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p 1 Rapid Level 3 Characterization of Biologics using a CESI 8000 Thermo Q-Exactive Platform Comprehensive Qualitative and Quantitative Analysis of Biopharmaceuticals Using CESI-MS Technology Bryan Fonslow and Andras Guttman SCIEX Separations, Brea, CA In the biopharmaceutical industry, comprehensive and reproducible characterization of monoclonal antibody therapeutics (mAbs) is crucial, as impurities and heterogeneity can impact safety and/or efficacy. mAbs are large glycosylated proteins (≈ 150 kDa) contain several other post-translational modifications (PTMs). Peptide mapping of mAbs can provide critical qualitative and quantitative information about impurities and heterogeneity. 1-2 Capillary electrophoresis (CE) in conjunction with mass spectrometry has exceptional separation efficiency and capabilities for peptide and glycopeptide mapping (Characterization Level 3) of mAbs. The application of an electrospray ionization (ESI) emitter integrating CE and ESI into a single dynamic process (CESI) has already proven useful for peptide and mAb analysis. 3-7 In this Technical Note, we present data for the Level 3 Characterization of a leading, representative monoclonal antibody, Trastuzumab (Herceptin). A Beckman Coulter CESI 8000 system, sold through SCIEX Separations, a part of AB SCIEX, coupled to a Thermo Q-Exactive mass spectrometer was used for the analyses. The results illustrate the benefits of the integration of CE and ESI in a single dynamic process coupled with high resolution mass spectrometry. Peptides, both large and small (3 65 amino acids), were identified, separated, and quantified. In particular, we identified and quantified degradative hotspots like aspargine-deamidation, methionine-oxidation, glutamic-acid-cyclization, and C-terminal lysine heterogeneity from a mere 100 fmol (15 ng) of sample. A glycosylation hotspot is highlighted by comprehensive characterization at Asn300 through separation, identification, and relative quantification of glycopeptides. Collectively, these results indicate the capabilities for CESI coupled with high resolution mass spectrometry to quickly and comprehensively characterize therapeutic mAbs using peptide and glycopeptide mapping from a small amount of sample with the use of a single protease, trypsin. Advantages of CESI 8000 for MS analysis Low flow rate. With CESI flow rates around 20 nL/min, ionization efficiency is maximized and ion suppression is dramatically reduced, allowing strong ionization of modified peptides and glycopeptides. 5,7 Since a single, open capillary tube is used for CESI, there are no difficult nanofluidic connections to make between other capillaries or pumps. Separation efficiency. The inherent separation efficiency of CE provides sharp peptide peaks for sensitive and reproducible identification as well as relative quantitation. Low sample requirements. CESI has small volume (< 250 nL) and mass (< 25 ng) requirements. Sample carry-over. Using an open tubular format with no solid support, all peptides elute from the capillary (both hydrophobic and hydrophilic), supporting comprehensive mAb sequence coverage and virtually eliminating sample losses as well as any carry-over. Comprehensiveness. Based on the 100% protein sequence coverage routinely attained with this approach, qualitative and quantitative analysis of mAb purity, stability, and glycoform heterogeneity is possible from a single CESI- MS run with the use of a single protease, trypsin. Reproducibility. Migration times vary less than 30 sec (< 1.5% RSD) over the 45 minute separation within triplicate runs. Relative abundance measurements of modified and unmodified peptides vary less than 2% among triplicate runs.

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Page 1: Rapid Level 3 Characterization of Biologics using a CESI 8000 … Biologi… · through separation, identification, and relative quantification of glycopeptides. Collectively, these

p 1

Rapid Level 3 Characterization of Biologics using a

CESI 8000 – Thermo Q-Exactive Platform

Comprehensive Qualitative and Quantitative Analysis of Biopharmaceuticals Using CESI-MS Technology Bryan Fonslow and Andras Guttman SCIEX Separations, Brea, CA

In the biopharmaceutical industry, comprehensive and

reproducible characterization of monoclonal antibody

therapeutics (mAbs) is crucial, as impurities and heterogeneity

can impact safety and/or efficacy. mAbs are large glycosylated

proteins (≈ 150 kDa) contain several other post-translational

modifications (PTMs). Peptide mapping of mAbs can provide

critical qualitative and quantitative information about impurities

and heterogeneity.1-2

Capillary electrophoresis (CE) in

conjunction with mass spectrometry has exceptional separation

efficiency and capabilities for peptide and glycopeptide mapping

(Characterization Level 3) of mAbs. The application of an

electrospray ionization (ESI) emitter integrating CE and ESI into

a single dynamic process (CESI) has already proven useful for

peptide and mAb analysis.3-7

In this Technical Note, we present data for the Level 3

Characterization of a leading, representative monoclonal

antibody, Trastuzumab (Herceptin). A Beckman Coulter CESI

8000 system, sold through SCIEX Separations, a part of AB

SCIEX, coupled to a Thermo Q-Exactive mass spectrometer was

used for the analyses. The results illustrate the benefits of the

integration of CE and ESI in a single dynamic process coupled

with high resolution mass spectrometry. Peptides, both large and

small (3 – 65 amino acids), were identified, separated, and

quantified. In particular, we identified and quantified degradative

hotspots like aspargine-deamidation, methionine-oxidation,

glutamic-acid-cyclization, and C-terminal lysine heterogeneity

from a mere 100 fmol (15 ng) of sample. A glycosylation hotspot

is highlighted by comprehensive characterization at Asn300

through separation, identification, and relative quantification of

glycopeptides. Collectively, these results indicate the capabilities

for CESI coupled with high resolution mass spectrometry to

quickly and comprehensively characterize therapeutic mAbs

using peptide and glycopeptide mapping from a small amount of

sample with the use of a single protease, trypsin.

Advantages of CESI 8000 for MS analysis

Low flow rate. With CESI flow rates around 20 nL/min,

ionization efficiency is maximized and ion suppression is

dramatically reduced, allowing strong ionization of modified

peptides and glycopeptides.5,7

Since a single, open

capillary tube is used for CESI, there are no difficult

nanofluidic connections to make between other capillaries or

pumps.

Separation efficiency. The inherent separation efficiency of

CE provides sharp peptide peaks for sensitive and

reproducible identification as well as relative quantitation.

Low sample requirements. CESI has small volume (< 250

nL) and mass (< 25 ng) requirements.

Sample carry-over. Using an open tubular format with no

solid support, all peptides elute from the capillary (both

hydrophobic and hydrophilic), supporting comprehensive

mAb sequence coverage and virtually eliminating sample

losses as well as any carry-over.

Comprehensiveness. Based on the 100% protein

sequence coverage routinely attained with this approach,

qualitative and quantitative analysis of mAb purity, stability,

and glycoform heterogeneity is possible from a single CESI-

MS run with the use of a single protease, trypsin.

Reproducibility. Migration times vary less than 30 sec (<

1.5% RSD) over the 45 minute separation within triplicate

runs. Relative abundance measurements of modified and

unmodified peptides vary less than 2% among triplicate

runs.

Page 2: Rapid Level 3 Characterization of Biologics using a CESI 8000 … Biologi… · through separation, identification, and relative quantification of glycopeptides. Collectively, these

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Orthogonality. CESI provides an orthogonal separation

mechanism to LC-MS-based methods for separation,

identification, quantification, and validation of sequence

variants as well as PTMs.

Experimental Design

Sample preparation: Trastuzumab (100 μg from 10 mg/mL

solution) was diluted and denatured using Rapigest followed by

reduction with DTT and alkylation by iodoacetamide. The sample

was then digested with trypsin at 37ºC overnight, dried, and

diluted three-fold with leading electrolyte (200 mM ammonium

acetate at pH 4), yielding a final concentration of 300 ng/μL of

the digested antibody.

CESI-MS/MS: CESI-MS/MS was performed using a Beckman

Coulter CESI 8000 system – sold through SCIEX Separations, a

part of AB SCIEX – connected to a Thermo Q-Exactive mass

spectrometer. 50 nL of the sample (equivalent to 100 fmol or 15

ng of digested antibody) in 100 mM leading electrolyte was

injected by pressure into the bare fused silica capillary column.

Transient-isotachophoresis (t-ITP) was applied to focus the

sample components prior to the electrophoretic separation. 10%

acetic acid was used as background electrolyte (BGE) and a

voltage of 20 kV was applied between the inlet vial and the CESI

sprayer. Samples were analyzed in triplicate.

Data-dependent acquisition (DDA) was performed on the mass

spectrometer consisting of a high resolution Orbitrap scan at 70K

followed by several high resolution MS/MS scans at 17.5K. The

DDA parameters were as follows: 120 msec Orbitrap MS survey

scan from 150 - 1800 m/z; 60 msec DDA scans on the top 10

most abundant ions above a threshold intensity of 3.3x104; HCD

fragmentation with 27% normalized collision energy. The total

MS cycle times were all 1 sec or less. Dynamic exclusion was

set to 15 sec to match the sharp peaks (~ 30 sec wide) inherent

to CESI.

Data Analysis: Data analysis was performed using AB SCIEX

ProteinPilot™ (Peptide and modified peptide identification),

ProteinMetrics Byonic (Glycopeptide identification), and Thermo

Xcalibur (Peptide Quantitation). Thermo .RAW files were

converted to .MGF files using ProteoWizard MS Convert.

Database searches were performed with the Trastuzumab,

common contaminants, and reversed protein sequences.

Glycopeptides were also extracted manually and the MS/MS

spectra were checked for diagnostic glycans ions.

The CESI process

Transient isotachophoresis capillary zone electrophoresis (t-ITP-

CZE) is the separation method used for CESI-MS analysis of

proteolytically-digested biologics. With CZE, peptides are

separated by their charge-to-frictional drag coefficient, also

conveyed as their charge-to-hydrodynamic volume ratio. Thus,

peptides that differ in charge and/or size, either due to sequence

or PTM differences, are regularly separated, 8 as will be

illustrated by representative peptides herein. Prior to the CZE-

based separation, approximately 10% of the inlet end of the

capillary tubing is pressure-loaded with the sample. Peptide

analytes are stacked during the t-ITP process based on their

mobilities within an electric field and mobility gradient zone. The

application of t-ITP improves sensitivity by approximately 10-fold

over normal CZE methods since 10-fold more sample can be

loaded onto the capillary without degrading the inherent high

separation efficiency of CZE.

Electroosmotic flow (EOF) is generated at the charged wall of

the capillary from the application of an electric field. Even at pH

4 from the 10% acetic acid BGE, the acidic silanol groups on the

inner capillary wall maintain a bulk negative charge, and drive

EOF towards the capillary outlet. Notably, unlike pressure-driven

flow techniques such as liquid chromatography with parabolic

flow profiles, EOF has a flat flow profile, which minimizes band

broadening, achieving exquisitely sharp peaks. EOF drives all of

the peptides as separated peaks out of the CESI tip for

electrospray ionization at a flow rate around 20 nL/min. At this

flow rate, the benefits of nanoelectrospray ionization (nESI) are

realized.

The electrospray ionization voltage is applied at the CESI tip

through a conductive liquid of 10% acetic acid. To fabricate the

CESI sprayer tip, the exterior polyimide coating has been

removed and the fused silica has been chemically etched until

small ions can pass through the capillary wall. The small ions,

and thus current, that can pass through the CESI tip facilitate

application of the ESI voltage. The ESI process then transfers

peptides from the liquid phase into the gas phase. Notably, the

only liquid that is electrosprayed with CESI is from the CE

separation (no sheath-flow condition), maximizing sensitivity at

low nanoliter per minute flow rates.3

CESI-MS analysis of peptides

Tryptic peptides from mAbs have a large distribution of sizes and

solution charge states making them quite amenable to CE

separation. Peptides from Trastuzumab are indeed visibly

separated and distributed by CESI from 25 to 38 minutes during

a CESI-MS run (Figure 1). Although not visible in the base peak

electropherogram (BPE), peptides were also identified as late as

42 min, particularly glycopeptides, which will be described in

more detail later. The triplicate BPEs are quite reproducible both

in a qualitative comparison of profiles (Figure 1A) and a

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p 3

quantitative comparison of migration times (Tables 1 & 2).

Generally, some correlations are observed between the peptide

mass-to-charge ratios (m/z’s) with mass spectrometry and their

charge-to-drag coefficients with CE (Figure 2B). However, the

distribution of peptide peaks throughout the migration time and

m/z separation spaces facilitate comprehensive identification and

quantification of peptides. A few representative extracted peptide

electrophoretic peaks illustrate the ability to simultaneously

separate peptides of both very small and very large sizes (Figure

1C). Additionally, these extracted peptides illustrate the sharp

peaks inherent to CE separation (15 – 30 sec in width) that

facilitate identification and quantification of peptides.

Figure 1: Representations of CESI-MS peptide separations. (A) Triplicate base peak electropherograms (BPEs) of trypsin-digested Trastuzumab run by CESI-MS with a Thermo Q-Exactive. (B) Peptide mass map and (C) extracted ion electropherograms (XIEs) of representative small and large peptides from single CESI-MS runs.

Achieving 100% sequence coverage

A number of factors can contribute to achieving 100% sequence

coverage of proteins in bottom-up proteomics. In addition to the

already described efficient peptide separation and electrospray

ionization with CESI-MS, a protein database search is an

essential step in the process. The Paragon algorithm within

ProteinPilotTM

allows for fast, user-friendly identification of

proteins and over 300 PTMs simultaneously,9 making it an

excellent comprehensive characterization tool for proteolytically-

digested biologics analyzed by CESI-MS. As indicated in Figure

2, the database search result yielded 100% sequence coverage

for heavy and light chains in all three replicates. The ability to

separate and recover both very small and very large, as well as

hydrophobic and hydrophilic peptides from a single trypsin

digestion mixture contributed to the reproducible 100% sequence

coverage of heavy and light chains.

130115_CEMS_Tras-20kv5psi60sec60mi_01_OK 1/15/2013 6:06:11 PM

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2nd longest peptide (25-mer)

Longest peptide

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B

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A

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p 4

Figure 2: Protein Pilot™ Software Database Search Results of CESI 8000 – Q-Exactive MS data reproducibly achieves 100% sequence coverage of Trastazumab heavy and light chains. The upper panel shows the 100% percent coverage of the identified heavy and light chains. The central panes show the two Protein Groups and representative peptide identifications that have been assigned to the heavy and light chains. The lower panes show the identified sequence coverages, which are color coded according to peptide identification confidence. Nearly 100% of the sequence coverage for the light chain is above 95% confidence (green). The lower confidence protein regions (yellow) inherent to two short peptides matches were further validated manually as both short peptides and missed cleavages on longer peptides. 100% sequence coverages were achieved for each of the CESI-MS analyses performed in triplicate.

Characterization Level 3 – Peptide Mapping

Peptide mapping of a therapeutic mAb with CESI-MS, similar to

LC-MS, involves identifying peptide sequences by tandem mass

spectrometry (MS/MS) and extracting peptide electrophoretic

peaks. The extracted ion electropherograms (XIEs) can be used

for sequence and PTM abundance comparison between different

biologics such as different mAb lots or between innovator,

biosimilar, and biobetter mAbs.

Both the BPEs and XIEs in Figures 1A and 1C illustrate the

characteristically sharp CE peaks that would be used for

comparative peptide mapping. That is, with the combined high

resolving powers of CESI in the separation dimension and high

resolution MS in the mass dimension, extracted peptide peaks

can be easily mapped between two or more samples. To best

illustrate the capabilities of peptide mapping with CESI-MS, the

identification and quantification of degradative hot spots on

peptides within Trastuzumab (Figure 3 and Table 1) are

highlighted. The common degradative PTMs that were identified

are N-terminal glutamate cyclization, methionine oxidation, C-

terminal lysine heterogeneity, asparagine deamidation,

tryptophan dioxidation, and lysine glycation. Additional less

abundant sites of the same degradative PTMs, among a few

others, were also identified by ProteinPilotTM

, but are not

described here.

The confident identification of these degradative PTMs are

illustrated by both well-matched high resolution MS/MS

fragmentation spectra in Figure 3 and very small precursor mass

errors (Table 1). For modifications with relatively large mass

shifts (i.e. oxidation, + 16 Da; dehydration, - 18 Da; glycation, +

162 Da, and C-terminal lysine loss, - 128 Da), these spectral

matches should provide unambiguous evidence for the PTMs.

For peptides containing small mass differences, such as

deamidated asparagine, which only differs in mass by one

Dalton, CESI can provide additional confidence to identifications.

The challenge of measuring a one Dalton mass shift on peptides,

even with high resolution MS, lies in the fact that isotopic

envelopes of unmodified and deamidated peptides can

overlap.10

Additionally, reverse-phase LC (RPLC) separations

often may not resolve these peptides since there is no change in

hydrophobicity from an asparagine to aspartic acid conversion;

however, there is a charge difference between Asn (neutral) and

Asp (acidic) that can be readily resolved by CE.11

Thus, the

solution-phase spatial resolution of the two peptides (Figure 3D)

created by the CE separation provides additional evidence that

the small mass differences measured by MS are from two unique

peptide species; and that the identification is not just form a

higher monoisotopic mass peak (from 13

C) within the same

isotopic envelope of the same unmodified peptide sequence.

Further, the small migration time shift (~ 30 sec) towards the

positively charged anode is expected from the change in peptide

charge from the neutral asparagine to the negatively charged

aspartic acid. That is, the more negatively charged peptide has a

stronger attraction towards the positively charged anode.

These separation advantages of capillary electrophoresis are

also observed with other PTMs that have more easily detectable

mass shifts by MS. For instance, the largest migration shift

observed for a degradative PTM was from the difference in

charge between the zwitterionic N-terminal glutamic acid

(positively charged under the acidic conditions used for CESI-

MS) to the neutral pyroglutamate (Figure 3A). The Glu to

pyroGlu migration shift was towards the anode, just as with

deamidation. Similarly, C-terminal lysine heterogeneity from the

loss of a positively charged lysine on the heavy chain can also

be confirmed by a large migration shift towards the anode

Table 1: Identification and relative quantification of Trastuzumab degradative post-translational modification hot spots

Modification Modified peptide sequence and associated mass shift

Monoisotopic mass [M+H]

+

Mass accuracy (ppm)

Average migration time (min)

1

Average relative abundance (%)

1,2

Methionine Oxidation K.DTLM(+15.9949)ISR.T 851.4291 0.33 37.95 ± 0.52 2.39 ± 0.98

Pyroglutamate formation -.E(-18.0106)VQLVESGGGLVQPGGSLR.L 1863.9923 0.95 31.41 ± 0.37 1.60 ± 0.19

Asparagine deamidation R.IYPTN(+0.9840)GYTR.Y 1085.5262 0.06 31.96 ± 0.37 15.57 ± 2.92

Lysine glycation K.VSNK(+162.0528)ALPAPIEK.T 1428.7944 -0.85 30.28 ± 0.33 0.28 ± 0.06*

Tryptophan dioxidation K.FNW(+31.9898)YVDGVEVHNAK.T 1709.7818 -1.62 30.32 ± 0.27 0.26 ± 0.03

C-terminal lysine loss K.SLSSPGK(-128.0950).- 660.3563 -0.80 34.39 ± 0.42 99.12 ± 0.10**

1Error was calculated as standard deviations from triplicate technical CESI-MS replicate runs.

2Relative abundances were calculated using peak areas of modified and unmodified peptides of the same sequence and charge state unless otherwise noted.

*The peak area of unmodified peptides K.APLPAPIEK.A was used for the relative abundance calculation. **The +1 charge state of the modified peptide and the +2 charge state of the unmodified peptide were used for the relative abundance calculation.

Page 5: Rapid Level 3 Characterization of Biologics using a CESI 8000 … Biologi… · through separation, identification, and relative quantification of glycopeptides. Collectively, these

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(Figure 3C). Importantly, all modified peptides identified had

very good migration time reproducibility, with standard deviations

less than 30 seconds. Thus, these migration time variations are

compatible with peptide mapping experiments. Of course these

separation capabilities are also applicable to PTM relative

quantification. Similar to the higher identification confidence of

deamidated peptides by CESI separation, quantification of

deamidated peptides is also improved. Quantification of peptides

relies on extracting ion masses over time. Since the isotopic

envelopes no longer overlap between unmodified and

deamidated peptides when spatially separated by CESI, their

XIEs also do not overlap in time.

Figure 3: Peptide mapping of Trastuzumab degradative PTM hotspots. MS/MS spectra identification and XIEs of modified and unmodified peptides for (A) N-terminal glutamate cyclization (pyroGlu), (B) methionine oxidation (MetOx), (C) C-terminal lysine heterogeneity, (D) asparagine deamidation, (E) tryptophan dioxidation, and (F) lysine glycation. XIE scales were adjusted to show the quantification of the lower abundance peptide electrophoretic peak. The amino acid site and type of PTM are shown in each panel for both modified and unmodified peptides.

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(%

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Time (min)

N-terminal GluN-terminal PyroGlu

Met255

Asn55

Asn55 deamidation

Trp280

Trp280

dioxidation

Lys329 glycation

Lys329

A

B

C

D

E

F

MetOx255

C-terminal Lys450

present

C-terminal Lys450

loss

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This is observed as two well-resolved XIE peaks for the

deamidated and unmodified peptides (Figure 3D). However,

even if peptides remain unresolved by CE, there are still benefits

for relative quantification over LC-based techniques. CESI is a

low flow rate method that essentially eliminates ion suppression.

Thus, unresolved modified and unmodified peptide peak areas

better represent their true relative abundances. Of the

degradative PTMs identified on Trastuzumab, this characteristic

is applicable to relative quantification of methionine, tryptophan

oxidation, and lysine glycation.

The relative abundances of the degradative PTMs are shown in

Table 1. Notably, relative abundances generally varied less than

0.2% among the triplicate CESI-MS runs. However, for

modifications that can also occur during sample preparation

(methionine oxidation and asparagine deamidation), their relative

abundances varied less than 3% among triplicate runs. Relative

quantification of these degradative PTMs was straightforward.

Quite simply, the peak area of the modified peptide was divided

by the sum of peak area of itself and the unmodified peptide.

The relative quantification of lysine glycation required a bit more

consideration. Trypsin is unable to cleave at the glycated version

of the lysine, but otherwise cleaves the non-glycated lysine as

expected. Thus, different peptide sequences were used to

perform relative quantification. The most abundant version of the

same portion of the glycated lysine peptide sequence was used

for the relative abundance calculation. However, lysine glycation

characterization would be more straightforward by peptide

mapping since differences in trypsin cleavage would not need to

be considered.12

Characterization Level 3 – Glycopeptide Mapping

Therapeutic mAb glycoforms are typically characterized at

Levels 1 and 2 through intact and reduced molecular weight MS

measurements, respectively, and at Level 4 as cleaved glycans

with LC or CE separations.13-14

N-glycosylation is usually

localized on a consensus asparagine residue in the IgG1 Fc

domain, but can also be localized in variable regions, non-

consensus regions, or even on glutamine.15-16

Thus, site specific

localization of glycans is becoming an essential mAb

characterization step. Characterization levels 1, 2, and 4 are not

well-suited for confirmation of N-glycosylation localization. Even

glycopeptides identified by LC-MS by Level 3 characterization

often remain unseparated by RPLC, leaving the potential for

misassignment of glycan structures and variability in relative

abundance measurements. With CESI-MS, comprehensive

glycoform analysis is achieved at characterization Level 3

through efficient separation and electrospray ionization of

glycopeptides.

Examples of the glycopeptide separations are shown in Figure

4A and B and their average migration times are listed in Table 2.

Migration times of glycopeptides were also reproducible, with the

majority having around 30 seconds of variability within triplicate

CESI-MS runs. As mentioned earlier, the CE separation is based

on the charge-to-frictional drag ratio of peptides. One of the keys

to glycopeptide separation and characterization with CESI-MS is

that each sugar unit adds enough addition frictional drag to the

corresponding glycoforms to be separated by CESI.

Glycopeptides with high mannose: M5 (also referred to as

Man5), afucosylated biantennary complex: A2, A2G1 and A2G2

(also referred to as G0, G1, G2), fucosylated biantennary

complex: FA2, FA2G1 and FA2G2 (also referred to as G0F,

G1F, G2F), and similar uncharged glycans are separated by this

mechanism. For the fucosylated (Figure 4A) and afucosylated

(Figure 4B) series, the separations are illustrated as resolved

peaks. Additionally, although not obvious from Figure 4, the

migration times between afucosylated and fucosylated versions

of the same glycan structures also have different migration

times. For instance, A2 and FA2 (also referred to as G0 and

G0F) had average migration times of 35.00 and 34.72 min (Table

2), respectively. In the instances of the A1, FA1, and FA2

glycoforms (also referred to as G0-GlcNAc, G0F-GlcNAc and

G0F), addition of 5-N-acetyl--neuraminic acid (Neu5Ac)

induces a larger migration shift due to changes in the

glycopeptide charge from the addition of the acidic Neu5Ac

residue. This mechanism is quite similar to the migration time

shifts observed with the degradative PTMs when the modified

peptide is more acidic than the unmodified peptide (Figures 3A,

C, & D).

As with other modified peptides, the XIEs of glycopeptides are

dependent on their identification through the combination of their

high mass accuracy precursor and fragment ion measurements.

The glycopeptides identified also have very high mass

accuracies (Table 2), all well below ± 5 ppm error. To illustrate

the quality of MS/MS spectra that were used to identify the

glycopeptides, the spectrum of the lowest abundance glycoform

identified as A2G2S1 and FA2G1 as the highest as shown in

Figure 4B. As with the majority of identified glycopeptide spectra,

a few peptide sequence-dependent b- and y-ions are matched,

but the spectrum matches mostly originate from glycan

signatures: an intact glycopeptide mass measurement, multiple

sugar unit losses from the glycopeptide, and multiple glycan

fragment ions. The fact that even the lowest abundance

glycopeptide has a high intensity, high quality matching spectrum

indicates the qualities of the other higher intensity spectra are

also high. The relative quantification values for the identified

Trastuzumab glycans are shown in Table 2. Most notably, the

A2G2S1 glycan was quantified at 0.06% relative abundance.

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Thus, between the most abundant FA2G1 glycoform at 54.49%

and the A2G2S1 glycoform, that represents three orders of

magnitude quantification of mAb glycosylation. Within this 1000-

fold dynamic range, 20 glycoforms were identified and quantified

as glycopeptides. Additionally, the majority of glycopeptide

relative abundance measurements were reproducible, with CVs

less than 10%. Thus, the combination of CESI and MS allow for

both separation and high confidence, high sensitivity

identification of glycopeptides, facilitating the confident

identification and quantification of mAb glycosylation.

Conclusions

We have presented a new platform combining CESI, a robust

and highly efficient separation and nanoelectrospray technique

connected on-line with a high accuracy and resolution mass

spectrometer for qualitative and quantitative analysis of

monoclonal antibody therapeutics. With this new CESI design,

peptides were separated and electrosprayed directly into the

mass spectrometer by making the terminal end of the separation

capillary a sprayer. The droplets formed during the

nanoelectrospray process in the CESI design are small and

result in highly efficient ionization. With flow rates around 20

nL/min, ion suppression was minimized, providing higher

sensitivity to analyze less abundant glycopeptides present in the

same sample. The sensitivity and dynamic range of the

approach for glycopeptides was illustrated by the relative

quantification of as low as 0.06% and three orders of magnitude

range of glycoform identification and quantification of 20

glycopeptides, respectively. Also notable, CESI-MS revealed

degradative PTM hotspots in the mAb down to 0.3% relative

abundance including methionine oxidation, asparagine

deamidation, cyclization of N-terminal glutamate to

pyroGlutamate, heavy chain C-terminal lysine heterogeneity,

tryptophan oxidation, and lysine glycation. The CESI-MS

analysis of Trastuzumab presented herein represents an

attractive workflow when rapid, comprehensive, reproducible

verification of a biologic’s primary sequence and glycosylation

profile are required, particularly when mass-limited.

Acknowledgments

We thank Dr. Alain Beck at Pierre Fabre, France for providing Trastazumab; Jean-Marc Busnel of Beckman Coulter, Anna Lou of SCIEX separations, and Zhiqi Hao and David Horn of Thermo Fisher Scientific for generation of the data; Chris Becker and Eric Carlson of ProteinMetrics for access to Byonic; and all for comments on the Technical Note.

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Figure 4: XIEs of glycopeptides identified by CESI-MS. From glycopeptide identifications by MS/MS, peaks were extracted for standard (A) fucosylated and (B) afucosylated mAb glycoforms for relative quantification. A representative glycopeptide MS/MS spectrum is shown for the lowest abundance A2G2S1 glycoform. Ions annotated in green represent glycan fragmentation. Further glycopeptide characterization is shown in Table 2. Glycan structure interpretation followed the CFG protocol.

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Table 2: Characterization of Trastuzumab heavy chain glycosylation hot spot N300 through identification and relative quantification. Glycan structure interpretation followed the CFG protocol.

Glycopeptides identified as R.EEQYN(Glycan)STYR.V

mAb glycan abbreviation

Glycan mass (Da)

Monoisotopic mass [M+H]

+ (Da)

Mass accuracy (ppm)

Average migration time (min)

1

Average relative abundance (%)

1,2

FA2G1 (G1F) 1606.5867 2796.0987 -0.41 34.92 ± 0.43 54.49 ± 1.45

FA2 (G0F) 1444.5339 2634.0459 0.25 34.72 ± 0.42 17.28 ± 0.97

FA2G2 (G2F) 1768.6395 2958.1515 -0.01 35.19 ± 0.43 8.59 ± 0.40

A2 (G0) 1298.476 2487.9880 0.67 35.00 ± 041 5.58 ± 0.31

A2G1 (G1) 1460.5288 2650.0408 0.93 34.88 ± 0.43 4.07 ± 0.09

FA1G1 1403.5073 2591.0037* 3.04 34.45 ± 0.41 1.52 ± 0.06

FA2G2S1 2059.7349 3249.2469 0.78 34.45 ± 0.42 1.41 ± 0.11

FA1 1241.4545 2430.9665 0.40 34.76 ± 0.44 1.25 ± 0.06

M5 (Man5) 1216.4228 2405.9349 0.09 38.18 ± 0.53 1.13 ± 0.66

FA2G1S1 1897.6821 3085.1785* 3.10 37.93 ± 0.52 1.02 ± 0.48

A1G1 1257.4494 2446.9614 0.44 38.03 ± 0.48 0.78 ± 0.40

FA1G1S1 1694.6027 2884.1147 -3.15 34.39 ± 0.42 0.57 ± 0.03

A2G2 (G2) 1622.5816 2812.0936 -2.77 34.71 ± 0.39 0.55 ± 0.03

A1 1095.3966 2284.9086 0.57 41.57 ± 0.64 0.51 ± 0.32

FA2G2S2 2350.8303 3540.3423 1.77 35.11 ± 0.44 0.44 ± 0.05

A1G1S1 1548.5448 2738.0568 -2.81 37.90 ± 0.51 0.32 ± 0.15

FA2BG1 1809.6661 2999.1781 -3.00 35.09 ± 0.44 0.17 ± 0.01

M4A1G1S1 1710.5976 2900.1096 -1.62 34.83 ± 0.43 0.14 ± 0.01

M5A1 1419.5022 2609.0142 -1.92 37.95 ± 0.52 0.14 ±.0.07

A2G2S1 1913.677 3103.1890 -1.74 38.05 ± 0.57 0.06 ± 0.03

1Error was calculated as standard deviations from triplicate technical CESI-MS replicate runs.

2Relative abundances were calculated using peak areas of glycosylated peptides of the same sequence and charge state (+3).

*[M+H]+ mass has a -18.0106 Da loss due to water neutral loss.

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

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Publication number: 10400614-01