Hydrogen exchange mass spectrometry for higher-order ... · Hydrogen exchange mass spectrometry for...

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