Structural elucidation of glycans as a tool to differentiate ......Toni Pascual 1 Structural...

Toni Pascual 1

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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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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• 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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• 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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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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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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• 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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• 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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• 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


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


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 ◘ 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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• 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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• 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

Structural elucidation of glycans as a tool to differentiate ......Toni Pascual 1 Structural...

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