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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
0.4
0.6
0.8
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
0.6
0.8
1.0
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 90 100
0
2
4
6
8
10
12
14
16
18
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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