Slides presented at SEP21 conference in Paris on Oct 6th 2021

1. Nouvelles approches en 2D-LC/MS
pour l’analyse de protéines thérapeutiques
dans un environnement régulé
Arnaud DELOBEL
Quality Assistance sa
Donstiennes, Belgique

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Arnaud Delobel – SEP 2021 – October 6th 2021
Quality Assistance sa
100% analytical services
100% (bio)pharmaceutical industry
214 highly-qualified employees
> 60% university graduates
69 worldwide R&D companies
& 15 projects (2020)
Product dedicated support
Customised project management
Compliance with EMA / FDA regulations
From discovery to market place
All laboratories on one site
5700 m²
35 years experience
~1.6 M € in machinery
& equipment (2020)
https://www.quality-assistance.com

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Arnaud Delobel – SEP 2021 – October 6th 2021
2D-LC/MS at Quality Assistance
• 2D-UPLC (H-Class Bio ; I-Class)
• Waters Xevo G2-XS QTOF
• Control by UNIFI / Full GMP compliance
• Multiple-heartcut mode
• At-column-dilution technology

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Arnaud Delobel – SEP 2021 – October 6th 2021
2D-LC: comprehensive (LCxLC) vs heart-cutting (LC-LC)
• Comprehensive HICxRP analysis of an ADC:
• Not robust enough for routine use
• Complex data interpretation
• Need for a specific software
solution
• Not ready for regulated
environments
Sarrut et al., J. Chromatogr. B (2016) - 10.1016/j.jchromb.2016.06.048

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Arnaud Delobel – SEP 2021 – October 6th 2021
2D-LC/MS and data integrity
• For the use in a regulated
environment:
▪ Need for qualified equipment
(IQ/OQ provided by the supplier)
▪ Need for validated software
(21 CFR Part 11 compliance)
▪ Transfer between software
should be avoided

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Arnaud Delobel – SEP 2021 – October 6th 2021
Heartcut 1 Heartcut 2
Multiple Heartcut 2D-LC in practice
Acquire 1st dim. chromatogram and determine fractions of interest
Slide courtesy of Waters Corp.

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Arnaud Delobel – SEP 2021 – October 6th 2021
Multiple Heartcut 2D-LC in practice
Start of 1st dimension analysis
Traps
SM-FTN
QSM/BSM
– Dim 1
PDA – Dim 1
BSM – Dim2
BSM-ACD
CM-A – Traps
CM-A - 2D
QTOF – Dim 2
1st dimension
2nd dimension
Waste
2 2

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Arnaud Delobel – SEP 2021 – October 6th 2021
Multiple Heartcut 2D-LC in practice
Transfer of Heartcut 1 to Trap 1
Traps
SM-FTN
QSM/BSM
– Dim 1
PDA – Dim 1
BSM – Dim2
BSM-ACD
CM-A – Traps
CM-A - 2D
QTOF – Dim 2
1st dimension
2nd dimension
Waste
2 1

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Arnaud Delobel – SEP 2021 – October 6th 2021
Traps
SM-FTN
QSM/BSM
– Dim 1
PDA – Dim 1
BSM – Dim2
BSM-ACD
CM-A – Traps
CM-A - 2D
Multiple Heartcut 2D-LC in practice
Send 1st dim. effluent to 1st dim. detector (fractions not of interest)
QTOF – Dim 2
1st dimension
2nd dimension
Waste
2 2

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Arnaud Delobel – SEP 2021 – October 6th 2021
Traps
SM-FTN
QSM/BSM
– Dim 1
PDA – Dim 1
BSM – Dim2
BSM-ACD
CM-A – Traps
CM-A - 2D
Multiple Heartcut 2D-LC in practice
Transfer of Heartcut 2 to Trap 2
QTOF – Dim 2
1st dimension
2nd dimension
Waste
2 1

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Arnaud Delobel – SEP 2021 – October 6th 2021
Multiple Heartcut 2D-LC in practice
Optional: desalting of all trap cartridges, compounds remain focused
Traps
SM-FTN
QSM/BSM
– Dim 1
PDA – Dim 1
BSM – Dim2
BSM-ACD
CM-A – Traps
CM-A - 2D
QTOF – Dim 2
1st dimension
2nd dimension
Waste
2 2

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Arnaud Delobel – SEP 2021 – October 6th 2021
Multiple Heartcut 2D-LC in practice
Analyze fractions in 2nd dimension (sequentially)
Traps
SM-FTN
QSM/BSM
– Dim 1
PDA – Dim 1
BSM – Dim2
BSM-ACD
CM-A – Traps
CM-A - 2D
QTOF – Dim 2
1st dimension
2nd dimension
Waste
1 2

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Arnaud Delobel – SEP 2021 – October 6th 2021
Our applications of 2D-LC/MS
Characterisation
of monoclonal
antibodies
Identification of
charge variants
Identification of
mass variants
Characterisation
of ADCs
Identification of
isomers
Identification of
small molecule
impurities
Quantification of free
drug and related
species

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Arnaud Delobel – SEP 2021 – October 6th 2021
WCX-HPLC profile of Adalimumab after basic stress (100 mM Tris, pH 8)
 Heart-cutting of each peak, desalting and MS analysis
Analysis of charge variants by 2D-LC/MS

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Arnaud Delobel – SEP 2021 – October 6th 2021
Analysis of charge variants by 2D-LC/MS
• Species identification after desalting & 2D-LC

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Arnaud Delobel – SEP 2021 – October 6th 2021
Application of HIC-RP 2D-LC/MS TO ADCETRIS®

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Arnaud Delobel – SEP 2021 – October 6th 2021
Advanced use of the 2D-LC/MS system with at-column dilution
technology
Ideas:
▪ Optimise the use of trap columns for advanced analyses
• Improve the peak shape in the second dimension (RP)
• Perform on-column reduction
▪ Use affinity columns or immobilised enzymes in the first
dimension
• Online sample purification
• Online sample preparation

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Arnaud Delobel – SEP 2021 – October 6th 2021
Optimisation of trap elution – influence of the temperature of
the trap column
Trap at RT
Trap at 80°C
mAb
Injection of Humira on trap column and elution on RP
Dimension 2 operated at 80°C
Elution is improved at high
trap column temperature

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Arnaud Delobel – SEP 2021 – October 6th 2021
Experimental setup - online reduction with TCEP
Injection of 10 µg Humira on trap column
LC HC
Trap at RT
Trap at 50°C
Trap at 80°C
Trap column
• Bioresolve RP mAb polyphenyl
• Reduction with 1 mM TCEP in 10/90 ACN/H2O
5 min at 0.5 mL/min
• Desalting with 0.1 % FA in H2O
10 min at 0.5 mL/min
Dimension 2
• Bioresolve RP mAb polyphenyl
• Mobile phases:
• 0.1 % FA in H2O
• 0.1 % FA in ACN
• Temperature: 80°C
Multiple peaks for both
light and heavy chains

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Arnaud Delobel – SEP 2021 – October 6th 2021
Setup online reduction – with DTT
Trap column
▪ Bioresolve RP mAb polyphenyl
▪ Reduction 10 mM DTT in ammonium bicarbonate
5 min at 0.5 mL/min
▪ Desalting 0.1% FA in H2O - 10 min at 0.5 mL/min
▪ Temperature: 80°C
Dimension 2
▪ Bioresolve (RP mAb polyphenyl)
▪ Mobile phases:
• 0.1 % FA in H2O
• 0.1 % FA in ACN
▪ Temperature: 80 °C
Only one peak of LC or HC
 all S-S bonds are reduced
LC HC
Injection of 10 µg Humira on trap column
LC
HC

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Arnaud Delobel – SEP 2021 – October 6th 2021
Use of Protein A affinity chromatography in 1st dimension
• Goal: Characterisation of mAbs present in complex mixtures
(eg. cell culture supernatants)
• Use of a protein A affinity column for purification purposes on dimension 1
▪ Step gradient for binding followed by elution

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Arnaud Delobel – SEP 2021 – October 6th 2021
Experimental setup – Characterisation of intact mAb
0.5 µg Humira
on column
Dimension 1 – Protein A
• MabPAC Protein A 35 x 4 mm (Thermo)
• Mobile phases:
▪ PBS, pH 7.5
▪ PBS, 12mM HCl, pH 2.4
Trap
• Bioresolve RP mAb Polyphenyl 30 x 2.1 mm (Waters)
• Desalting: 10 min at 0.15 mL/min – 0.1 % FA in H2O
• Column temperature: 80°C
Dimension 2 - RP
• Bioresolve RP mAb Polyphenyl 50 x 2.1 mm (Waters)
• Mobile phases:
▪ 0.1 % FA in H2O
▪ 0.1 % FA in ACN
• Column temperature: 80°C

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Arnaud Delobel – SEP 2021 – October 6th 2021
Analysis of mAbs in cell culture media
No influence of medium on results
0.4 µg Humira
(Drug Product Formulation)
0.4 µg Humira
spiked in cell culture medium

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Arnaud Delobel – SEP 2021 – October 6th 2021
Experimental setup – Characterisation of mAb with online
reduction
Dimension 1 – Protein A
• MabPAC Protein A 35 x 4 mm (Thermo)
• Mobile phases:
▪ PBS, pH 7.5
▪ PBS, 12mM HCl, pH 2.4
Trap
• Bioresolve RP mAb Polyphenyl 30 x 2.1 mm (Waters)
• Reduction with 10 mM DTT in 50 mM ABC (5 min at 0.5 mL/min)
• Desalting - 0.5 mL/min with 0.1 % FA in H2O
• Column temperature: 80°C
Dimension 2 - RP
• Bioresolve RP mAb Polyphenyl 50 x 2.1 mm (Waters)
• Mobile phases:
▪ 0.1 % FA in H2O
▪ 0.1 % FA in ACN
• Column temperature: 80°C

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Arnaud Delobel – SEP 2021 – October 6th 2021
Characterisation of mAb with on-trap DTT reduction
Light chain
Heavy chain 0.2 µg Humira injected
Light Chain
Heavy Chain

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Arnaud Delobel – SEP 2021 – October 6th 2021
IdeS digestion of mAbs (FabRICATOR® enzyme – Genovis)
www.genovis.com

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Arnaud Delobel – SEP 2021 – October 6th 2021
Setup – Dimension 1 – FabRICATOR® HPLC column
• Initial test with PBS 1x as eluent
• Chosen conditions: 200 mM ammonium acetate
200 mM ammonium acetate
Nivolumab
Humira
Adcetris

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Arnaud Delobel – SEP 2021 – October 6th 2021
Dimension 1
▪ FabRICATOR HPLC column
▪ Eluent: 200 mM ammonium acetate
▪ Isocratic for ~3 CV at 0.025 mL/min
Trap
▪ Bioresolve RP mAb polyphenyl, 30 x 2.1 mm
▪ Temperature: 80°C
Dimension 2
▪ MAbPac RP 50 x 3 mm, 4 µm
▪ Eluent A: 0.05 % FA + 0.05 % TFA in H2O
▪ Eluent B: 0.05 % FA + 0.05 % TFA in ACN
▪ Temperature: 80 °C
Experimental conditions – mAb subunit analysis
The two expected species are obtained by digestion on
the FabRICATOR column and separated on RP (Dim 2)
Fc/2
F(ab’)2
5 µg Humira
on column

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Arnaud Delobel – SEP 2021 – October 6th 2021
Automated subunit analysis – Humira – ESI-Qtof MS spectra
Fc/2
F(ab’)2

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Arnaud Delobel – SEP 2021 – October 6th 2021
Dimension 1
▪ FabRICATOR HPLC column
▪ Eluent: 200 mM ammonium acetate
▪ Isocratic for ~3 CV at 0.025 mL/min
Trap
▪ Bioresolve RP mAb polyphenyl, 30 x 2.1 mm
▪ Reduction: 10 mM DTT in ABC – 5 min at 0.5 mL/min
▪ Desalting 0.05 % FA + 0.05 % TFA in H2O
10 min at 0.5 mL/min
▪ Temperature: 80°C
Dimension 2
▪ MAbPac RP 50 x 3 mm, 4 µm
▪ Mobile phase A: 0.05 % FA + 0.05 % TFA in H2O
▪ Mobile phase B: 0.05 % FA + 0.05 % TFA in ACN
▪ Temperature: 80 °C
Experimental conditions – mAb subunit analysis
with online reduction
Fc/2
LC
Fd
1 µg Opdivo
on column

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Arnaud Delobel – SEP 2021 – October 6th 2021
Automated subunit analysis of Nivolumab with online
reduction
Fc/2
LC
Fd

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Arnaud Delobel – SEP 2021 – October 6th 2021
Case study: monitoring of mAb oxidation at the subunit level
by automated IdeS digestion and online reduction
• Oxidative stress of mAb Nivolumab by incubation with H2O2 at two
levels for 45 minutes.
• Stopped by addition of methionine
• Injection on 2D-LC/MS system with FabRICATOR column for IdeS
digestion, online reduction and RP elution

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Arnaud Delobel – SEP 2021 – October 6th 2021
Online subunit analysis of Nivolumab at different oxidation levels
(IdeS digested and DTT reduced)
Fc/2
LC Fd
Non-oxidized
Ox-1
Ox-2
Ox-1
Ox-1
Non-oxidized
Non-oxidized
Control
0.075 % H2O2
0.2 % H2O2

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Arnaud Delobel – SEP 2021 – October 6th 2021
Automated identification for Fc/2 subunit species
at different oxidation levels
Automatic identification of the different species and calculation of the
percentage of modification within UNIFI
Control
0.075 % H2O2
0.2 % H2O2
No H2O2 0.075 % H2O2 0.2 % H2O2

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Arnaud Delobel – SEP 2021 – October 6th 2021
What’s next?

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Arnaud Delobel – SEP 2021 – October 6th 2021
What’s next?

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Arnaud Delobel – SEP 2021 – October 6th 2021
Take-home messages
• 2D-LC/MS can be used in regulated environments for the
characterisation of biotherapeutics
• The at-column dilution technology offers new possibilities for on-line
sample reduction
• Affinity columns or immobilised enzymes can be used in the first
dimension for automated sample pre-treatment
 automated in-process control or QC analyses at subunit levels (MAM)
• New developments (3D, 4D-LC) will open up even greater perspectives

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Arnaud Delobel – SEP 2021 – October 6th 2021
Acknowledgements
Camille ALLAIN
Claire BUTRÉ
Isabelle FRANÇOIS
(Chromlsa)

39. Special Issue "Early Career Stars in Analysis of Natural
Products and Pharmaceuticals"
Special Issue Editor
Dr. Irina Ielciu
Department of Pharmaceutical Botany, Iuliu
Haţieganu University of Medicine and
Pharmacy, 400337 Cluj-Napoca, Romania
ielciu_irina@yahoo.com
Dr. Arnaud Delobel
Strategy & Innovation, Quality Assistance sa,
Thuin, Belgium
arnaud.delobel@quality-assistance.be
For further reading, please follow the link to the Special Issue Website at:
https://www.mdpi.com/journal/separations/special_issues/Products_Pharmaceutical
Keywords
• natural products
• pharmaceuticals
• separation techniques
• chemical analysis
Contact: separations@mdpi.com
or ethan.xu@mdpi.com

40. Editor-in-Chief
Prof. Dr. Frank L. Dorman
The Pennsylvania State University,
University Park, PA 16802, USA
E-mail: separations@mdpi.com
Twitter: @Sep_MDPI
2.777
Impact Factor
SCIE-WoS
3.4
CiteScore
Scopus
Indexing
Sections
• Analysis of Food and Beverages
• Analysis of Fragrances
• Analysis of Energies
• Analysis of Natural Products and
Pharmaceuticals
• Bioanalysis/Clinical Analysis
• Environmental Analysis
• Forensics/Toxins
• Materials in Analysis
Associate Editor
Prof. Dr. Victoria Samanidou
Aristotle University of Thessaloniki,
Thessaloniki, Greece

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Arnaud Delobel – SEP 2021 – October 6th 2021
arnaud.delobel@quality-assistance.be
+32 71 53 47 81
www.quality-assistance.com
Technoparc de Thudinie, 2
B-6536 Donstiennes (Belgium)
Thank you
for your
attention!