High-throughput Biophysical Analysis of Protein Stability: Comparability Assessments and Formulation Development

David B. Volkin

Department of Pharmaceutical Chemistry,Macromolecule and Vaccine Stabilization Center

2nd I t ti l S i Hi h O d St t2nd International Symposium on Higher-Order Structure of Protein Therapeutics

February 2013February 2013

Outline of Presentation

Protein Stability and Comparability AssessmentsIntroduction

Case study highlighting challenges and opportunities of high-throughput biophysical analysisp y y

Protein Stability and Formulation DevelopmentIntroduction

Case studies utilizing high-throughput biophysical analysis-

• Albumin-fusion protein

• Pentameric recombinant plasma glycoprotein

Biopharmaceutical Comparability Approaches and Analytical Challengesy g

Biochemical and biophysical testing:QC analytical testsyAnalytical characterization tests for structure or activityStability profile and degradation profile

B l l d l Biological and animal testing:Biological assays that are linked to mechanism of actionAnimal pharmacology & toxicology studies if appropriate

Clinical testing:Human PK studies where good correlates with clinical activity are known I h ll f h b l h ff d/ In the event all of the above are inconclusive, human efficacy and/or safety studies may be needed

Analytical challenge: y gNeed for improved methodologies to examine higher-order structural integrity and conformational stability

Comparability Assessments-Need for New Analytical Approaches to Evaluate

Higher-Order Structure and StabilityHigher Order Structure and Stability

1 One approach is to use higher-resolution 1. One approach is to use higher resolution biophysical techniques:

X-ray CrystallographyNMRNMRIon Mobility Mass SpectrometryHD-Exchange Mass Spectrometry

2. Are there alternative approaches to obtain better biophysical characterization data with protein drugs???

Large amounts of lower-resolution dataHigh throughput screeningData analysis and visualization

Empirical Phase Diagrams-Visualize Large Amounts of Biophysical Data

(Developed by Middaugh Lab; Applied to Formulation of Proteins and Vaccines)

0 3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

340

341

342

343

344

345

346A

Rel

ativ

e M

E at

222

nm

B

Rel

ativ

e Fl

. pea

k in

tens

ity

C

Fl. p

eak

posi

tion

(nm

)

Experimental Data

(Developed by Middaugh Lab; Applied to Formulation of Proteins and Vaccines)

0 10 20 30 40 50 60 70 80 900.1

0.2

0.3

0 10 20 30 40 50 60 70 80 90

0.0

0.1

0 10 20 30 40 50 60 70 80 90338

339

20 30 40 50 60 700.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

0 10 20 30 40 50 60 70 80 90

0.0

0.2

0.4

0.6

0.8

1.0

0 10 20 30 40 50 60 70 80 90496

498

500

502

504

506

508

510

512

514

516

518

520

522

524

0.6 1.0 16

Temperature (oC)

R

Temperature (oC) Temperature (oC)

D

Rel

ativ

e he

lix c

onte

nt

Temperature (oC)

E

Rel

ativ

e AN

S Fl

. pea

k in

tens

ity

Temperature (oC)

F

ANS

Fl. p

eak

posi

tion

(nm

)

Temperature (oC)

G H I

Experimental Data

Visualization

0 10 20 30 40 50 60 70 80 90

0.0

0.1

0.2

0.3

0.4

0.5

0 10 20 30 40 50 60 70 80 90

0.0

0.2

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0 10 20 30 40 50 60 70 80 90 100

6

8

10

12

14

Opt

ical

den

sity

at 3

50 n

m

Temperature (oC)

Rel

ativ

e Fl

. lig

ht s

catte

ring

Temperature (oC)

Figure 1

Exce

ss M

HC

Temperature (oC)

0.9

1.0

1.1

0.9

1.0

1.1

348

349

350

351

A B C

Interpretation0 10 20 30 40 50 60 70 80 90

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 10 20 30 40 50 60 70 80 90

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 10 20 30 40 50 60 70 80 90

339

340

341

342

343

344

345

346

347

348

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

0.2

0.4

0.6

0.8

1.0

502

504

506

508

510

512

514

516

518

520

522

524

Rel

ativ

e M

E at

222

nm

Temperature (oC)

Rel

ativ

e Fl

. pea

k in

tens

ity

Temperature (oC)

Fl. p

eak

posi

tion

(nm

)

Temperature (oC)

D

Rel

ativ

e he

lix c

onte

nt

E

Rel

ativ

e AN

S Fl

. pea

k in

tens

ity

F

ANS

Fl. p

eak

posi

tion

(nm

)

Interpretation

20 30 40 50 60 701.0

0 10 20 30 40 50 60 70 80 900.0

0 10 20 30 40 50 60 70 80 90

500

0 10 20 30 40 50 60 70 80 900.0

0.2

0.4

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0 10 20 30 40 50 60 70 80 90

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0 10 20 30 40 50 60 70 80 90 100

0

2

4

6

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10

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20

Temperature (oC) Temperature (oC) Temperature (oC)

G

Opt

ical

den

sity

at 3

50 n

m

Temperature (oC)

H

Rel

ativ

e Fl

. lig

ht s

catte

ring

Temperature (oC)

Figure 2

I

Exce

ss M

HC

Temperature (oC)

REVIEW: Maddux NR et al, J. Pharm. Sci. 100, 4171-97 (2011)

Typical Procedure for Constructing EPD

…Fluorescence Emission

Circular Dichroism UV AbsorbanceTechniques:

Preprocessing:

Buffer Subtraction Buffer Subtraction Buffer Subtraction

Peak Picking/Tracing

2nd Derivative / Smoothing

Select one or twoWavelength(s)ep ocess g Picking/Tracing SmoothingWavelength(s)

AverageMultiple Runs

AverageMultiple Runs

Peak Picking/Tracing

pAverage

Multiple RunsNormalization Normalization

Input Matrix PreparationInput Matrix Preparation

Normalization

Singular Value DecompositionSingular Value Decomposition

C l M i

EPD Processing:

Color Mapping

Empirical Phase DiagramEmpirical Phase Diagram

Case Study:Fibroblast Growth Factor (FGF1)

Alsenaidy MA, Wang T et al, P t i S i 21 418 32 (2012)Protein Science 21, 418-32 (2012)

16 kDa, heparin-binding protein unstable at room temperature

Multifunctional protein involved in angiogenesis, wound healing, embryonic development

Possible therapeutic agent for the treatment of ischemic diseases

Collaboration with Michael Blaber, Florida State University looking at 10 different mutants

Bernett MJ, Somasundaram T, Blaber M,Proteins: Structure, Function, and Bioinformatics 57,

626-634 (2004)

The Empirical Phase Diagram (EPD) Summarizes and Visualizes Biophysical Datap y

Empirical Phase Diagram pH 3 pH 4 pH 5 pH 6 pH 7 pH 8

of FGF1 Wild TypeANSFluorescence

80

90

atur

e (°

C)Intrinsic

Fluorescence

50

60

70

Tem

peraLight

Scattering

20

30

40

pH

CircularDichroism

pH3 4 5 6 7 8

10

Spectroscopic data: pH 3-8, 10-90°C, FGF1- WT

WT FGF1 + HeparinEmpirical Phase Diagram of WT

80

90WT FGF1

pera

ture

(o C)

2

50

60

70

Tem

120

30

40

pH3 4 5 6 7 8

10

Empirical Phase Diagram of K12V-P134V-C117V90

FGF1- Mutant H Empirical Phase Diagram of SYM690

FGF1- Mutant J

C)

2

60

70

80

o C)

3

60

70

80

Tem

pera

ture

(o C

130

40

50

Tem

pera

ture

(o

1

30

40

50

pH

1

3 4 5 6 7 810

20

30

pH3 4 5 6 7 8

10

20

Ongoing Work: Possible Use of EPDs for Biopharmaceutical Comparability?p p y

• Can we extend results from FGF-1 mutant study to examine same molecule with different post-translational modifications?

Clustering analysis helps identifySimilar color regions represent similar

p

• How to define structural regions more quantitatively?

70

80

90

2C) )

270

80

90

Clustering analysis helps identify regions computationally

Similar color regions represent similar conformational behaviors

2

30

40

50

60

0 2

Tem

p (°

C

Tem

p (°

C 2

130

40

50

60

2

13 4 5 6 7 8

10

20 1pH

T

pH

T 1

3 4 5 6 7 810

201

• Currently assessing EPDs using an IgG1 mAbs with varying glycosylation patterns

Alternative Data Visualization Approaches (BSA):Structural Stability by EPDs, Radar Charts, Chernoff Face Diagrams

Kim et al, Protein Science 21, 1540–53 (2012)

22

Protein Stability:Challenges During Formulation Developmentg g p

Protein molecule• Unique sequence and physicochemical properties

S ifi bi l i l ti iti• Specific biological activities• Major changes due to small differences:

Alter amino acid residue or glycosylation pattern

Environment around the protein• Solution pH, ionic strength• Different classes of pharmaceutical excipients

Diff i i• Different primary containers

Stresses on this combination• Temperature and time (storage)• Agitation, freeze/thaw, light, lyophilization, etc.• Formulation design space:

protein structure vs formulation vs environmental protein structure vs. formulation vs. environmental stresses

http://www.cartage.org.lb/en/themes/sciences/lifescience/generalbiology/physiology/LymphaticSystem/Antibodymediated/Antibodymediated.htmhttp://www.sciencedirect.com/science/article/pii/S157096390600286X

http://www.americanpharmaceuticalreview.com/Featured-Articles/37325-Implementation-of-a-Platform-Approach-for-Early-Biologics-Development/

Protein Stability and Formulation Development-Case Study with Recombinant Pentameric Glycoproteiny y p

Characterization and t bili ti f bi t

Backgroundstabilization of recombinant

human protein pentraxin (rhPTX-2)

PTX-2 functions as part of innate immune system as soluble pattern recognition receptor.

Lui J et al, J Pharm Sci 102, 827-41 (2013)

Unique structure with two sides: • Calcium mediated ligand binding • Fcγ receptor bindingg

Receptor binding initiates biological responses including regulation of monocytes populationsmonocytes populations.

rhPTX-2 has anti-fibrotic activity in vivo and has entered clinical trials.

Initial Biophysical Characterization:Pentameric Pentraxin Protein (rhPTX-2)

SDS-PAGE

SV-AUC SEC

15

20

25

/o C)

DSC

0

5

10

Cp

(kca

l/mol

e/cIEF

20 30 40 50 60 70 80 90 100 110 120-5

Temperature (oC)

Biophysical Characterization of rhPTX-2:Higher-Order Structural Changes as Function of Temp and pH

CD CD216nmMelts

OD350nmMelts

T Fl T FlTrp Fluor. Trp Fluor. Int. Melts

Trp Fluor. λ Melts

ANS Fluor. I M l

ANS Fluor.

Int Melts

ANS Fluor. λ Melts

Aggregation of rhPTX-2:Effect of Excipients

SEC analysis after 24 hours at 65°C at pH 7.5

controlstabilizers destabilizerst=0

Protein Stability and Formulation Development-Case Study with Albumin Fusion Proteiny

Biophysical characterization d t bili ti f th

Backgroundand stabilization of the

recombinant albumin fusion protein sEphB4-HSA

EphB4 is tyrosine kinase receptor overexpressed in variety of epithelial cancers.

Shi S et al, J Pharm Sci 101, 1969-84 (2012)

Binding of EphB4 to EphrinB2 ligand binding signals cell adhesion, migrationmigration.

sEphB4 (extracellular domain of protein) is effective antagonist of i li I hibit t th isignaling. Inhibits tumor growth in

animal models.

sEphB4-HSA shows activity with Subramanian GM et al, Nature Biotechnology 25, 1411 - 1419 (2007)

p yextended pK in animals

Initial Biophysical Characterization ofsEphB4, HSA, sEphB4-HSA

Size Analysis Stability Analysis

DSC DLS

SDS-PAGE

DSC DLS

5 S l

DS/DP 3

4

5 sEphB4-HSA sEphB4 HSA

ibut

ion

SV AUC

S values5.73.84.4

DS/DP Process Changes

and Comparability1

2

c(s)

dis

tri SV-AUC

2 4 6 8 10 12 14 16 18 200

Sedimentation Coefficient (s)

Biophysical Characterization of sEphB4 and sEphB4-HSA:Higher-Order Structural Changes as Function of Temp and pH

sEpHB4

-2.0E+06

-1.0E+06

0.0E+00

1.0E+06

ellip

ticity

(deg

•cm

2 /dm

ol)

pH 3pH 4pH 5pH 6H 7

3.0E+05

4.0E+05

5.0E+05

6.0E+05

7.0E+05

8.0E+05

fluorescence intensity pH 3

pH 4

pH 5

pH 6

pH 7

pH 8

1.0E+06

1.5E+06

2.0E+06

2.5E+06

3.0E+06

fluorescence intensity pH 3

pH 4

pH 5

pH 6

pH 7

pH 8

-3.0E+06

200 210 220 230 240 250 260

Wavelength (nm)

Mol

ar pH 7

pH 8

5.0E+06

mol

)

0.0E+00

1.0E+05

2.0E+05

400 450 500 550 600

ANS

Wavelength (nm)

2.0E+06 pH 3

0.0E+00

5.0E+05

305 325 345 365 385 405

Trp

Wavelength (nm)

2.5E+06

pH 3

DS/DP -2.0E+07

-1.5E+07

-1.0E+07

-5.0E+06

0.0E+00

200 210 220 230 240 250 260

Mol

ar e

llipt

icity

(deg

•cm2 /d

m

pH3pH4pH5pH6pH7pH8

0.0E+00

5.0E+05

1.0E+06

1.5E+06

400 450 500 550 600

ANS fluorescence intensity pH 3

pH 4

pH 5

pH 6

pH 7

pH 8

0.0E+00

5.0E+05

1.0E+06

1.5E+06

2.0E+06

Trp fluorescence intensity pH 3

pH 4

pH 5

pH 6

pH 7

pH 8

DS/DP Process Changes

and Comparability

sEpHB4 HSA

Wavelength (nm)400 450 500 550 600

Wavelength (nm)305 325 345 365 385 405

Wavelength (nm)

Circular Dichroism (CD) Trp fluorescence ANS fluorescence

sEpHB4-HSA

Conformational Stability sEphB4-HSA:Effect of NaCl and Disaccharides

Trp fluorescence peak position vs. temperature (center of mass method)

DS/DP Effect on Thermal Onset Temperature (Tonset)DS/DP

Process Changes and

Comparability

Protein (pH 7): ~ 55°C

+ 150 mM NaCl ~ 70°C+ 10% sugar ~ 58-60°C+ NaCl and sugar ~ 75°C

Aggregation of sEphB4-HSA:Effect of NaCl and Disaccharides

SE-HPLC analysis after 20 hours at 48˚C at pH 7

NaCl accelerates protein aggregationSucrose inhibits Effect of NaCl less pronounced with sugar

Excipient Sugar (%)

Aggregate ± SD (%) Excipient Sugar

(%)Aggregate ± SD (%)

DS/DP

( ) ( ) ( ) ( )+ 150 mM NaCl No NaCl

Sucrose

0 35.9 ± 0.0

Sucrose

0 24.9 ± 0.25 20.1 ± 0.1 5 14.3 ± 0.1

10 10 1 ± 0 2 10 7 6 ± 0 1DS/DP Process Changes

and Comparability

Sucrose Sucrose10 10.1 ± 0.2 10 7.6 ± 0.115 5.6 ± 0.1 15 5.4 ± 0.120 4.0 ± 0.1 20 4.0 ± 0.10 36.5 ± 0.1 0 25.0 ± 0.15 19 5 ± 0 2 5 13 4 ± 0 1

Trehalose Trehalose5 19.5 ± 0.2 5 13.4 ± 0.1

10 9.6 ± 0.2 10 7.2 ± 0.215 6.1 ± 0.0 15 5.3 ± 0.120 4.0 ± 0.0 20 4.0 ± 0.1

Correlation Analysis of EphB4-HSA with ~25 Excipients:Aggregation Rate by SEC vs. Conformational Stability by DSC

100100R2 = 0.7923

60

80

100

(%

)

R2 = 0.0175

60

80

100

e (%

)

20

40

60

Aggr

egat

e

20

40

Aggr

egat

e

DS/DP

052.0 54.0 56.0 58.0 60.0 62.0

Tm of sEphB4 domain of sEphB4-HSA (oC)

060.0 64.0 68.0 72.0 76.0 80.0

Tm of HSA domain of sEphB4-HSA (oC)DS/DP Process Changes

and Comparability

Tm of sEphB4 domain of sEphB4 HSA ( C)p ( )

Structural stability of EphB4 domainStructural stability of EphB4 domain mediates aggregation and overall instability

of fusion protein EphB4- HSA

Summary-Protein Stability and Therapeutic Protein Development

Formulation DevelopmentDevelopment

Analytical

Protein Stability

Comparability Analytical Methods

ComparabilityAssessments

Acknowledgements

C th bli h d t d• Co-authors on published papers presented

• Russ Middaugh and Sangeeta Joshi at KU

• Mike Blaber, Florida State University

• Financial support from VasGene Therapeutics and Promedior

KU Macromolecule and Vaccine Stabilization Center

Macromolecular and Vaccine Stabilization Center at KU:

Unique and innovative center specializing in the characterization and stabilization of vaccines as well as protein and DNA based pharmaceuticals.

http://mvsc.ku.edup

Thank you for your attention !Thank you for your attention !

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