ULTRA-LOW ESI-MS FLOW RATES MAKE A
DIFFERENCE IN THE ANALYSIS OF
BIOTHERAPEUTICS
Gabor Jarvas1,2; Bryan Fonslow1,3; John R. Yates III1; and Andras Guttman2,3
1The Scripps Research Institute, La Jolla, CA; 2Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Hungary; 3Sciex, Brea, CA(For Research Use Only. Not for use in diagnostic procedures)

ABSTRACT
Biopharmaceuticals, especially therapeutic monoclonal antibodies,
have emerged as a very promising new generation of proteindrugs, but concomitantly represent new bioanalytical challenges
for the field. While these large biomolecules requirecomprehensive structural characterization, their heterogeneity andcomplexity in most instances are beyond the application domain of
the analytical techniques available today. Integration of capillaryelectrophoresis with electrospray ionization in a single dynamicprocess (termed CESI) coupled with high resolution mass
spectrometry holds the promise to fulfill this gap, even at the intact
protein level. Some of the main advantages of CESI-MS are theability to produce stable electrospray at ultra-low flow rates (5-20nL/min range) in a robust and reliable manner. In this presentation,
the effect of CESI flow rate on ionization efficiency, ion
suppression and detection sensitivity will be discussed. Our intact
therapeutic antibody analysis results demonstrated that thesensitivity of CESI-MS was increased by an order of magnitudewith the decrease of the flow rate from 250 nL/min to 20 nL/min.
On the other hand, ultra-low flow rates significantly (2.5x) reduced
the ion suppression effect in respect to samples containing both
highly and weakly ionizable analytes of biotherapeutic interest.

First the ion suppression phenomenon was systematically studied
using a well-defined oligosaccharide – peptide mixture.
Maltotetraose represented an uncharged oligosaccharide, which isconsidered a weakly ionizable analyte. Neurotensin, on the otherhand, is an easily protonated, 13 amino acid peptide. Figure 1
shows the signal intensity ratios as the function of the increasing
flow rate. As one can observe, the lower the flow rate the lower theneurotensin / maltotetraose intensity ratio, i.e., lower the ionsupression. As a first approximation we consider that lower flow
rates produce smaller initial droplet size thus the formed free ionshave a more diluted chemical environment where analyte – analyteinteractions are less pronounced. Consequently, the inherent chargeof neurotensin has no such a significant effect on the individual
ionization efficiency of the maltotetraose molecules.

SAMPLE PREPARATION
Mixtures of maltotetraose (M = 684.12 g/mol) and neurotensin (M= 1674.04 g/mol) were prepared in equimolar concentration of bothanalytes at 10-5 mol/L in a mixture of 10 mM aqueous ammonium
acetate and methanol (1:1 by volume). For the intact protein
analysis Humira was analyzed at a concentration of 3 µM in 5%
formic acid solution.

Figure 1 Signal intensity ratios at different flow rates between neurotensin (NT)and maltotetraose (MT) calculated as (NT+1 + NT+2 +NT+3) / MT+1.

Figure 3 Humira infusion at 20 nL/min (A) and 250 nL/min (B) flow rate. 20
min integration was used to generate MS spectra.

SENSITIVITY
The sensitivity of the CESI-MS setup for intact protein analysis
was evaluated by comparing the MS spectra measured atdifferent flow rates. The qualitative analysis of the spectra inFigure 3 revealed no significant differences between the observed
spectra at 20 and 250 nL/min flow rates. Assuming same infusion
times, lower flow rate requires less sample amount, which isimportant when the sample availability is limited. Furthermore, thedecreased flow rate dramatically increased the sensitivity of theanalysis. Figure 4 shows the observed counts relative to the unity(one mole) amount of the infused analyte. As the figure depicts, at20 nL/min flow rate the detection sensitivity increased by an orderof magnitude compared to the higher flow rate of 250 nL/min.

INSTRUMENTATIONCESI 8000 conditions
Uncoated capillary
Pressure infusion
Capillary i.d.: 30 µm
Capillary length: 91 cmFigure 2 Characteristic MS spectra of a 1:1 neurotensin and maltotetraose
mixture at 9.12 nL/min flow rate.
Table 1. Measured signal intensity ratios of neurotensin and maltotetraose atdifferent flow rates.

ION SUPPRESSION

8µ𝐿𝑉∆𝑃 =𝜋𝑟 4where ∆P is the pressure difference, µ is the dynamic viscosity, L
is the total length of the capillary, V is the volumetric flow rate andr is the radius of the capillary.

Conclusions
Increased sensitivity. CESI-MS at a flow rate of 20nL/min showed increased sensitivity by an order ofmagnitude compared to higher flow rate (250 nL/min).
Reduced sample requirement. CESI-MS at a flow rate of
20 nL/min produces the same spectrum quality at theintact protein level as at a flow rate of 250 nL/min.
Decreased ion suppression. Ultra-low flow rates (< 20nL/min) significantly (2.5x) reduced the ion suppression
effect in respect to samples containing both highly andweakly ionizable analytes.

CESI 8000 was hyphenation with Thermo LTQ and Q-Exactive
mass spectrometers, respectively.

The liquid flow rate in electrospray ionization (ESI)
determines the initial droplet size, thus plays an essential
role in the efficiency of the spray process. It has also beenreported that genuine nano-ESI, where ion suppression is
sufficiently low or even negligible, is only available under agiven flow rate limit of 20 nL/min 1. While previous studies
focused on the determination of such a flow rate limit1 withregular ESI spray settings, this is the first study toinvestigate this effect with the porous sprayer (CESI setup).
CESI is the integration of capillary electrophoresis (CE) andelectrospray ionization (ESI) into a single dynamic
process2. In this presentation, flow rates were controlled by
the built-in pump of a CESI 8000 instrument and were
accurately determined using the Hagen-Poiseuilleequation:

Figure 4 Normalized signal intensities at 20 nL/min (left) and 250 nL/min(right) flow rates. Sample: 3 µM Humira.

Intact mAb analysis
Robust, comprehensive and reproducible characterization of
monoclonal antibody therapeutics is crucial in thebiopharmaceutical industry. This can represent a challenge
when sample availability is limited3, 4. MAbs are subject to coand post-translational modifications, which results in e.g.,
glycosylation micro-heterogeneity that may affect antigenicity
and immunogenicity5. MAb analysis is usually performed at
the peptide level after tryptic digestion (level 3 analysis) andrequires complex sample preparation steps, which could
hinder some of the structural details6. CESI offers mAb
analysis at the intact protein level (level 1 analysis) using
only very small amounts of samples. Figure 3 compares theMS spectra of the analysis of an intact protein therapeutics
Humira, by simple infusion using the CESI sprayer. The
spectra in Figure 3A was obtained by infusing 3 µM Humira
sample with a flow rate of 20 nL/min. Figure 3B shows thesame but at 12.5 fold higher flow rate (250nL/min).
Deconvolution of the spectra was accomplished by an inhouse developed script.

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