Ulttra-sennsitive hosthceell prote
ein deteection using C
CESI-MMSwitth SWAATH® acquisitationSubb-ppm HCP sensitivitysthrought
an orthogonal
al CE separaation and reeduced ion ssuppressioon
Brya
an Fonslow1 and Eric Joh
hansen2
1
SCIIEX Separattions, Brea, CA;
C 2SCIEX,X, Redwood City,CCA
KEY BENEFITS
D
Detection of host cell proteiins from limite
ed sample
qquantities in th
he early development phaseeO
Orthogonal seeparation mechanism for addditionalm
method develoopment or dettection validattion

Im
mprovement in
i sensitivity throught
reduc
ced ions
suppression anda higher ionization efficiency at ultra-loow
fflow rates

Introoduction
Host cell proteins (HHCPs) are undeesired impuritie
es in biologic
prepa
aration process
ses and can neegatively affect biotherapeutic
qualitty, potency, andd safety. Identification and quuantification ofHCPss within therape
eutic monoclon
nal antibody (mmAb)
prepa
arations remainn a challenge withw a need for improved
sensittivity and specificity. Mass sp
pectrometry, pa
articularly dataindep
pendent acquisition such as provided by SW
WATHacquisition, provides
s a robust and sensitive meanns to quantify
HCPss in the parts-per-million (ppmm) range. The in
ntegration ofcapillaary electrophorresis (CE) and electrospray io
onization (ESI)
into oone process (CESI) presents thet possibility tot improve thesensittivity of HCP quantitation throough reduced io
on suppression
nand immproved ioniza
ation efficiency at ultralow nan
noliter per
minutte flow rates.
We demonstrate thee use of CESI-M
MS with SWATTH acquisition
for thee ultrasensitivee detection of hosthcell proteinns (HCPs) in a
representative mAb preparation. To
T simulate HCPs, we spiked amAb digest with digested protein standards
sover a concentratio
onrangee of sub-ppm too parts per thouusand (ppth) co
oncentrations.
HCP proteins at pptth concentrations were identiffied with a CES
SIMS innformation-dependent acquisiition (IDA) run tot generate anion libbrary for protein
ns/peptides. Id
dentical CESI separations
sweereperforrmed with the HCP
H
dilution seeries using SWWATHacquisition. The ion library and SW
WATH data werre used toscreeen for peptides and fragment ions
iwhich bes
st represent thee
HCP concentrations
s. The concenttration-indicativ
ve peptides anddions w
were then used
d to generate calibration curve
es for the HCPPs.
HCPss were quantifie
ed down to thee low ppm range and in somecasess even into the sub-ppm range, representing
g detection ofHCPss over three ord
ders of magnitu
ude.

CESI 80000 Plus High PPerformance Se
eparation Modulle coupled to a®
TripleTTOF 5660 Syste
em via a Nanosp
pray® III sourcee, CESI adapter,
ptiMS Silica Surfrface Cartridge
and OpExpeerimental
 Sam
mple Preparattion: A represeentative mAb and 17 protein
stanndard mixture were digested
d separately too peptides at 1
mg/mhour digestion protocol with R
RapiGest, DTTT,mL using a 4-hiodo
oacetamide, an
nd trypsin. Th
he digested prrotein standard
dmixtture was spiked
d into the digessted mAb throu
ughout the ppthppmm, and ppb raange to repre
esent contamiinant host cell
protteins. The pep
ptide preparatio
ons were then diluted to 250
mL in 125 mM ammonium ace
etate, pH 4.
µg/m
 CES
SI Conditions:: CESI experim
ments were carrried out with aSCIEEX CESI 8000
0 Plus system (P/N A98089) eequipped with atemp
perature contro
olled auto sampler and a pow
wer supply withthe aability to delive
er up to 30 kV. An OptiMS Siliica Surface
Carttridge (P/N B077367) with a po
orous tip was uused forinfussion and peptidde mapping exxperiments. Solutions of 10%acettic acid were employed as thee background eelectrolyte(BGGE) and conducctive liquid. Afteer pressure inje
ection of ~25
p1

ng peptides, sam
mple stacking wasw performed usingutransientotachophoresis (t-ITP). CESI separationss
weere performed ata
iso20 kV. The samee CESI separatiion conditions area used for IDDA
entification and SWATH acquisition quantitation runs.
ide®
 MSS/MS Conditio
ons: A SCIEX TripleTOFT56000+ system witth®
aNNanoSpray III source and CE
ESI adapter (P//N B07366)conntrolled by Anaalyst® TF 1.7 So
oftware were used.
uHCPideentification wass performed witth information dependent
dacq
quisition (IDA) with 15 MS/MS
S cycles (100 anda 50 ms
acccumulation times for MS and MS/MS, respeectively). HCP
dettection and qua
antification werre performed using dataind
dependent SWA
ATH acquisition with 30 constant windowwid
dth scans (30 m/z
m with 1 m/z overlaps) from 300 – 1200 m/zmusiing 150 and 500 ms accumulattion times for MSM and SWATHH
scaans, respectiveely.

S spectra at low
wer concentrations. ProteinPiilot search
MS/MS
results from the IDA eexperiment is sshown in Figure
e 2. Thee fragment ionss from protein aand peptides ddatabase
peptidematchees can later be
e evaluated as ggood quantitatiive
represeentatives of a H
HCPs concentrration from the SWATH
acquis ition data.

 Da
ata Analysis: HighH
resolution
n MS and MS/M
MS spectra weere®
anaalyzed using SCIEX ProteinPilot™, PeakView
P, anndMu
ultiQuant™ softtware.

Figure 2. Identificationn of representattive mAb and prrotein standardssTMESI-MS IDA run
ns using ProteinnPilot . Peptides shown infrom CEgreen w
within the prote
ein sequence rep
present the bes
st candidates forTM
quantittation using SW
WATH acquisittion since they hhave the best®
e peptides are immported into th
he PeakView
MS/MS signals. These
H microapp for eevaluation.
SWATHFigure
e 1. Raw data frrom a CESI-MS IDA run on the 1 parts per
thoussand protein staandard and mAbb sample preparration. The raw
data iss represented asa (A) a heat ma
ap of the peptide
es selected for
MS/MS, (B) a represeentative peptide MS/MS spectraa, and (C) a tota
alectropherogramm (TIE) of the CE
ESI peptide sepparation.
ion eleRessults

Figure 3. Calculation of variable SWA
ATHTM acquisittion window
ast lysate and (B) 1 parts perwidths based on IDA rruns of a (A) yea
thousa nd HCP/mAb prreparation.

The first step for a hosthcell protein
n experiment iss identifying
proteiins with an IDAA experiment whilew
present att higher
conceentrations. In practice, this exxperiment wouldd be performed
don a ppartially purified mAb preparaation. Here we simulated thepartia
ally purified preparation with protein
pstandardds spiked in at 1
parts per thousand byb mass relativ
ve to the mAb. Representativeedby PeakView in IDA
A Explorer is
CESI-MS IDA data displayed
CIDA runsr
allow for id
dentification ofshown in Figure 1. CESI-MS
des within representative hosst cell proteins whichw
yield gooodpeptidSWAT H acquisition ccan be performed with differen
nt m/z windowurations to focu
us on informatio
on-rich regionss of the m/z
configudomainn. We investiga
ated the use off constant and vvariablewindow
w widths on thee detection of H
HCPs using CE
ESI-MS.
Peptidees are generally most abunda
ant in the 400 – 800 m/z
range, so one of the vvariable window
w configuration
ns tested wasated from LC-M
MS IDA analysiss of a common
n peptide digest
generafrom yeeast lysate. The other variable window conffiguration wep2evalua ted was generrated from the rreplicate CESI--MS IDA runs
he HCP/mAb preparation as rrepresented in Figure 1. Bothfrom thvariabl e window conffigurations are shown in Figurre 3. Triplicate
eormed on the ddilution series ofo
SWAT H acquisition runs were perfo
nstrative host cell proteins. Fiigure 4 shows representative
demon
total io n electrophero
ograms from thee replicate qua
antitation runs.

Figuree 4. Representa
ative total ion electropherogram
ms (TIEs) fromTM
CESI-MS SWATH acquisition
aruns including blank and HCP-spikkedpth concentratio
on range.
runs iin the ppb to ppFiguree 5. (A) Extracte
ed fragment ion electropherogrrams formyoglobin peptide VEADIAGHGQEVVVLIR y7 and y13
3 ions. (B)hing SWATH MS
S spectra (blue) to previously id
dentifiedMatchmyoglobin peptide frragment ions (pink). (C) Fragm
ment ion peak arrea
based on HCP co
oncentration froom triplicate runns.
plot bdate peptides aand fragment io
ons can be sele
ected forCandidquantittation using SW
WATH Processing in PeakVieew. Figure 5
shows representativee SWATH data selected for quuantitation ofd fragment ion electropherograms forHCPs. Two extracted
obin peptide VE
EADIAGHGQE
EVLIR (Figure 55A) have themyoglosame mmigration profil e indicating theey are from thee same peptidee.
These two ions were matched to thoose found in th
he peptide
library from the IDA rrun (Figure 5B). Additionally, when theent ions are con
nsidered betwe
een triplicate ru
uns of thefragme
calibra tion curve, the appropriate quuantitative trend is observede 5C). Collectivvely these threee pieces of datta illustrate thee(Figurecapabi lity of quantifying HCP surrog
gate peptides bbased on a feww
ent ions.
fragmeFigure 6. Calibration ccurves from triplicate CESI-SWATH MS runs ofobin peptides (AA) VEADIAGHGQQEVLIR b2 ion and (B)myogloHGTVVVTALGGLK y7 ioon.

p3

Figuree 7. Extracted fragmentfion ele
ectropherogram
ms for myoglobin peptide NDIA
AAK in the conce
entration range
e from 0.1 ppm tto 100 ppmTM
using MultiQuant .

Due tto the very low concentrations
s of host cell prroteins within
therappeutic mAb preeparations, the sensitivity of massm
specttrometric metho
ods are genera
ally pushed to the limit,
poten
ntially into the lo
ow S/N levels. With the reduc
ced ionsupprression of CES
SI run at ultra-lo
ow flow rates (~~20 nL/min), it’s
possible to extend th
he dynamic ran
nge and sensitivity of HCP
ected
detecction. Figures 6 illustrates the common, expe
sensittivities and dyn
namic ranges foor the peptidess identified usinng
SWATTH acquisition,, previously shoown with LC-M
MS methods.1
That is, from the canndidate peptide
e ion surrogate
es for HCPs
ATH Processinng from CESI-M
MS runs, two
identified using SWA
ue myoglobin peptides could beb quantified down to low ppm
muniqulevelss (Figure 6). Hoowever, there werew
exampless of peptides wiith
even greater sensitivity and dynammic range in our analysis. To
e S/N ratios forr one of these quantifiedq
speciffically show thepeptid
des we generated extracted fragmentfionelectrropherograms forf myoglobin peptide
pNDIAAAK shown inFiguree 7. Even at 0.1 ppm peptide NDIAAK has a S/N ratio of~20, ffacilitating deteection of this reepresentative HCP
Hat the sub-ppm llevel. Notably, the blank sam
mple run had ann intensity of ~1
10
at thee noise level, whilew
the 0.1 ppm sample had an intensity off~200..

Concclusions
An inteegrated CESI-M
MS workflow is presented that facilitates theultra-seensitive detectiion of represen
ntative host cell proteins in amAb p reparation. Thee combined use of CESI with SWATH
educed ion supppression and ddataacquis ition exploits reendent analysiss to create a po
owerful tool for host cell
indepeproteinn quantitation. TThis new appliccation adds to the versatility
and effficiency of openn tube capillaryy electrophoressis for biologics
scharaccterization and complements tthe detection oof HCPs by our
high- aand mirco-flow LC-MS methodds.

References1.

Juustin Blethrow aand Eric Johannsen, High senssitivity host cell
prootein quantitatiion in an IgG1 monoclonal anntibody
preeparation via ddata-independe
ent acquisition with aTrripleTOF® Systeem, SCIEX Technical Note, PPublication
Nuumber 7460213
3-01.

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