High-throughput screening modeling & novel analytical
methods to link surface properties to protein aggregation
root cause of mAbs and polyconjugate vaccines
July 2014 Workshop on Protein Aggregation and Immunogenicity
Jean-Bernard Hamel,
R&D Pharmaceutical technology Senior manager – BDM - PS
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Presentation layout
• Introduction
– Drug/Devices development & Drug/Device interaction Challenges
• BD approach to early container-drug interaction rapid screening
– Representative components/simplified system
• Case study on predicting stability dependence of monoclonal
antibodies from container surface
• Case study on conjugated vaccines sensitivity to silicone oil:
Accelerated protocol and Interfacial Tension as predictor
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Cross-functional expertise needed
Formulation
Science
Mechanical
Engineering
Protein Analysis
Combo Product
QbD
DRUG
Fill-Finish
Operations
DEVICE
Human Factors
Health
Economics
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Device-Related Challenges Developing
Robust Combination Products
AUTOINJECTOR
PFS
POTENTIAL DEGRADATION PATHWAYS
Effects of Surfaces and Leachables on the Stability of Biopharmaceuticals
JARED S. BEE & al, April 2011 DOI 10.1002/jps.22597
Interface incompatibilities:
•
Air bubble, polymers, silicone oil, stopper
•
Soluble aggregates, particles, adsorption losses,
unfolded protein
Physical/chemical instabilities caused by leachables
CARTRIDGE
•
Tungsten, Fe-ions from needle, stopper leachables
•
Heterogeneous particles, chemical modifications,
particles, soluble aggregates, unfolded protein
Device-Specific:
•
4
Oxygen permeability, glass delamination,
needle clogging, light exposure
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Integration into thecustomer development process
Learn from the past
Phase I
Phase II
Formulation freeze
GMP batches
Device/Drug
Combination freeze
20002005 2006
Needle
Tungsten
Silicone
Phase
III
adhesive
Formal ICH stability
Device/drug Compatibility
Need to be conducted at early stage
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Device Selection and Implementation
Crucial factors for the development of injectable biopharmaceuticals
Device Evaluation
Combination Product
Development
Device selection
Drug/Device
interaction
Leachables
Simulated studies
Early extractables
BD product development
6
Placebo
extractables
Components
extractables
Available as customized
bio-analytical services
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BD approach
for rapid customer
screening
Multiple
analysis
DOE
Selection
Verification
Accelerated
Incubation
4 days
Drug
Formulations
7
PFS simplified system
microplate
Stat.
analysis
High throughput
Analysis
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Verification
Proposed
Accelerated
Combination
ICH stability
Microplates as simplified system
Phase II: BD PFS adaptation to 96 well plates
BD syringe simulation - 10 plate technologies were characterized
Particulates
E&L Migration
Surface
topography
-BD Hypak™
-BD XSi™
-Bare Glass
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Surface energy
PDMS microplates
XSi™- microplates
Hydrophilic microplates
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Microplate characterization
Plates/Syringes Subvisible particulates comparison
SbVP (2-100Cm)
120
100
SbVP (2-100Cm)
100
% from BD Hypak
% from PDMS coated plates
120
80
60
40
20
80
60
40
20
Standard PFS
0
0
PDMS Microplate XSi™-Microplate
BD Hypak™
BD XSi™
Cross-linked silicone chain networking
leads to improved layer structure.
Part. level is reduced in XSi™ wells
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BD XSi™ syringe
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Microplate characterization
Plates/Syringes Subvisible particulates comparison
SbVP (2-100Cm)
120
100
80
60
40
20
80
60
40
20
0
0
PDMS Microplate XSi™-Microplate
BD Hypak™
Part. level is reduced in XSi™ wells
10
SbVP (2-100Cm)
100
% from BD Hypak
% from PDMS coated plates
120
BD XSi™ syringe
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BD XSi™
Microplate characterization
Plates / Syringes comparison surface energy
Low surface energy
High surface energy
=
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Need for multiple analytical techniquesGG.
• Adapt existing techniques to rapid
•
•
•
•
•
•
screening
BCA method for protein adsorption evaluation
SEC techniques for monomer lost detection - UPLC
DLS techniques for oligomers detection
MFI techniques for aggregation detection
Fluorescence probe assays for monitoring protein aggregation
Develop
exploration techniques for protein-surface interactions:
• SPR for protein adsorption kinetic evaluation
• Zeta potential for surface charge measurement
• Interfacial Tension
12
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Putting it all together: case study on mAb
Phase I
• Identification of critical stability parameters
• Selection of pilot conditions
Phase II
•
Development of a tool predicting protein
aggregation in contact with the material
•
Generation of the experimental results on the
critical stability parameters
•
Selection of two extreme conditions
Phase III
•
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Validation of the extreme conditions on real
containers
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mAb stability: microplate screening and predictions
Hydrophilic
Low particle
Hydrophilic
Hydrophobic
High Hydrophobic particles
Full Factorial Plan 211 x 3 =6144 Cond.
Fractional DOE with 32 conditions
Reduced prediction model in simplified system gives a good
estimation of interactions confirmed by formal ICH stabilities
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Hydrophobic
Low particle
Advanced analytics
Zeta potential of surface
Principle
Microplate
Cell
Zeta Analyser
Time saved on screening approach allows to further
explore specific interactions – Charge interactions
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Advanced analytics
mAb stability: an unexpected correlation
SEC measurment
5
4,5
Predicted Ln (Monomer lost) at 100mv
mAb PI = 8.5
PDMS
High part pH8
4
3,5
3
PDMS
High part PH5
Hydrophobic
Low Part, pH8
2,5
Hydrophobic
Low part, pH5
2
1,5
Hydrophilic
1
pH5
Hydrophilic
pH8
0,5
0
-60
-50
-40
-30
-20
-10
0
Zeta charges (mV)
mAb stability increase if surfaces charges decrease and protein
pH
charges increase. The effect is dependent on the conductivity.
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Hydrophilic LP
Hydrophiilic HP
Hydrophobic HP
Hydrophobic LP
Silicone-oil incompatibility
%. And prophilactic vaccines ?
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Stressed conditions PFS – polyconjugated vaccine
Accelerated study design
• Polyconjugated vaccine – fixed concentration
• Accelerated protocol in different primary packaging:
–
–
–
–
Free silicone oil (standard and oversiliconized )
Baked-in silicone
Cross-linked silicone (XSi™)
Plastic non-lubricated
T0
T3d
T5d
T7d
T28d
2 days agitation
Storage: accelerated conditions for refrigerated products: ICH 25°C/60%RH
Reference are stored in vials at 4°C and in the same conditions as the samples
60rpm agitation
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Drug product analysis ( Visual inspection, MFI, DLS, Intrinsic Fluorescence)
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Stressed conditions PFS – polyconjugated vaccine
MFI and visual inspection
100000 Particle (>1um, #/mL) by MFI
28288776
T0
80000
60000
19333721
T3days
T7days
40000
1066720
20000
0
Drastic increase upon shaking in
siliconized PFS, EXCEPT for BD
HypakTM XSiTM PFS, which remains
low.
Increase upon shaking in SVPs for reference vials due to large air/water interface.
No visible particulates were observed for reference vials, non
siliconized and XSi™ PFS, while turbidity was observed for
standard, and high silicone PFS
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Stressed conditions PFS – polyconjugated vaccine
intrinsic fluorescence
Siliconized
Ref vial 5°C
Standard
TM
XSi
Bare glass
RFU (Ex:280nm)
3000
2500
2000
High Si
Baked
Plastic
Ref vial 25°C
Non-siliconized
1500
1000
500
0
-500
Em:330nm
Em:360nm
• Largest difference from reference vial @ 5°C was observed for
all siliconized PFS EXCEPT BD HypakTM XSiTM PFS.
• BD HypakTM XSiTM PFS performs similar to reference vial.
20
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Bibliography ref.: B. Bolgiano et al. : Vaccine 19 (2001)
3189–3200
3500
Polyconjugated Vaccine Intrinsic Fluorescence
(T3d -T7d)
Advanced analytics
Interfacial tension (IFT) measurement
Principle
Equipment
Software
IFT
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Advanced analytics – adsorption on silicone
CRM197 carrier protein
CRM197 Therapeutic interest
Poly-conjugate vaccines:
45
41
PI : 4.6
Antigen 1
Antigen 2
Antigen 3
Antigen 4
L.
G
Buffer
I
39
Covalent binding of protein & one or many antigen(s)
Protein
II
37
crm197 5µg/mL in
buffer
III
35
crm197 10µg/ml in
buffer
33
crm197 20µg/ml in
buffer
31
crm197 50µg/ml in
buffer
29
crm197 100µg/mL
in buffer
27
25
G
0
100
200
300
400
500
Time [s]
• Concentration-dependent adsorption of the CRM197 carrier
protein at the PDMS/water interface
22
PH : 6.6
43
IFT [mN/m]
• Used in 10 conjugate vaccines
against: Haemophilus influenzae, S.
Pneumoniae, N. meningitidis
• Proven to be efficient in monocyte
targeting or brain targeting carrier
CRM 197 carrier protein adsorption at
PDMS/water interface
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600
Advanced analytics – adsorption on silicone
Alum Adjuvants – carrier protein binding
45
45
43
41
41
39
39
37
CRM
35
-
+
AH
33
31
27
37
CRM
35
200
27
Time (s)
300
400
500
0
100
200
Time (s)
300
400
CRM197 (carrier protein for polyconjugated vaccine) adsorption
on silcone-oil is strongly dependant on adjuvant type and
concentration
23
AP
Formulation Buffer
AlPO4-placebo dil4
AlPO4-placebo std
CRM50µg/mL- buffer
CRM50µg/mL-AlPO4 dil4
CRM50µg/mL-AlPO4 std
25
100
-
33
29
25
0
-
31
AlOH3 - placebo
formulation buffer
CRM50µg/mL-AlOH3 std
CRM50µg/mL-AlOH3 dil4
CRM50µg/mL-buffer
29
IFT (mN/m)
IFT (mN/m)
43
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500
Advanced analytics – adsorption on silicone
Alum adjuvant binding through IFT measurements
45
43
43
41
41
39
39
IFT [mN/m]
IFT [mN/m]
Conjugate vaccine 2
Conjugate vaccine 1
45
37
35
37
35
33
33
Vaccine 1 placebo
Vaccine 1 + Al(OH)3
31
29
Isolate Al(OH)3
Vaccine 1
Vaccine 2 Placebo
Vaccine 2 + Al(OH)3
31
29
Isolate Al(OH)3
Vaccine 2
27
27
25
25
0
100
200
300
400
500
600
0
Time [s]
100
200
300
Conjugate vaccine 1:
Antigen X grafted to CRM197 carrier protein
preferentially adsorbed on Alum over
drug/PDMS interface
Conjugate vaccine 2:
Antigen Y grafted to CRM197 carrier protein
adsorption at the drug/PDMS interface is
enhanced by the presence of Al(OH)3
Polyconjugated vaccines adsorption on silcone-oil is strongly
dependant on the antigens nature
24
400
Time [s]
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500
Conclusions
• Evaluation of drug/device interaction needs to be assessed
at the early stage of drug development.
• Promote a design space where a science-based, multidisciplinary effort is shared across pharma companies and
BD.
• Rapid screening may help to select the appropriate
drug/device system, to help streamline combination product
development.
25
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Acknowledgments
BD team
• Tzvetelina Chevolleau
• Neli Nencheva
• Benoit Duroux
• Frederique Crozet
• Franck Gratier, Perrine Roux
• Cynthia Fuentes
• Julie Berube
• Paolo Mangiagalli
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Q&A session
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