Post on 20-Dec-2020
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Structural elucidation of glycans as a tool to differentiate recombiant from endogenous glycoproteins. José A. (Toni) Pascual Deputy Head WADA-accredited aD Laboratory. Barcelona
Developments and Challenges in the Detection of Doping with Peptide Hormones and Related Substances Rome, 15-16 June 2011
WADA Scientific Symposium
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Structural elucidation of glycans as a tool to differentiate recombiant from endogenous glycoproteins
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Structural elucidation of glycans as a tool to differentiate recombiant from endogenous glycoproteins
basic area
endogenous area
acidic area
NESP (AranespTM)
rEPO αβ (BRP std)
uEPO (NIBSC std)
anode +
cathode -
1 2
3 4
5
Band id.
6
rEPO ω (RepotinTM)
Band id.
C B A
D
ε
CERA (MirceraTM)
Band Id.
α β γ δ
TD2009EPO IEF analysis
rEPO δ (DynepoTM)
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Structural elucidation of glycans as a tool to differentiate recombiant from endogenous glycoproteins
• How can we unequivocally differentiate exogenous from endogenous ?
• Which and Where are the differences ?
• Are they “presence” vs “absence” of a particular moiety?
• Is the “presence” of a particular moiety in the exogenous or the endogenous substance?
◘ Relative abundances are much more difficult to demonstrate and defend in Court
◘ “Absence” is a much lesser evidence
• What is the required sensitivity ? ◘ Different applicable tools: detection separation CE LC GC PAGE IA…
MS FD SPR … CLD
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Structural elucidation of glycans as a tool to differentiate recombiant from endogenous glycoproteins
• Differences in AA ? ◘ Antibodies can be raised against a specific moiety in the recombinant
species.
COMPARISON OF AA SEQUENCES:
1 APPRLICDSR VLERYLLEAK EAENITTGCA EHCSLNENIT VPDTKVNFYA 51 WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVNSSQPWEP LQLHVDKAVS 101 GLRSLTTLLR ALGAQKEAIS PPDAASAAPL RTITADTFRK LFRVYSNFLR 151 GKLKLYTGEA CRTGDR
EPO
1 APPRLICDSR VLERYLLEAK EAENITTGCN ETCSLNENIT VPDTKVNFYA 51 WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVNSSQVNET LQLHVDKAVS 101 GLRSLTTLLR ALGAQKEAIS PPDAASAAPL RTITADTFRK LFRVYSNFLR 151 GKLKLYTGEA CRTGDR
NESP
Gimenez, 2005
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Structural elucidation of glycans as a tool to differentiate recombiant from endogenous glycoproteins
• Differences in AA ? ◘ Antibodies can be raised against a specific moiety in the recombinant
species.
SELECTION OF THE PEPTIDES:
LVNSSQPWEPLQLHVC-NH2 EPO peptide (15 AA): 81
QVNETLQLHVDKAVSGLRSC-NH2 NESP peptide (19 AA): 86
IMMUNIZATION: KLH
ON
O
OS
H2N ONH + adjuvant (MPL: monophosphoryl lipid A)
PEPTIDE
days 0 14 28 35 42
1st Nº boost 2nd 3rd 4th
49 14 days 14 days 7 days 7 days 7 days
monthly +7 days
5th......
...
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Structural elucidation of glycans as a tool to differentiate recombiant from endogenous glycoproteins
• Differences in AA ? ◘ Antibodies can be raised against a specific moiety in the recombinant
species.
IN-GEL RECOGNITION: IEF-PAGE rEPO NESP rEPO NESP
0.3 ng 0.12 ng 750 ng 250 ng 250 ng 750 ng
Ab Anti-pepEPO Ab Anti-pepNESP Ab clone: AE7A5
rEPO NESP
Gimenez, 2005
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Structural elucidation of glycans as a tool to differentiate recombiant from endogenous glycoproteins
• Differences in Glycans ? ◘ Glycans are not DNA coded and their final composition depend on many
parameters: host cells, culture conditions, purification…
◘ Biosimilars may always be produced which will difficult the establishment of “relative criteria” in the absence of an unequivocal difference.
Eprex Eprex Schellekens, 2004
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Structural elucidation of glycans as a tool to differentiate recombiant from endogenous glycoproteins
• Differences in Glycans ? ◘ Differences in IEF behaviour of EPOs are located in the carbohydrates.
SIALIDASE TREATMENT
rEPO NESP uEPO
Std Std Std partial total partial total partial total
Belalcazar, 2004
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Structural elucidation of glycans as a tool to differentiate recombiant from endogenous glycoproteins
• Differences in Glycans ? ◘ Differences in IEF behaviour of EPOs are located in the carbohydrates.
Reichel, 2010
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ASIALO-GLYCOPROTEIN ANALYSIS
Substances Heterogeneity Mw glycoprotein
Mw after sialidase SAcalculated
LacNAc Repeats
rEPO δ ++ ∼29.485 Da 26.022 Da 11-12 +
rEPO αβ +++ ∼29.129 Da 26.034 Da 10-11 +++
α 2-R Sialidase
17000 23000 29000 35000 Mass (m/z)
26034
26400 26766 27128 27495
25669
25305
15000 24000 33000 42000 Mass (m/z)
29.485
29.129
rEPO δ
Asialo-Glycoprotein
rEPO δ
rEPO αβ rEPO αβ
3 x
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Ser
15000 24000 33000 42000
O-glycans rEPO αβ rEPO δ
3 % 6 %
6 % 3 %
58 % 29 %
33 % 62 %
Ser Ser
Ser
Ser
17000 19000 21000
29.129
Ser
Ser
Ser
Ser
29.485
18172
O-Glycoprotein
PNGase F
O-GLYCOPROTEIN ANALYSIS
rEPO δ
rEPO αβ
SA mean 1.2 1.5
N-acetylgalactosamine
Galactose
Sialic acid
19120
18829
18538
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N 1 2 3 4
N 1 2 3 4
FETUIN
0 1 2 3 4 Nº SA 2AB
(AranespTM) NESP
(BRP Std) rEPOαβ
(from Teknika) (not purified)
rEPO raw
N-Glycan pool: WAX HPLC-FLD
rEPO δ
rEPO αβ
0 1 2 3 4
(Charge Heterogeneity)
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(After sialidase digestion)
Minutes
0.00 5.00 10.00 15.00 20.00 25.00
2AB
(from Teknika) (not purified)
rEPO raw
(AranespTM) NESP
(BRP Std) rEPOαβ
0
Neuraminidase α 2-3,6,8,9 (from C. Perfringens)
N-Glycan pool: WAX HPLC-FLD
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(Structure Complexity)
FETUIN
Minutes20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00
(AranespTM) NESP
(BRP Std) rEPOαβ
(from Teknika) (not purified)
rEPO raw
Minutes 20 20 20 20 20 20 40 40 40 40 40 40 60 60 60 60 60 60 80 80 80 80 80 80
rEPO δ
rEPO αβ
N-Glycan pool: NP HPLC-FLD
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RAAM: Array of Exoglycosidases
Minutes 20 30 40 50 60 70 80 90 10
{ ∆ α 2- R }
{ β 1- 4 } { ∆ α 2- R }
{ β 1- R } { β 1- 4 } { ∆ α 2- R }
1
2
3
{ β 1- 4 }
PROCEDURE:
NEURAMINIDASE α 2-3,6,8,9
GALACTOSIDASE β 1-4
{ ∆ α 2- R }
N-ACETYL GLUCOSAMINIDASE
β 1-R { β 1- R }
core
899.0 1619.4 2339.8 3060.2 3780.6 4501.0
Mass (m/z)
1200.2437
2AB [ M+Na ]+
1
2
3
NP HPLC-FLD
(BRP Std) rEPOαβ
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RAAM: Array of Exoglycosidases
NP HPLC-FLD
(DynepoTM) rEPOδ
rEPO δ total
rEPO δ + sialidase
rEPO δ + sialidase + galactosidase
rEPO δ + sialidase + galactosidase + N-acetylglucosaminidase
1000 1500 2000 2500 3000
2200 2900 3600 4300
2200 2900 3600 4300
1400 1820 2240 2660 3080
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N-Glycan pool: MALDI-TOF-MS (By band after 2D PAGE)
IEF SDS
rEPO αβ
2000 2600 3200 3800 4400 5000
1
2
3
4
5
6
7
α 1 2 3 4 5 6 7
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Structural elucidation of glycans as a tool to differentiate recombiant from endogenous glycoproteins
• Structural elucidation of Glycans ◘ Neu5Gc in EPO will unequivocally discriminate between exogenous and
endogenous EPO !
GLYCOPROTEIN
TFA hydrolysis
Sialic Acid pool
DMB NH2
NH2
O
O
capHPLC-FLD HPLC-TOF-MS ChipLC-QqQ
O
OH
HONH
HO OHO
OH
HO
O
H3C
O
OH
HONH
HO OHO
OH
HO
OHO
capHPLC-FLD
(BRP Std) rEPOαβ
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(SA speciation) Sialic Acid pool: capHPLC-FLD
rEPO δ
rEPO αβ
(DynepoTM)
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(SA speciation)
min 20 22.5 25 27.5 30 32.5 35 37.5
Sialic acids ref. mixture
Negative plasma
Negative plasma + Neu5Gc
Suspicious plasma
Neu5Gc Neu5Ac
Sialic Acid pool: capHPLC-FLD
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Sialic Acid pool: ChipLC-QqQ
Percentage of Neu5Gc with respect to Neu5Ac
<
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Structural elucidation of glycans as a tool to differentiate recombiant from endogenous glycoproteins
• Conclusions ◘ Direct approaches can be devised, but we still lack the sensitivity to
apply them to human urine or serum samples.
◘ Indirect approaches based on the differential interaction with lectins or antibodies have also been succesfully assayed as screening methods.
◘ Specific features found in the endogenous hormone may not be as useful for glycoproteins as the multiplicity of isoforms is a confounding factor.
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Structural elucidation of glycans as a tool to differentiate recombiant from endogenous glycoproteins
• Acknowledgements ◘ This results were obtained thanks to the tireless work of the
members of the Biological Research Group at IMIM.
- Dr. Viviana Belalcazar - Dr. Esther Llop - Dr. Joaquim Mallorquí - Dr. Ricardo Gutierrez - Dr. Jordi Segura
◘ And other partners:
- Dr. Carme de Bolòs [Cancer Research Program, IMIM] - Dr. Hans Kammerling [UNiversity of Utrecht] - Dr. Gerrit Gerwig [University of Utrecht]
We acknowledge the financial support from WADA