Glycan Structural Analysis Throughout Biotherapeutic Development

GLYCAN STRUCTURAL ANALYSIS THROUGHOUTBIOTHERAPEUTIC DEVELOPMENT

Dr Richard EastonTeam Leader Carbohydrate AnalysisTeam Leader, Carbohydrate Analysis

SGS Life Science ServicesWokingham, UK

What is glycosylation? What is glycosylation?

What aspects of glycosylation are key? What aspects of glycosylation are key?

H th b i ti t d? How can these be investigated?

2

N GLYCOSYLATIONN-GLYCOSYLATION

Attached to Asn in the consensus Attached to Asn in the consensus sequence …Asn-X-Ser/Thr…where X is any AA except Pro

Initiated in ER by en bloc transfer of a pre-formed lipid-anchored conserved glycan

Mature glycans formed by trimming (in ER and Golgi) and elongation (Golgi)

Found on mammalian, insect, fungal, bacterial and plant cell proteins

3

O GLYCOSYLATIONO-GLYCOSYLATION

Occurs on Ser and ThrOccurs on Ser and Thr

No consensus sequence but some “rules” eg nearby proline, tandem repeats of Ser/Thr

Initiated in Golgi by addition of a single sugar - usually GalNAc in mammals

Additi l dd d ti ll t f th Additional sugars are added sequentially to form the core sequence and then the antennae

Cores 1 & 2 are very common in many glycoproteinsy y g y p

Other monosaccharides may act as linkers depending on the protein structure eg O-linked Fucose in EGF domains

Cores 1 & 2 are very common in many glycoproteins

Found on mammalian, insect, fungal, bacterial and plant cells

4

MONOSACCHARIDE STRUCTURESMONOSACCHARIDE STRUCTURES

5

EUKARYOTIC N GLYCAN BIOSYNTHESISEUKARYOTIC N-GLYCAN BIOSYNTHESIS

6

IN YEAST HIGH MANNOSE GLYCANS ARE MADE LARGER BY POLYMANNOSYLATIONLARGER BY POLYMANNOSYLATION

7

PLANTS AND INVERTEBRATES DECORATE THEN-GLYCAN CORE IN MORE DIVERSE WAYS THAN MAMMALS

8

N-GLYCOSYLATION IN FISH AND BIRDSN-GLYCOSYLATION IN FISH AND BIRDS

some fish glycans are the mostdecorated of all known glycans

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decorated of all known glycans

SELECTED MAMMALIAN N GLYCAN STRUCTURESSELECTED MAMMALIAN N-GLYCAN STRUCTURES

(i)(iii)

(ii)

ErythropoietinGlycodelin S

Gl d li A

(iv)

(v)+/-

(vi)

Glycodelin A

+/-

Tamm-Horsfall glycoprotein

M kidNeutrophil

10

Mouse kidney

O LINKED OLIGOSACCHARIDES CORES 1 AND 2O-LINKED OLIGOSACCHARIDES CORES 1 AND 2

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ANALYSIS OF GLYCOPROTEINSANALYSIS OF GLYCOPROTEINS

12


13


14


15


16


17


18


19


20

MONOSACCHARIDE ANALYSIS – BOVINE FETUINO OS CC S S O U

Is my molecule glycosylated?

• Is the glycosylation N linked and if so what type (complex high mannose)?M-Scan Ltd.Sample: Fetuin standard

Job: 12849 Sample No: Acquired on: 29-Sep-2000 at 13:37:38 by:

100

%

FETST299 Scan EI+ 298

6.30e421.83

N-Acetylneuraminic acid

• Is the glycosylation N-linked and if so what type (complex, high mannose)?• Is there evidence of O-glycosylation?

Is there evidence of carbohydrate contamination coming through the process?

• High levels of glucose may indicate breakdown of, for example, gel filtration beads

100

0

%

FETST299 Scan EI+ 173

1.56e516.98

N-Acetyl

g g y , p , g

100

0

%

16.38 21.8317.13

FETST299 Scan EI+ 20411.32 Galactose

N-Acetylgalactosamine

yglucosamine

0

100

%

3.69e512.08

12.65 13.12

13.48 16.98 21.83

FETST299 Scan EI+

MannoseGalactose

Glucose

100

%

FETST299 Scan EI+ TIC

3.99e610.28

7.727.00

21.8311.32 12.08

11.4716.98

13.1212.65 13.50

Arabitol (IS)

21

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00Time0

7.72

DMB SIALIC ACID ANALYSISDMB SIALIC ACID ANALYSIS

Neu5Ac Neu5Gc

Sialic acid reference panel

Neu5,9Ac2

Neu5,7Ac2

Neu5Gc9Ac

KDNReagentpeak

Neu5,7(8),9Ac3Gc

ReagentReagentpeak

22

PERMETHYLATIONPERMETHYLATION

Population analysis by MALDI MS Population analysis by MALDI-MS

Identification of glycan compositions and an initial assessment of the types of structures presentassessment of the types of structures present

Electrospray analysis

A determination of glycan antennae present in the sample through fragmentation of the glycans.

Identification of unwanted epitopes eg GalαGalp p g

GC-MS

Identification of monosaccharide linkages in the population

23

MALDI MS ANALYSIS OF N GLYCANSMALDI-MS ANALYSIS OF N-GLYCANS

Secretory glycoprotein

Antibody

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FRAGMENTATION IN ELECTROSPRAY IONISATIONFRAGMENTATION IN ELECTROSPRAY IONISATION

O

OO O

CH2ORO

CH2OR

+OO O

OR OR OR ORH

H NA

A-type oxonium ion cleavage

HexNAcorSialic Acid

OCH2OR

+

O

OR OR

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OR OR

NANOSPRAY MS/MS ANALYSIS (Q-TOF)NANOSPRAY MS/MS ANALYSIS (Q-TOF)

Fetuin permethylated N-glycansMS/MS 1791 ([M+2H]2+)

Consistent with the compositionNeuAc Hex HexNAc

NeuAc+ - methanol NeuAcHexHexNAc+ - methanol

344 2

MS/MS 1791 ([M+2H]2 ) NeuAc3Hex6HexNAc5

NeuAcHexHexNAc+100344.2

825.5

NeuAc+

%%376.2

793.5 1792.0

200 400 600 800 1000 1200 1400 1600 1800m/z0

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200 400 600 800 1000 1200 1400 1600 1800

MS/MS OF THE SIGNAL AT M/Z 895 5MS/MS OF THE SIGNAL AT M/Z 895.5

298.2

CH2OMeCH2OMeMeOCH2

CH

CHNMeAc

CHOMe

CHOMe

OHC

CC

MeOCHH

H

C OH

OMeOC

MeOCH

H

COOMe

AcNMeC

CH

O

MeOCH

CHOMe

CH2OMe

OMeOCH

H

H

CHC

OMe

AcNMeC

620.3

520.3

376 2/398 2376.2/398.2

NeuAcHexHexNAcitol + Na+

27

MS/MS OF THE SIGNAL AT M/Z 895 5MS/MS OF THE SIGNAL AT M/Z 895.5

298.2298.2

CH2OMeCH2OMeMeOCH2

CH

CHNMeAc

CHOMe

CHOMe

OHC

CC

MeOCHH

H

C OH

OMeOC

MeOCH

H

COOMe

AcNMeC

CH

O

MeOCH

CHOMe

CH2OMe

OMeOCH

H

H

CHC

OMe

AcNMeC


28

MS/MS OF THE SIGNAL AT M/Z 1256.6

CH2OMe

CH

CHNMeAc

OC OH

CH2OMe

M OCH

MeOCH

MeOCH2

620.3

CH

CHOMe

CHOMe

CH

OHC

CC

MeOCHH

HH

OMeOC

MeOCH

H

H

H

COOMe

CHC

AcNMeC

CH

O

CH2HOMe

MeOCH

MeOCH2

881.5659.3376.2/398.2

OC

MeOCH

H

H

H

COOMe

CHC

AcNMeC

CH

O

284.2H

OMe 506.3

376.2/398.2


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MS/MS OF THE SIGNAL AT M/Z 1256.6

CH2OMe

CH

CHNMeAc

OC OH

CH2OMe

M OCH

MeOCH

MeOCH2

CH

CHOMe

CHOMe

CH

OHC

CC

MeOCHH

HH

OMeOC

MeOCH

H

H

H

COOMe

CHC

AcNMeC

CH

O

CH2HOMe

MeOCH

MeOCH2659.3

OC

MeOCH

H

H

H

COOMe

CHC

AcNMeC

CH

O

659.3 HOMe

659.3


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LINKAGE ANALYSIS

45 GC/EI-MS analysis – Spectrum of 1,2-linked Mannose

LINKAGE ANALYSIS

OAc

OAc

CH3O

CH2OCH3

H

45

H OAc

CH2OCH3

117

161306

129OAc

OCH3 OAc

D

H OCH3

H OCH3161

205190

234129

130

129

130

CHDOAc

H OAc 277

74

146

161 74

190

161

190

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LINKAGE ANALYSIS

73 GC/EI-MS analysis – spectrum of 1,3,6-linked Mannose

LINKAGE ANALYSIS

OA

CH2OAc

73

H OAc

CH2OAc

145

189306

129

OAc

OAc

CH3OH

D H OAc

H OCH3189

261190

234118 129

OAc OCH3

CHDOAc

H OCH3305

74

118129

74

234

189

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LINKAGE ANALYSIS

45 GC/EI-MS analysis – spectrum of 1,4-linked N-Acetylglucosamine

LINKAGE ANALYSIS

OAc

OAc

AcO

CH2OCH3

H

45

H OAc

CH2OCH3

117

189347

OAc

OCH3 NCH3Ac

D

OCH3H

H OAc 189

233203

275117

CHDOAc

H NCH3Ac305

74

159117

74

159

233

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LINKAGE ANALYSISLINKAGE ANALYSIS

10016.752 117+118+129+159

3.33e6RT

4-GlcNAc

%

6 G l14.001

t-Gal

2-Man3-Gal

6-Gal

2,4-Man4,6-GlcNAc

10.000 11.000 12.000 13.000 14.000 15.000 16.000 17.000 18.000 19.000 20.000rt0

13.171

13.471 14.941

t-Gal3,6-Man

,

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STEREOSPECIFICITY OF LINKAGE

3363.3

90

100

Voyager Spec #1=>SM3=>AdvBC(32,0.5,0.1)[BP = 2041.4, 4588]

681.5

Mixture of Gal1-3Gal1-4Gal1-3Gal (Mass 885Da)and Gal1-4GlcNAc1-3Gal1-4Glc (Mass 926 Da)Incubated with -Galactosidase

681.5

50

60

70

80

% In

tens

ity

926.7926.7

00

10

20

30

40

%

599.0 719.2 839.4 959.6 1079.8 1200.0Mass (m/z)

00

2728 6100

Voyager Spec #1=>SM3=>AdvBC(32,0.5,0.1)[BP = 1836.2, 4445]

885.5722 4

Mixture of Gal1-3Gal1-4Gal1-3Gal (Mass 885 Da)and Gal1-4GlcNAc1-3Gal1-4Glc (Mass 926 Da)885.5722 5 2728.6

60

70

80

90

100 722.4 and Gal1 4GlcNAc1 3Gal1 4Glc (Mass 926 Da)Incubated with -Galactosidase

885.5722.5

20

30

40

50

% In

tens

ity

35

599.0 719.2 839.4 959.6 1079.8 1200.0Mass (m/z)

00

10

2 AB STACKED CHROMATOGRAMS2-AB STACKED CHROMATOGRAMS

Five separate Mab N glycan preparationsFive separate Mab N-glycan preparations

G0F G1F G2FRun 1 0.59 0.32 0.09Run 2 0.58 0.32 0.09Run 3 0.59 0.32 0.09Run 4 0.58 0.33 0.09Run 5 0 58 0 32 0 09Run 5 0.58 0.32 0.09Average 0.584 0.322 0.09SD 0.0055 0.0045 0.00%CV 0.94 1.40 0.00

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2 AB STACKED CHROMATOGRAMS2-AB STACKED CHROMATOGRAMS

Diff i t f M b tiDiffering amounts of Mab preparations

100μg

10μg

50μg

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TAKE HOME MESSAGESTAKE HOME MESSAGES

Mass spectrometric analysis provides detailed structural information on composition, antennal structures and linkage.

Chromatography provides a unique glycan profile due to the precise structure and associated interactions of the glycans with the column

t i Thi fil b d f i t hi h thmatrix. This profile can be used as a reference against which other batches can be compared. Useful for characterisation, stability, forced degradation studies.

The use of LC/ES-MS of proteolytic digests allows sites of glycosylationto be isolated and identified. These sites can then be analysed using the b t h i t d t i th t f th l t h itabove techniques to determine the nature of the glycans at each site.

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CARBOHYDRATES MAKE IT ON TV!CARBOHYDRATES MAKE IT ON TV!

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THANK YOU FOR YOUR ATTENTIONTHANK YOU FOR YOUR ATTENTION

Life Science Services Dr Andrew J ReasonGroup Manager SGS M-Scan Europe

SGS M Scan Ltd Phone: +44 (0) 118 989 6940

Life Science Services Dr Richard EastonTeam Leader, Carbohydrate Analysis

SGS M S Ltd Ph 44 (0) 118 912 1190

+ 41 22 739 9548 SGS M-Scan Ltd Phone: +44 (0) 118 989 69402-3 Millars Business Centre, Fax: +44 (0) 118 989 6941Fishponds Close, E-mail : [email protected], RG41 2TZ, UK Web : www.sgs.com/biopharma

SGS M-Scan Ltd Phone: +44 (0) 118 912 11902-3 Millars Business Centre, Fax: +44 (0) 118 912 1191Fishponds Close, E-mail : [email protected] E-mail : [email protected], RG41 2TZ, UK Web : www.sgs.com/biopharma

+ 1 866 SGS 5003+ 65 637 90 111

+ 33 1 41 24 87 87 + 1 877 677 2667

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1 877 677 2667

QUESTIONS ?QUESTIONS ?

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Healthcare

Glycan Structural Analysis Throughout Biotherapeutic Development