Characterization of new antibody-derived therapeutics at ... · ECL1 ECL2 ECL3 monovalent nanobody bivalent nanobody ~15 kDa ~30 kDa 35GS linker-(GGGGS) 7-~100 kDa G-protein coupled

Characterization of new antibody-derived therapeutics at the intact and middle-up level of analysis using sheathless CE-MS

AT EUROPE

DUBLIN, 13-MARCH-2019

Elena Dominguez-Vega

Center for proteomics and metabolomics

Monoclonal antibodies

13/03/20192 E.Dominguez-Vega | [email protected]

➢ Most selling pharmaceuticals: eg. Adalimumab, Infliximab, Rituximab, Trastuzumab

Analytical characterization

Macroheterogeneity

• Aggregation

• Free chains (e.g. free LC, free HC)

• Partial cleavage

• ...

SECIEX CE-SDSCIEFHILICRPLC

MS

Microheterogeneity

• N-glycan variability

• Post-translational modifications (PTMs)

• incomplete disulfide bond formation

• …

New antibody formats


Antibody fragments Bispecific antibodiesFusion proteins

CE for the separation of intact proteins


• high separation efficiency

• small diffusion coefficient is advantage

• reveal subtle protein differences/changes

M

zC

r

zelectro

6

time (min)

•no stationary phase; free solution, no interaction

• denaturing and native separation

CE provides good opportunities to characterize new antibody formats

Versatility

Intact protein analysisAssessment of protein heterogeneity

CE-MS of intact proteins


1844.68

2012.28

2213.38

2459.22

2766.47

1600 2000 2400 2800 m/z

CE–ESI-MS

time (min)

efficient intact protein separation protein assignment

capillary electrophoresis mass spectrometry

• high separation efficiency• small diffusion coefficient is advantage

• reveal subtle protein differences/change• allows native conditions

•mass loadability of CE is limited

• accurate mass information on intact proteins• difficult ionization of intact proteins

• ESI of intact protein yields number of multiply charged ions

Sheathless CE-MS (CESI-MS)

• improves ionization efficiency by nanospray

• no additional liquid:

- avoids dilution (increased sensitivity)

- decreases noise

- allows native MS (conformation studies)

E.Dominguez-Vega | [email protected] 13/03/20196

Avoiding protein adsorption in CE(-MS)


pH BGE < pI protein

- - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - -

+ -

EOF

- - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - -

- ++ + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + +

- - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - -

+ -

No EOF

EOF

Capillary coatings for CESI-MS

Neutral coatings

Positive coatings: PEI

CESI-MS systems for intact protein analysis


0 2 4 6 8 10 12 14 16 18 Time (min)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Inte

nsit

y x

10

6

Neutrally-coated

PEI-coated capillary

Cytochrome C

Lysozyme

RNaseA

RNaseA

Lysozyme

Cytochrome C

• Protein test mixture (RNaseA, Lysozyme and Cytochrome C, 0.2 g/L each)• BGE 50 mM ammonium acetate pH 3.0

Outline

9

FUSION PROTEINS

BISPECIFIC ANTIBODIES

NANOBODIES


Nanobodies


NH2

COOH

ECL1 ECL2 ECL3

monovalent nanobody

bivalent nanobody

~15 kDa ~30 kDa

35GS linker-(GGGGS)7-

~100 kDa

G-protein coupled receptors

Cameloid antibodies

Common modifications of nanobodies:

Heterogeneity • Non-glycosylated• PTMs, eg. deamidation, pyroglutamate

Degradation products• Truncated forms (e.g. Myc-His tag)

Multivalent nanobodies• Improper linkage (monovalent nanobody)

CESI-MS of nanobody-1

11 E.Dominguez-Vega | [email protected]

2 4 6 8 10 12 14 16 18 Time (min)0

1

2

31

2

peak mass (Da) assignment

1 14590.2 Nb1

2 14591.2 deamidated Nb1

Inte

nsit

y x

10

6

Monovalent Nb1

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

+ -

No EOF

BGE, 50 mM ammonium acetate (pH 3.0).1 µM sample (10 ng/µL)

13/03/2019



2 4 6 8 10 12 14 16 18 Time (min)0

1

2

31

2

2 4 6 8 10 12 14 16 18 Time (min)0

1

2

3

Inte

nsit

y x

10

6


1 14590.2 Nb1


3 15527.7 Nb1+(GGGGS)3?

4 28835.4 Nb1-35GS-Nb1

5 28836.4 deamidatedNb1-35GS-Nb11

2

4

5

3

Inte

nsit

y x

10

6

Monovalent Nb1

Bivalent Nb1


1 14590.2 Nb1


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

+ -

No EOF

BGE, 50 mM ammonium acetate (pH 3.0).1 µM sample (10 ng/µL)

35GS linker



0.00

0.25

0.50

0.75

1.00

1.25

8 10 12 14 16 18 Time (min)

Inte

nsit

y x

10

6 1

2

3

4

5

7

8

9

10

11

12

13

14

15

16

17

1819

206


1 15498.5 10A10

2 15499.5 deamidated 10A10

3 1952.9 L126-H142

4 15499.5 deamidated 10A10

5 15500.5 bisdeamidated 10A10

6 15500.5 bisdeamidated 10A10

7 1953.9 deamidated L126-H142

8 15087.4 10A10-(H140-H142)

9 1953.9 deamidated L126-H142

10 15088.3 deamidated M10A10-(H140-H142)


11 13561.6 10A10-(L132-H142)

12 13874.8 10A10-(E130-H142)

13 14004.9 10A10-(E129-H142)

14 13305.5 10A10-(Q124-H142)

15 13434.5 10A10-(K125-H142)

16 14604.2 10A10-(A136-H142)

17 14605.2 deamidated 10A10-(A136-H142)

18 14605.2 deamidated 10A10-(A136-H142)

19 14606.2 bisdeamidated 10A10-(A136-H142)

20 14606.2 bisdeamidated 10A10-(A136-H142)

Monovalent Nb2

Myc-His Tag

Resolution of isomeric forms


8 10 12 14 16 Time (min)

0

1

2

3

3

7

9

EIE 651.9 ± 0.5 m/z

Mw, 1952.9 Da

Inte

nsit

y x

10

6

651.966

652.294

652.294

651.5 652.0 652.5 653.0 653.5 m/z

L126-H142peak 3

peak 7

peak 9

L126-H142+ 1 deamidation

L126-H142+ 1 deamidation

Outline

15

FUSION PROTEINS


NANOBODIES


Bispecific antibodies


two different binding sites (different targets)

Heavy Chain dimer

Common side products

anti-Target 1 antibody

anti-Target 2 antibody

half antibodies

¾ antibody

Complementary CH3 mutation

free LC and LC dimers (LC Homo- and Heterodimers)

Looking at microheterogeneity: Middle-up approach


reduction

2x Fd’ 2x Lc

2x Fc/2

IdeS cleavage

Monospecific mAb

K

Middle-up approach: Domain specific analysis of microheterogeneity

F(ab)2

2x Fc/2

CESI-MS of digested monoclonal antibodies


6 8 10 12 14 16 18 20 Time (min)

50 mM ammonium acetate pH 3.0

F(ab)2+Fc/2+50 kDa fragments

Fc/2

5% acetic acidF(ab)2

50 kDa fragments

F(ab)27% acetic acid + 10% Isopropanol

Fc/2

IdeS cleavage

F(ab)2

~ 100 KDa

2x Fc/2~ 25 KDa

Charge heterogeneity of Fc/2 fragments

13/03/2019

19

E.Dominguez-Vega | [email protected]

non-glycosylated+K

+K+K

N→D

+K

N→D

+K+K

14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 Time [min]0.00

0.25

0.50

0.75

1.00

1.25

Inte

nsit

y x

10

6

1.50

1.75

19

BGE, 6% Acetic Acid+10%IPA

Middle-up of bispecific antibodies


reduction

6 FRAGMENTS

3 FRAGMENTS

2x Fd’ 2x Lc

2x Fc/2

Fd’1 Fd’2

Lc2

Fc2/2Fc1/2

IdeS cleavage

Lc1

Monospecific mAb

Bispecific mAb

CE-FTICR-MS of a bispecific mAb


- - - - - - - - - - - - - - - - -

- ++ + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + - - - - - - - - - - - - - - - - - -

EOF

BPE

1

2

3

4

Inte

nsit

y x

10

8

26 27 28 29 30 31 32 Time [min]

Fc/ 2

2Fc/2

1

26 27 28 29 30 31 32 Time [min]

0.5

1.0

1.5

2.0

2.5

Inte

nsit

y x

10

9

CE-FTICR-MS of a bispecific mAb


BPE

1

2

3

4

Inte

nsit

y x

10

8

26 27 28 29 30 31 32 Time [min]

*Deconvoluted mass spectra

Da

Da

Da

Da

Da

Da

12T FTICR

CESI

Nano-ESIInterface

CE-FTICR-MS of a bispecific mAbs


29.0 30.0 31.0 32.0Time [min]

0

1

2

3

4

5

Inte

ns

ity x

10

8

Da

29.0 30.0 31.00

1

2

3

Inte

ns

ity x

10

8

Time [min]32.0

4

Da

Fc/ 2

2

Fc/2

1

N→D

N→D

BGE, 10% Acetic Acid + 20% IPASeparation, -20 kV, 20 ⁰C

- - - - - - - - - - - - - - - - -

- ++ + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + - - - - - - - - - - - - - - - - - -

EOF

Outline

24

FUSION PROTEINS


NANOBODIES


Fusion protein with multiple glycosylation sites


New biological entity (NBE)Bifunctional fusion protein

Asn1

Asn2

Asn1

Asn3

Asn2

Asn3

➢ 6 potential glycosylation sites

➢ Elongated heavy chain (> 180 kDa)

➢ Reduction of -S-S- bridges results in two main fragments

• Fragment A: non glycosylated

• Fragment B: 3 potential glycosylation sites,

Asn1, Asn2 and Asn3

o Asn1 and Asn2 fully occupied

o Asn3 occupancy 50%

CESI-MS of protein with multiple glycosylation sites



4 6 8 10 12 14 16 18 Time (min)

0.00

0.25

0.50

0.75

1.00

1.25

Inte

ns

ity x

10

5

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

+ -

No EOF

BGE, 10% Acetic Acid

Reduction

DTT

2X + 2XAsn1

Asn2

Asn1

Asn3

Asn2

Asn3

Asn1

Asn2

Asn3

10 12 14 16 18 20 Time (min)

0.0

0.5

1.0

1.5

Inte

ns

ity x

10

4

CESI-MS of protein with multiple glycosylation sites



Reduction

DTT

2X + 2XAsn1

Asn2

Asn1

Asn3

Asn2

Asn3

PyroE

Asn1

Asn2

Asn3

Asn3

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

+ -

No EOF

BGE, 10% Acetic Acid

Glycoforms containing glycans at Asn1 and Asn2

28 E.Dominguez-Vega | [email protected]

365

291

365291

162

1

2

3

4

5

Inte

nsit

y x

10

3

291

Asn2

Asn1Asn1

Asn2 Asn2

Asn1Asn1

2x

Asn1 Asn1

Asn2 Asn2 Asn2

Asn1

3x

Asn2 Asn2

Asn1

162+

+

Asn1

Asn2

Asn3

Da

13/03/2019

291364

364

291

291

0.5

1.0

1.5

2.0

2.5

Da

Glycoforms containing glycans at Asn1, Asn2 and Asn3


291

Inte

nsit

y x

10

3

+

+

Asn2

Asn1

Asn2

Asn1

Asn3Asn3

3x

2x

Asn2

Asn1

Asn3

Asn1

Asn2

2xAsn3

Asn1

Asn2

3xAsn3

Asn1

Asn3

Asn2Asn2

Asn1

Asn3

3x

-1SiAAsn1

Asn2

Asn3

Da

Conclusions


➢efficient protein separations

➢ sensitive detection

➢ suitable for proteins with very different characteristics

➢ assessment of micro and macroheterogeneity

Sheathless CE-MS allows characterization of different newantibody-derived therapeutics

Perspectives

- study of antibody interactions

- analysis of human immunoglobulins

Acknowledgements

CPM – Glycomics and Glycoproteomics

Christoph Gstöttner

Steffen Lippold

Simone Nicolardi

Manfred Wuhrer BioAnalytical Chemistry

Rob Haselberg

Govert W. Somsen

Medicinal Chemistry

Raimond Heukers

Martine Smit

Documents

Characterization of new antibody-derived therapeutics at ... · ECL1 ECL2 ECL3 monovalent nanobody bivalent nanobody ~15 kDa ~30 kDa 35GS linker-(GGGGS) 7-~100 kDa G-protein coupled