Multiple Heartcut 2D-LC/MS: a powerful tool for biopharma analysis in a regulated environment (WEBAF, Dublin 2019) Visit www.quality-assistance.com for more information

1. 1Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Multiple Heartcut
2D-LC/MS: a powerful tool for
biopharma analysis in a regulated
environment
Arnaud Delobel, PhD
Director, R&D
Waters European Biopharma Analytical Forum – Dublin – October 16th 2019

2. 3Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Back to WEBAF 2019: conclusions of my talk
• Current situation:
▪ MassLynx (without Security Pack!!) to
control 2D-LC with Xevo G2-XS QTOF
and perform data acquisition
▪ Data transfer to UNIFI
▪ Data processing and reporting within
UNIFI
But compliance is OK when 2D-LC is used with MassLynx on a triple-quad system

3. 4Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Back to WEBAF 2019: conclusions of my talk
Manchester, April 6th 2016 …
Future developments :
▪ Implement the use of UNIFI for
2D-LC/MS analyses for
GxP-compliant work
▪ Switch from single to multiple-
heart-cutting to improve
throughput

4. 5Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Agenda
• Multiple-heartcut 2D-LC: what is it used for, how does it work?
• Case study: Characterisation of antibody-drug-conjugates
• Using the capabilities of 2D-LC with At-Column Dilution technology:
presentation of the work in progress

5. 6Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Multiple Heartcut 2D-LC/MS
at Quality Assistance
Introduction

6. 7Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
2D-LC/MS systems at Quality Assistance sa
• Characterisation:
▪ Waters Xevo G2-XS QTOF
▪ 1D or 2D-UPLC (H-Class Bio ; I-Class)
▪ Control by UNIFI (1D and 2D)
▪ Full GMP compliance
• Quantitative analyses:
▪ Waters Xevo TQS
▪ 2D-UPLC (H-Class Bio ; I-Class)
▪ Control by MassLynx
▪ Full GMP compliance

7. 8Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
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

8. 9Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
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
environmentsSarrut et al., J. Chromatogr. B (2016) - 10.1016/j.jchromb.2016.06.048

9. 10Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Trapping columns and At-Column Dilution
 Short column packed with stationary phase
 Typical dimensions of trapping column:
 20 or 30 mm L x 2.1 mm ID x 5 or 10 µm dp
 Stationary phase selection is dependent on
application:
 Good starting point: Dim 2 stationary phase
 For ADC: C4 / Polyphenyl (Bioresolve RP)
 Other applications: C8, C18
 When higher retentivity is required: more
retentive material (HSS T3, Oasis HLB)
 User has full flexibility and decides how to use the
system
 Patented technology
 Empty storage loop
 Loop size determines maximum volume of the
fraction to transfer
 Loop size also determines injection volume to Dim 2
– solvent effects and peak fronting!!
 Once the loop is installed, the user has little flexibility
in system operation
Interface with trapping columnsInterface with storage loops

10. 11Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
SM-FTN
QSM-Dim1
TUV
BSM-Dim2
CM-A – 2D
ISM
Single heartcut 2DLC
One injection on 2D system
Transfer of one fraction to Dim 2
PDA
SM-FTN
PDA
CM-A – 2D
CM-A -Traps
Multiple heartcut 2DLC
One injection on 2D system
Transfer of up to seven fractions to Dim 2
QSM-Dim1
BSM-Dim2
ISM
TUV
Heartcut
Heartcut 3Heartcut 1 Heartcut 2 ...
Single vs multiple heartcut 2D-LC
Slide courtesy of Isabelle François, Waters Corp.

11. 12Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Heartcut 1 Heartcut 2
Multiple Heartcut 2D-LC in practice
Acquire 1st dim. chromatogram and determine fractions of interest
Slide courtesy of Isabelle François, Waters Corp.

12. 13Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Dimension 1
1
3
5
2
4
6
VL
1
3
5
2
4
6
VR
Traps
Dimension 2
PH
PH
Waste
1
SM-FTN
QSM/BSM –
Dim 1
PDA – Dim 1
BSM – Dim2
ISM/BSM-ACD
TUV – Dim 2
CM-A – Traps
CM-A - 2D
2 1
22
Multiple Heartcut 2D-LC in practice
Start of 1st dimension analysis
Slide courtesy of Isabelle François, Waters Corp.

13. 14Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Dimension 1
1
3
5
2
4
6
VL
1
3
5
2
4
6
VR
Traps
Dimension 2
PH
PH
Waste
SM-FTN
QSM/BSM –
Dim 1
PDA – Dim 1
BSM – Dim2
ISM/BSM-ACD
TUV – Dim 2
CM-A – Traps
CM-A - 2D
2 2
Multiple Heartcut 2D-LC in practice
Send 1st dim. effluent to 1st dim. detector (fractions not of interest)
Slide courtesy of Isabelle François, Waters Corp.

14. 15Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Dimension 1
1
3
5
2
4
6
VL
1
3
5
2
4
6
VR
Traps
Dimension 2
PH
PH
Waste
2
SM-FTN
QSM/BSM –
Dim 1
PDA – Dim 1
BSM – Dim2
ISM/BSM-ACD
TUV – Dim 2
CM-A – Traps
CM-A - 2D
2 1
33
Multiple Heartcut 2D-LC in practice
Transfer of Heartcut 2 to Trap 2
Slide courtesy of Isabelle François, Waters Corp.

15. 16Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Dimension 1
1
3
5
2
4
6
VL
1
3
5
2
4
6
VR
Traps
Dimension 2
PH
PH
Waste
1
2
SM-FTN
QSM/BSM –
Dim 1
PDA – Dim 1
BSM – Dim2
ISM/BSM-ACD
TUV – Dim 2
CM-A – Traps
CM-A - 2D
2 2
Multiple Heartcut 2D-LC in practice
Optional: desalting of all trap cartridges, compounds remain focused
Slide courtesy of Isabelle François, Waters Corp.

16. 17Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Dimension 1
1
3
5
2
4
6
VL
1
3
5
2
4
6
VR
Traps
Dimension 2
PH
PH
Waste
SM-FTN
QSM/BSM –
Dim 1
PDA – Dim 1
BSM – Dim2
ISM/BSM-ACD
TUV – Dim 2
CM-A – Traps
CM-A - 2D
1 2
1
2
Multiple Heartcut 2D-LC in practice
Analyse fractions in 2nd dimension
Slide courtesy of Isabelle François, Waters Corp.

17. 18Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Characterisation of
Antibody-Drug Conjugates
(ADCs) by HIC-RP/MS
Case study

18. 19Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Benefits of 2D-LC/MS for the characterisation of ADCs
• Hydrophobic Interaction Chromatography (HIC) is a method of choice for the
characterisation of ADCs
▪ Determination of naked antibody for Lys-linked conjugates (or site-specific conjugation technologies)
▪ Determination of DAR and drug load distribution for Cys-linked conjugates
• Due to high salt content in mobile phase, it is impossible to hyphenate the
chromatography to MS for peak identification
• Fraction collection and desalting is time-consuming and is not easily amenable to low
concentration species (due to dilution effects)
2D-LC/MS is the solution for a quick identification
of peaks observed on HIC chromatograms

19. 20Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
1st dimension – HIC separation
DAR calculation based on UV280 signal
Calculated DARUV280 = 4.0
0
2 4
6
8
#drugs

20. 21Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
1st dimension – HIC separation – 5 Heartcuts
Analysis id 1067

21. 22Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
• Low signal → poor quality deconvolution
Heartcut #1 - DAR 0
Expected mass: 148079 Da

22. 23Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Heartcut #2 - DAR 2 – Identification of sub-species
Light chain + 1 drug Light chain + 2 heavy chains + 1 drug

23. 24Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Heartcut #3 - DAR 4 – Isoform 1
Light chain
+ heavy chain + 2 drugs
2 heavy chains + 2 drugsLight chain + 1 drug

24. 25Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Heartcut #3 - DAR 4 – Isoform 2
Light chain + heavy chain + 2 drugs

25. 26Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Heartcut #4 – DAR 6
Heavy chain + 3 drugs
Light chain +
heavy chain
+ 2 drugs
Light chain
+ 1 drug

26. 27Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Heartcut #5 - DAR 8
Light chain + 1 drug Heavy chain + 3 drugs

27. 28Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
DARaverage (HIC/UV) = 4.0
HIC-RP analysis of Adcetris®

28. 29Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Implementation of new
workflows using 2D-LC/MS
for biotherapeutics
development
Work in progress

29. 30Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Analysis of in-process samples using 2D-LC/MS
• Aim of the test: to be able to characterise monoclonal antibodies in
complex in-process samples, without sample pre-treatment
• 1st dimension: protein A affinity column
• 2nd dimension: reversed-phase for desalting of the sample before MS
analysis

30. 31Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
• 1st D – Protein A
▪ Column Protein A (Thermo)
▪ Eluent A: 1X PBS, pH 7.5
▪ Eluent B: 1X PBS, 12mM HCl, pH 2.4
▪ Flow rate: 0.3 mL/min
• 2nd D - RP
▪ Column Bioresolve RP mAb polyphenyl
▪ Eluent A: 0.1% FA in H2O
▪ Eluent B: 0.1% FA in ACN
• Trap (x3)
▪ Xbridge C4
▪ Eluent: 0.1% FA in H2O
▪ 1 mL/min for 10 min for each trap column
Analysis of in-process samples using 2D-LC/MS
Experimental conditions
Time (min) 0 1 1.02 3.5 3.55 9
%A 100 100 0 0 100 100
%B 0 0 100 100 0 0
Time (min) 0 2 3 13 15 17 19 21 23
%A 95 95 80 10 10 90 10 95 95
%B 5 5 20 90 90 10 90 5 5
Flow rate (mL/min) 0.3 0.2 0.2 0.2 0.3 0.3 0.3 0.3 0.3

31. 32Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Analysis of in-process samples using 2D-LC/MS
Preliminary results

32. 33Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Going further for analysis in the second dimension …
• Analysis of the intact antibody does not allow to detect everything
▪ Analysis of 150 kDa species is not ideal
▪ Resolution is limited
▪ Mass accuracy is limited
• What if we could generate sub-units before 2nd dimension?
• Idea:
▪ Use the trap columns to generate the sub-units
▪ On-column reduction using TCEP to generate light and heavy chains

33. 34Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
On-column reduction: preliminary tests
• Injection of Humira on the trap column (BEH300 C4)
• Reduction
▪ Trap at 50°C
▪ TCEP in 10% ACN at 0.5mL/min for 5-10 min
• Rinsing step
▪ 0.1% FA in H2O at 0.5 mL/min for 10min
• 2nd D - RP
▪ Eluent A: 0.1% FA in H2O
▪ Eluent B: 0.1% FA in ACN

34. 35Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
On-column reduction: preliminary results
Light chain
Heavy chain

35. 36Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Quantification of free drugs in ADCs
• Quantification of free drug in ADCs is critical for batch release and
during stability studies
• Cytotoxic payloads are extremely toxic and low limit of quantifications
should be achieved
• MS is often required to reach low LOQ, and the protein has to be
removed (offline or online) before the analysis
• 2D-LC/MS is a way to meet all those criteria

36. 37Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Experimental conditions
• 1st D – on-line SPE
▪ Oasis MAX 20*2.1 mm, 30 µm
▪ Eluent (A) 2% FA in H2O / (B) 2% FA in ACN
• 2nd D - RP
▪ Eluent (A) 0.1 % FA in H2O / (B) 0.1 % FA in ACN
▪ Column BEH C18 50x2.1 mm, 1.7 µm (QA ref UPLC.1.7)
• Trap
▪ Xbridge C18
▪ 0.1 % FA in H2O
▪ 0.3 mL/min for 10 minutes
• DM1 solubilised at 1 mg/mL in ACN and further diluted in eluent A (from 10 to 0.05 µg/mL)
• MS analysis in positive mode
• Full MS (m/z 50-2500)
• SIR on m/z 738.3
0 1 6 14 14.5 16
% A 90 90 50 10 90 90
% B 10 10 50 90 10 10
Flow rate 0.3 mL/min
Flow rate 0.1 mL/min

37. 38Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Preliminary results

38. 39Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
What’s next?
• Online digestion using:
▪ Immobilised IdeS (FaBRICATOR®) followed by on-column reduction and RP-LC
separation for subunit analysis
▪ Immobilised trypsin followed by RP-LC separation for fully automated peptide mapping
• Qualification of the 2D-LC system for full GMP compliance
Interested in a collaboration?

39. 40Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
Acknowledgements
Camille ALLAIN
Claire BUTRÉ
Isabelle FRANÇOIS

40. 41Arnaud Delobel, PhD – Waters European Biopharma Analytical Forum – Dublin – October 16th 2019
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
Any question?