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Hydrogen exchange mass spectrometry for higher-order structure determination in therapeutic protein discovery and development
David D. WeisDepartment of ChemistryAdams Institute for Bioanalytical ChemistryUniversity of Kansas
DISCLAIMER: THIS SEMINAR IS PRESENTED FOR INFORMATIONAL PURPOSES ONLY AND DOES NOT REPRESENT AN ENDORSEMENT BY THE UNIVERSITY OF KANSAS.
Citations and links for this work
Arora, J; et al. Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody. mAbs 2015, 7, 525-539.
http://www.tandfonline.com/doi/abs/10.1080/19420862.2015.1029217?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed#.VeRtuZdGwl8
Manikwar, P; et al. Correlating excipient effects on conformational and storage stability of an IgG1 monoclonal antibody with local dynamics as measured by hydrogen/deuterium-exchange mass spectrometry. J Pharm Sci 2013, 102, 2136-2151.
http://onlinelibrary.wiley.com/doi/10.1002/jps.23543/abstract;jsessionid=8285294C29C018B7BFA9F6CAD972C2D6.f03t03
Majumdar, R; et al. Effects of Salts from the Hofmeister Series on the Conformational Stability, Aggregation Propensity, and Local Flexibility of an IgG1 Monoclonal Antibody. Biochemistry 2013, 52, 3376–3389.
http://pubs.acs.org/doi/abs/10.1021/bi400232p
Majumdar, R; et al. Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life. mAbs 2015, 7, 84-95.
http://www.tandfonline.com/doi/abs/10.4161/19420862.2014.985494?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed#.VeRt65dGwl8
49 biologics
Major Histocompatibilty Complex (MHC) protein, http://www.youtube.com/watch?v=Y79Xl0LfYI4
Higher order structure is essential for function
Applications of hydrogen exchange (HX)Protein interactions
Epitope mapping
Ligand binding Formulations
Comparability
Disordered proteins
Amide hydrogens serve as backbone sensors.
Leu Ala Pro Lys Ser
Noexchange
Toofast
millisecondsto days
Hydrogen exchange reports on protein conformation and dynamics.
opobs ch
cl
kk k
k
Conformationand dynamics
Chemical
D2O
H2O
kop
kclkch
H/D exchange kinetics probes backbone dynamics.
D2O D2O
Flexible regions exchange rapidly
Rigid regions exchange slowly
MS approach uses quench and proteolysis.
Undeuterated
Quench0 °C
pH 2.5
LabelingD2O
Deuterated
ProteolysisPepsin
Mass Analysis
676 680 684
m/z
Undeuterated
5 sec
4 hr
Peptides progressively gain mass.
Deuterium uptake curve
MassIncrease
(Da)
D2O Exposure (s)100 101 102 103 104
0
3
6
9
Bound
Free
Efficient and robust platformsare now available Informatics
QTOF-MS
HPLC
Automation
Year
Days
Applications of hydrogen exchange (HX)
Epitope mapping
Protein interactionsLigand binding Formulations
Comparability
Disordered proteins
Ricin as a vaccine target• Extremely toxic
(~1 µg/kg inhaled)• Multiple organ failure• Lethal within 72 hrs• Potential terror agent
Vaccine discovery
Recover antibodies
Functional assays
Map the epitope
Immunize animals
antigen
Eliciting neutralizing antibodies
• Location?• Mechanism?• Rational design?
RTA peptic peptide map gives 100% coverage
1 10 100 1000 10000 1000000
1
2
3
4
5
6
#D2O
(Da)
Labeling Time (s)
RTA* alone RTA* + cPB10
1 10 100 1000 10000 1000000
1
2
3
4
5
6
7
#D2O
(Da)
Labeling Time (s)
RTA* alone RTA* + cPB10
1 – 12: AIFPKQYPIINF 94 – 108: FHPDNQEDAEAITHL
Epitope mapping with hydrogen exchange
RTA
RTA +antibody
Hyd
roge
n ex
chan
ge (D
a)
Hyd
roge
n ex
chan
ge (D
a)
Exchange time (s) Exchange time (s)
bound freem m m
-5-4-3-2-101
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120
Peptide Number
Rel
ativ
e M
ass
Diff
eren
ce (
Da)
10 s
102 s
103 s
104 s
24 hr
Epitope mapping by HX protection
-5-4-3-2-101
-5-4-3-2-101
-5-4-3-2-101
-5-4-3-2-101
PDB:3SRT
Location of PB10 epitope by HX-MS
HX-MS agrees with Pepscan
180˚
180˚
PB10
other epitopes
Pepscan
HX-MS
Applications of hydrogen exchange (HX)Protein interactions
Epitope mapping
Ligand binding Formulations
Comparability
Disordered proteins
mAbs are the Cadillacs of biotherapeutics.
• 150 kDa• IgG1• Glycosylated• 12 disulfide bonds• 50 mg/mL, pH 6
Antigenbinding
SSSS
Antigenbinding
VH
VL
CL
CH1
CH2
CH3
VH
VL
CL
CH1
CH2
CH3
glycan glycan
40 mg mAb0.8 mL
Large dose, small volume
Pre-filled syringe
Reversible self-association
ViscosityLoss of efficacyImmunogenicity
40 mg in 0.8 mL
mAb C undergoes reversible self-associationH
ydro
dyna
mic
dia
met
er (n
m)
Protein concentration (mg/mL)H
ydro
dyna
mic
dia
met
er (n
m)
Salt concentration (mM)
Promoted by high pH and sulfate
300 mM Na2SO44 °C
300 mM NaCl4 °C
Sulfate promotes association
Visc
osity
(cP
)
Protein concentration (mg/mL)
Mapping protected interface with HX-MS
Time (seconds)
Mas
s in
crea
se (D
a)
mAb-C Heavy 11-22 (CL) mAb-C Heavy 135-140 (CH2) mAb-C Heavy 166-172 (VH)
mAb-C Heavy 45-59 (VH)mAb-C Heavy 36-54 (VL)mAb-C Heavy 48-70 (VL)
mAb-C (5 mg/mL)mAb-C (60 mg/mL)
highlow
Mechanism of RSA
Applications of hydrogen exchange (HX)Protein interactions
Epitope mapping
Ligand binding Formulations
Comparability
Disordered proteins
Maintaining the physical stability ofprotein therapeutics is a critical problem.
Solution: Develop a stabilizing formulation.
Loss of efficacy
Immunogenicity
Conformationalinstability
Aggregation
Citrate
Acetate Histidine
Phosphate
Arginine
Glycine
Proline
Lysine
Methionine
Sucrose
Trehalose
Sorbitol
Glutamate
Glycerol
Urea
Mannitol
Glucose
Lactose
Albumin
Gelatin
PVP
PLGA
PEG
Sodium chloride
Potassium chloride
Sodium sulfate
Polysorbate
EDTA
DTPA
Ethanol
m-cresol Tris
Benzylalcohol Rational formulation requires
mechanistic understanding.
thiocyanatearginine
Thermal stability Aggregation Backbone dynamics
Does backbone flexibility correlate with stability?
chloride sucrosesulfate
mAb-BIgG1
The connection between flexibility and stability was not obvious.
Stabilizers Destabilizers
Faster dynamics
Slower dynamics
Loss of stability correlated with increased flexibility in CH2 domain.
Nocorrelation
Sulfate slowed aggregation.Thiocyanate accelerated aggregation.
0 2 4 6 8 10 12
30
40
50
60
70
80
90
100
Monomer(%)
4 C25 C
Months
NaSCN
Na2SO4
Control
40 °C
Stabilizers and destabilizersacted as expected.
∆Tm1 Aggregationthiocyanate −9.0 °C Faster (++)arginine −1.9 °C Faster (+)chloride +0.3 °C Negligiblesucrose +1.5 °C Slower (−)sulfate +1.8 °C Slower (−)
How do these excipients workat the molecular level?
The effects of excipients are not uniform.
∆m
CH1 CH2 CH2
CH2 VL VL
Arg, 0.5 MNaCl, 0.1 Msucrose, 0.5 M
The effects are excipient-dependent.
Peptide (N to C)
±0.59 Da (99% CI)120 s
103 s
104 s
105 s
Differentialdeuteriumuptake (Da)
arginine, 0.5 M sucrose, 0.5 M
Arg+SCN−
sucrose SO
faster
slowerno effect
no data
The correlation between stability andaltered H/D exchange is not obvious.
Homology model based on [Saphire, 2001] 1HZH
SCN− SO
Destabilizer Stabilizer
Extremes have nearly-identical effects.
Arg+SCN−
sucrose SO
faster
slowerno effect
no data
The correlation with altered hydrogen exchange is not obvious.
Arg+SCN−
DestabilizerDestabilizer
Destabilizers have very different effects.
A hydrophobic segment of the CH2 domainmay mediate aggregation.
CH3 CH3
CH2 CH2VFLFPPKPDTLMI
Destabilizers and oxidation increased backbone flexibility. [Houde, 2010]
Protein A binding inhibits aggregation. [Zhang, 2012]
Disulfide bond increased thermal stability. [Gong, 2009]
Summary
Epitope mapping
ricin antibody
Protein interactions
mAb self-association
Formulations
aggregation hotspots
University of Kansas: David Volkin & Russ MiddaughWadsworth Center, NY State Dept. of Health: Nick MantisMedImmune: Hardeep Samra, Hasige Sathish, Reza Esfandiary, Steven Bishop,
Prakash Manikwar