h/d exchange mass spectrometry: applications in antibody

David Weis, University of Kansas NIH 5/7/14

For informational purposes only. 1

H/D Exchange Mass Spectrometry: Applications in Antibody Aggregation and Disordered Proteins

David Weis

Department of Chemistry

Instrumentation

Bio-pharmaceuticsCrowding

McGuffee, & Elcock, PLoSComput Biol 2010, 6, e1000694.

Disordered proteins

Ligand discovery



H/D exchange as an enabling technology

Backbone flexibility contributes toprotein function and dysfunction.

Disordered proteins

Aggregation hotspots

H/D exchangemass spectrometry

Amide hydrogens serve as backbone sensors.

Leu Ala Pro Lys Ser

Noexchange

Toofast

millisecondsto days



Major Histocompatibilty Complex (MHC) proteinhttp://www.youtube.com/watch?v=Y79Xl0LfYI4

H/D exchange reports on backbone conformation and dynamics.

O NH O O

kop

kcl

OD−

OH−

kint

opobs int

cl

kk k

k

Kaj Linderstrøm-LangCarlsberg Laboratory

Conformationand dynamics

Intrinsic



H/D exchange kinetics probes backbone dynamics.

D2O D2O

Flexible regions exchange rapidly

Rigid regions exchange slowly

MS approach uses quench and proteolysis.

Undeuterated

Quench

0 °CpH 2.5

Labeling

D2O

Deuterated

ProteolysisPepsin

Mass Analysis



Pepsin digestion gives localized coverage.

Average length: 14 residuesTotal coverage: 85%

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

TOF-MS

HPLC

Automation

Year

Weeks

Disordered proteins






Acid blobsNegative noodles

Intrinsically disorderedIntrinsically unstructured

Natively denaturedNatively unfolded

Mostly unstructuredFloppy

Dynamic

The disorder continuum

PONDR:Predictor Of Natively Disordered Regions

Romero, P.; Obradovic, Z.; Li, X.; Garner, E. C.; Brown, C. J.; Dunker, A. K. Proteins 2001, 42, 38-48. www.pondr.com

Spa32 (S. flexneri)

Residue Number

0 50 100 150 200 250 300

PO

ND

R s

core

0.0

0.2

0.4

0.6

0.8

1.0

Disordered

Ordered



How does coupled binding and folding work?

Disorderedstate

Foldedconformer Folded

complex

CBPco-activator

binding domain

ACTRactivation domain

Kd = 38 nM

+

Unstructured Molten globule

A model system for coupled binding and folding

Transiently structured

Transiently unfolded

Peter Wright, Scripps



The speed limit of H/D exchange

R A L L D Q L

0

1

2

3

kint

(s−1)

int

ex

kPF

kProtection factor:

maximum rate

measured rate

D2O Exposure (s)

10-3 10-2 10-1 100 101 102 103 104

Deu

teri

um

Lev

el (

Da)

0

1

2

3

4

5

Kinetic analysis of H/D exchange

CBP ACTR

free

bound

1 ktD t N e



Mapping protection factors

G T Q N R P L L R N S L D D L V G P P S N L E G Q S D E R A L L D Q L H T L L S N T D A T G L E E I D R A L G I P E L V N Q G Q A L E P K Q D

1-12 36-47 60-7113-22 48-59

Aα1 Aα2 Aα3

33-42

29-3533-47

00 0

33-47

36-4729-35

33-42

48-5960-711-12

13-22

P N R S I S P S A L Q D L L R T L K S P S S P Q Q Q Q Q V L N I L K S N P Q L M A A F I K Q R T A K Y V A N Q P G M Q

Cα1

1-14 30-38 43-5931-40

431-10 17-30 40-

Cα2 Cα3000

1-141-10 17-30

30-38 43-5931-40

40- 43

<33-9

9-2020-50

>50

PF

Free

Complex

Complex

Free

CBP

ACTR

CBP is a molten globule

NMR secondary shifts

Ebert, Bae, Dyson, & Wright Biochemistry 2008, 47, 1299.

Free CBP

Kjaergaard, Teilum, & Poulsen, PNAS 2010, 107, 12535 (2KKJ)

Structure by NMR

∆Gu = 1.5 kcal mol−1

~8% Unstructured



CBP transiently populates an unfolded state.

Can H/D exchange reveal transiently foldedstates?



Exchange is too fast

10−3 10−2 10−1 100 101 102 103 104

Exchange time (sec)

Millisecond quench-flow H/D exchange

40-3500 msec



Kinetic analysis of exchangeby an unstructured protein

Exchange (s)

0.0 3.5

Deu

tera

tio

n (

%)

0

50

100

1017-10241029-10381039-1048 1045-10621059-10631059-10631064-10661072-1080 1081-1087

t50%

Peptide-level half-life reveals residual structure

α-helix α-helixabsentSecondary structure in complex



Localizing structure by residue-level averaging

it

2

2

j jn

ij

n

t Lt

L

weighted half-life

normalization factor

t1t2t3t4

Localized half-life correlates well withresidual structure.

Kjaergaard, 2010

α-helix α-helixabsent

H/D exchange

NMR

Helical propensity AGADIR



H/D exchange can capture both extremes of disorder

ACTR

CBP

Molten globule

Unstructured

Disordered proteins






49 biologics

There has been rapid growth in the useof therapeutic monoclonal antibodies.

0

31

1985 2011

$20 billionin sales

FDA approvedmAbs



mAbs are the Cadillacs of biotherapeutics.

• 150 kDa• IgG1• Glycosylated• 12 disulfide bonds• 50 mg/mL, pH 6

Antigenbinding

SS

SS

Antigenbinding

VH

VL

CL

CH1

CH2

CH3

VH

VL

CL

CH1

CH2

CH3

glycan glycan

Maintaining the physical stability ofprotein therapeutics is a critical problem.

Solution: Develop a stabilizing formulation.

Loss of efficacy

Immunogenicity

Conformationalinstability

Aggregation



Development of a stabilizing formulation is essential.

GenerallyRegardedAsSafe

Formulation Active ingredient

Excipients

mAb

Excipients can alter mAb stability.

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

Do local dynamics correlate with stability?

chloride sucrosesulfate

mAb-BIgG1

Sucrose increased thermal stability.Arginine decreased thermal stability.

Temperature (°C)

Cp

(cal

mol

−1

K−

1)

sucrose+6 °C

Temperature (°C)

arginine−4 °C



mAb-B aggregated at 40 °Cunder accelerated storage conditions.

8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 min

Ab

sorb

ance

(28

0 n

m)

Dimer

Monomer

FragmentsMultimer

0.5 M NaSCN40 °C

initial

2 months

Sulfate slowed aggregation.Thiocyanate accelerated aggregation.

0 2 4 6 8 10 12

30

40

50

60

70

80

90

100

Monomer(%)

4 C

25 C

Months

NaSCN

Na2SO4

Control

40 °C



Stabilizers and destabilizersacted as expected.

∆Tm1 Aggregation

thiocyanate −9.0 °C Faster (++)

arginine −1.9 °C Faster (+)

chloride +0.3 °C Negligible

sucrose +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 M

NaCl, 0.1 M

sucrose, 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 H/D exchangeis not obvious.



Arg+SCN−

DestabilizerDestabilizer

Destabilizers have very different effects.

A hydrophobic segment of the CH2 domainmay mediate aggregation.

CH3 CH3

CH2 CH2

VFLFPPKPDTLMI

Destabilizers and oxidation increased backbone flexibility. [Houde, 2010]

Protein A binding inhibits aggregation. [Zhang, 2012]

Disulfide bond increased thermal stability. [Gong, 2009]



New insights into function and dysfunction

Increased flexibility accelerates aggregation

H/D exchange

Detecting transient states in disordered proteins

CAREERMCB-1149538

University of Kansas: David Volkin & Russ Middaugh

MedImmune: Hardeep Samra, Hasige Sathish, Steven Bishop, Prakash Manikwar

h/d exchange mass spectrometry: applications in antibody

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