Native and IM-MS characterization of ADCs Alain VAN DORSSELAER

43 Pages·2015·4.44 MB·English

Native and IM-MS
characterization of ADCs
Alain VAN DORSSELAER

BioOrganic Mass Spectrometry Laboratory, IPHC
University of Strasbourg, CNRS
Strasbourg - France
ASMS 2015 Waters User’s meeting 1
mAbs as therapeutics
 mAbs : a very important class of human therapeutics
- > 50 mAbs & derivatives currently approved (EMA/FDA)
- indications: inflammation, autoimmunity, cancer, transplantation,…
- > 400 mAbs & derivatives in pre-clinical development and clinical trial
 mAbs are complex therapeutic proteins
(150 kDa glycoproteins)
 anti-cancer mAbs in clinical studies
- 84 unmodified IgG (51%)
- 10 bispecific (6%)
A single shot gun
- >35 ADC (15%)
- 17 engineered (10%)
- 16 fragments (10%)
 Next generation empowered mAbs :
Bispecific mAbs (bsAbs) and Antibody-Drug-Conjugates (ADCs)
+ GSH
+
Double-barreled shotgun:
mAb + cytotoxic A highly
two antigens targetted 2 different half mAbs
cytotoxic drug
IgG4 IgG4 Bispecific mAb (bsAb)
(natalizumab) (6F4-2) (natalizumab/6F4-2)
mAb characterization is a nightmare
Peptide mapping Fragment analysis Intact mAb Higher Order Structure
(700-7000 Da) (25-100 kDa) analysis Aggregates
(150 kDa) mAb/Ag complexes
(>150 kDa)
Size
The MS
LC-MS
toolbox
Native MS (nano)LC-MS/MS
enzymatic digestion
Native IM-MS Top-down MS
(FT-ICR + Orbital trap)
HDX-MS Crosslinking MS
Native MS: a brief history
2012
Conformation
High
resolution
Binding native MS
2006
2000
Ion Mobility MS
Synapt
Automated
G2
1996 nanoESI Synapt
1991
nanoESI G1
Native
Increase in size,
MS
nb of partners
and heterogeneity
1991 2002 2006 2013
Holo-myoglobine Haemocyanins Ribosomes Bacteriophage HK97
17.5 kDa 2.3 MDa 2.3 MDa capsid
18 MDa
110000 1100447744..1199 xx1100 xx2200
1100223366..4411
1144008888..0066
1133887700..6688
1144331133..5588
1100772233..2233 1133665599..9900 1166770022..77112266991122..7722
%%
1166449966..9933
1100000088..8844 1177112266..6644
1133445555..1133 1166229988..8877 1177334499..1111
44442222..3333 1166110011..2288 1199440044..0000
44117733..9911 1100998822..66441133225555..2211 1188771199886699..99880077..1111 1199661155..7766
33994499..9999 44669966..4499 99779911..4422 1155991144..7788 119922880033000055..3355..6611992211449933..6644 Snijder et al. Angew. Chem 2013
2200000000 44000000 66000000 88000000 1100000000 1122000000 1144000000 1166000000 1188000000 2200000000 2222000000 2244000000 mm//zz
McKay et al. JACS 2006
Katta et al. JACS 1991 Sanglier et al. JASMS 2002
4
Native MS analysis
Native MS gives information on assemblies maintained
by noncovalent interactions
ESI-MS
instrument
3000 3500 m/z
Gas phase
Solution

Sample preparation step Instrumental setting
Data interpretation
= desalting step optimizations
- Buffer exhange - Control of the energy - Binding stoichiometries
- Use of volatile aqueous communicated to the - Binding specificity
buffer at neutral pH ions in the 1st pumping - Solution affinities
- Ammonium buffer stage region of the - Dynamics of
(0-1M) with controled pH instrument assembly/disassembly
(Vc, Pi adjustment)
Be carefull !
Native Mass Spectrometry: a recursive question
We dream to get from Native MS and IM-MS experiments :
• Accurate mass measurements, stoichiometries,…
• Information on interactions forces
• Subunit spatial arrangement, structural information, shape, conformation changes,…
… but all this information concerns molecules in water solution
1- Electrospray mecamism
salts
2- Evaporation in interface
Dried, in vacuo
In water solution
Specific
non-covalent interactions
?
6
Toutankhamon
Native MS workflow for mAb analysis
For mAbs minimal and easy sample preparation are well descibed
Purified
G1F/G0F
147239 144190
mAb, bsAb or ADC G1F/G1F
G2F/G0F
147077 147401 PNGase-F
G0F/G0F
G2F/G1F
G1F/G0F 147563
147239 144190
G1F/G1F Deglycosylation Step If necessary
G2F/G0F Mass Mass
147077 147401 PNGase-F (PNGase F, IgZero, etc.) 146700 147200 147700 148200 143700 144000 144300 144600
G0F/G0F
G2F/G1F
147563
Buffer Exchange
Mass Mass
146700 147200 147700 148200 143700 144000 144300 144600 (spin columns, microconcentrators,
SEC etc.)
Native MS and IM-MS
(with ESI, needles or Chip-based introduction)
Data Interpretation
Synapt G2 HDMS (Waters)
+ nanoMate Triversa (Advion)
How do we jumped into the mAb field?
 2008-2012: Reference Monoclonal Antibodies
(mAb)
- mAb native MS
- mAb/Ag binding stoichiometries
- Native IM-MS
 2013: towards bispecific mAbs
(bsAbs)
- Native MS for monitoring of bsAb formation
- IM-MS for bsAb
- Real-time IM-MS to monitor bsAb formation
 2014: immunoconjugate analysis
(ADC)
- Native MS mandatory for ADCs’ analysis
- Native MS for drug load profiles, average
DAR
- IM-MS for ADC heterogeneity assessment
- IM-MS for drug load profiles, average DAR
8
1. Native MS and IM-MS
for reference mAb analysis
2. Native MS and IM-MS
for bsAb formation
3. Native MS and IM-MS
for ADC formation
9
Intact mAb analysis : benefits of native MS for intact mAb analysis
 Denaturing Conditions
Tramstu IgAGZerbo de n2at 2uµMM in H2O:ACN:FA (50:50:1) 45+ Deconvoluted
mass spectrum
100
Non covalent dimer
Full MS spectrum
not detected
%
1000 1500 2000 2500 3000 3500 4000 4500 m/z
0 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 m/z
 Native Conditions
mAb 5µM in AcNH4 150mM pH7.5
25+
trastu 5uM ACNH4 150mM pH7.5 Viva6 120v 6mbar Deconvoluted
In Native conditions mass spectrum
100
sensitivity and mass accuracy
are as good as
Full MS spectrum
in Denaturing conditions.
But spectra are more simple,
and you see interactions !!!
%
dimer
3%
2000 3000 4000 5000 6000 7000 8000 9000 m/z
10000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 1000m0/z
Beck et al. J Mass Spectrom. 2015 Feb;50(2):285-97.