i An update to this article is included at the end

Journal of Chromatography B 1058 (2017) 73–84

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/jchromb

Protocols for the analytical characterization of therapeutic monoclonal MARK

antibodies. I – Non-denaturing chromatographic techniques
Alexandre Goyona, Valentina D’Atria, Balazs Bobalya, Elsa Wagner-Roussetb, Alain Beckb,
⁎
Szabolcs Feketea, Davy Guillarmea,
a
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Centre Médical Universitaire (CMU) – Rue Michel-Servet 1, 1206 Geneva, Switzerland
b
Center of Immunology Pierre Fabre, 5 Avenue Napoléon III, BP 60497, 74160 Saint-Julien-en-Genevois, France

A R T I C L E I N F O A B S T R A C T

Keywords: Size-, charge- and hydrophobicity-related variants of a biopharmaceutical product have to be deeply
Size exclusion chromatography characterized for batch consistency and for the assessment of immunogenicity and safety effects. Size exclusion
Ion exchange chromatography chromatography (SEC) and ion exchange chromatography (IEX) are considered as the gold standard for the
Hydrophobic interaction chromatography analysis of high molecular weight species (HMWS) and charge-related variants, respectively. Hydrophobic
Therapeutic monoclonal antibodies
interaction chromatography (HIC) has drawn renewed attention to monitor the small drug payload distribution
Antibody-drug conjugates
Protocol
in the cysteine-linked antibody-drug conjugates (ADC). These three chromatographic techniques, namely SEC,
HIC and IEX, are historical, non-denaturing and robust approaches widely used for the characterization of
biopharmaceutical proteins. Despite the broad spectrum of monoclonal antibodies (mAbs) structures, isoelectric
points (pIs) and hydrophobicities, generic protocols can be applied to separate their size-, charge- and
hydrophobicity-related variants, using the last generation of chromatographic columns and appropriate mobile
phase conditions. Straightforward protocols are described in this manuscript with representative chromatograms
of ten distinct Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved
therapeutic mAb products to illustrate the performance of the SEC, IEX and HIC methods.

1. Introduction stability studies [7–9].

To date, a significant number of mAbs have been marketed and this
trend is likely to continue, as over 470 mAbs are currently under
clinical development [1,2]. In 2013, biopharmaceutical proteins ac-
counted for a total sales value of $140 billion representing approxi-
mately 20% of the total pharmaceutical market [3]. Monoclonal
antibodies represent roughly 50% of those sales and are considered as
the fastest growing class of therapeutics, with sales grown from $39
billion in 2008 to almost $75 billion in 2013, a 90% increase [4]. In
addition, due to the patent expiry of blockbuster mAbs, many pharma-
ceutical companies are also entering the biosimilar market [5].
Currently, 140 biosimilars are in development for five mAb blockbus-
ters, namely adalimumab, infliximab, rituximab, bevacizumab and
trastuzumab that account for $44.2 billion sales in the US in 2014
[5]. To demonstrate biosimilarity to a reference product, analytical
methodologies should assess the aggregation levels and other potential
variations such as protein deamidation and oxidation, according to the
FDA guidance for industry [6]. For this purpose, SEC and IEX are still
considered as the gold standards, and they have also been used in many
The characterization of biopharmaceutical proteins comprised a
wide range of analytical and mass spectrometric techniques [10]. The
theory and practice of SEC, IEX and HIC is well documented in the
literature [11–13]. SEC separates proteins into three major species: high
molecular weight species (HMWS), main peak (predominantly the
monomeric form), and low molecular weight species (LMWS). Basic
variants induced by aspartic acid isomerization, N- and C-terminal
modifications as well as acidic variants generated by deamidation and
glycation are analyzed by ion exchange chromatography (IEX). IEX can
be divided into two sub types; cation exchange chromatography (CEX)
and anion exchange chromatography (AEX). Proteins are eluted using
salt- or pH-gradients. Fekete et al. have shown that salt-gradient was
more effective to separate the charge-related variants of the mAbs when
compared to the pH-gradient method [14]. In HIC, the retention
mechanism is solely based on the interactions between the amino acids
located at the surface of the molecule and the stationary phase.
Therefore, differences in retention times between hydrophobicity-
related variants may result from conformational change. SEC, IEX and
HIC are valuable for protein analysis at intact level due to the use of an

⁎
Corresponding author.
E-mail address: davy.guillarme@unige.ch (D. Guillarme).

http://dx.doi.org/10.1016/j.jchromb.2017.05.010
Received 14 February 2017; Received in revised form 9 May 2017; Accepted 10 May 2017
Available online 11 May 2017
1570-0232/ © 2017 Elsevier B.V. All rights reserved.
A. Goyon et al.
aqueous mobile phase prepared at physiological pH and temperature
with medium – to high salt concentrations (from 0.1 M to 2.0 M). The
three analytical methods are also non-destructive and can be of
particular interest for the biological assessment of collected fractions.
ADCs represent another important class of biopharmaceutical
proteins. Even if only two ADC products were marketed, namely
trastuzumab emtansine and brentuximab vedotin, over 60 ADCs were
in clinical trial phases in 2017 [1,15] including 8 in late stages [16].
The drug load distribution is a critical quality attribute (CQA) of an
ADC, since an unconjugated antibody will act as a competitor for the
target antigen, whereas an ADC possessing a high drug payload appears
to be unstable in plasma/serum and may release its cytotoxic drugs too
early [17]. The drug-to-antibody ratio (DAR) defined as the average
number of drugs conjugated to the antibody also accounts for the
quality assessment of an ADC. The drug load distribution and average
DAR can be measured using HIC but also native MS methodologies
[17–19].
This article aims to provide some generic analytical conditions for
the analysis of a wide range of mAbs and ADCs in SEC, IEX and HIC, by
using the last generation of stationary phases and adapted mobile
phases. This protocol focuses on the intact protein level of analysis
under non-denaturing conditions. Appropriate solutions are given to
manage possible hurdles during these analyses. Ten commercial mAbs
were injected in SEC, IEX and HIC to illustrate the performance of the
proposed analytical methods. In addition, the commercial ADC, bren-
tuximab vedotin, was injected in HIC to highlight the cytotoxic
molecule payloads distribution.
2. Experimental
2.1. Chemical and reagents
Commercially available salts should be of the highest available
chemical grade.
Gradient grade methanol for HPLC (art. 34860), potassium chloride
(art. 60128), potassium phosphate monobasic (art. 60218), potassium
phosphate dibasic (art. 60353), sodium chloride (art. 73575), 2-(N-
morpholino)ethanesulfonic acid (MES) monohydrate (art. 69889), MES
sodium salt (art. M5057), ammonium sulfate (art. 09978) were
purchased from Sigma-Aldrich (Buchs, Switzerland). Commercially
available FDA approved mAbs and ADCs were kindly provided by the
Center of Immunology Pierre Fabre (Saint-Julien en Genevois, France).
Table 1 lists biopharmaceutical proteins and their physico-chemical
properties found in previous studies [20,21].
2.2. Chromatographic media
2.2.1. SEC columns
TSKgel UP-SW3000 column (2.0 μm, 150 mm × 4.6 mm, 250 Å)
from Tosoh (art. 0023449, Tokyo, Japan) or alternatively
AdvanceBioSEC column (2.7 μm, 150 mm × 4.6 mm, 300 Å) from
Agilent Technologies (art. PL1580-3301, Wilmington, DE, USA) were
used as these columns are able to minimize non-specific interactions
[22]. The columns lifetime was estimated to be in the range of 300–500
Table 1
Properties of the commercial therapeutic mAbs used in the protocols.
Class mAb
Protein adalimumab belimumab bevacizumab denosumab infliximab ofatumumab
MW (kDa) 148 147 149 147 149 149
pIa 8.8 8.6 8.5 8.8 7.6 8.8
Hydrophobicityb Very low Low High Very low Low Low
a
Isoelectric points (pI) were experimentally determined by cIEF [19].
b
Hydrophobicities were experimentally compared using HIC [20].
74
Journal of Chromatography B 1058 (2017) 73–84
injections.
2.2.2. IEX columns
BioPro SP-F non-porous strong cation exchange column (5.0 μm,
100 mm × 4.6 mm) supplied by YMC (art. SF00S05-1046WP, Kyoto,
Japan). Alternatively, the Bio MAb NP5 column (5.0 μm,
250 mm × 2.1 mm) can be purchased from Agilent (art. 5190-2411,
Wilmington, DE, USA). The BioPro SP-F column typically lasts for at
least 1 000 injections.
2.2.3. HIC columns
MAbPac HIC-10 column (5.0 μm, 250 × 4.6 mm, 1000 Å) was
purchased from Thermo Fisher Scientific AG (art. 088481, Sunnyvale,
CA) or alternatively the TSKgel Butyl-NPR column (2.5 μm,
100 mm × 4.6 mm) from Tosoh (art. 42168, Tokyo, Japan) may be
chosen. The columns lifetime was estimated to be in the range of
500–800 injections.
After use, SEC, IEX and HIC columns should be conditioned with
water/methanol (85:15, v/v) to prevent any algal or bacterial growth.
2.3. Laboratory device
Samples were homogenised using a vortex mixer Genie 2 (art. SI-
0236, Scientific Industries, New York, USA). Water was obtained from a
Milli-Q Purification System from Millipore (Bedford, MA, USA). The
mobile phases were filtered through a 0.22 μm polyethersulfone (PES)
membrane filter (art. 12.8900, Sartorius, Göttingen, Germany) and pH
was measured with a SevenMulti pH Meter S40 (Mettler Toledo,
Greifensee, Switzerland). Mobile phase was stored in amber Schott-
Duran bottles (Wertheim/Main, Germany) to prevent algal or bacterial
growth and should be used within three days. 1.5-mL HPLC vials with
200-μL conical glass insert (31 × 5 mm, tip: 9 mm) were purchased
from BGB Analytik Vertrieb GmbH (art. 110501, Rheinfelden,
Germany). Alternatively, plastic inserts can also be used. Special
attention should be paid to the shape of conical insert as they may
affect SEC measurements of HMWS [23].
2.4. Instrumentation
Experiments were performed on a Waters ACQUITY UPLC™ H-Class
system equipped with a quaternary solvent delivery pump, an auto-
sampler, a fluorescence (FL) detector (excitation at 280 nm, emission at
340 nm for aqueous mobile phases, 5 Hz, 0.2-s time constant) and a
photodiode array (PDA) detector to acquire UV spectrum from 220 nm
to 320 nm for the confirmation of brentuximab vedotin HIC peaks.
Fluorescence detection is more specific to proteins and more sensitive
than UV detection, therefore FL detector may be preferred for biophar-
maceuticals analysis. The Waters ACQUITY UPLC™ H-Class system
includes a 10 μL flow-through-needle injector, a 0.5 μL UV detector
flow-cell and a 2 μL FL detector flow-cell.
2.5. Software
Data acquisition and instrument control were performed by
ADC
palivizumab pertuzumab rituximab trastuzumab brentuximab
vedotin
148 148 145 146 153
9.0 9.0 9.1 8.8 NA
Low Low Medium Low Very high
A. Goyon et al. Journal of Chromatography B 1058 (2017) 73–84

Empower Pro 3 software (Waters). Data transferring was achieved by
using Excel (Microsoft).
3. Protocols
3.1. General aspects
Experiments should be performed in accordance with relevant
national and international guidelines and regulations. Material Safety
Data Sheets (MSDS) should be available for all chemicals, inherent risks
and corresponding safety precautions shall be identified. Cytotoxic
drugs of the antibody-drug conjugates are highly toxic; therefore, all
necessary precautions should be taken especially when working with
concentrated solutions.
The mobile phase has to be filtered due to the high salt content.
Aqueous mobile phase should be stored in amber Duran bottles to
prevent any algal or bacterial growth. If available, bio-inert LC systems
may be preferred to traditional LC systems to avoid possible protein
adsorption on the system. In particular, inert materials such as titanium
should be used preferentially compared to the very hydrophobic
polyether ether ketone (PEEK) for chromatography tubing and injection
needles. Recently, manufacturers have produced Bio UHPLC instru-
ments dedicated to the analysis of biomolecules [24]. Table 2 lists the
issues already encountered in our laboratory and solutions as well as
advices to prevent such problems.
3.2. Samples handling
Biopharmaceutical products should be protected from light and
stored between 2 °C and 8 °C.
When analyzing a large number of samples, a stability study should
be performed within a reasonable timescale (24 h is appropriate in most
cases) to check any degradation of the therapeutic proteins stored in the
autosampler, especially when they are diluted.
3.3. Preparation of analytical reagents and samples
3.3.1. Preparation of mobile phase and other reagents
3.3.1.1. SEC mobile phase. Two independent solutions of 50 mM
potassium phosphate monobasic and 50 mM potassium phosphate
dibasic are first prepared. For this purpose, weight 3.40 g ± 0.03 g
of potassium phosphate monobasic into a weighing boat. Transfer this
powder into a 500-mL volumetric flask. Complete the flask with milli-Q
water and homogenize the solution. Similarly, weight 4.35 g ± 0.04 g
of potassium phosphate dibasic into a weighing boat. Transfer this
powder into a 500-mL volumetric flask. Complete the flask with milli-Q
water and homogenize the solution.
Mobile phase: water containing 50 mM of potassium phosphate and
250 mM potassium chloride, pH = 6.8.
Weight 9.32 g ± 0.09 g of potassium chloride into a weighing
boat. Transfer this powder into a 500-mL volumetric flask. Complete
to volume by adding, with stirring, the potassium phosphate monobasic
and potassium phosphate dibasic solutions (40:60, v/v). Check the pH
of the mobile phase to be 6.8 ± 0.1 with a pH meter. Filter through
PES 0.22 μm membrane filters before use.
3.3.1.2. IEX mobile phase. Two independent solutions of 10 mM MES
sodium salt and 10 mM MES monohydrate are first prepared. Weight
2.17 g ± 0.02 g of MES sodium salt into a weighing boat. Transfer this
powder into a 1-L volumetric flask. Complete the flask with milli-Q
water and homogenize the solution. Similarly, weight 2.13 g ± 0.02 g
of MES monohydrate into a weighing boat. Transfer this powder into a
1-L volumetric flask. Complete the flask with milli-Q water and
homogenize the solution.
Mobile phase A: water containing 10 mM of MES, pH = 6.0.
Into a 500-mL amber Duran, add, with stirring, the MES sodium salt
and MES monohydrate solutions (47:53, v/v). Check the pH of the
mobile phase A to be 6.0 ± 0.1 with a pH meter. Filter through PES
0.22 μm membrane filters before use.
Mobile phase B: water containing 10 mM of MES and 1 M of sodium
chloride, pH = 6.0.
Weight 29.2 g ± 0.2 g of sodium chloride into a weighing boat.

Table 2
Troubleshooting and prevention of problems during chromatographic analysis of mAb and ADC samples using non-denaturing techniques.

Problem Cause Solution Prevention

Sample syringe Salt clogging in some parts of the
overpressure autosampler, preferentially in
the injection valve.
Carry over Protein adsorption onto the
system or onto the column.
Absence of peaks. Protein adsorption to the active
sites of the column.
Column overpressure Salt clogging, protein adsorption
to column frits.
Column leak Column fitting body is
unscrewed from the internal nut.
If allowed by the local rules in place, remove the Wash the system after every batch of samples with water:
injection valve from the LC system. Prepare a mixture methanol (85:15, v/v) for 60 min at 0.3 mL/min.
of water: methanol (50:50, v/v) and insert the injection When the system is not used for a longer period (typically
valve in a beaker containing the water: methanol during vacations), purge and wash all solvent lines with
mixture. Place the beaker in an ultrasonic bath and water for 30 min at 0.3 mL/min to remove salts. Then wash
sonicate for around 30 min. Remove the injection valve the system with water: methanol (85:15, v/v) for 30 min at
from the beaker and connect it again to the LC system. 0.3 mL/min to avoid any bacterial or algal growth.
HPLC columns should be first replaced by a “zero Use a Bio-Inert LC System. Inert fluorescence and ultraviolet
volume” union connector to determine whether the detector cells should also be used.
problem comes from the column or the system. After
washing the system with water to remove salts, a
strong system wash may be performed using a “zero
volume” union connector and using a mixture of water:
acetonitrile: methanol: isopropanol (25:25:25:25, v/v/
v/v) at 0.2 mL/min for 60 min.
Inject ten times a concentrated protein sample to coat When using the column for the first time, inject ten times a
the silica and condition the column in order to prevent concentrated protein sample to feed the column.
protein adsorption. As example, 5 μL of a basic protein
such as cytochrome C at 5 mg/mL may be injected 10
times.
First, wash the column with water at 0.2 mL/min for Filter the mobile phase. Wash and store the columns
60 min. If this washing is not sufficient, then wash the following the supplier recommendations.
column in reversed flow mode at 0.1 mL/min with
water for 60 min. Replace the column if the procedure
failed.
Screw the fitting body to the column nut. Replace the Avoid high-pressure rise when equilibrating the column.
column if the procedure failed.

75
A. Goyon et al. Journal of Chromatography B 1058 (2017) 73–84

Transfer this powder into a 500-mL volumetric flask. Complete to

B: water containing 100 mM potassium phosphate buffer, pH = 6.8

A: water containing 100 mM potassium phosphate buffer and 2 M
volume by adding, with stirring, the MES sodium salt and MES

100
60
monohydrate solutions (53:47, v/v). Check the pH of the mobile phase

0
B to be 6.0 ± 0.1 with a pH meter. Filter through PES 0.22 μm
membrane filters before use.

45.2
100
0
3.3.1.3. HIC mobile phase. Two independent solutions of 100 mM

100
potassium phosphate monobasic and 100 mM potassium phosphate

45

Thermo Fisher MAbPac HIC-10 column

0
dibasic are first prepared. Weight 13.6 g ± 0.1 g of potassium

(5.0 μm, 250 × 4.6 mm, 1000 Å)

phosphate monobasic into a weighing boat. Transfer this powder into

100
a 1-L volumetric flask. Complete the flask with milli-Q water and

ammonium sulfate, pH = 6.8

40
(5 for brentuximab vedotin)

0
homogenize the solution. Similarly, weight 17.4 g ± 0.1 g of
potassium phosphate dibasic into a weighing boat. Transfer this mass

100
into a 1-L volumetric flask. Complete the flask with milli-Q water and

0
homogenize the solution.

Time (min)
Mobile phase A: water containing 100 mM of potassium phosphate
buffer and 2 M of ammonium sulfate, pH = 6.8.

A (%)
B (%)
HIC

0.5

5.0

0.8
Weight 66.1 g ± 0.5 g of ammonium sulfate into a weighing boat.

25

35
Transfer this powder into a 500-mL volumetric flask. Complete to

YMC BioPro SP-F non-porous strong cation exchange column (5.0 μm,
volume by adding, with stirring, the potassium phosphate monobasic

B: water containing 10 mM MES and 1 M sodium chloride, pH = 6.0

100
30
and potassium phosphate dibasic solutions (20:80, v/v). Check the pH

0
of the mobile phase A to be 6.8 ± 0.1 with a pH meter. Filter through
PES 0.22 μm membrane filters before use.

21.2
100
Mobile phase B: water containing 100 mM of potassium phosphate

0
buffer, pH = 6.8.
Into a 500-mL amber Duran, mix under stirring the potassium

100
21
0
phosphate monobasic and potassium phosphate dibasic solutions

A: water containing 10 mM MES, pH = 6.0

(50:50, v/v). Check the pH of the mobile phase to be 6.8 ± 0.1 with
a pH meter. Filter through PES 0.22 μm membrane filters before use.

20.2

100
0
20
80
20
3.3.1.4. Injection wash solvent. Prepare wash solvents for the injection
system by mixing water/methanol 85:15 (v/v) for post injection washes

100 mm × 4.6 mm)

and for pre-sample injection system washes. 100
Time (min) 0

3.3.2. System suitability reference standard

A (%)
B (%)

In 2016, the United States Pharmacopeia (USP) released the

IEX

0.5

1.0

0.6
25

15
monograph < 129 > entitled Analytical Procedures for Recombinant
Therapeutic Monoclonal Antibodies [25]. A “universal” monoclonal anti-
Water containing 50 mM potassium phosphate

body was introduced by the USP as a System Suitability Reference

and 250 mM potassium chloride, pH = 6.8

Standard (SSRS). To our knowledge, such procedure is solely available

for the SEC analysis of mAbs. Therefore, and similarly to the USP
(2.7 μm, 150 mm × 4.6 mm, 300 Å)

monograph 〈129〉, we proposed hereafter a system suitability proce-

Water: methanol (85:15, v/v),6 s
Agilent AdvanceBioSEC column

dure for the three historical chromatographic techniques (SEC, IEX and
Summary of the generic SEC, IEX and HIC conditions for mAbs analysis.

HIC) using pertuzumab or trastuzumab as SSRS. Based on our experi-

ence and the literature, pertuzumab or trastuzumab may represent
alternative candidates to the USP universal mAb, due to their enhanced
stability [26].
Before use, allow pertuzumab or trastuzumab samples to reach
280; 340

room temperature. Vortex gently. Try to avoid any dilution because it

0.35

can affect protein stability. However if dilution is required, dilute the

SEC

0.5

0.5
NA

25

60

biopharmaceutical product to 0.5 mg/mL with the blank formulation

into a 200-μL conical glass insert placed into a 1.5-mL HPLC vial. The
Concentration (mg/mL)

First equilibration time

Injection volume (μL)

system suitability solution should be used within 24 h after dilution and

Excitation; emission
Flow rate (mL/min)

Post injection wash

temperature (°C)

temperature (°C)

stored between 2 and 8 °C during analysis.

Mobile phase

Column oven

Autosampler
Gradient list

(nm, nm)
Column

(min)

3.3.3. Preparation of the samples

Before use, allow the samples to reach room temperature. Vortex
each ample gently. Try to avoid any dilution because it can affect
LC conditions.

FL conditions
Autosampler
Techniques

protein stability. If required, dilute the mAb product to 0.5 mg/mL with
Samples

the blank formulation into a 200-μL conical glass insert placed into a
Table 3

1.5-mL HPLC vial. A blank should also be prepared using the

biopharmaceutical product formulation.

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A. Goyon et al. Journal of Chromatography B 1058 (2017) 73–84

Table 4 3.5. Data analysis

Drug load distribution and DAR calculation of brentuximab vedotin, based on HIC
analysis.
3.5.1. Expression of results for size, charge and hydrophobicity variants of
Drug load Percentage peak Weighted peak area (drug mAb samples
area (%) load × percentage peak area (%)) Ideally, peaks should be integrated using automatic integration
rather than manual integration to minimize human operator error. Any
0 6.0 0
peaks that are present in the blank formulation should be excluded from
1 1.1 1.1
2 25.4 50.8 integration. Protein-related peaks that elute before the main peak are
3 3.0 9.0 classified as HMWS, acidic variants, and less hydrophobic variants in
4 36.3 145.2 SEC, IEX and HIC, respectively. On the opposite, protein-related peaks
6 20.8 124.8 that elute after the main peak are classified as LMWS, basic variants,
8 7.5 60
and more hydrophobic variants in SEC, IEX and HIC, respectively.
Weighted average 3.9
DAR Relative quantitation of the size-, charge- and hydrophobic-related
variants are expressed in percentage related to the total peak area. The
result obtained for a biopharmaceutical product should be reported in
3.4. Analysis percentage, with an accuracy of one decimal place. Based on our
experience, non-detected amount must be expressed as < 0.1%.
3.4.1. Preparation of the analytical system
Create the LC method by setting all the chromatographic para-
3.5.2. Drug load distribution and drug-to-antibody ratio (DAR) calculation
meters: gradient elution program, equilibration time, mobile phase flow
of ADC samples by HIC
rate, column temperature, injection volume, washing cycles and auto-
Calculate the peak areas of all the different drug-loaded species as a
sampler temperature. Load the LC method and equilibrate the system
percentage of the total peak area and transcribe the information in a
for ten column volumes. Table 3 lists the SEC, IEX and HIC generic
table [19,27]. The percentage peak area (%) represents the drug load
conditions used in our laboratory. In SEC, proteins are eluted in the
distribution.
window between the exclusion volume and the permeation volume of
Then, multiply the percentage peak area by the corresponding drug
the column that is typically around 2 min and 6 min with
load, in order to calculate the weighted peak area and report the
4.6 mm × 150 mm columns at 0.35 mL/min.
information in the same table.
The IEX and HIC generic elution gradient can be optimized. a)
Finally, the weighted average DAR can be calculated by summing
Determine the mobile phase composition at the limits of the elution
the weighted peak area column and dividing the sum by 100 as follows:
window of the protein-related species under generic conditions. b) The
start and end compositions of the new gradient should be respectively weightedaverageDAR = ∑ (Weightedpeakarea )/100.
set close to the start and end limits of the elution window obtained with
the generic gradient. c) Final gradient composition is experimentally For example, the drug load distribution and weighted average DAR
adjusted by decreasing the start composition in case proteins elute of the ADC brentuximab vedotin were reported in Table 4.
before the double void column volume or/and increasing the end
composition in case proteins are not all eluted. This approach allows 3.5.3. Confirmation of HIC peaks with an ADC
improving the selectivity at constant analysis time. For example, The identification of the peaks observed on the HIC profile of an
gradients starting from 5 to 7.5% mobile phase B to 9.5–10% mobile ADC sample, can be confirmed by monitoring the UV spectrum from
phase B were successively applied in this protocol for the separation by 220 to 320 nm in the case where the drug/linker of the ADC is UV
IEX of charge-related variants of infliximab and pertuzumab. Similarly, active [18,27,28]. Individual UV spectrum of each HIC peak should be
gradients starting from 15 to 20% mobile phase B to 35–60% mobile normalized to the maximum absorbance of the antibody at 280 nm.
phase B were applied for the analysis by HIC of hydrophobicity-related Normalized UV spectrums are differentiated at the maximum absor-
variants of infliximab and pertuzumab. bance of the drug-linker species which is around 250 nm for brentux-
imab vedotin. The relative absorbance increase with the drug load
enables the identification of HIC peaks. For example, the individual UV
3.4.2. Sequence set up spectrum of each HIC peak normalized to the maximum absorbance of
According to the USP monograph < 129 > , injections of the brentuximab vedotin at 280 nm were represented in Fig. 1.
biopharmaceutical samples should be bracketed minimally with single
injections of the blank formulation and the system suitability mAb
(defined in Section 3.3.2). Inject samples in the following order: blank
formulation, system suitability mAb sample, blank formulation, bio-
pharmaceutical samples, blank formulation, system suitability mAb
sample and blank formulation again. In addition, a sequence containing
a blank formulation, a system suitability mAb and a blank formulation
should be injected after each 10 biopharmaceutical samples.

3.4.3. Analysis time

Sample preparation is minimal when analyzing biopharmaceutical
protein products as only dilution may be performed prior to injection
onto the LC system. Therefore, analysis time mostly relies on the system
equilibration and chromatographic analysis time. Following the SEC,
IEX and HIC protocols summarized in Table 3, and considering the
analysis times, 200, 30 and 15 biopharmaceutical products can be
analyzed within 24 h in SEC, IEX and HIC, respectively.
Fig. 1. UV spectra of brentuximab vedotin drug-loaded species.

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A. Goyon et al. Journal of Chromatography B 1058 (2017) 73–84

Fig. 2. SEC (A), IEX (B) and HIC (C) chromatographic profiles of pertuzumab. The chromatograms were obtained using the generic conditions described in Table 3, except the IEX and
HIC gradients that were optimised.

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A. Goyon et al. Journal of Chromatography B 1058 (2017) 73–84

Fig. 3. SEC chromatographic profiles of pertuzumab (A), adalimumab (B), belimumab (C), bevacizumab (D), denosumab (E), infliximab (F), ofatumumab (G), palivizumab (H), rituximab
(I), trastuzumab (J). The SEC chromatograms were obtained using the generic conditions described in Table 3.

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A. Goyon et al. Journal of Chromatography B 1058 (2017) 73–84

Fig. 4. IEX chromatographic profiles of pertuzumab (A), adalimumab (B), belimumab (C), bevacizumab (D), denosumab (E), infliximab (F), ofatumumab (G), palivizumab (H), rituximab
(I), trastuzumab (J). The IEX chromatograms were obtained using the generic conditions described in Table 3.

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Fig. 5. IEX chromatographic profile of infliximab. The chromatogram was obtained using the generic conditions described in Table 3, except the gradient that was optimised.
3.5.4. Data quality assessment
The three suitability criteria defined in the USP monograph 〈129〉
for suitability requirements deal with a) chromatogram, b) chromato-
gram similarity and c) quantitative criteria. These criteria should be
applied with the USP mAb if available. In this protocol paper, a generic
data quality assessment is proposed for SEC, IEX and HIC using
pertuzumab as SSRS.
a) SEC, IEX and HIC chromatograms of the SSRS should be
consistent with the chromatograms of pertuzumab shown in Fig. 2A–C.
b) Chromatographic profiles of the pertuzumab SSRS injections that
bracket injections of the biopharmaceutical products should be con-
sistent with each other and with the typical chromatogram profile
depicted in Fig. 2A–C. All protein-related peaks in the reference
chromatogram should be present, well resolved and in the same elution
order as that in the pertuzumab SSRS chromatograms.
c) Quantitative criteria
When dealing with the relative quantification of protein variants,
bracketing pertuzumab SSRS solutions should meet the following
quantitative criteria in order to validate a batch of samples. Based on
our experience, in-house criteria for pertuzumab are given hereafter to
serve as an example but solely the USP certificate for the USP mAb
should prevail.
In SEC, the percent peak area of the HMWS in the pertuzumab SSRS
solution must range between 0.1% and 0.3%. The percent peak area of
the main peak in the system suitability solution must be 99.3%–99.8%.
The percent peak area of the LMWS in the system suitability solution
must be no more than 0.3%.
In IEX, the percent peak area of the acidic variants in the
pertuzumab SSRS solution must range between 16% and 20%. The
percent peak area of the main peak in the system suitability solution
must be 73%–79%. The percent peak area of the basic variants in the
system suitability solution must range between 5% and 7%.
In HIC, the percent peak area of the hydrophilic variants in the
pertuzumab SSRS solution must range between 0.1% and 0.4%. The
percent peak area of the main peak in the system suitability solution
must be 98.5%–99.3%. The percent peak area of the hydrophobic
variants in the system suitability solution must range between 0.6% and
1.1%.
4. Application
The ten therapeutic mAbs listed in Table 1 and covering a wide
range of hydrophobicities and pIs were analysed in SEC, IEX and HIC
81
Journal of Chromatography B 1058 (2017) 73–84
following the above-described protocols. The ADC sample, namely
brentuximab vedotin, was also injected in HIC to measure the different
drug-loaded species present in the biopharmaceutical sample. All the
analytical conditions are given in Table 3.
4.1. SEC
The SEC separations of ten commercial mAbs is shown in Fig. 3. As
expected, HMWS and LMWS were separated from the main peak.
Except for the HMWS of rituximab (Fig. 3I) and ofatumumab (Fig. 3G),
baseline resolutions (Rs) between the HMWS or LMWS and the main
peak were always achieved (Rs ranged from 1.7 to 2.3). For rituximab
and ofatumumab, partially denatured monomers may be unresolved
from the main peak as it has already been reported by Philo for some
biopharmaceutical proteins [29]. The tailing factor for the main peak
was well below 1.5 (USP tailing factor comprised between 1.16 and
1.29), which is considered suitable. Highly comparable SEC profiles
were obtained with the ten mAbs, thus confirming the genericity of the
SEC method.
4.2. IEX
Generic IEX conditions were applied for the separation of acidic and
basic variants from the main peak, as depicted in Fig. 4. The IEX profiles
of adalimumab (Fig. 4B), infliximab (Fig. 4F), pertuzumab (Fig. 4A),
rituximab (Fig. 4I) and trastuzumab (Fig. 4J) were in agreement with
those published in the literature [26,30–32]. In comparison to other
mAb products, belimumab product heterogeneity or secondary inter-
actions with the stationary phase may account for the tailed peaks
observed in Fig. 4C.
In the case of IEX, generic conditions do not allow a sufficient
resolving power to separate charge variants. Therefore, the IEX
separation can be further optimized on the basis of the generic
chromatograms reported in Fig. 4, and taking into account the elution
window and gradient conditions. An example of such optimization is
provided for infliximab, based on the generic chromatogram obtained
in Fig. 4F. With this generic gradient, various species of infliximab
eluted from 8 to 13 min, therefore the composition of mobile phase B
was adjusted from 5% to 10%B in 20 min, rather than 0–20%B in
20 min. As shown in Fig. 5, the separation of the acidic and basic
variants from the main peak of infliximab was significantly improved
using the optimized gradient. The typical two major peaks eluting after
the main peak at 14 min and 16 min in Fig. 5 were also observed by
A. Goyon et al. Journal of Chromatography B 1058 (2017) 73–84

Fig. 6. HIC chromatographic profiles of pertuzumab (A), adalimumab (B), belimumab (C), bevacizumab (D), denosumab (E), infliximab (F), ofatumumab (G), palivizumab (H), rituximab
(I), trastuzumab (J). The HIC chromatograms were obtained using the generic conditions described in Table 3.

82
A. Goyon et al. Journal of Chromatography B 1058 (2017) 73–84

Fig. 7. HIC chromatographic profile of infliximab. The HIC chromatogram was obtained using the generic conditions described in Table 3, except the gradient that was optimised.

Fig. 8. HIC chromatographic profile of the ADC sample, brentuximab vedotin. The HIC chromatogram was obtained using the generic conditions described in Table 3.

Jung et al. and identified as charge-related variants possessing one C- other ones were much more heterogeneous, such as infliximab (Fig. 6F)
terminal lysine and two C-terminal lysine, respectively [30]. or rituximab (Fig. 6I). With the generic gradient, various species of
infliximab elute from 16 to 22 min, therefore the composition of mobile
phase B was adjusted from 15% to 35%B in 40 min, rather than 0–100%
4.3. HIC
B in 40 min. As shown in Fig. 7, the separation of the hydrophilic and
hydrophobic variants from the main peak of infliximab was improved
Separations of the less and more hydrophobic variants from the
using the optimized gradient.
main peak of the 10 commercial therapeutic mAbs are shown in Fig. 6.
As previously described, HIC is the reference technique for ADC
Unfortunately, these HIC profiles cannot be directly compared to
characterization. The separation of the distinct drug-loaded species of
published results. Indeed, the recent review of Haverick et al. explains
brentuximab vedotin is shown in Fig. 8. The composition of mobile
that most of the findings related to the analysis of mAb variants in HIC
phase B was adjusted from 20% to 100%B in 40 min, rather than
were from pharmaceutical companies and the name of the mAb was
0–100%B in 40 min. The HIC profile of brentuximab vedotin was very
kept confidential [33]. As shown in Fig. 6, some of the tested mAbs do
similar to the ones already published [18,27]. In addition, two peaks
not possess a significant amount of hydrophobic variants. This is for
attributed to DAR 4 isomeric species were observed. Confirmation of
example the case of belimumab (Fig. 6C), denosumab (Fig. 6E),
the HIC peaks was performed by recording the UV spectra of the ADC
palivizumab (Fig. 6H) or trastuzumab (Fig. 6J). On the contrary, some

83
A. Goyon et al.
drug-loaded species from 220 to 320 nm, as reported in Fig. 1. The
difference of relative absorbance at about 250 nm of the HIC peaks
confirmed the peak assignment of the various drug-loaded ADC species.
Finally, the drug load distribution and the weighted average DAR value
of 3.9, listed in Table 4, were both consistent with the literature
[19,27].
5. Conclusions
To answer the growing number of novel biopharmaceutical pro-
ducts (particularly mAbs) entering in clinical trials and the significant
attention drawn to biosimilars, there is need for highly efficient and
robust analytical methods. This protocol paper describes SEC, IEX and
HIC methodologies, which are considered as key methods for the
analysis of therapeutic mAb and ADC samples. Generic protocols were
described at the intact protein level and using non-denaturing chroma-
tographic conditions. To achieve a sufficient level of performance with
such complex samples, the latest generation of SEC, IEX and HIC
columns has to be employed, using suitable mobile phase conditions.
The performance of these three “historical” techniques was illustrated
with ten commercial mAbs and a cysteine-linked ADC, brentuximab
vedotin. Practical advices and troubleshooting were also provided to
ensure the robustness of the analytical methods. Limitations of these
procedures that would require additional method development relate to
the analysis of acidic proteins with IEX and non-cysteine-linked ADCs
with HIC. Additional analytical strategies related to the use of enzymes
for the analysis of biopharmaceutical proteins at the bottom-up and/or
middle down levels with denaturing techniques (RPLC and HILIC)
combined with high resolution mass spectrometry (HRMS) are provided
in two other protocol papers from the same authors.
6. Perspectives
Chromatographic peaks identification is challenging in IEX, SEC and
HIC, because of the use of non-volatile salts, which prevent direct
coupling to mass spectrometry (MS). Yet, these techniques are mostly
used in quality control laboratories, in a comparative way to a reference
product and they do not intend to provide such information in routine
analysis. However, in the presence of unknown species during product
development, various strategies exist to assign chromatographic peaks.
Isolation and characterization of variants is commonly performed in off-
line mode. Otherwise, on-line characterization can be achieved by using
the second dimension of two dimensional liquid chromatography (2D-
LC) as a desalting step before injection onto MS [34,35] or using in-line
fraction collection device [36].
Acknowledgements
Authors wish to thank the master students Amarande Murisier and
Tatiana Sofia Marciano Dias for performing the experiments in agree-
ment with the protocols. Jean-Luc Veuthey from the University of
Geneva is acknowledged for his support and discussions.
Davy Guillarme wishes to thank the Swiss National Science
Foundation for support through a fellowship to Szabolcs Fekete
(31003A159494).
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 Update
Journal of Chromatography B
Volume 1067, Issue , 1 November 2017, Page 89–90

DOI: https://doi.org/10.1016/j.jchromb.2017.10.003
 Journal of Chromatography B 1067 (2017) 89–90

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/jchromb

Corrigendum

Corrigendum to “Protocols for the analytical characterization of therapeutic MARK

monoclonal antibodies. I—Non-denaturing chromatographic techniques” [J.
Chromatogr. B 1058 (2017) 73–84]
Alexandre Goyona, Valentina D’Atria, Balazs Bobalya, Elsa Wagner-Roussetb, Alain Beckb,
⁎
Szabolcs Feketea, Davy Guillarmea,
a
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d’Yvoy 20, 1211 Geneva 4, Switzerland
b
Center of Immunology Pierre Fabre, 5 Avenue Napoléon III, BP 60497, 74160 Saint-Julien-en-Genevois, France

The authors regret that an error occurred in Section 3.3.1.3 and
Table 3. The correct HIC mobile phase A contains 100 mM of potassium
phosphate buffer and 1 M of ammonium sulfate, pH = 6.8 (ammonium
sulfate concentration is not 2 M as written in the original version of the
manuscript).
In section 3.3.1.3, the correct composition of the HIC mobile phase
A is “Mobile phase A: water containing 100 mM of potassium phosphate
buffer and 1 M of ammonium sulfate, pH = 6.8. the HIC.” In addition,
the correct version of Table 3 is appended below.
The authors would like to apologise for any inconvenience caused.

Techniques SEC IEX HIC

Samples Concentration 0.5 0.5 0.5 (5 for brentuximab vedotin)

(mg/mL)
LC Mobile phase Water containing 50 mM potassium A: water containing 10 mM MES, A: water containing 100 mM
condi- phosphate and 250 mM potassium pH = 6.0 potassium phosphate buffer and 1 M
tions chloride, pH = 6.8 ammonium sulfate, pH = 6.8.
B: water containing 10 mM MES and B: water containing 100 mM
1 M sodium chloride, pH = 6.0 potassium phosphate buffer,
pH = 6.8.
Gradient list NA Time (min) 0 20 20.2 21 21.2 30 Time (min) 0 40 45 45.2 60
A (%) 100 80 0 0 100 100 A (%) 100 0 0 100 100
B (%) 0 20 100 100 0 0 B (%) 0 100 100 0 0
Injection 0.5 1.0 5.0
volume (μL)
Column Agilent AdvanceBioSEC column YMC BioPro SP-F non-porous strong Thermo Fisher MAbPac HIC-10
(2.7 μm, 150 mm × 4.6 mm, 300 Å) cation exchange column (5.0 μm, column (5.0 μm, 250 × 4.6 mm,
100 mm × 4.6 mm) 1000 Å)
Column oven 25 25 25
temperature
(°C)
Flow rate 0.35 0.6 0.8
(mL/min)
First 60 15 35
equilibration
time (min)

DOI of original article: http://dx.doi.org/10.1016/j.jchromb.2017.05.010

⁎
Corresponding author.
E-mail address: davy.guillarme@unige.ch (D. Guillarme).

http://dx.doi.org/10.1016/j.jchromb.2017.10.003

Available online 10 October 2017

1570-0232/
A. Goyon et al. Journal of Chromatography B 1067 (2017) 89–90

Autosampler Post injection Water: methanol (85:15, v/v), 6 s Water: methanol (85:15, v/v), 6 s Water: methanol (85:15, v/v), 6 s
wash
Autosampler 8 8 8
temperature
(°C)
FL Excitation; 280; 340 280; 340 280; 340
condi- emission (nm,
tions nm)

90