Glycosylation is a key structural and functional element found on a wide variety of biotherapeutics. As such, alterations in glycan profile can significantly affect the efficacy of a drug through, for example, half life in the bloodstream or biological activity as well as being a potential source of immunogenicity. The glycan profile can be selected and controlled through the choice of cell line as well as control of bioreactor conditions. The use of analytical techniques that provide structural data on this type of post translational modification are vital in the development and characterisation of biologics. Techniques in glycan structural characterisation are discussed in this presentation.

1. GLYCAN STRUCTURAL ANALYSIS THROUGHOUT
BIOTHERAPEUTIC DEVELOPMENT
Dr Richard Easton
Team Leader Carbohydrate AnalysisTeam Leader, Carbohydrate Analysis
SGS Life Science Services
Wokingham, UK

2.  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

3. 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

4. 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

5. MONOSACCHARIDE STRUCTURESMONOSACCHARIDE STRUCTURES
5

6. EUKARYOTIC N GLYCAN BIOSYNTHESISEUKARYOTIC N-GLYCAN BIOSYNTHESIS
6

7. IN YEAST HIGH MANNOSE GLYCANS ARE MADE
LARGER BY POLYMANNOSYLATIONLARGER BY POLYMANNOSYLATION
7

8. PLANTS AND INVERTEBRATES DECORATE THE
N-GLYCAN CORE IN MORE DIVERSE WAYS THAN
MAMMALS
8

9. N-GLYCOSYLATION IN FISH AND BIRDSN-GLYCOSYLATION IN FISH AND BIRDS
some fish glycans are the most
decorated of all known glycans
9
decorated of all known glycans

10. SELECTED MAMMALIAN N GLYCAN STRUCTURESSELECTED MAMMALIAN N-GLYCAN STRUCTURES
(i)
(iii)
(ii)
Erythropoietin
Glycodelin S
Gl d li A
(iv)
(v)
+/-
(vi)
Glycodelin A
+/-
Tamm-Horsfall glycoprotein
M kid
Neutrophil
10
Mouse kidney

11. O LINKED OLIGOSACCHARIDES CORES 1 AND 2O-LINKED OLIGOSACCHARIDES CORES 1 AND 2
11

12. ANALYSIS OF GLYCOPROTEINSANALYSIS OF GLYCOPROTEINS
12

13. ANALYSIS OF GLYCOPROTEINSANALYSIS OF GLYCOPROTEINS
13

14. ANALYSIS OF GLYCOPROTEINSANALYSIS OF GLYCOPROTEINS
14

15. ANALYSIS OF GLYCOPROTEINSANALYSIS OF GLYCOPROTEINS
15

16. ANALYSIS OF GLYCOPROTEINSANALYSIS OF GLYCOPROTEINS
16

17. ANALYSIS OF GLYCOPROTEINSANALYSIS OF GLYCOPROTEINS
17

18. ANALYSIS OF GLYCOPROTEINSANALYSIS OF GLYCOPROTEINS
18

19. ANALYSIS OF GLYCOPROTEINSANALYSIS OF GLYCOPROTEINS
19

20. ANALYSIS OF GLYCOPROTEINSANALYSIS OF GLYCOPROTEINS
20

21. 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
6.30e4
21.83
N-Acetyl
neuraminic 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
1.56e5
16.98
N-Acetyl
g g y , p , g
100
0
%
16.38 21.83
17.13
FETST299 Scan EI+
20411.32 Galactose
N-Acetyl
galactosamine
y
glucosamine
0
100
%
3.69e512.08
12.65 13.12
13.48 16.98 21.83
FETST299 Scan EI+
Mannose
Galactose
Glucose
100
%
FETST299 Scan EI+
TIC
3.99e6
10.28
7.72
7.00
21.83
11.32
12.08
11.47
16.98
13.12
12.65
13.50
Arabitol (IS)
21
8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00
Time0
7.72

22. DMB SIALIC ACID ANALYSISDMB SIALIC ACID ANALYSIS
Neu5Ac Neu5Gc
Sialic acid reference panel
Neu5,9Ac2
Neu5,7Ac2
Neu5Gc9Ac
KDN
Reagent
peak
Neu5,7(8),9Ac3Gc
ReagentReagent
peak
22

23. 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

24. MALDI MS ANALYSIS OF N GLYCANSMALDI-MS ANALYSIS OF N-GLYCANS
Secretory glycoprotein
Antibody
24

25. FRAGMENTATION IN ELECTROSPRAY IONISATIONFRAGMENTATION IN ELECTROSPRAY IONISATION
O
OO O
CH2OR
O
CH2OR
+
OO O
OR OR OR OR
H
H NA
A-type oxonium ion cleavage
HexNAc
or
Sialic Acid
O
CH2OR
+
O
OR OR
25
OR OR

26. NANOSPRAY MS/MS ANALYSIS (Q-TOF)NANOSPRAY MS/MS ANALYSIS (Q-TOF)
Fetuin permethylated N-glycans
MS/MS 1791 ([M+2H]2+)
Consistent with the composition
NeuAc Hex HexNAc
NeuAc+ - methanol NeuAcHexHexNAc+ - methanol
344 2
MS/MS 1791 ([M+2H]2 ) NeuAc3Hex6HexNAc5
NeuAcHexHexNAc+
100
344.2
825.5
NeuAc+
%%
376.2
793.5 1792.0
200 400 600 800 1000 1200 1400 1600 1800
m/z0
26
200 400 600 800 1000 1200 1400 1600 1800

27. MS/MS OF THE SIGNAL AT M/Z 895 5MS/MS OF THE SIGNAL AT M/Z 895.5
298.2
CH2OMe
CH2OMeMeOCH2
CH
CHNMeAc
CHOMe
CHOMe
OHC
CC
MeOC
HH
H
C O
H
OMeOC
MeOCH
H
COOMe
AcNMeC
C
H
O
MeOCH
CHOMe
CH2OMe
OMeOC
H
H
H
C
H
C
OMe
AcNMeC
620.3
520.3
376 2/398 2376.2/398.2
NeuAcHexHexNAcitol + Na+
27

28. MS/MS OF THE SIGNAL AT M/Z 895 5MS/MS OF THE SIGNAL AT M/Z 895.5
298.2
298.2
CH2OMe
CH2OMeMeOCH2
CH
CHNMeAc
CHOMe
CHOMe
OHC
CC
MeOC
HH
H
C O
H
OMeOC
MeOCH
H
COOMe
AcNMeC
C
H
O
MeOCH
CHOMe
CH2OMe
OMeOC
H
H
H
C
H
C
OMe
AcNMeC
NeuAcHexHexNAcitol + Na+
28

29. MS/MS OF THE SIGNAL AT M/Z 1256.6
CH2OMe
CH
CHNMeAc
O
C O
H
CH2OMe
M OCH
MeOCH
MeOCH2
620.3
CH
CHOMe
CHOMe
CH
OHC
CC
MeOC
HH
H
H
OMeOC
MeOCH
H
H
H
COOMe
C
H
C
AcNMeC
C
H
O
CH2H
OMe
MeOCH
MeOCH2
881.5
659.3376.2/398.2
OC
MeOCH
H
H
H
COOMe
C
H
C
AcNMeC
C
H
O
284.2
H
OMe 506.3
376.2/398.2
NeuAcHexHexNAcitol + Na+
29

30. MS/MS OF THE SIGNAL AT M/Z 1256.6
CH2OMe
CH
CHNMeAc
O
C O
H
CH2OMe
M OCH
MeOCH
MeOCH2
CH
CHOMe
CHOMe
CH
OHC
CC
MeOC
HH
H
H
OMeOC
MeOCH
H
H
H
COOMe
C
H
C
AcNMeC
C
H
O
CH2H
OMe
MeOCH
MeOCH2
659.3
OC
MeOCH
H
H
H
COOMe
C
H
C
AcNMeC
C
H
O
659.3 H
OMe
659.3
NeuAcHexHexNAcitol + Na+
30

31. 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
161
306
129OAc
OCH3 OAc
D
H OCH3
H OCH3
161
205
190
234129
130
129
130
CHDOAc
H OAc 277
74
146
161 74
190
161
190
31

32. LINKAGE ANALYSIS
73
 GC/EI-MS analysis – spectrum of 1,3,6-linked Mannose
LINKAGE ANALYSIS
OA
CH2OAc
73
H OAc
CH2OAc
145
189
306
129
OAc
OAc
CH3O
H
D H OAc
H OCH3
189
261
190
234
118 129
OAc OCH3
CHDOAc
H OCH3
305
74
118
129
74
234
189
32

33. 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
189
347
OAc
OCH3 NCH3Ac
D
OCH3H
H OAc
189
233
203
275117
CHDOAc
H NCH3Ac305
74
159117
74
159
233
33

34. LINKAGE ANALYSISLINKAGE ANALYSIS
100
16.752 117+118+129+159
3.33e6
RT
4-GlcNAc
%
6 G l
14.001
t-Gal
2-Man
3-Gal
6-Gal
2,4-Man
4,6-GlcNAc
10.000 11.000 12.000 13.000 14.000 15.000 16.000 17.000 18.000 19.000 20.000
rt0
13.171
13.471 14.941
t-Gal
3,6-Man
,
34

35. 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 Gal1-3Gal1-4Gal1-3Gal (Mass 885Da)
and Gal1-4GlcNAc1-3Gal1-4Glc (Mass 926 Da)
Incubated with -Galactosidase
681.5
50
60
70
80
%Intensity
926.7
926.7
00
10
20
30
40
%
599.0 719.2 839.4 959.6 1079.8 1200.0
Mass (m/z)
00
2728 6100
Voyager Spec #1=>SM3=>AdvBC(32,0.5,0.1)[BP = 1836.2, 4445]
885.5722 4
Mixture of Gal1-3Gal1-4Gal1-3Gal (Mass 885 Da)
and Gal1-4GlcNAc1-3Gal1-4Glc (Mass 926 Da)885.5722 5 2728.6
60
70
80
90
100 722.4 and Gal1 4GlcNAc1 3Gal1 4Glc (Mass 926 Da)
Incubated with -Galactosidase
885.5722.5
20
30
40
50
%Intensity
35
599.0 719.2 839.4 959.6 1079.8 1200.0
Mass (m/z)
00
10

36. 2 AB STACKED CHROMATOGRAMS2-AB STACKED CHROMATOGRAMS
Five separate Mab N glycan preparationsFive separate Mab N-glycan preparations
G0F G1F G2F
Run 1 0.59 0.32 0.09
Run 2 0.58 0.32 0.09
Run 3 0.59 0.32 0.09
Run 4 0.58 0.33 0.09
Run 5 0 58 0 32 0 09Run 5 0.58 0.32 0.09
Average 0.584 0.322 0.09
SD 0.0055 0.0045 0.00
%CV 0.94 1.40 0.00
36

37. 2 AB STACKED CHROMATOGRAMS2-AB STACKED CHROMATOGRAMS
Diff i t f M b tiDiffering amounts of Mab preparations
100μg
10μg
50μg
37

38. 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 glycosylation
to 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.
38

39. CARBOHYDRATES MAKE IT ON TV!CARBOHYDRATES MAKE IT ON TV!
39

40. THANK YOU FOR YOUR ATTENTIONTHANK YOU FOR YOUR ATTENTION
Life Science Services Dr Andrew J Reason
Group Manager SGS M-Scan Europe
SGS M Scan Ltd Phone: +44 (0) 118 989 6940
Life Science Services Dr Richard Easton
Team 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 6940
2-3 Millars Business Centre, Fax: +44 (0) 118 989 6941
Fishponds Close, E-mail : andrew.reason@sgs.com
Wokingham
Berkshire, RG41 2TZ, UK Web : www.sgs.com/biopharma
SGS M-Scan Ltd Phone: +44 (0) 118 912 1190
2-3 Millars Business Centre, Fax: +44 (0) 118 912 1191
Fishponds Close, E-mail : uk.biopharma@sgs.com
Wokingham E-mail : richard.easton@sgs.com
Berkshire, 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
40
1 877 677 2667

41. QUESTIONS ?QUESTIONS ?
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