Stress testing and physico-chemical characterization of monoclonal antibodies : The approach of an...

European leader in analytical sciences

Written by : Géry Van Vyncht

R&D Director, S&I Department

24/09/2015

STRESS TESTING AND

PHYSICO-CHEMICAL

CHARACTERIZATION OF

MONOCLONAL

ANTIBODIES :

THE APPROACH OF AN

ANALYTICAL CRO

Speed up people’s access to new medicines

THE COMPANY

Updated: 7th September 2015

100% analytical services

100% (bio)pharmaceutical industry

Over 30 years (since 1982)

148 highly-qualified employees

> 60% university graduates

90 worldwide R&D companies (2014)

All laboratories on 1 site

Product dedicated support

Customised project management

Compliance with EMA / FDA regulations

From discovery to market place

5 200 m² on 1 site

~2 M € in machinery & equipment (2015)

2


Accelerated stress studies for biotech products :

Main degradation pathways / Stability

Validation / specificity assessment of stability indicating chromatographic methods (IEX, SEC)

Peak identification for purity / impurity profiling

Efficient generic approach on mAbs using state-of-the-art analytics :

Oxidative stress and deamidation (and PTMs)

IEX and 2D-UPLC/MS on the intact protein (isoforms, DP)

Middle-up information on sub-units (enzymatic digestion followed by RP-HPLC/MS)

Amino acid level by peptide mapping (RP-UPLC/MSE)

INTRODUCTION / CONTENT

3


Therapeutic mAbs undergo numerous post-translational modifications and degradations

Heavy chain N-terminal pyroglutamic acid (-17/-18)

N-glycosylation

C-terminal lysine truncation (-128 Da)

Deamidation (+1/-18 Da)

Oxidation (+16 Da)

MABS : PTMS & DEGRADATION

Figure adapted from Beck et al. Anal. Chem. 2012, 84, 4637−4646

-NSTY-

4


INTACT LEVEL ANALYSIS

2D-LC/MS (IEX X RP) OF HUMIRA

(ADALIMUMAB)

5


Autoblend optimization / WCX (1/2)

Minutes

18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00 32.50 33.00 33.50 34.00 34.50 35.00 35.50 36.00

6.56.76.97.1

pH

AU

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.32

0.34

0.36

0.38

0.40

0.42

0.44

Minutes

18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00

[MOPS]

50 mM 40 mM 20 mM


6


AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

Minutes

18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00 32.50 33.00 33.50 34.00 34.50 35.00 35.50 36.00 36.50 37.00 37.50 38.00 38.50 39.00 39.50 40.00

NaCl gradient

Minutes

20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00 32.50 33.00 33.50 34.00 34.50 35.00 35.50 36.00 36.50 37.00 37.50 38.00

Buffer

MOPS MES

a

b

c

d

e


Autoblend optimization / WCX (2/2)

7


Final IEX (WCX) conditions

Final conditions allow the efficient resolution of both acidic and basic species :

MOPS, 50 mM

pH 7.1

Gradient b

0.750 ml/min

25 - 100 µg injection

Amenable to 2D-LC/MS


8


AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

Minutes

20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

0.110

0.120

Minutes

20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00 32.50

Initial

Carboxypeptidase b digestion


C-Terminal Lys isoforms

9


AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

Minutes

20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00

0

5

10

15

0 10 20

%A

rea

Time (h)

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

0.065

0.070

0.075

0.080

0.085

0.090

Minutes

20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00 32.50

* **

Initial

Stressed

(100 mM Tris, pH 8)


Basic pH stress

10


Heartcutting to trap & 2D

WCX elution profiles equivalent with and without deglycosylation (slight RT shift only)

Higher quality MS spectra after deglycosylation

Targeted heartcuts (6 s)

Focalisation and desalting (> 20 min ; H2O / HCOOH) on a trap column (Waters X-Bridge BEH300 C4 ; 30 mm, 3.5 µm)

Injection on a RP-UPLC second dimension column for on-line MS analysis (Waters BEH300 C4 ; 50 x 2.1 mm, 1.7 µm)


11


Main Basic 1 Basic 2Acid 2Acid 1

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

Minutes

20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00


Heartcutting to trap & 2D

12


Species identification after desalting & 2D

Main

Basic 1

Basic 2

Acid 2

Acid 1

+ 1 Lys

+ 2 Lys

Deamidations

Deamidations

and/or Oxidation

Raw data Deconvoluted (centroid)


13


SUB-UNIT LEVEL ANALYSIS

RP-LC/MS OF HUMIRA (ADALIMUMAB)

14


Large range of RP columns tested

Desalting,sub-unit separation, with glycosylation and lysine truncation detection:

Waters MassPrep Micro Desalting (HPLC)

Waters BEH300 C4 (UPLC)

Phenomenex Aeris Widepore C4 (Core shell)

Subunit separation with detection of additional post-translational modifications:

Agilent PLRP-S (HCOOH or TFA)

Thermo Scientific MAbPac RP (50 x 3 mm ; 5 µm ; 1500 Å ; TFA)


15


Separation of isoforms, PTMs and degradations

IdeS(30 min; with / without

buffer exchange)

1 µg mAb

Reduction(DTT)

Oxidation PyroGlu

ox

Intact

F(ab’)2

Fc/2

Fd

Oxidation PyroGlu

No glycosylation± Lys

Fc/2 LC


16


H2O2 ↗

0 %

Fc/2 F(ab’)2Non oxidized

OxOx

Ox x 2 Non oxidized

Ox

Ox

Ox x 2

0.2 %

Oxidative stress (45 min)


17


RT ↗ Fc/2 G0F

Fc/2 G1F

Fc/2 Oxidized G0F

Fc/2 Oxidized G1F

Fc/2 2 x Oxidized G0F

Fc/2 2 x Ox G1F+TFA


Oxidative stress / MS identification

18


0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1

% m

od

ifie

d

% H2O2

Fc/2 ox1

Fc/2 ox2

F(ab')2 ox1

F(ab')2 ox2

Oxidative stress


19


RP-UPLC-ESI/MSE PEPTIDE MAPPING

OF HUMIRA (ADALIMUMAB)

20


High confidence sequence coverage

UPLC gradient : 85 min, using ACN / H20 / TFA ; 1-10 µg injected

Fully integrated analysis, process and reporting (Unifi)

Fast, total sequence coverage at 10 ppm, regardless of the column used

≥ 93% of sequences found with < 5 ppm error and confirmed by at least 2 b/y fragment

PEPTIDE / AMINO ACID LEVEL ANALYSIS

21

normal stringent

BEH300 C18 100 93

CSH130 C18 100 94

Aeris XB-C18 100 93

30

40

50

60

70

80

90

100

% C

ove

rag

e


PTMs and degradations

Robust quantification of C-terminal lysine truncation, Pyroglutamic acid, N-glycosylations, oxidations, and deamidations


22

0.0

20.0

40.0

60.0

80.0

100.0

BEH300 CSH130 Aeris XB-C18

%m

od

ifie

d -Lys C-term

G0F N

G1F N

PyroGlu E

0.0

10.0

20.0

30.0

40.0

50.0

N31 (CDR L1)

N137/138 (LC)

N152/158 (LC)

N54 (CDR H2)

N280/290 (HC)

N319 (HC)

N388/393/394 (HC)

LC

DIQMTQSPSS LSASVGDRVT ITCRASQGIR NYLAWYQQKP GKAPKLLIYA ASTLQSGVPS RFSGSGSGTD FTLTISSLQP

EDVATYYCQR YNRAPYTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ

ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

HC

EVQLVESGGG LVQPGRSLRL SCAASGFTFD DYAMHWVRQA PGKGLEWVSA ITWNSGHIDY ADSVEGRFTI SRDNAKNSLY

LQMNSLRAED TAVYYCAKVS YLSTASSLDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV

SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG

GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG

KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP

VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K

CDRs

Typical modifications N-Deamidations (no stress)

22

Need for trypsin “low pH”


Stressed mAb (100 mM Tris, pH 8)

0

20

40

60

80

0 10 20 30 40

%m

od

ifie

d

Stress time (min)

D1/17 (LC)

Q199 (LC)

N152 (LC)

0

1

2

3

0 10 20 30 40

%m

od

ifie

d

Stress time (min)

Q3/6 (LC)

Q55 (CDR L2)

Q79/89 (LC/CDR L3)

Q124 (LC)

Q3/Q13 (HC)

D30/31 (CDR H1)

Significant deamidation Marginal deamidation

23


23

LC




HC







CDRs


Oxidative stress / Methionine

Robust quantification of methionine oxidation

CDRs

0.0

20.0

40.0

60.0

80.0

0 0.05 0.1 0.15 0.2

%o

xid

atio

n

%H2O2 (45 min)

M4 (LC)

M34 (CDR H1)

M83 (HC)

M256 (HC)

M432 (HC)

0

20

40

60

80

0 0.05 0.1 0.15 0.2

%O

xid

atio

n M

256

%H2O2 (45 min)

BEH300

CSH130

Aeris XB-C18

24


24

LC




HC







CDRs


- 64 Da

Peptide DTLM256ISR

Confirmation of oxidation site by MSE


25


Intact level2D-LC/MS(IEX x RP)

Sub-unit levelRP-LC/MS

Peptide/AA levelPeptide mappingRP-UPLC/MSE

IdeS

Reduction (DTT)

PeptidesReduction (DTT, IAC) / Trypsin

DIQMTQSPSSLSASVGDR

DSTYSLSSTLTLSKEAK

LLIYAASTLQSGVPSR

ASQGIR

Substructures

Intact

Figure adapted from Beck et al. Anal. Chem. 2012, 84, 4637−4646

Deglycosylation

(Rapid PNGase, with or

without reducing buffer)

F(ab’)2

Fc / 2

Fd

LC

Fc / 2

26

GENERAL APPROACH


Three levels of analysis

Intact, substructure, peptide/amino acid: various degrees of easyness / precision

Optimized workflows

Fast

Quick sample preparation, low amounts required

Efficient LC separation and heartcutting (Waters 2D-UPLC & Xevo-G2XS-Q/TOF)

Semi-automated data processing and reporting (MassLynx, Unifi)

CONCLUSIONS AND PERSPECTIVES

27



28

Optimized workflows (ctd)

Versatile

WCX and/or RP separation for different modifications detection

Autoblend development for quick adjustments to new samples

Large range of columns for different needs / samples

Large range of enzymes for various substructure generation

Reliable

High resolution MS detection for best precision and sensitivity

2D-LC/MS for fast identification confirmation

High repeatability


Detection and quantification of post-translational modifications at each level

Information on key mAb elements (CDRs) / CQA

Ideal for stressed / aged samples monitoring

Batch-to-batch consistency

In-depth studies of isoforms / DP with 2D-LC/MS

Stability studies & formulation optimization

ADCs (in progress)

DAR and Payload distribution

Conjugation sites, PTMs, isoforms


29


Arnaud DELOBEL, R&D Team Manager

Eric LARGY, R&D Scienist

Anicet CATRAIN, Scientist

Région Wallonne / FEDER

30

ACKNOWLEDGEMENTS

Date post:	13-Jan-2017
Category:	Healthcare
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Stress testing and physico-chemical characterization of monoclonal antibodies : The approach of an...

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