For Research Use Only. Not for use in diagnostic procedures.

The effect of ultra-low flow on the ionization of biotherapeutics
Gabor Jarvas1,2; Bryan Fonslow 1,3; John R. Yates III,1 and Andras Guttman2,3
1The Scripps Research Institute, La Jolla, CA, 2Horvath Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Hungary; 3Sciex, Brea, CAABSTRACT
Biotherapeutics, especially monoclonal antibodies have emerged as very promising new generation protein
drugs. However, their comprehensive analysis represents new challenges in the field. These large biomoleculesrequire full structural characterization but their heterogeneity and complexity in most cases are not easily
addressable with the analytical techniques at hand today. The advent of the integration of capillaryelectrophoresis with electrospray ionization in a single dynamic process (termed CESI1) coupled with highresolution mass spectrometry holds the promise to fulfill this need at each levels of the characterization ofbiotherapeutics (level 1: intact; level 2: reduced; level 3: bottom up; level 4: glycosylation). Traditionally all levels
suffer from the ion suppression phenomenon, which is systematically investigated in this presentation. Some ofthe main advantages of CESI-MS are the ability 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 the sensitivity of CESI-MS was increased by an order of magnitude with the decreaseof 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 analytesof biotherapeutic interest.

SAMPLE PREPARATION
Mixtures of maltotetraose (M = 684.12 g/mol) and neurotensin (M = 1674.04 g/mol) were prepared in equimolar
concentration of both analytes 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.

ION SUPPRESSIONSENSITIVITY
Table 1. Measured signal intensity ratios of neurotensin and maltotetraose at different flow rates.

The liquid flow rate in ESI determines the initial droplet size, thus plays an essential role in the efficiency of thespray process. It has also been reported that genuine nano-ESI, where ion suppression is sufficiently low oreven negligible, is only available under a given flow rate limit of 20 nL/min2. While previous studies focused on
the determination of such a flow rate limit2 with regular ESI spray settings, this is the first study to investigate
this effect with the porous sprayer (CESI setup). In this presentation, flow rates were controlled by the built-inpressure system of a CESI 8000 instrument and were estimated determined using the Hagen-Poiseuilleequation:

The sensitivity of the CESI-MS setup for intact protein analysis was evaluated by comparing the MS spectra
measured at different flow rates. The qualitative analysis of the spectra in Figure 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 is important when the sample availability is limited.
Furthermore, the decreased flow rate dramatically increased the sensitivity of the analysis. Figure 4 shows theobserved counts relative to the unity (one mole) amount of the infused analyte. As the figure depicts, at 20nL/min flow rate the detection sensitivity increased by an order of magnitude compared to the higher flow rate of
250 nL/min.

where ∆P is the pressure difference, µ is the dynamic viscosity, L is the total length of the capillary, V is thevolumetric flow rate and r is the radius of the capillary.
First the ion suppression phenomenon was systematically studied using a well-defined oligosaccharide –peptide mixture. Maltotetraose represented an uncharged oligosaccharide, which is considered a weakly
ionizable analyte. Neurotensin, on the other hand, is an easily protonated 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 higher
the maltotetraose / neurotensin intensity ratio, i.e., lower the ion suppression. As a first approximation weconsider that specific surface-to-volume ratios of the droplets play an important role. Charge carriers aresituated at the interface surface (of the droplet), so molecules with inherent positive charges (neurotensin) repel
each other and accumulate in the bulk phase of the droplet. Thus, assuming equal initial concentration of bothanalytes, neutral molecules (like maltotetrose which does not ionize as readily) will be more highly represented
at the surface of the droplet, where they will be better ionized.

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 co- and 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) and requires complex sample preparation steps, which could hinder some of thestructural details6. CESI offers mAb analysis at the intact protein level (level 1 analysis) using only very small
amounts of samples. Figure 3 compares the MS 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 the same but at 12.5 fold higher flow rate
(250nL/min). Note that the MS conditions were not optimized, as our interest was to demonstrate thecapabilities and advantages of CESI-MS analysis with ultra-low flow rates.

INSTRUMENTATION
Flow rate experiments were carried out with online hyphenation of a CESI 8000 (Beckman Coulter, soldthrough SCIEX, Brea, CA) capillary electrophoresis unit and an LTQ mass spectrometer (Thermo, Bremen,Germany). The total length of the bare fused silica separation capillary was 91 cm with 30 μm inner diameter.
Typical instrument parameters of the LTQ system were: positive polarity, 200°C capillary temperature, 35 V
capillary voltage, 1.8 kV spray voltage, 100 V tube lens voltage, 400-2000 m/z scan range,1 micro scan, 3e5AGC target, 10 ms max injection time. A Thermo Q-Exactive mass spectrometer was applied during the intact
protein analysis experiments with the following set up: positive polarity, 350°C capillary temperature, 1.6 kVspray voltage, 25 V S-lens voltage, 100 S-lens RF level, 2000-4000 m/z scan range, 140K resolution, 10 microscan, 3e6 AGC target, and 60 ms max injection time. All experiments were carried out as infusions (pressureinjection) thus electroosmosis did not need to be considered.

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

CONCLUSIONS•••

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

REFERENCES
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L. Sun, M. D. Knierman, G. Zhu and N. J. Dovichi, Analytical chemistry, 2013, 85, 5989-5995.
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Figure 1. Signal intensity ratios at different flow rates between maltotetraose (MT) and neurotensin (NT)calculated as MT+1 / (NT+1 + NT+2 +NT+3).

TRADEMARKS/LICENSING
Figure 3. Humira infusion at 20 nL/min (A) and 250 nL/min (B) flow rate. 20 min integration was used togenerate MS spectra.
Figure 2. Characteristic MS spectra of a 1:1 neurotensin and maltotetraose mixture at 9 nL/min flow rate.

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