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

®
Ultra-sensitive host cell protein detection using CESI-MS with SWATH Acquisition
Edna Betgovargez1; Bryan Fonslow1,2; Eric Johansen2
1SCIEX, Brea, CA; 2SCIEX, 1201 Radio Rd., Redwood City, CA
The same CESI separation conditions are used for IDA identification and SWATH TM acquisition quantitation runs.
Figure 3 shows some representative total ion electropherograms from quantitation runs.

ABSTRACTRESULTSWe demonstrate the use of CESI-MS with SWATHTM acquisition for the ultrasensitive detection of host cell
proteins (HCPs) in a representative mAb preparation. To simulate HCPs, we spiked a mAb digest with digested
protein standards over a concentration range of ppb to parts per thousand (ppth) concentrations. mAb andHCP proteins at ppth concentrations were identified with a CESI-MS information-dependent acquisition (IDA)
run to generate an ion library for proteins/peptides. Identical CESI separations were performed with the HCP
dilution series using SWATHTM acquisition. The ion library and SWATH data were used to screen for peptides
and fragment ions which best represent the HCP concentrations. The concentration-indicative peptides andions were then used to generate calibration curves for the HCPs. HCPs were quantified down to the low ppm
range and in some cases even into the high ppb range, representing detection of HCPs over three orders ofmagnitude.

The first step for a host cell protein experiment is identifying proteins with an IDA experiment while present at
higher concentrations. In practice, this experiment would be performed on a partially purified mAb preparation.
Here we simulated the partially purified preparation with protein standards spiked in at 1 parts per thousand bymass relative to the mAb. CESI-MS IDA runs allow for identification of peptides within representative host cell
proteins which yield good MS/MS spectra at lower concentrations. These peptide fragment ions are evaluatedas good quantitative representatives of a HCPs concentration.
Figure 1. Raw data from a CESI-MS IDA run on the1 parts per thousand standard protein and mAbsample preparation. The raw data is represented as(A) a heat map of the peptides selected for MS/MS,(B) a representative peptide MS/MS spectra, and(C) a total ion chromatogram (TIC) of the CESI
peptide separation.

BA

INTRODUCTION
Host cell proteins are undesired impurities in biologic preparation processes and can negatively affectbiotherapeutic quality, potency, and safety. Identification and quantification of HCPs within therapeutic
monoclonal antibody (mAb) preparations remain a challenge with a need for improved sensitivity and specificity.
Mass spectrometry, particularly data-independent acquisition such as provided by SWATH™ acquisition,provides a robust and sensitive means to quantify HCPs in the parts-per-million (ppm) range. The integration ofcapillary electrophoresis (CE) and electrospray ionization (ESI) into one process (CESI) presents the possibility
to improve the sensitivity of HCP quantitation through reduced ion suppression and improved ionization
efficiency at ultralow nanoliter per minute flow rates.

Figure 4. Representative
total ion electropherograms(TIEs) from CESI-MS
SWATHTM acquisition runs
including blank and HCPspiked runs in the ppb toppth concentration range.

MATERIALS AND METHODSAFigure 2. Identification of representative mAb and HCPs fromCESI-MS IDA runs using ProteinPilotTM. Peptides shown ingreen within the protein sequence represent the best
candidates for quantitation using SWATHTM acquisition since
they have the best MS/MS signals and are imported into thePeakView® SWATH microapp.

CESI Conditions:
CESI experiments were carried out with a SCIEX CESI 8000 system equipped with a temperature controlled
autosampler and a power supply with the ability to deliver up to 30 kV. A commercial bare fused-silica capillary
cartridge with a porous tip was used for infusion and peptide mapping experiments. Solutions of 10% acetic
acid were employed as the background electrolyte (BGE) and conductive liquid. After pressure injection of ~25
ng peptides, sample stacking was performed using transient isotachophoresis (t-ITP). CESI separations wereperformed at 20 kV.
MS/MS Conditions:A SCIEX TripleTOF® 5600+ system with a NanoSpray® III source and CESI adapter controlled by Analyst ® TF
1.7 Software were used. HCP identification was performed with information dependent acquisition (IDA) with15 MS/MS cycles (100 and 50 ms accumulation times for MS and MS/MS, respectively). HCP detection andquantification were performed using data-independent SWATHTM acquisition with 30 constant window widthscans (30 m/z with 1 m/z overlaps) from 300 – 1200 m/z using 150 and 50 ms accumulation times for MS andSWATH scans, respectively.

Blank

1 ppm

C

Sample Preparation:
A representative mAb and 17 standard protein mixture were digested separately to peptides at 1 mg/mL using a4-hour digestion protocol with RapiGest, DTT, iodoacetamide, and trypsin. The digested standard protein
mixture was spiked into the digested mAb throughout the parts per thousand, ppm, and ppb range to represent
contaminant host cell proteins. The peptide preparations were then diluted to 250 mg/mL in 125 mM ammonium
acetate, pH 4.

Due to the very low concentrations of host cell proteins within therapeutic mAb preparations, the sensitivity ofmass spectrometric methods are pushed, potentially into the low S/N levels. With the reduced ion suppressionof CESI run at ultra-low flow rates (~20 nL/min), it’s possible to extend the dynamic range and sensitivity of HCP
detection. Figures 5 and 6 illustrated the common, expected sensitivities and dynamic ranges for the peptides
identified. However, there were examples of peptides with even greater sensitivity and dynamic range in ouranalysis. To specifically show the S/N ratios for one of these quantified peptides we generated extracted
fragment ion electropherograms for myoglobin peptide NDIAAK shown in Figure 6. Even at 0.1 ppm peptide
NDIAAK has a S/N ratio of ~20, facilitating detection of this representative HCP over 3 orders of magnitude.

Figure 5. (A) Extracted fragment ion electropherograms formyoglobin peptide VEADIAGHGQEVLIR y7 and y13 ions.
(B) Matching SWATH MS spectra (blue) to previously
identified myoglobin peptide fragment ions (blue). (C)Fragment ion peak area plot based on HCP concentration.

33 ppm0.1 ppm

0.3 ppm

3.3 ppm

10 ppm100 ppm

Figure 6. Calibration curves from triplicate CESI-SWATH
runs of myoglobin peptides (A) VEADIAGHGQEVLIR b2ion and (B) HGTVVTALGGLK y7 ion.

AFigure 7. Extracted fragment ion electropherograms for myoglobin peptide NDIAAK in the concentration range from 0.1
ppm to 100 ppm using MultiQuantTM.

SWATHTM acquisition can be performed with different m/z window configurations to focus on information-richBregions of the m/z domain. We investigated the use of constant and variable window widths on the detection ofHCPs using CESI-MS. Peptides are generally most abundant in the 400 – 800 m/z range, so one of thevariable window configurations tested was generated from LC-MS IDA analysis of a common peptide digest
from yeast lysate. The other variable window configuration we evaluated was generated from the replicate
CESI-MS IDA runs from the HCP/mAb preparation as shown in Figure 1. Both variable window configurations
are shown in Figure 2.
A

10 ppm100 ppm

CONCLUSIONS
NoiselevelBC

An integrated CESI-MS workflow is presented that facilitates the ultra-sensitive detection of representative hostcell proteins in a mAb preparation. The combined use of CESI with SWATH TM acquisition exploits reduced ion
suppression and data-independent analysis to create a powerful tool for host cell protein quantitation. This newapplication adds to the versatility and efficiency of open tube capillary electrophoresis for biologics
characterization.

B

TRADEMARKS/LICENSING

3 ppm
Data Analysis:High resolution MS and MS/MS spectra were analyzed using SCIEX ProteinPilot TM, PeakView®, andMultiQuantTM softwares.

100 ppm
Noiselevel
Figure 3. Calculation of variable SWATHTM acquisition window widths based on IDA runs of a (A) yeast lysate and (B)
1 parts per thousand HCP/mAb preparation.

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100 ppb

1 ppm

10 ppm100 ppm

Concentration (ppm)

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