characterizing monoclonal antibodies and antibody–drug...

Monoclonal antibodies (mAbs) have

emerged as important therapeutics

for the treatment of life-threatening

diseases, including cancer and

autoimmune diseases (1,2,3). Today,

more than 40 mAbs are marketed

in the United States and Europe, of

which 18 display blockbuster status;

six have sales greater than $6 billion

(Humira, Remicade, Enbrel, Rituxan,

Avastin, and Herceptin) and over 50

are in late-stage clinical development.

mAbs are currently considered

to be the fastest growing class of

therapeutics with sales more than

doubled since 2008 (1,2,3).

The successes of mAbs have

triggered the development of various

next generation formats, such as

antibody–drug conjugates (ADC),

bispecific mAbs, mAb mixtures,

polyclonal antibodies, antibody

fragments (Fab, Fc, nanobodies),

Fc fusion proteins, brain penetrant

mAbs, and glyco-engineered

mAbs. In oncology, ADCs are

particularly promising because they

synergistically combine a specific

mAb linked to a biologically active

cytotoxic drug using a stable linker

(4–6). The promise of ADCs is that

highly toxic drugs can be selectively

delivered to tumour cells thereby

substantially lowering side effects

as typically experienced with

classical chemotherapy. Two ADCs

are currently marketed, namely

brentuximab vedotin (Adcetris)

and ado-trastuzumab emtansine

(Kadcyla), and over 30 are in clinical

trials (4–6).

With the top-selling mAbs evolving

out of patent there has been a

growing interest in the development of

biosimilars (7). In 2013, we witnessed

the European approval of the first

two mAb biosimilars (Remsima and

Inflectra), which both contain the

same active substance, infliximab (8).

In April 2016, Inflectra also reached

marketing authorization in the US and

a third infliximab biosimilar (Flixabi)

was recently approved in Europe.

Remicade, infliximab’s blockbuster

originator, reached global sales of

$8.9 billion in 2013 (3).

Structural ComplexityTogether with a huge therapeutic

potential, these products come with

a structural complexity that drives

state-of-the-art chromatography and

mass spectrometry to its limits and

this is exactly where two-dimensional

liquid chromatography (2D-LC)

comes into play. mAbs are tetrameric

immunoglobulin G (IgG) molecules

with a molecular weight (MW) of

150 kDa (± 1300 amino acids)

composed of two light (Lc – 25 kDa)

and two heavy (Hc – 50 kDa)

polypeptide chains connected

through interchain disulphide

bridges (Figure 1). Twelve intrachain

disulphide bridges, four within

each Hc and two within each Lc,

furthermore guarantee its structural

integrity. The structure can also

be divided in the antigen binding

fragment (Fab) and the crystallizable

fragment (Fc). Antigen binding is

mediated by the Fab fragment,

while the Fc fragment is responsible

for the effector function, that is,

antibody dependent cell-mediated

cytotoxicity (ADCC) and complement

dependent cytotoxicity (CDC). All

mAbs are glycoproteins and have two

conserved N -glycosylation sites in

the Fc region that can be occupied

with complex and high mannose type

N -glycans.

These glycan structures are known

to play a role, amongst others, in the

effector function and are the subject

of extensive research towards the

development of glyco-engineered

mAbs with improved effector

functions. A variety of other chemical

and enzymatic modifications

(wanted and unwanted) taking place

during expression, purification, and

long-term storage further shape the

mAb and give rise to a substantial

heterogeneity. Modifications and

Characterizing Monoclonal Antibodies and Antibody–Drug Conjugates Using 2D-LC–MSKoen Sandra, Isabel Vandenheede, Mieke Steenbeke, Gerd Vanhoenacker, and Pat Sandra, Research Institute for

Chromatography, Kortrijk, Belgium

Two-dimensional liquid chromatography (2D-LC) has in recent years seen an enormous evolution, and with the introduction of commercial instrumentation, the technique is no longer considered a specialist tool. One of the fi elds where 2D-LC is being widely adopted is in the analysis of biopharmaceuticals, including monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs). These molecules come with a structural complexity that drives state-of-the-art chromatography and mass spectrometry (MS) to its limits. Using practical examples from the authors’ laboratory complemented with background literature, the possibilities of on-line 2D-LC for the characterization of mAbs and ADCs are presented and discussed.

149www.chromatographyonline.com

BIOPHARMACEUTICAL PERSPECTIVES

variants often encountered include

(but are not limited to) asparagine

deamidation, aspartate isomerization,

succinimide formation, N -terminal

pyroglutamate formation, C-terminal

lysine truncation, methionine or

tryptophan oxidation, glycation,

cysteine variants, and sequence

variants (9). Even though only a single

molecule is cloned, thousands of

possible variant combinations may

exist for one given mAb and they all

contribute to the safety and efficacy

of the product.

Compared to naked mAbs,

ADCs further add to the complexity

because the heterogeneity of the

initial antibody is superimposed with

the variability associated with the

conjugation strategy. Conjugation

typically takes place on the amino

groups of lysine residues or on the

sulphydryl groups of interchain

cysteine residues as is the case

in, respectively, ado-trastuzumab

emtansine and brentuximab vedotin

(b)(a)

1D-Column 1D-Column

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2D-Column

2D-Pump

2D-Pump

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OUT

Fill-direction

Fill-direction

Analyze-direction

Analyze-direction

Loop

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Figure 2: (a) Modern modulators for comprehensive LC×LC and (b) for multiple heart-cutting LC (mLC–LC).

(a)

(b) (c)

FabF(ab’)2

N-ter

C-ter

DM1 Thioether linker

Trastuzumab Brentuximab

Proteasecleavable linker MMAE

Light chain (Lc)

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C-ter

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Hc

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Figure 1: Schematic representation of mAb and ADC. (a) Anatomy of humanized IgG1 trastuzumab (Herceptin). Primary sequence of the light and heavy chain and schematic representation of a functional monoclonal antibody. Subunits that can be formed following chemical or enzymatic treatment are shown: light chain (Lc), heavy chain (Hc), crystallizable fragment (Fc), fragment antigen binding (Fab), Fc/2, and F(ab’)2. Asparagine at position 300 in the heavy chain is occupied with bi-antennary N-glycans. (b) Anatomy of the ADC ado-trastuzumab emtansine (Kadcyla), which combines the trastuzumab (a) with the cytotoxic microtubule-inhibiting maytansine derivative DM1 conjugated to lysine residues via a non-reducible thioether linker. DM1 is conjugated with an average of 3.5 drugs per antibody. (c) Anatomy of the ADC brentuximab-vedotin (Adcetris), which combines the antibody brentuximab with the antimitotic drug monomethylauristatin E (MMAE) conjugated to interchain cysteine residues via a cathepsin cleavable valine-citrulline linker. MMAE is conjugated with an average of 4 drugs per antibody.

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BIOPHARMACEUTICAL 1&341&$5*7&4

(Figure 1). With 80–100 lysine and only eight interchain

cysteine residues available, lysine conjugation yields a

more heterogeneous mixture of species compared to

cysteine conjugation.

On top of the described primary structure, mAbs and

ADCs also have distinct higher order structures dictating

their function, which might be influenced by the above

described modifications, and can appear as dimers or

aggregates, which have the potential to induce immune

responses.

Two-Dimensional Liquid ChromatographyAn emerging tool to tackle this complexity is 2D-LC

(10–13). In 2D-LC, two different chromatographic

separation mechanisms are combined and peaks eluting

from the first column are further separated on a second

column with an orthogonal separation mechanism. The

aim is to substantially increase the separation power

for complex samples or to make the first-dimension

separation, in case it makes use of non-volatile salts,

compatible with mass spectrometry (MS). Orthogonal

behaviour is typically governed when combining the

following chromatographic modes: affinity chromatography

(Protein A), anion or cation exchange chromatography

(AEX, CEX), hydrophilic interaction liquid chromatography

(HILIC), hydrophobic interaction chromatography

(HIC), reversed-phase liquid chromatography (LC), and

size-exclusion chromatography (SEC).

Peaks can be transferred from one dimension to

the other dimension in either an on-line or an off-line

approach. Historically, off-line transfer has been the

method of choice and is still widely applied. With robust,

on-line 2D-LC instrumentation made commercially

available in recent years, however, on-line 2D-LC should

no longer be considered a specialist tool and is finding

its way to mainstream laboratories. This article focuses on

on-line 2D-LC.

In on-line 2D-LC one makes use of a first-dimension

pump, injector, column, and, optionally, a first-dimension

detector, which is combined with a second pump, a

second injector—now termed modulator, a second

column, and a detector. The aim of the modulator is to

efficiently transfer peaks from one dimension to the other

without compromising the separation (Figure 2). In cases

where the modulator transfers all first-dimension peaks to

the second dimension, this is known as comprehensive

2D-LC or LC×LC. The modulator is composed of a valve

with two loops installed (Figure 2[a]). While one loop

is being filled with peaks from the first dimension, the

second loop is being analyzed on the second dimension

column. This second-dimension separation is typically

very fast (in the order of 30 s) to facilitate a high sampling

frequency and maintain the first-dimension separation. In

cases where the modulator is programmed to transfer one

or a couple of peaks from the first dimension to the second

dimension, the method is termed (multiple) heart-cutting

2D-LC or (m)LC–LC. A modulator that allows one to store

up to 12 first dimension fractions is pictured in Figure 2(b).

Loops are then analyzed on the second dimension one

by one and the first and second dimension run times are

decoupled, which means that there is no requirement

to perform fast separations. The following section will

highlight how both modes are being applied in the analysis

of mAbs and ADCs.

(Multiple) Heart-Cutting LC–LC(m)LC–LC is typically applied for the analysis of intact

mAbs or large fragments thereof (for example, Lc, Hc, Fab,

Fc, F[ab’]2, Fc/2, Fd), which can be generated following

chemical reduction or enzymatic digestion, with the aim of

increasing resolution (for mAbs and ADCs, peaks typically

contain many variants) and allowing an in-depth product

characterization and to make separations compatible with

MS (11,12).

Birdsall et al. showed the potential of an on-line

HIC–reversed-phase LC–UV–MS setup for the

characterization of interchain cysteine conjugated ADCs

(14). HIC separates the ADC based on the number of

drugs conjugated for each antibody and as such allows

the drug-to-antibody ratio (DAR) and drug distribution

to be determined. HIC maintains mAbs in their native

state, so it is especially well suited for interchain cysteine

conjugated ADCs. The inherent problem of HIC is its

incompatibility with MS detection as a result of the use

of non-volatile salts. However, applying heart-cutting

2D-LC facilitates the combination of HIC and MS using

a reversed-phase LC desalting step prior to MS. The

denaturing reversed-phase conditions give rise to

dissociation of the ADC into the respective subunits and

as such this heart-cutting setup provides unambiguous

identification of positional isomers. The downside of the

described approach is that the setup only allows the


BIOPHARMACEUTICAL PERSPECTIVES

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second dimension reversed-phase

LC–MS analysis of one heart-cut,

even though the HIC chromatogram

is heavily populated with peaks.

Characterizing all peaks, therefore,

requires several re-injections.

This issue is alleviated in multiple

heart-cutting LC–LC where the

modulator design allows the storage

of multiple first dimension fractions.

Figures 3 and 4 show the mLC–LC

analysis of cetuximab (Erbitux).

Compared to other mAbs, which

have one N -glycosylation site in

the conserved region of the Hc,

cetuximab contains an additional

glycosylation site in the variable

region of the heavy chain (15).

Both the conserved and second

N -glycosylation site are occupied

with different types of sugars. While

the former contains the typical

mAb bi-antennary complex glycans

G0F, G1F, and G2F, the latter site is

occupied with a diverse mixture of

glycans containing a large amount

of α-(1J3) linked galactose residues

next to N -glycolylneuraminic (NGNA)

acid terminating glycans. This unique

glycosylation pattern stems from

the fact that these antibodies are

produced in murine SP2/0 opposed

to CHO cell lines. In this mLC–LC

example (Figures 3 and 4), IdeZ

digested cetuximab was analyzed

on the orthogonal combination

CEX–reversed-phase LC hyphenated

to UV and QTOF-MS detection.

An immunoglobulin-degrading

enzyme from Streptococcus equi

ssp zooepidemicus (IdeZ) cleaves

mAbs underneath the hinge region

thereby generating a F(ab’)2

and Fc/2 fragment and as such

separates out the two glycosylation

sites. Six CEX heart-cuts were

transferred to reversed-phase LC–

MS. Chromatographic conditions

are described in Sandra et al. (16).

CEX peaks 1, 2, and 3 correspond

to the 100 kDa F(ab’)2 fragment and

from the corresponding MS data it is

shown that these peaks differ in their

NGNA degree with peak 1 carrying

NGNA terminating N -glycans on

both F(ab’)2 arms, peak 2 carrying

one NGNA terminating glycan on

one F(ab’)2 arm and a neutral glycan

on the other F(ab’)2 arm, and peak

3 carrying neutral glycans on both

arms. N -glycolylneuraminic acid

renders the protein more acidic,

which explains the earlier elution on

CEX. Peaks 4, 5, and 6 correspond

to the Fc/2 fragments and differ in

C-terminal lysine (K) truncation with

peak 6 carrying two lysine residues,

peak 5 one, and peak 4 being fully

truncated. Lysine is a basic amino

acid explaining the increased

retention time on CEX. Both Fc/2

fragments, which are no longer

connected via interchain disulphide

bridges, remain associated

under the native CEX conditions.

Denaturation, however, occurs during

reversed-phase LC–MS analysis,

which explains the detection of

25 kDa fragments using MS (Figure 4)

and the double peaks (5a: Fc/2 + K

and 5b: Fc/2 without K) observed

on reversed-phase LC (heart-cut 5,

Figure 3[b]).

An identical CEX–reversed-phase

LC(–UV)–MS setup has recently been

used to identify the main isoforms

of the mAb rituximab (17) and to

characterize the antibody–drug

conjugate (ADC) ado-trastuzumab

emtansine (16). Instead of storing

first dimension fractions in loops,

Alvarez et al. designed a setup that

allowed six peaks of interest from

a CEX or SEC separation to be

analyzed and identified by trapping

and desalting the fractions onto a

series of reversed-phase cartridges

with subsequent MS analysis (18).

An on-line disulphide reduction step

was furthermore incorporated into

the workflow, allowing more detailed

characterization of mAbs.

The examples described in this

section offer a small taste of what

2.0 3.0 4.0

0

100

200

300

400

500

2

3

15b

5a

64

15 17.5 20 22.5 25 27.5

Time (min)

(a)

(b)

Fc/2

F(ab’)2

Time (min)

mA

Um

AU

30 32.5 35 37.5

0

10

20

30

40

50

60

70

1

2

3

4

65

Figure 3: mLC–LC data of IdeZ treated cetuximab. CEX–UV (214 nm) chromatograms with (a) the heart-cuts and (b) the representative reversed-phase LC–UV (214 nm) chromatograms. Deconvoluted QTOF-MS spectra associated with the annotated peaks are shown in Figure 4. Fractions were transferred from one dimension to the other using the modulator shown in Figure 2(b). Experimental conditions can be obtained from reference 16.



can be achieved using (m)LC–LC.

In various vendor application notes

and non-peer-reviewed journals,

more setups and applications can

be found (19,20,21); for example,

implementing Protein A followed by

SEC, CEX, or reversed-phase LC–MS

with the aim of selecting the optimal

mAb-producing clones based on

various critical attributes, such as

titer, charge variants, aggregation,

molecular weight, and high level

modifications (glycosylation).

LC–LC has not only been applied

to characterize the drug substance

(mAb or ADC). In a recent study

by Li et al., SEC was coupled to

reversed-phase LC in a heart-cutting

2D-LC setup for identification and

quantification of unconjugated small

molecule drugs and related small

molecule impurities in ADC samples

(22). These free drug species can

increase the risk to patients and

reduce the efficacy of the ADC. The

SEC column in the first dimension

provided information on the size

variants (dimers, aggregates) of

the ADC and separated the small

molecular species from the ADC,

which were then analyzed by

reversed-phase LC. Both small

molecule degradation products

and aggregation of the ADC were

observed in stability samples. The

authors subsequently used the same

setup to investigate the low recovery

of the spiked linker drug in the free

drug assay for an ADC (23). It could

be concluded that the spiked linker

drug reacts with residual reactive

groups on the ADC. Birdsall et al.

described an alternative approach

based on mixed-mode solid-phase

extraction (SPE) combined with

reversed-phase LC and MS for the

measurement of free drug species.

The SPE step removes the ADC while

it captures the free drug species for

transfer to the second dimension. The

authors claim that the method is two

orders of magnitude more sensitive

than UV-based methods (24).

LC–LC combining SEC with

mixed-mode chromatography has

been used to quantitate mAbs and

excipients such as sucrose, Na+,

K+, histidine, Cl-, succinate, and

polysorbate 80 in drug products

including mAbs, ADCs, and vaccines

(25). Proteins were directed to a

UV detector while excipients were

monitored by evaporative light

scattering detection (ELSD). Heart

cutting 2D-LC with charged aerosol

detection (CAD) and MS detection

was also implemented to study the

molecular heterogeneity and stability

of polysorbate 20 in the presence of a

mAb formulation sample matrix (26).

A mixed-mode column was used in

the first dimension to separate the

polysorbate esters from the protein

in the formulation sample, and the

esters were further separated by a

reversed-phase LC method in the

second dimension to quantify the

multiple ester subspecies. A second

2D-LC method using CEX in the first

dimension and reversed-phase LC in

the second dimension was used for

the analysis of degradation products

of polysorbate 20.

Comprehensive LC×LCIn biopharmaceutical analysis,

comprehensive LC×LC has

predominantly been used in peptide

mapping studies for the detailed

characterization and comparability

assessment of innovator and

biosimilar mAbs and to assess drug

conjugation sites in ADCs (27–29).

The use of the technology beyond

R&D in a routine environment (QA or

QC) has also been suggested (27).

Trypsin digestion of a monoclonal

antibody will easily generate

over 100 peptides with varying

physicochemical properties

all present in a wide dynamic

concentration range. The complexity

associated with these digests

demands the best in terms of

separation power. Compared

to one-dimensional separations

(1D-LC), LC×LC will drastically

increase peak capacity if the two

dimensions are orthogonal and

the separation obtained in the first

dimension is maintained upon

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N-acetvlglucosamine N-glycolylneuraminic acidMannose Galactose Fucose

100500 101000 101500 102000 102500 103000

Heart-cut#1RPLC peak 1




Heart-cut#5RPLC peak 5a

Heart-cut#5RPLC peak 5b


0

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24600 25000 25400 25800 26200

+K

+K

G0F

7:9

7;9

Figure 4: Deconvoluted QTOF spectra associated with seven reversed-phase LC peaks observed in six CEX heart-cuts shown in Figure 3.



transfer to the second dimension.

Orthogonal combinations for

LC×LC-based peptide mapping

are strong cation exchange (SCX)

× reversed-phase LC, hydrophilic

interaction liquid chromatography

(HILIC) × reversed-phase LC, and

reversed-phase LC × reversed-phase

LC at different pHs in the two

dimensions (27). Note that in the

above combinations, reversed-phase

LC is always used in the second

dimension. The reasons for that

are the commercial availability of

high-quality, “fast” reversed-phase

LC second dimension columns and

their compatibility with MS. The

highest orthogonality is obtained

with SCX × reversed phase LC

and HILIC × reversed phase LC

because the separation mechanisms

are completely different. However,

reversed-phase LC × reversed-phase

LC is the preferred approach

because (i) it offers the highest

ruggedness from the excellent solvent

compatibility in both dimensions,

(ii) the peak capacity is very high as

a result of the high plate numbers

in the individual dimensions, and

(iii) orthogonality is acceptable as

a result of the zwitterionic nature of

peptides resulting in major selectivity

differences when performing

separations using reversed-phase LC

at pH extremes (pH 2 versus pH 10).

Vanhoenacker et al. recently

described the potential

of reversed-phase LC ×

reversed-phase LC combined with

UV and MS detection for the detailed


assessment of a trastuzumab innovator

(Herceptin) and candidate biosimilar

(27). A wealth of information was

provided in the peptide map allowing

identity, purity, and comparability to

be assessed. Striking similarities were

noticed when comparing the peptide

maps, but when looking in more detail

differences could be found and these

were shown to reside in modifications

(C-terminal lysine truncation,

asparagine deamidation).

To further illustrate the

attractiveness of LC×LC for detailed


assessment of mAbs, Figure 5

compares the reversed-phase LC

× reversed-phase LC tryptic peptide

maps of an infliximab originator

and candidate biosimilar for which

apparently, the recombinant

expression in Chinese Hamster

Ovary (CHO) cells was going

wrong. Both plots are very similar

but some striking differences are

noted when the MS data is taken into

consideration. The spots SLSLSPG

and SLSLSPGK clearly present in

the originator are replaced by one

spot SLSLSPGI in the biosimilar,

which according to the mass spectral

data corresponds to the addition

of an isoleucine (I) to SLSLSPG

or the replacement of lysine (K)

by isoleucine in SLSLSPGK at the

C -termini of the heavy chain.

The origin of the two spots,

SLSLSPG and SLSLSPGK, in the

originator mAb can be explained

by the knowledge that heavy

chains are historically cloned with

a C -terminal lysine but during

cell culture production, host cell

carboxypeptidases act on the

antibody resulting in the partial

removal of these lysine residues.

A very small retention shift was

also noted in another spot that

could be attributed to a threonine

to serine substitution in heavy

chain peptide NYYGS(TJS)

Figure 5: Reversed-phase LC × reversed-phase LC–QTOF-MS peptide mapping of infliximab originator and candidate biosimilar and drawing of the mAb with annotation of the modifications. First and second dimension separation consist of reversed-phase LC operated at high and low pH, respectively. Fractions were transferred from one dimension to the other using the modulator shown in Figure 2(a). First dimension peaks were sampled 3–4 times. Experimental conditions can be obtained from reference 16.



YDYWGQGTTLTVSSASTK. Two

point mutations are at the origin of

this wrong recombinant expression

(Figure 5). These mutations were

confirmed with reverse transcription

polymerase chain reaction (RT-PCR)

and DNA sequencing. According

to US and European regulatory

authorities, identical primary

sequence is primordial to similarity,

thereby ruling out this candidate

biosimilar from further development.

Very recently, the possibilities of

LC×LC-based peptide mapping

in the characterization of ADCs

have been demonstrated (16). The

methodology was shown to be

particularly valuable to assess drug

conjugation sites. Figures 6(a) and

6(b) display the reversed-phase LC

× reversed-phase LC–MS tryptic

peptide maps of naked trastuzumab

(Herceptin) and ado-trastuzumab

emtansine (Kadcyla). The latter

combines trastuzumab with the

cytotoxic microtubule-inhibiting

maytansine derivative DM1

conjugated to lysine residues via

a non-reducible thioether linker.

Since Herceptin and Kadcyla have

the same amino acid sequence,

most of the peptide map is

identical. Differentiating spots are,

nevertheless, observed and are in

Figure 6: Reversed-phase LC × reversed-phase LC–MS peptide maps of (a) Herceptin and (b) Kadcyla and reversed-phase LC × reversed-phase LC all ions MS/MS peptide maps of (c,d) Herceptin and (d,f) Kadcyla showing extracted ion at m/z 547.22056 (extracted at 20 ppm mass accuracy). Collision energy was, respectively, 20 (c,d) and 40 eV (e,f). The identity of the annotated spots is shown in Table 1. First and second dimension separation consist of reversed-phase LC operated at high and low pH, respectively. Fractions were transferred from one dimension to the other using the modulator shown in Figure 2(a). First dimension peaks were sampled 3–4 times. Experimental conditions can be obtained from reference 16.

(a)

(c)

(e)

(b)

(d)

(f)

Table 1: A selection of conjugated peptides corresponding to the annotated spots in

the LC×LC peptide map shown in Figures 6(d) and (f). The conjugated lysine residues

are marked.

ChainSequence Location

Sequence

1 Lc 184–190 ADYEKHK

2 Hc 292–295 TKPR

3 Hc 413–419 LTVDKSR

4 Hc 222–251 SCDKTHTCPPCPAPELLGGPSVFLFPPKPK(1)

5 Hc 20–38 LSCAASGFNIKDTYIHWVR

6 Hc 305–325 VVSVLTVLHQDWLNGKEYKCK(1)

7 Hc 259–291 TPRVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK

(1) Two potential conjugation sites.



the upper right corner of the plot of

Kadcyla, which corresponds to the

most hydrophobic part. Since the

conjugation of the drugs makes the

peptides more hydrophobic, these

spots are expected to correspond

to the conjugated peptides. The

fragmentation spectra associated

with DM1 conjugated peptides are

heavily populated by fragments

originating from the drug, for

example, at m/z 547.2206. This

ion can be used to recognize

conjugated peptides in LC×LC–

MS maps. One can operate the

QTOF-MS system in the all ions MS/

MS mode, which means that the

quadrupole is operated in the RF

only mode thereby transferring all

peptides to the collision cell where

collision-induced dissociation takes

place.

As illustrated in Figures 6(c–f),

when extracting from the data

the DM1 specific fragment ion at

m/z 547.2206 at high mass accuracy,

the upper right corner of the 2D

peptide map lights up for Kadcyla

in comparison to Herceptin, making

this LC×LC methodology extremely

powerful to highlight conjugated

peptides. The latter plot is virtually

empty illustrating the selectivity that

is offered by this all ions MS/MS

functionality. Figures 6(c) and (d)

display the all ions MS/MS data

obtained at a collision energy of

20 eV, Figures 6(e) and (f) the data

at a higher collision energy of 40 eV.

Complementary information is

obtained when acquiring the data at

two different collision energy values.

A selection of identified conjugated

peptides is shown in Table 1. Since

the cytotoxic drug has a maximum

UV absorbance at 252 nm, this

wavelength can also be used to

confirm the positioning of conjugated

peptides in the peptide map as an

alternative to the all ions MS/MS

approach—evidently with a lower

specificity.

A similar strategy was applied

to reveal the conjugation sites on

brentuximab-vedotin (Adcetris),

the interchain cysteine conjugated

ADC. With only eight residues (four

on each half antibody) available for

conjugation, Adcetris is much simpler

compared to Kadcyla. Figure 7(a)

displays the reversed-phase LC

× reversed-phase LC–MS tryptic

peptide map of Adcetris. In analogy

to DM1 conjugated peptides,

MMAE conjugated peptides also

contain specific ions originating

from the cytotoxic molecule, that

is, at m/z 718.5113. As shown in

Figures 7(b) and (c), these ions can

be extracted from all ions MS/MS

data allowing conjugated peptides

in the 2D plots to be recognized. A

selection of identified conjugated

peptides is shown in Table 2. Of

particular interest is the detection

of conjugated intrachain cysteine

residues here exemplified with heavy

chain peptide NQVSLTCLVK (peak 3

in Figure 7[c]). These intrachain

residues are not targeted during

1

23

4

5

1

23

4

5

2D

, R

PLC

, 2

4s

2D

, R

PLC

, 2

4s

1D, RPLC, 45 min

(a)

(b)

(c)

1D, RPLC, 45 min

2D

, R

PLC

Figure 7: Reversed-phase LC × reversed-phase LC–MS peptide map of (a) Adcetris and (b,c) reversed-phase LC × reversed-phase LC all ions MS/MS peptide map showing extracted ion at m/z 718.5113 (extracted at 20 ppm mass accuracy). Collision energy was at 20 eV. (b) Full plot, (c) Zoomed intensified plot. The identity of the annotated spots is shown in Table 2. First and second dimension separation consist of reversed-phase LC operated at high and low pH, respectively. Fractions were transferred from one dimension to the other using the modulator shown in Figure 2(a). First dimension peaks were sampled 3–4 times. Experimental conditions can be obtained from reference 16.



ADC manufacturing. Apparently the

TCEP reduction step applied to the

mAb prior to conjugation results

in collateral intrachain disulphide

bridge reduction next to interchain

reduction (the latter links are more

labile). The resulting free thiol groups

can then react with the protease

cleavable linker-MMAE intermediate.

These unwanted side products

are only detected at trace levels

and illustrate the sensitivity of the

presented methodology.

It is clear that LC×LC holds

great potential in peptide mapping.

Recently, this 2D-LC mode has been

used at the protein level to resolve

intact mAbs or large fragments

thereof. Sorensen et al. demonstrated

the use of CEX × reversed-phase

LC coupled to MS to compare mAb

originators and biosimilars following

IdeS digestion thereby generating

F(ab’)2 and Fc/2 fragments and

subsequent reduction generating Lc,

Fd’, and Fc/2 (30). 2D-LC enabled

direct identification of charge variants

separated by the CEX column by

indirect coupling of the separation

to MS detection through the second

dimension reversed-phase LC

separation. The authors concluded

that the richness of the data enables

facile assessment of the degree of

similarity between mAbs. In a series

of papers, Sarrut et al. described the

analysis of the interchain cysteine

conjugated ADC brentuximab-vedotin

using HIC × reversed-phase

LC combined with UV and MS

detection (31,32). This setup

simultaneously allowed the DAR and

the predominant positional isomers

to be determined. The authors

furthermore suggested the use of this

methodology in quality control when

operated in UV-only mode.

AcknowledgementThe authors acknowledge Sonja

Schneider and Udo Huber

(Agilent Technologies, Waldbronn,

Germany), Maureen Joseph

(Agilent Technologies, Wilmington,

USA), and our colleagues from the

biopharmaceutical industry.

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Koen Sandra is Scientific Director

at the Research Institute for

Chromatography (RIC, Kortrijk,

Belgium).

Isabel Vandenheede is a Protein

Analyst at RIC.

Mieke Steenbeke is LC Lab Technician

at RIC.

Gerd Vanhoenacker is LC Product

Manager at RIC.

Pat Sandra is President of the RIC and

Emiritus Professor at Ghent University

(Ghent, Belgium).

Table 2: A selection of conjugated peptides corresponding to the annotated spots

in the LC×LC peptide map shown in Figure 7(b) and (c). The conjugated cysteine

residues are underlined. Payload: MMAE + linker. Carbamidomethylation results from

alkylation of free cysteine residues with iodoacetamide.

ChainSequence Location

Sequence

1 Lc 216-218 GEC + payload

2 Hc 219-222 SCDK + payload

3 Hc 361-370 NQVSLTCLVK + payload

4 Hc 223-248THTCPPCPAPELLGGPSVFLFPPKPK + 1* payload + 1*

carbamidomethylation

5 Hc 223-248 THTCPPCPAPELLGGPSVFLFPPKPK + 2* payload



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