advances in lc/ms methodologies for the characterisation of complex glycoproteins - case study:...

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ADVANCES IN LC/MS METHODOLOGIES FOR THECHARACTERISATION OF COMPLEX GLYCOPROTEINS

CASE STUDY ON ETANERCEPTWritten by :

Arnaud Delobel – R&D Director

“Challenges and Opportunities in Protein Analytics”– Brussels, March 10th

2017

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CHALLENGES FOR GLYCOSYLATION ANALYSIS IN BIOPHARMA

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Glycosylation is usually a critical quality attribute of glycoproteins

Glycosylation can have a huge impact on protein activity, stability and/or safety

Glycosylation pattern must be thoroughly characterised during product development

Glycosylation must be controlled for batch release (and sometimes during stability studies)

Reliable and robust methods must be used. These methods must be validated according to international guidelines to be used for batch release.

Advances in LC/MS methodologies allow the fine characterisation of N- and O-glycosylation in a regulated environment.

PLATFORM USED FOR GLYCOSYLATION ANALYSIS

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Xevo G2-XS QTOF with

UPLC (H-Class Bio)

Control by UNIFI with

full GMP compliance

OVERVIEW OF ANALYTICAL METHODS FOR GLYCOSYLATION ANALYSIS AT QUALITY

ASSISTANCE

CETUXIMAB (ERBITUX®)

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Anti-EGFR chimeric monoclonal antibody.Glycosylation sites on both Fc and Fab moieties.

Cetuximab is an EGFR inhibitor used for the treatment of metastatic colorectal cancer, metastatic non-small cell lung cancer and head and neck cancer.

ETANERCEPT (ENBREL®)

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TNF receptor

Human Fc

N-glycans

N-glycans

O-glycans

TNF receptor 2 - Fc fusion protein, ~ 150 kDa. 6 N-glycosylation sites, > 10 O-glycosylation sites

Approved in the US and EU to treat rheumatoid arthritis, juvenile rheumatoid arthritis and psoriatic arthritis, plaque psoriasis and ankylosing spondylitis

It reduces the effect of naturally

present TNF, and hence is a TNF

inhibitor, functioning as a decoy

receptor that binds to TNF.

METHODS USED FOR THE CHARACTERISATION OF ETANERCEPT AND CETUXIMAB GLYCOSYLATION

PATTERNS

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N-glycosylation• N-glycans profiling (at glycan and subunit levels)• N-glycans sialylation profiling• Site specific analysis

O-glycosylation• O-glycans profiling (at glycan and subunit levels)• O-glycosylation sites discovery

Quantification and profiling of sialic acids

PRINCIPLE OF N-GLYCAN PROFILING ANALYSIS

Dextran ladder injection

RT > GU

Sample injection

RT-based candidate identification

MS-based identification confirmation

database

N-GLYCANS PROFILING OF ETANERCEPT

RapiFluor labellingAcquity UPLC BEH Glycan (Waters)

150 x 2.1 mm, 1.7µm

N-GLYCANS PROFILING OF CETUXIMAB

RapiFluor labellingAcquity UPLC BEH Glycan (Waters)

150 x 2.1 mm, 1.7µm

N-GLYCANS PROFILING – 2-AB VS RAPIFLUOR-MS

Injection of 10x less material

Similar FLR signal

(i.e. 10x sensitivity gain)

10x increase in MS

(i.e. 100x sensitivity gain)

FLR 0.6% relative intensity

z = 3z = 2

N-GLYCANS SIALYLATION PROFILING BY AEX-RP

Mixed-mode AEX-RP column

GlycanPac AXR-1 (Thermo Scientific)

150 x 2.1 mm, 1.9 µm

2-AB labelling

Fetuin

Etanercept

neutral mono di tri tetra penta

dye

dye

N-GLYCANS SIALYLATION PROFILING BY AEX-HILIC

3 independent preparations 1.4% mean RSD

Mixed-mode AEX-HILIC column

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

Minutes9.5 10.0 10.5 11.0

Minutes10.0 10.5 11.0 11.5

neutral

mono

di

tri

tetra

penta

Etanercept Fetuin

neutral

mono

di

RapiFluor-MS labelling

SITE-SPECIFIC N-GLYCANS PROFILING - ETANERCEPT Peptide mapping methodology (reduction / alkylation,

enzymatic digestion)o Trypsin is used for most proteinso Triple digestion (trypsin, Asp-N, Glu-C) is used

for complex proteins, e.g. Etanercept

Separation of tryptic peptides using LC in HILIC mode

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













G0FG2F

S1G1F

G2S1

G2F G2 M5

G2FS2 G0

G1FS1

G2S2

0%

5%

10%

15%

20%

N149 (TNF)

N171 (TNF)

N317 (Fc)

Trypsin

65 aa, 2 sites

8 aa, 1 site

Trypsin + Glu-c + Asp-N

19 aa, 1 site

20 aa, 1 site

7 aa, 1 site












NVFSCSVMHE ALHNHYTQKS LSLSPGK 3 sites

N-GLYCANS ANALYSIS AT SUBUNIT LEVEL - CETUXIMAB

Generic mAbs gradient

Optimized gradient

Cetuximab

ACQUITY UPLC Glycoprotein Amide 300 Å (Waters )

150 x 2.1 mm, 1.7 µm

LC

Fd’

Fc/2

Intrinsic fluorescence

N-GLYCANS ANALYSIS AT SUBUNIT LEVEL - CETUXIMAB

O-GLYCANS PROFILING

No enzyme available for O-glycans release!

N-glycan released and filtered out

O-glycan release by reductive b-elimination (optimized protocol for minimal peeling)

LC separation

o Hypercarb, 100 x 1.0 mm, 3 µm column

o 1 µL injection (equivalent to 2.5 µg starting protein)

MS detection in negative-ion mode

O-GLYCANS PROFILING OF ETANERCEPT

Hypercarb (Thermo Scientific)

100 x 1.0 mm, 3 µm

peeling

Low energy High energy

In-source fragments

O-GLYCOSYLATION SITES DISCOVERY - ETANERCEPT

LPAQVAFTPY APEPGSTCRL REYYDQTAQM CCSKCSPGQH AKVFCTKTSD TVCDSCEDST YTQLWNWVPE

CLSCGSRCSS











LPAQVAFTPY APEPGSTCRL REYYDQTAQM CCSKCSPGQH AKVFCTKTSD TVCDSCEDST YTQLWNWVPE

CLSCGSRCSS










NVFSCSVMHE ALHNHYTQKS LSLSPGK89 putative O-glycosylation sites (bold) (19% of the whole sequence!)

Trypsin: 65 aa, 29 aa

De-N-glycosylation

Desialylation

Triple digestion

O-GLYCOSYLATION SITES DISCOVERY - ETANERCEPT

Glycopeptide confirmation (MSE

)Glycopeptides discovery

5 peptides with 1 to 7 core 1

ETD MS/MS spectrum of m/z = 520.6 (z = 3) DAVCTSTSPTR

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* Rz+1 T S T A Dz+2 A D

624.666z 2

624.666z+1 2

626.427z+2 2

303.204c 3

304.178c+1 3

463.275c 4

462.336c-1 4

464.404c+1 4

809.417z+1 4

810.489z+2 4

564.264c 5

565.345c+1 5

910.445z+1 5

651.382c 6

650.930c-1 6

652.408c+1 6

997.427z+1 6

752.365c 7

751.389c-1 7

753.387c+1 7

1097.460z 7

1098.455z+1 7

1099.429z+2 7

936.453c 9

934.927c-1 9

937.553c+1 9

1357.720z+1 9

1358.499z+2 9

1402.690c 10

1400.802c-1 10

1403.684

c+1 10

1428.730z+1 10

1429.703z+2 10

1559.781c+1 11

1543.702z+1 11

1544.690z+2 11 (772.85 2+)

(772.85 2+)z+2 11 1544.690

ETANERCEPT O-GLYCOSYLATION MAPPING

LPAQVAFTPY APEPGSTCRL REYYDQTAQM CCSKCSPGQH AKVFCTKTSD TVCDSCEDST

YTQLWNWVPE CLSCGSRCSS











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

ANALYSIS OF O-GLYCOSYLATION AT SUBUNIT LEVEL

N-deglycosylated Etanercept subunits

ANALYSIS OF O-GLYCOSYLATION AT SUBUNIT LEVEL

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 13Number of O-glycans

R² = 0.9867

0

2

4

6

8

10

12

29 31 33 35 37 39 41 43

Num

ber o

f O-g

lyca

ns

Retention time (min)

Mean O-glycansMean C1S(3)1 Mean C1S(3,6)2

31 identified glycoforms

Sialic acids can be critical for safety and/or efficacy of biopharmaceuticals Sialic acids are realeased by mild acidic hydrolysis and derivatised with DMB Analysis is performed by UPLC/FLR

QUANTIFICATION AND PROFILING OF SIALIC ACIDS

Acquity UPLC BEH C18 (Waters)

100 x 2.1 mm, 1.7 µm

TAKE-HOME MESSAGES

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Glycosylation is a critical quality attribute of biopharmaceuticals and must be thoroughly characterised.

The use of orthogonal techniques is mandatory to get a complete view of the glycosylation pattern.

Recent advances in liquid chromatography and mass spectrometry now allow a full characterisation of the glycosylation, even in complex glycoproteins.

All the methods described are robust and show satisfactory precision and accuracy.

Most of these methods can be validated and used in a regulated environment for batch release or stability testing.

For more information …

“Orthogonal LC/MS methods for the comprehensive characterization of therapeutic glycoproteins, from released glycans to intact protein level”

E. Largy, F. Cantais, G. Van Vyncht, A. Beck and A. Delobel

Journal of Chromatography A, in press – doi:10.1016/j.chroma.2017.02.072

AKNOWLEDGEMENTS

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Eric LARGY Anicet CATRAIN Fabrice CANTAIS

Speed up people’s access to new medicines

[email protected]

+32 71 53 47 81

www.quality-assistance.com

Technoparc de Thudinie, 2

B-6536 Donstiennes (Belgium)

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