elsa wagner-rousset slides -...

1

Emerging Mass Spectrometry based methods

for therapeutic mAbs, biosimilars and ADCs

deep structural characterization.

Elsa Wagner-Rousset, PhD

Centre d’Immunologie Pierre Fabre, FRANCE

Titre

CASSS AT Europe 2015, E. Wagner-Rousset 2

Outline

• ADCs : Present and Future

• Case studies from the ADC’s analytical toolbox

• LC-MS following IdeS digestion

• Native MS & Ion Mobility MS

• Biosimilarity assessment of mAbs

• CESI-MS/MS

• Summary

Titre


• 2 ADCs recently approved by FDA and EMA :

- brentuximab vedotin (Adcetris®, Seattle Genetics)

- ado-trastuzumab emtansine (Kadcyla®, Genentech):

• > 30 in clinical trials

Titre


Antibody Drug Conjugate structure

* Panowski et al., mAbs 6:1, 34-45, 2014.

A cytotoxic drug is conjugated to a monoclonal antibody by means of a select linker

Titre


Delivery of drugs by ADCs to cancer cells

* Panowski et al., mAbs 6:1, 34-45, 2014.

1. ADC selective binding to a cell-surface tumor antigen

2. Internalization of the ADC-antigen complex

3. Traffic to lysosomal compartments : degradation

4. Releasing of the active cytotoxic drug inside the cell > cell death

Titre


ADC’s analytical characterization

• Challenging when compared to unconjugated mAbs.

3 levels of complexity:

1. mAb heterogeneity

2. linker & payload

3. Conjugation chemistry

N-term. blocking(Gln/Glu =>

PyroGlu: -17/-18 Da)

C-term. clipping (- Lys: -128 Da)C-term. amidation (OH => NH2: -1 Da)

GlycosylationHeterogeneity

(Fab & Fc :+1445, 1607, 1769 Da…)

Disulfide shufflingFree thiol groups

Cysteinylation (+119 Da)Thioether (-32 Da)

Oxidation (Met-O, Trp-O: +16 Da)Deamidation (Asn => Asp: +1 Da)

Isomerization (Asp => IsoAsp: 0 Da)Succinimide (Asn/Asp => Suc: -17 Da)

Glycation (Lys-Glc: +162 Da)

Dimers, aggregatesIgG fragments (H2L, H2, HL, H, L)

Misfolded forms

CH3

CH2

CH1

VH

CL

VL

Hi

IgG1 = H2L2(148 000-149 000 Da)

* Beck et al., Anal Chem, 2013 , 15;85(2):715-36.

Titre



2. Linker & payloads

* P.D. Senter, Current Opinion in Chem. Biol., 2009; 13:1-10.

* Wakankar et al., mAbs, 2011 ; 3:2; 161-172.

• Contain labile bonds leading to drug release.

• Stability issue

• Other optimized combination of drug/linker to come.

Titre


A B C


3. Conjugation chemistry

* Lin et al., Pharm Res, 2012 ;29(9):2354-66.

• (A) Trastuzumab emtansin

• (B) Brentuximab vedotin

• (C) Thio-trastuzumab

• ADC production using A) or B) results in high heterogeneity in DAR and

location of conjugation site

Titre


Brentuximab vedotin manufacturing

Wakankar A et al, AAPS, 2010 (Genentech)

Le LN et al, Anal Chem, 2012 (Genentech)

Valliere-Douglas JR et al, Anal Chem 2014 (Seagen)

• Mixture of covalent/ non-covalent IgGs

• Need of specific analytical methods

• (1) “Denaturing” = non-covalent interchain

bonds (L-H, H-H) are disrupted

• (2) “Native” = non covalent interchain bonds

(L-H, H-H) are maintained

Titre


The ADC’s analytical tool box

DAR

Drug Load

Profile

Un-conjugated

mAb

Conjugation sites

Drug related

impurities

Higher order

structure

Denaturing methods•SDS-PAGE

•CE-SDS

•Peptide mapping

•LC-MS (+/- Red ; IdeS)

Native methods•UV

•HIC

•SEC

•Native MS

•Ion mobility MS

Nature Reviews, A. Mullard, 12, 2013.

Titre


Case study 1

Titre


Antibody Fluorophore Conjugates (AFCs)

NNH

HN

O

OO

O

NH

NH2O

NH

O O N

HN

O

O

NN

O

OCH3O OCH3O

NH

HOS

Maleimide Caproic acid

Maleimidocaproyl Valine Citruline

PABC MethylValine

Valine Dolaisoleucine Dolaproline Norephedrine

MMAE

Attachment groupProtease cleavable

linkerMMAE

Cytotoxic drug

NNH

HN

O

OO

O

NH

NH2O

NH

O O N

HN

O

O

NN

O

OCH3O OCH3O

NH

HOS



PABC MethylValine

Valine Dolaisoleucine Dolaproline Norephedrine

MMAE

Attachment groupProtease cleavable

linkerMMAE

Cytotoxic drug

NNH

HN

O

OO

O

NH

NH2O

NH

O O N

HN

O

O

NH

SO

ONS



PABC MethylValine

Fluorochrome

NNH

HN

O

OO

O

NH

NH2O

NH

O O N

HN

O

O

NH

SO

ONS



PABC MethylValine



PABC MethylValine

Fluorochrome

A

B

Non toxic Dansyl Sulfonamide Ethyl Amine (DSEA)-linker maleimide:

• designed and synthetized to mimic Brentuximab vedotin and most of ADCs in

clinical trials

• conjugated on interchain cysteines of trastuzumab

Titre


Size-exclusion chromatography (SEC)

• Trastuzumab-mc_DSEA displayed 2 populations by SEC, multimeric and

monomeric species isolated and characterized by :

• SDS-PAGE

• CE-SDS

• HIC

• Native Mass Spectrometry

• LC-MS after IdeS digestion and reduction

• Relationship between aggregation of the AFC and average Dye to Antibody

Ratio (DAR)?

Monomers

Multimers

20.0

10.0

0.0

30.0

40.0

50.0

60.0

mAU

0.0 50.0 100 150 200 250 300 mL

69.5%

30.5%

Monomers

Multimers

20.0

10.0

0.0

30.0

40.0

50.0

60.0

mAU

0.0 50.0 100 150 200 250 300 mL

69.5%

30.5%

Titre


SDS-PAGE

• Non Reducing

• H2L, H2, HL, H, L + payloads

• Monomeric versus multimeric profiles : different payload distributions

• Reducing

• Increased MW (multimers versus monomers)

• Higher conjugation level for multimers

250

150

100

75

50

37

2520

tras tu

zumab

AFC mon

o mer

s

AFC mult

imer

stra

s tuzu

mabAFC m

ono m

ers

AFC mult

imer

s

NR R

Titre


2,95

3

4,47

7

5,78

4

6,09

0

6,56

5

8,73

0

10,2

57

AU

-0,10

0,00

0,10

0,20

0,30

0,40

0,50

0,60

0,70

0,80

0,90

Minutes2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00 10,00 11,00 12,00

Hydrophobic Interaction Chromatography (HIC)

• Monomeric trastuzumab-mc_DSEA• homogeneous HIC distribution centered on D4

• Mutimeric fraction• contains high loaded forms

BA

U

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

Minutes2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Monomeric

0PL

2PL 4PL

6PL

8PL

Av. DAR: 4.3

Multimeric

Titre


Native MS

• Native desalting conditions (200mM NH4Ac, pH7), maintain intact the bivalent structure of AFC allowing :

• Relative distribution of drug loaded species

• Direct av. DAR determination

1491971953

1471851052

145173114

1512081252

153219488

2 %

26 %

2 Payloads

22 %

40 %

10 %

0 Payload

4 Payloads

6 Payloads

8 Payloads

Av. DAR: 4.4

« monomeric » trastuzumab-mc_DSEA

Titre


IdeS for ADC/AFC characterization

AFC mixture(150 kDa)

LC(25 kDa)

Fc/2(25 kDa)

1) IdeS

2) DTT

Fd(25 kDa)

Fd0 Fd1 Fd2 Fd3

L0 L1

IdeS• Immunoglobulin-degrading enzyme of Streptococcus pyogenes

• FabRICATORTM (www.genovis.com)

• 3 fragments of ≈25kDa providing LC and MS resolution

Titre


LC-UV-MS Trastuzumab (IdeS + reduction)

A

Time10.00 20.00 30.00 40.00

AU

0.0

5.0e-3

1.0e-2

1.5e-2

2.0e-2

2.5e-2 Range: 1.6e-2

17.5011.28

6.37

13.62

mass25000 25200 25400 25600 25800

%

0

100 4.03e425237

2521825091

25399

2525525560

mass23200 23400 23600 23800

%

0

100 6.53e423443

23462

mass25000 25200 25400 25600 25800

%

0

100 4.84e425383

25364 25401

B Fc/2 at 11.3 min LC at 13.6 minC Fd at 17.5 minD

• 3 fragments identified according to their MS profiles :

• Fc/2 carrying glycosylation : G0F, G1F + G0 and G2F (CHO prod.)

• Light Chain

• Fd

1) Non-conjugated mAb

Fc/2

L0

Fd0

Titre


Time10.00 20.00 30.00

AU

0.0

5.0e-3

1.0e-2

1.5e-2

2.0e-2

2.5e-2Range: 2.65e-211.48

16.64

14.25

20.52

25.8921.34

30.00

mass25000 25250 25500 25750

%

0

100 2.82e425235

2521625089

25397

2525525418 25559

mass25000 25250 25500 25750

%

0

100 2.82e425235

2521625089

25397

2525525418 25559

mass23200 23400 23600 23800

%

0

100 3.68e423442

23463

mass23200 23400 23600 23800

%

0

100 3.68e423442

23463

mass24000 24250 24500 24750 25000

%

0

100 3.80e424447

24468

mass24000 24250 24500 24750 25000

%

0

100 3.80e424447

24468

mass25000 25250 25500 25750

%0

100 1.92e425383

25362 25404

mass25000 25250 25500 25750

%0

100 1.92e425383

25362 25404

mass26000 26250 26500 26750

%

0

100 2.83e426388

26368 26408

mass26000 26250 26500 26750

%

0

100 2.83e426388

26368 26408

mass27000 27250 27500 27750

%

0

100 1.18e427392

27373 27413

mass27000 27250 27500 27750

%

0

100 1.18e427392

27373 27413

mass28000 28250 28500 28750

%

0

100 3.17e328397

2837828418

28455

mass28000 28250 28500 28750

%

0

100 3.17e328397

2837828418

28455

Fc/2 at 11.5 min

L0 at 14.3 min

L1 at 16.6 min

Fd1 at 20.5 min

Fd0 at 18.1 min

Fd2 at 25.9 min

Fd3 at 30.0 min

BA

C

D E

F

G

H

• Un-conjugated & loaded IdeS fragments well separated

• Fd1 and Fd2 split in 2 peaks : positional isomers

• The number of payloads fits with the number of possible free thiol

residues oxidized during AFC preparation : interchain cys residues

LC-UV-MS AFC (IdeS + reduction)2. Monomeric fraction

Fc/2

L0Fd0

L1Fd1

Fd2

Fd3

Titre


Time10.00 20.00 30.00 40.00

AU

0.0

5.0e-3

1.0e-2

1.5e-2

2.0e-2

2.5e-2 Range: 2.127e-2

11.47

5.00

16.34

14.06

29.28

25.4219.70 34.51

mass27000 27200 27400 27600 27800

%

0

100 1.07e427393

27371 27416

mass25000 25200 25400 25600 25800

%

0

100 9.60e425236

2521425089

25398

252582556125420

mass28000 28200 28400 28600

%

0

100 3.38e428398

2837828418

mass29200 29400 29600 29800

%

0

100 4.84e329402

29422

mass25400 25600 25800 26000 26200 26400

%

0

100 7.42e325452

25472

26387

mass24000 24200 24400 24600 24800 25000

%

0

100 8.62e424448

24469

mass23200 23400 23600 23800

%

0

100 2.54e423443

23465

Fc/2 at 11.5 min

L0 at 14.1 min

L1 at 16.3 min Fd4 at 34.5 min

BA

C

D E

F

G

HL2 & Fd1 at 19.7-21.1 min

Fd3 at 29.3 min

Fd2 at 24.5-25.4 min

• Qualitatively & quantitatively different from monomeric fraction

• Highly loaded fragments

• Higher conjugation rate than expected

• L2 and Fd4 reveal extra-conjugation sites

LC-UV-MS AFC (IdeS + reduction)3. Multimeric fraction

Fc/2

L0Fd0

L1

Fd1Fd2

Fd3

Fd4

Titre


• DAR (LC) = Σ[nALn / ΣALC]

• DAR (Fd) = Σ[nAFdn / ΣAFd]

• Av.DAR= 2 x [DAR (LC) + DAR (Fd)]

AFC : Average DAR

DAR is more than doubled in the multimeric fraction

10.00 15.00 20.00 25.00 30.00 35.00

AU

0.0

1.0e-2

2.0e-2

L0Fc/2 Fd0

10.00 15.00 20.00 25.00 30.00 35.00

AU

0.0

1.0e-2

2.0e-2

L0Fc/2 Fd0

Time10.00 15.00 20.00 25.00 30.00 35.00

AU

0.0

1.0e-2

2.0e-2

LCT-13-0911-OC 2: Diode Array Range: 2.105e-2

L012%

Fd13%

Fd216%

Fd372%

Fd49%

L175%

Fc/2

L213%

Time10.00 15.00 20.00 25.00 30.00 35.00

AU

0.0

1.0e-2

2.0e-2

LCT-13-0911-OC 2: Diode Array Range: 2.105e-2

L012%

Fd13%

Fd216%

Fd372%

Fd49%

L175%

Fc/2

L213%

Av. DAR = 3.8

Av. DAR = 7.8

A

B

C

10.00 15.00 20.00 25.00 30.00 35.00

0.0

1.0e-2

2.0e-2

Range: 2.645e-2

L032%

L168%

Fd153%

Fd016%

Fd226% Fd3

5%

Fc/2

10.00 15.00 20.00 25.00 30.00 35.00

0.0

1.0e-2

2.0e-2

Range: 2.645e-2

L032%

L168%

Fd153%

Fd016%

Fd226% Fd3

5%

Fc/2

Monomeric AFC

Multimeric AFC

Trastuzumab

Titre


Case study 1 - conclusions

• IdeS for characterization of AFC/ADCs by LC-

UV-MS

• Sub-unit analysis reduces sample complexity

• Fast sample preparation

• 25 kDa fragments easily ionized and analyzed by ESI-TOF-MS

• Payload distribution and DAR

• Monitor variants and PTMs

• C-ter lysine truncation, pyroglutamation, oxidation, degradation

• Fc/2 > N-glycosylation

• Highly conjugated mAb tend to aggregation

Titre


Case Study 2 : Native MS of ADCs

Native MS gives information on assemblies maintained

by noncovalent interactions

ESI-MSinstrument

3000 3500 m/z

Gas phaseSolution

Instrumental setting

optimizations

Control of the energy

communicated to the ions

in the 1st pumping stage

region of the instrument

(Vc, Pi adjustment)

Data interpretation

- Binding

stoichiometries

- Binding specificity

- Solution affinities

- Dynamics of

assembly/disassembly

Sample preparation

= desalting step

- Buffer exchange

- Volatile aqueous buffer

- Ammonium buffer

(0-1M) with controled pH

* Cianferani S., ASMS, 2014.

Titre


1008040 6020 120 140 Mass (kDa)

145,910 Da

123,500 Da101,072 Da

75,586 Da

25,042 Da53,177 Da

• Non-covalent interactions are

maintained in the gas phase

• ADC is detected as an intact

molecule

• DAR can be assessed

6000 7000 m/z1000 3000 500040002000

6000 7000 m/z1000 3000 500040002000

Native ConditionsIgGZero treated ADC @ 5 µM in 150 mM AcONH4 pH7.5

Denaturing conditionsIgGZero treated ADC @ 2 µM in H2O:ACN:FA (50:50:1)

Native MS for cysteine -linked ADCs

37+

15+

37+

• Non-covalent interactions are

disrupted

Titre


Comparison of HIC & Native MS

Minutes3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

D0

D2 D4

D6

D8

∑

∑=

8

0

8

0

n

n

DAR

DAR

A

nADAR = 4.0

D0

D2

D4

D6

D8

D826+

D425+

D024+

D625+

D224+

b)

1008040 6020 120 mass (kDa)D2

D424+

D224+D6

25+

D023+

D825+ D0

D2 D4

D6D8

d)

e)

1.HIC

3.Native MS

(deglycosylated)

2.Native MS

* Vallière-Douglass J.F., Anal Chem, 2012.

* Debaene F. et al., Anal Chem 2014; in press.

6.1%

25.2% 34.8%

24.0%

8.3%

6.4%

40.8% 38.5%

9.4%5.0%

6.4%

26.7%

34.5%

21.9%

7.2%

Brentuximab Vedotin :

Drug load distribution and average DAR

are identical when calculated from HIC or

native MS after deglycosylation

DAR = 4.0

DAR = 3.3

DAR = 4.0

Titre


Native MS

146 152 mass (kDa)145 150148147 151149 153

D0

D1

D3

D2

D4

Av. DAR = 1.8

Advantages of native MS over HICExample of a hinge cys-linked ADC

HIC

2 3 4 5 67

8 9 10 11 min.

D0

D2?

D4?D1?

Av. DAR = 2.0• Broad peaks

• Ambigous DAR assessment

• Odd DAR not expected

• Unambigous DAR assessment

• Confidence in average DAR

determination

Titre


Brentuximab Vedotin

Off line coupling HIC & Native MS of ADC

Tim

e (m

in.)

34

56

78

910

D0

D4

D2

D6

D8

Mass (kDa)145 150 155

145904 ± 2 DaD0

148539 ± 1 DaD2

151173 ± 1 DaD4

153815 ± 2 DaD6

156457 ± 3 Da

D8

a) b)

•HIC peak collection and

subsequent fractions analyzed

by native MS

• Drug load confirmation

•Native MS of ADC as support

for HIC method development.

* Debaene F. et al., Anal Chem 2014, in press.

Titre


Ions separation takes place according to Ion Mobility

• Drift times can be related to collisional cross sections (CCS)

• CCS : projection of the area of complexes measured in the gas phase

• Information on ion gas phase conformation

Ion Mobility cell

2+ 2+1+

Drift time+

1+Ion separation according to ion mobility

Size, shape Charge

Compactness ��

Drift time ��

z ��

Drift time ��2+ 1+ 2+ 1+

Drift time

N2

z

x

y

z

x

y

z

x

y

z

x

y

x

y

zx

y

z

Combining Ion Mobility and Mass Spec

* Cianferani S., ASMS, 2014.

Titre


What’s Ion Mobility able to perform for ADCs?


• Native IM-MS :

• ADC heterogeneity in drug binding is observed on native IM-MS plots

Deglycosylated Parent mAbO25596FD E.raw : 1

24+

12 16 20 22 tD (ms)

7500

7000

6000

5500

m/z

181410

6500

5000

23+

21+

IgGZero ADC

O23564FDE.raw : 1

12 16 20 22 tD (ms)

7500

7000

6000

5500

m/z

181410

6500

5000

Deglycosylated ADC

Titre


What’s Ion Mobility able to perform for ADCs?


• Native IM-MS :

• DAR are separated from each other in the IM cell

• Relative quantitation for average DAR determination

• Collision cross section determination

Deglycosylated Parent mAb Deglycosylated ADC

16 17 18 19 tD (ms)

6700

6600

6500

6400

m/z

23+

22+

16 17 18 19 tD (ms)

6700

6600

6500

6400

m/z

DO 23+

D0 22+

D2 23+

D6 24+

D4 23+

D8 24+

Titre


Average DAR from semi quantitative native MS and IM-MS

MaxEnt1 deconvolution

Quantitation based on ion

intensities

n=8

D0

D2D4

D6

D8

DAR

= 3.9 ± 0.1DAR

= 3.7 ± 0.1DAR

= 4.0

Number of

drug load

Calculation based on area

measured under fited gaussian

of charge states extracted ions

n =3

Minutes3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

D0

D2 D4

D6

D8

Native MS

HIC

Average DAR determination and drug-load profiles obtained

from native IM-MS are in good agreement with HIC & native MS data

146 Mass (kDa)150148 154152 158156

D4

D2

D6

D8D0

Native IM-MS


Titre


Native IM-MS of intact cysteine-linked ADC

• Drug binding induces constant and reproducible ∆∆∆∆tD and ∆∆∆∆CCS differences

• Very slight conformational changes are induced on the parent mAb upon drug binding

Parent mAb ADC

- DO D2 D4 D6 D8

Drift Time 24+

(ms)14.2 ± 0.0 14.2 ± 0.1 14.9 ± 0.1 15.6 ± 0.1 16.3 ± 0.1 17.0 ± 0.1

∆(drift time) (ms) - - + 0.7 + 0.7 + 0.7 +0.7

CCS 24+ (nm²) 68.0 ± 0.0 68.1 ± 0.1 68.8 ± 0.1 69.5 ± 0.1 70.3 ± 0.1 71.1 ± 0.1

∆CCS (nm²) - - + 0.7 + 0.7 + 0.8 +0.8

resolving power of IM cell

tD/∆tD at FWHM : 16.4 ± 0.8

D0

14.2

D2

14.9

D4

15.6

D6

16.3

D8

17.0

13 14 15 16 17 18 19 tD (ms)66

67

68

69

70

71

72

73

74

75

76

77

22 23 24 25 26 27 28 29

DAR8

DAR6

DAR4

DAR2

DAR0

Charge states

Measured CCS (nm 2)

Titre


Case study 3 : biosimilarity using CESI-MS/MS

Sheathless Capillary Electrophoresis – tandem mass spectrometry (CESI-MS/MS)

• CE-ESI-MS Coupling :

• CE is a miniaturized technique performing ultra-low flow rates

• Decreasing the flow allows for increased sensitivity in the ESI-MS*

« Ultra-low flow » CESI-MS

• MS/MS : structural informations

*Wilm, Mann International Journal of Mass Spectrometry 1994, 136, 167–180

Titre


CESI-MS: mAbs characterization workflow

In-solution tryptic digestionAnalysis by

t-ITP CESI-MS/MS

Amino acid sequence

characterization

PTMs hot spots

characterization

Glycosylations

(structure)

CESI8000 coupled to 5600 TripleTOF MS

Primary structure characterization workflow based on bottom-up proteomics strategy

• Single shot (200 fmoles of digest)

• Peptide mapping including amino acid sequence coverage with 1 enzyme

(Trypsin)

• Characterization of PTM hot spots and glycosylation

Titre


CESI-MS/MS of trastuzumab

• Sequence coverage of trastuzumab by CESI-MS/MS

100% sequence coverage achieved in a single injection through

only purely tryptic unmodified peptides

Gahoual R. et al., Anal. Chem., 2014 (86), 9074-9081

variable domain

complementarity determining region

constant domain

identified peptides

Titre


Systematically >90% of the y/b ions could be retrieved from the CESI-MS/MS data

Amino acid sequence characterization (trastuzumab)



Titre


MS/MS spectrum of digested peptides LT04

APK

(m/z 315.2039 ; 2+)

MS/MS spectrum of digested peptides HT15

DYFPEPVTVSWNSGALTSGVHTFPAVLQS

SGLYSLSSVVTVPSSSLGTQTYICNVNHKP

SNTK

(63 amino acids ; m/z 1119.898 ; 6+)

Implementation of CE allows separation and successful detection of a larger variety of peptides

than classical RP-LC


Titre


PTMs hot spots

N-terminal glutamic acid cyclization characterization

• CE mechanism separates peptide with N-terminal glutamic acid cyclization (1.8%) from the unmodified

peptide (98.2%)

Results suggest partial

modification of sample

Favorable conditions to estimate

sample modification level

HT01

pyroglu - HT01

Extracted ion electropherograms of peptides HT01 and modified HT01


Titre


Methionine oxidation

• Methionine oxidation causes peptide mass shift (+15.99 Da) leading to the separation of the

modified peptide in CZE

confirmed by MS/MS spectra


Methionine (M)

Methioninesulfoxide (oxiM)

EIEs and MS/MS spectra of peptides HT21 (intact and modified)


PTMs hot spots

Titre


Asparagine (N) Aspartic acid (deaN)

• Deamidation (+ 0.98 Da) involves mobility change in CZE enabling the separation of the unmodified peptide

CE separation of deamidated peptides eases the

identification of the modification by MS

EIEs and MS/MS spectra of peptides LT04 (intact and modified)

Asparagine deamidation



PTMs hot spots

Titre


Aspartic acid isomerization

CE separation prior to MS analysis allows in this particular case to include

aspartic acid isomerization in the overall characterization workflow

HT23 (-D283-)

HT23 (-isoD283-)

EIEs and MS/MS spectra of peptides HT23 (intact and modified)


PTMs hot spots


Titre


Trastuzumab versus biosimilar

• Complete sequence coverage obtained for

trastuzumab

• Biosimilar candidate sequence could be

successfully identified except HC K217

Suggesting an amino acid substitution

between the two samples

Gahoual R. et al., MAbs, 2014, 6:6, 1464-1473.

Amino acid sequence similarity

Titre


Glycoforms characterization

• Fc/2 glycosylation site characterization

Heterogenous glycoforms could

be identified

Difference in glycoforms

distribution could be observed

Cetuximab possess two different N-

glycosylation sites

Significant number of glycans could

be characterized

Cetuximab versus biosimilar


Titre


• Fd glycosylation site characterization

Glycoforms exhibited by the candidate biosimilar are significantly different from cetuximab

Rejected as biosimilar� Capped gal-α1,3-gal glycans significantly reduced

� 30 % of glycans contains N-acetylneuraminic acid

Cetuximab versus biosimilar

Glycoforms characterization


Titre


Conclusions

• For mAbs & ADCs, development & analytical characterization are challenging

• High heterogeneous products

• Mass spectrometry has a golden place in the analytical pipeline of

biopharmaceutical companies

• Analysis key points

• Sample preparation (ex: IdeS digestion & Reduction)

• Hyphenation of Capillary Electrophoresis

• Emergent MS methods are becoming trend

• Native MS

• Payload distribution - av. DAR determination of « hinge cys-linked » ADCs

• Binding stoichiometries mAb/Ag

• Ion Mobility-MS

• Conformational differences between DAR allow separation

• Orthogonal method to determine av. DAR

Titre


THANK YOU

Alain BECK

Elsa WAGNER-ROUSSET

Marie-Claire JANIN-BUSSAT

Christine KLINGUER-HAMOUR

Olivier COLAS

Laura MOREL-CHEVILLET

Mélissa EXCOFFIER

Thierry CHAMPION

Sarah CIANFERANI

François DEBAENE

Julien MARCOUX

Guillaume TERRAL

Johann STOJKO

Alain VAN DORSSELAER

Emmanuelle LEIZE-WAGNER

Yannis FRANCOIS

Rabah GAHOUAL

Michael BIACCHI

Armelle CHARRIE

Pierre ALBRECHT

Noelle POTIER

Cécile PERRET

Na-Thi NGUYEN

elsa wagner-rousset slides -...

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