Plus instrument and were estimated using the HagenPoiseuille equation:

Experimental
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO)(1)¬unless otherwise noted. Mixtures of maltotetraose (M = 684.12g/mol) and neurotensin (M = 1674.04 g/mol) were prepared inwhere ∆P is the pressure difference, μ is the dynamic viscosity,equimolar concentration of both analytes at 10–5 mol/L in a 1:1

L is the total length of the capillary, V is the volumetric flow rate

mixture of 10 mM aqueous ammonium acetate and methanol

and r is the radius of the capillary.

(J.T. Baker, Center Valley, PA). At the intact protein level, Humira,also called adalimumab (Abbott Laboratories, Abbott Park, IL)
was analyzed (3 μM in 3% formic acid).
Flow rate experiments were carried out with online hyphenationof a CESI 8000 Plus High Performance Separation-ESI Module(Beckman Coulter, sold through SCIEX, Brea, CA) capillaryelectrophoresis unit and an LTQ mass spectrometer (Thermo,Bremen, Germany). The total length of the separation capillary
was 91 cm with 150 μm outer and 30 μm inner diameter. Typicalinstrument parameters of the LTQ system were: positive polarity,
200 °C capillary temperature, 35 V capillary voltage, 1.8 kVspray voltage, 100 V tube lens voltage, 400–2000 m/z scan
range, 1 micro scan, 3e5 AGC target and 10 ms max injection
time. A Thermo Q Exactive mass spectrometer was used during
the intact protein analysis experiments with the following setup:positive polarity, 350 °C capillary temperature, 1.6 kV sprayvoltage, 25 V S-lens voltage, 100 S-lens RF level, 2000–4000
m/z scan range, 140 K resolution, 10 micro scan, 3e6 AGC targetFigure 2. Measured signal intensity ratios at different flow rates
between maltotetraose (MT) and neurotensin (NT) calculated as
MT+1/(NT+1 + NT+2 +NT+3). Maltotetraose and neurotensin were infusedwith an equimolar mixture.

and 60 ms max injection time.

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Results and Discussion
Ion suppression: The ion suppression phenomenon was
systematically studied using a well-defined oligosaccharide/peptide mixture. Maltotetraose is a hydrophilic oligosaccharide
with no charge and is considered weakly ionizable. Neurotensin
is a 13 amino acid peptide that is easily protonated and ionized.
Figure 2 depicts the calculated maltotetraose/neurotensin signal
intensity ratios at increasing flow rates from 10 nL/min to >300 nL/min. As observed, lower flow rates resulted in a higher-

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 themajority of the charges are transferred from the droplet tothe analyte, but the molecular dynamics of ESI are still notcompletely understood.12 Our experiments attempted to shedlight on the nature of ESI phenomena from a physical chemistry
point of view.

intensity ratio, generally following an exponential trend. The

As a first approximation, we considered that specific surface-to-higher ratio is the result of an increased maltotetraose signal fromvolume ratios play an important role during the ion suppression

the reduced ion suppression at the lower flow rates. In practice,phenomena. For a binary system, where aqueous droplets arefor biotherapeutic molecules characterized by CESI-MS either as

dispersed in the air, the liquid phase (εrl, relative permittivity ~80)

sugars or those containing sugars, they will be more effectively

acts as a conductor, while air acts as an insulator (εr, relative

ionized, detected and quantified, and their signals will more

permittivity ~ 1). Thus charge carriers are situated at the interface

closely represent their actual abundance.

surface, i.e., an electrospray droplet carries a net positive charge

We then plotted the normalized signal intensities as a functionof flow rates for the maltotetraose and neurotensin (the latter in+1, +2 and +3 charge states), as depicted in Figure 3. Please
note that an equimolar mixture of maltotetraose and neurotensin
were infused. Figure 3 further verifies that ion suppression waspractically negligible at a ~20 nL/min flow rate. Normalized

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’slaw). Consequently, assuming equal initial concentration ofboth analytes, neutral molecules will have a partially higher
concentration at the surface since they

signal intensities (i.e., per mole) converge to a saturation regime

do not repel each other. This can be considered a valuable

starting at around 20 nL/min, comparable to earlier publishedcompetitive advantage for charges during the droplet fission, as aresults.6 This is notable since they fit their data to 3 different

higher surface-to-volume ratio ensures a higher concentration oftheoretical ESI models based on scaling laws relating to initial

neutral compounds in the surface layer. We also considered thatdroplet size and flow rate. Thus the benefits of using CESI are

the inherent charge of neurotensin had no significant effect onconsistent with those of nano-ESI. Additionally, since the CESI

the individual ionization efficiency of the maltotetraose molecules.

capillary has an etched, electrically conductive tip instead of ametal-coated, pulled tip, it does not suffer from typical clogging
issues or loss of conductive, metal coating.

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 demandfor high-performance and sensitive bioanalytical methods ispushed to its limits. In addition to molecular complexity, otherchallenges may arise when sample availability is limited,13such 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 andimmunogenicity.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

Figure 3. Normalized signal intensities at different flow rates ofmaltotetraose and neurotensin (+1, +2, and +3 charge states).
Maltotetraose and neurotensin were infused with equimolar mixture.

as modification stoichiometry and heterogeneity, is lost.15 CESI
provides mAb analysis at the intact protein level (level 1) evenp3

from very small amounts of samples. The sensitivity of theconsideration, a notable advantage in sensitivity was observed

CESI-MS setup for intact protein analysis was evaluated bythat was not previously shown with other protein molecules

comparing the MS spectra measured at different flow rates.

[2, 8]. By integrating both spectra over the same infusion times,

Figure 4 compares the MS spectra from the analysis of an intact

a lower flow rate required significantly less sample to obtain thetherapeutic monoclonal antibody (Humira) by simple infusion

same information. In this case, the signal intensity (in counts perusing the CESI sprayer at ultra-low (A: 20 nL/min) and low (B:mole) was 12.5 times greater at 20 nL/min than at 250 nL/min.

250 nL/min) flow rates. Please note that the MS conditions wereThis ratio was proportional to the flow rate and clearly illustrated

not optimized, as our interest in this work was to demonstrate thethe benefits of performing nano-ESI mAb analysis using

capabilities and advantages of CESI-MS analysis with ultra-low

CESI-MS, in terms of sensitivity at ultra-low flow rates. Whileflow rates. The spectra in Figure 4A were obtained by infusing

this is not only an interesting attribute of CESI-MS, it is also

a non-desalted Humira sample (3 μM in 3% formic acid) withmost applicable when sample availability is limited. For instance,

a 20 nL/min flow rate. Figure 4B shows the same experimentover the 20 min course of the 2 different flow rate analyses, onlywith a higher flow rate (250 nL/min). Although qualitative

173 ng of Humira was used at 20 nL/min, while 2.16 μg wascomparison of the spectra in Figure 4 revealed no significant

used at 250 nL/min.

differences between 20 and 250 nL/min flow rates, upon furtherConclusions
The systematic study of this work demonstrated the effect of low
flow rate on ionization efficiency, ion suppression and detection
sensitivity using CESI-MS for molecules of biotherapeutic
relevance. The results of this technical information bulletinreconfirm that there is an exponential drop in ion suppression
at ultra-low flow rates. Throughout the flow rate experiments,
CESI enabled stable and reproducible electrospray even at< 20 nL/min. The ion suppression effect was reduced by 2.5-foldfor the weakly ionizable oligosaccharide in the presence of aneasily ionizable peptide at the ultra-low flow level.
Infusion experiments of a therapeutic mAb with CESI-MS at
the flow rates of 20 nL/min (typical electroosmotic flow in CE)
and 250 nL/min (typical flow rate in nanoLC and sheath-flow
CE-MS) demonstrated enhanced sensitivity, i.e., an order ofmagnitude better and proportional to the flow rate ratio. It wasalso demonstrated that using a 20 nL/min flow rate in CESI
resulted in apparently the same spectrum quality at the intact
protein level as the higher flow rate of 250 nL/min, and thus
facilitated the analysis of mass-limited samples. In summary,
our infusion experiments illustrated a valuable advantage in
CESI-MS sensitivity over sheath-flow CE-MS approaches, which
typically operate at similar flow rates as nanoLC. By combining
the capabilities of a CE separation with ultra-low flow rate nanoESI conditions, CESI-MS provides a powerful tool for sensitiveand comprehensive characterization biotherapeutics.

Figure 4. Humira infusion at 20 nL/min (A) and 250 nL/min (B) flow rates.
20 min integration was used to generate the MS spectra.

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Acknowledgement
The support of the Momentum grant #97101 of the Hungarian
Academy of Sciences (MTA-PE Translational Glycomics) is
gratefully acknowledged.

9.

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