Stratégies orthogonales pour la caractérisation de glycoprotéines thérapeutiques par LC/MS (SEP, Paris 2019)

1
Stratégies orthogonales pour la
caractérisation de
glycoprotéines thérapeutiques
par LC/MS
Arnaud Delobel, PhD
SEP 2019, Paris – 28 mars 2019

2
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mAbs expertise
(2018)

3
• Numerous drugs are
glycoproteins
▪ mAbs (adalimumab, infliximab ,
rituximab, trastuzumab, …)
▪ Recombinant-DNAengineered proteins
(Etanercept, EPO,
lenograstim – rhG-CSF, …)
• Glycosylation is critical for the
efficacy and safety of the drug
▪ Solubility
▪ Stability
▪ Pharmacokinetics and dynamics
▪ Conformation/binding→ biological
activity!
Glycosylation of therapeutic proteins
• Two main types of glycosylation
▪ N-glycosylation: Asn-X-Ser and Asn-X-
Thr (X ≠ Pro)
▪ O-glycosylation: Ser and Thr
• Organism-dependent heterogeneity
▪ Micro-heterogeneity: glycan nature for a
given glycosylation site
▪ Macro-heterogeneity: number and
position of glycosylation sites

4
Glycosylated proteins
Beck et al. Anal. Chem. 85, 715–736 (2012).

6
Model compounds: adalimumab and etanercept

7
Analysis at the
glycan level

8
Use of RapiFluor-MS for
quick sample prep and high
sensitivity in both FLR and
MS
N-glycans release and derivatisation
Adapted from Waters poster « Rapid preparation of released N-glycans
for HILIC analysis using a novel fluorescence and MS-active labeling reagent » (2015)

9
Dextran ladder injection
RT > GU
Sample injection
RT-basedcandidate identification
MS-based identification confirmation
database
Identification principle for RFMS-derivatised glycans

10
Analysis of released N-glycans in HILIC mode
Acquity UPLC BEH Glycan (Waters)
150 x 2.1 mm, 1.7µm
Monoclonal antibody standard
RapiFluor-MS labeling
3 independent sample preparations

11
Analysis of released N-glycans in HILIC mode
Acquity UPLC BEH Glycan (Waters)
150 x 2.1 mm, 1.7µm
Etanercept
RapiFluor-MS labeling

12
Gain in sensitivity using RapiFluor-MS vs 2-AB
FLR
Injection of 10x less material
Similar FLR signal (i.e. 10x sensitivity gain)
10x increase in MS (i.e. 100x sensitivity gain)
Low energy MSE
High energy MSE
0.6% relative intensity
RFMS
z = 3
2-AB
z = 2

13
Analysis of released N-glycans in AEX-RP
mixed-mode chromatography
GlycanPac AXR-1 (Thermo Scientific)
150 x 2.1 mm, 1.9 µm
Fetuin
Etanercept
neutral mono di tri tetra penta
dye
dye

14
Analysis of released N-glycans in AEX-HILIC
mixed-mode chromatography
3 independentpreparations 1.4% mean RSD
GlycanPac AXH-1 (Thermo Scientific)
250 x 2.1 mm, 1.9 µm
EU
0.0
25.0
50.0
75.0
100.0
125.0
150.0
175.0
Minutes
9.5 10.0 10.5 11.0
Minutes
10.0 10.5 11.0 11.5
neutral
mono
di
tri
tetra
penta
EtanerceptFetuin
neutral
mono
di

15
• No universal enzyme for O-glycans release
• An optimised chemical release was used
▪ Alkaline β-elimination under reducing conditions
▪ Limited peeling side-reaction
▪ No labeling
• Separation on porous graphitic carbon (PGC) stationary phase on a 1 mm
column
• Detection by ESI-QTOF/MS in negative ionisation mode
• Customised workflow within UNIFI to identify the O-glycans based on MS
and MSE data
Challenges of O-glycans analysis

16
Analysis of O-glycans by PGC-MSE
Hypercarb (Thermo Scientific)
100 x 1.0 mm, 3 µm
Fetuin
Etanercept
peeling
Low energy
In-source fragments
High energy

17
Analysis at the
peptide level

18
Analysis in reversed phase mode
XIC @ m/z 204.1
on high energy
function
(fragment ion characteristic of
glycosylated peptides)

19
Analysis in HILIC mode
ACQUITY UPLC Glycoprotein Amide 300 Å (Waters )
150 x 2.1 mm, 1.7 µm
Glycopeptides
Reference
N-deglycosylated

20
Analysis in HILIC mode
ACQUITY UPLC Glycoprotein Amide 300 Å (Waters )
150 x 2.1 mm, 1.7 µm
Glycopeptides
Reference
N-deglycosylated

21
Analysis of Etanercept O-glycosylated peptides
LPAQVAFTPY APEPGSTCRL REYYDQTAQM CCSKCSPGQH
AKVFCTKTSD TVCDSCEDST YTQLWNWVPE CLSCGSRCSS
DQVETQACTR EQNRICTCRP GWYCALSKQE GCRLCAPLRK
CRPGFGVARP GTETSDVVCK PCAPGTFSNT TSSTDICRPH
QICNVVAIPG NASMDAVCTS TSPTRSMAPG AVHLPQPVST
RSQHTQPTPE PSTAPSTSFL LPMGPSPPAE GSTGDEPKSC
DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT
CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK
GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE
WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG
NVFSCSVMHE ALHNHYTQKS LSLSPGK
89 putative O-glycosylation sites (bold) (19% of the whole sequence!)
Trypsin: 65 aa, 29 aa
De-N-glycosylation
Desialylation
Triple digestion

22
Glycopeptides discovery
5 peptides with 1 to 7 core 1
Glycopeptide confirmation (MSE)

23
Trypsin + Glu-C + Asp-N:
• 5 glycopeptides
• 24 remaining sites

24
ETD MS/MS spectrum of m/z 520.6 (z = 3)
D A V C T S T S P T R Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg
Ion 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11
c D A V C T S T S P T* R 133.061 204.098 303.166 463.197 564.245 651.277 752.324 839.356 936.409 1402.589 1558.690
c-1 D A V C T S T S P T* R 132.053 203.090 302.158 462.189 563.237 650.269 751.317 838.349 935.401 1401.581 1557.682
c+1 D A V C T S T S P T* R 134.069 205.106 304.174 464.205 565.252 652.284 753.332 840.364 937.417 1403.597 1559.698
z D A V C T S T S P T* R 158.092 624.272 721.325 808.357 909.405 996.437 1097.484 1257.515 1356.584 1427.621 1542.648
z+1 D A V C T S T S P T* R 159.100 625.280 722.333 809.365 910.413 997.445 1098.492 1258.523 1357.591 1428.628 1543.655
z+2 D A V C T S T S P T* R 160.108 626.288 723.341 810.373 911.420 998.452 1099.500 1259.531 1358.599 1429.636 1544.663
11 10 9 8 7 6 5 4 3 2 1 Arg Thr Pro Ser Thr Ser Thr Cys Val Ala Asp
m/z200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
50
100
150
200
250
300
350
400
Abs. Int. * 1000
c C T S T T*
c-1 T T*
c+1 C T S T T* R
z+1 T S T A D
z+2 A D
624.666
z 2
624.666
z+1 2
626.427
z+2 2
303.204
c 3
304.178
c+1 3
463.275
c 4
462.336
c-1 4
464.404
c+1 4
809.417
z+1 4
810.489
z+2 4
564.264
c 5
565.345
c+1 5
910.445
z+1 5
651.382
c 6
650.930
c-1 6
652.408
c+1 6
997.427
z+1 6
752.365
c 7
751.389
c-1 7
753.387
c+1 7
1097.460
z 7
1098.455
z+1 7
1099.429
z+2 7
936.453
c 9
934.927
c-1 9
937.553
c+1 9
1357.720
z+1 9
1358.499
z+2 9
1402.690
c 10
1400.802
c-1 10
1403.684
c+1 10
1428.730
z+1 10
1429.703
z+2 10
1559.781
c+1 11
1543.702
z+1 11
1544.690
z+2 11 (772.85 2+)
(772.85 2+)
z+2 11 1544.690
• Regular MS/MS (CID) does not allow the localisation of glycosylationsites
• Accurate localisation of O-glycosylation sites by ETD fragmentation
DAVCTSTSPTRDAVCTSTSPTRDAVCTSTSPTR

25
8.5% 71% 95%
98%
2.1%
8.9%
100%
100%
100%
58.3%
100%
94.5%
100%
O-glycosylated
amino-acids: 13
< 15% of putative sites

26
Analysis at the
subunit level

27
• IdeS Protease:
▪ derived from Streptococcus
pyogenes.
▪ Engineered, recombinant
protease overexpressed
in E. coli.
Subunit analysis using IdeS or IdeZ enzymes
• IdeZ Protease:
▪ derived from Streptococcus
equi subspecies zooepidemi
cus.
▪ Engineered, recombinant
protease overexpressed
in E. coli.
▪ Significantly improved
activity against mouse
IgG2a and IgG3 compared
to IdeS
https://www.genovis.com/applications/antibody-characterization/antibody-oxidation/

28
Subunit analysis of Adalimumab by HILIC
LC Signal G0F G1F G2F
Intact RP MS 0.70 0.30 0.00
IdeS RP MS 0.75 0.23 0.02
IdeS /DTT RP MS 0.75 0.23 0.02
IdeS /DTT
HILIC
widepore
Intrinsic
fluorescence
0.74 0.24 0.02
Peptide
mapping
RP UV/MS 0.75 0.23 0.02
Released
Glycans
HILIC Fluorescence 0.74 0.24 0.02
ACQUITY UPLC Glycoprotein Amide 300 Å (Waters)
150 x 2.1 mm, 1.7 µm
Intrinsic fluorescence

29
Subunit analysis of Etanercept by HILIC
N-deglycosylated Etanercept subunits

30
Subunit analysis of Etanercept by HILIC
0.0% 0.1% 0.0%
1.4%
9.3%
34.6%
47.2%
7.3%
0.2% 0.0%
0%
10%
20%
30%
40%
50%
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Numberof O-glycans
R² = 0.9867
0
2
4
6
8
10
12
29 31 33 35 37 39 41 43
NumberofO-glycans
Retention time (min)
Mean O-glycans
Mean C1S(3)1
Mean C1S(3,6)2
31 identified glycoforms

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• Glycosylation is critical for the safety
and efficacy of biotherapeutics.
• Orthogonal methods have to be
applied for characterisation studies
and QC testing.
• LC/MS is a valuable tool for
glycosylation analyses.
• All these methods can be applied in
a regulated environment.
Take-home messages
Largy et al. (2017), J. Chromatogr. A 1498, 128-146
10.1016/j.chroma.2017.02.072

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Acknowledgments
• Eric LARGY
• Anicet CATRAIN
• Fabrice CANTAIS

33
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?

Stratégies orthogonales pour la caractérisation de glycoprotéines thérapeutiques par LC/MS (SEP, Paris 2019)

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