Post on 02-Apr-2021
Developability Evaluation During Early Pre-formulation: The Meeting Point Between Research and Development
Bernardo Perez-Ramirez, MS, Ph.D. Senior Scientific Director BioFormulations Development, Global BioTherapeutics One the Mountain Rd. Framingham MA 01701, USA bernardo.perez@sanofi.com
Stability of Biopharmaceuticals: Getting the Chemistry Right. 4th Annual MIBIO Conference. Tuesday, September 30th, 2014. Downing College, Cambridge-UK.
Challenges Facing Protein Formulation and Product Development ● Time and budget allotted for developing and implementing formulations. ● Regulatory requirements. ● Requirements to work closely across sites and divisions as well as with
external suppliers. ● Need for quality, innovation and rapid time to market.
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Dev
elop
men
t Tr
ansi
tion
Product Development Timeline
● Need time for robust formulation development ● Insufficient window for real-time data ● Start formulation development early
(integration with research)
Phase 1/2 Formulation Development Timeline
Purification
Cell Culture
Formulation
Characterization & Analytical
Manufacturing/
Clinical Trials
Cell Culture
Formulation
Characterization & Analytical
Purification Purification
Cell Culture
Formulation
Characterization & Analytical
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Principles of the Formulation Developability Assessment: The Interface with Discovery Research
● Intrinsic Quality of the Constructs ● Can it handle the stresses associated with development and
manufacturing process? ● What are the degradation “pathways” of the protein? ● Rank candidates
● Information on Proper Handling ● Reduce false negative results due to incorrect handling
● Very Early Information on Protein Solution Behaviour ● Accelerates the formulation development process ● Foundation for QbD
● Great Return on Investment ● Cost ~20 mg and 4-8 weeks.
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Developability Evaluation During Early Pre-Formulation
Drug Discovery
Approach to Developability
Development
Manufacturing
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Protein Expression Clone stability Growth properties Titer Production Variability
Cellular Immunology In vitro characterization In-silico response
Formulation Stability of process intermediates Protein Stability
Purification Process Development Impurity Profile Yields Stability
Development Candidate Risk Assessment – Synergistic Collaboration
“Developability” Assessment
Candidate Selection
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Formulations Role in Developability Assessment
Developability Evaluation
Relevant Process Stresses Freeze/thaw Shear/Mechanical stress -Viscosity, injectability, solubility Mock viral inactivation
Forced Decomposition Degradation (pH 5-8) Temperature (45 °C)
• Desired results: Sufficient degradation to understand protein profile and differentiate candidate molecules
• Desired results: No change
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Stability and Solution Behavior Concerns
• Oxidation • Deamidation • Isomerization • Fragmentation • Disulfide exchange
Physical Stability • Soluble aggregation • Self association • Insoluble aggregation • Particle formation • Solubility • Viscosity
Chemical Stability
Function/binding
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Analytics
A280 (concentration) Turbidity Circular dichroism (secondary and tertiary structure) CE-SDS (purity) SDS-PAGE (purity) SEC (aggregation, fragmentation) N-terminal sequencing (identity) AUC (aggregates/oligomerization)
cIEF (charge variants) Peptide mapping Oxidation ( methionines in CDR) Isomerization (aspartic acid in CDR) Deamidation (PENNY peptide)
Physical Stability
Chemical Stability
Functional Stability Surface Plasma Resonance (Biacore)
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CASE STUDY: Which candidate has the best chance of making it through development and manufacturing…?
* *
* *
Construct 1
Construct 2
Construct 3
Construct 4
* *
* *
* *
* *
Mouse IgG2
Human IgG1 1
Human IgG1 2
CDR Grafting
Sequence Optimisation
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More thermal stability in Constructs 1 and 2 Less secondary structure in Construct 3
General Properties
Wavelength (nm)
210 220 230 240 250C
D S
igna
l
-10
0
10
Construct 1Construct 2Construct 3Construct 4
Construct 1 Construct 2 Construct 3 Construct 4
Mel
ting
Tem
pera
ture
(o C)
74
76
78
80
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Wavelength (nm)
210 220 230 240 250
CD
Sig
nal
-10
0
10
Construct 4 post-viral inactivationConstruct 4 time 0
Wavelength (nm)
210 220 230 240 250
CD
Sig
nal
-10
0
10
Construct 3 post-viral inactivationConstruct 3 time 0
Wavelength (nm)
210 220 230 240 250
CD
Sig
nal
-10
0
10
Construct 2 post-viral inactivationConstruct 2 time 0
Wavelength (nm)
210 220 230 240 250
CD
Sig
nal
-10
0
10
Construct 1 post-viral inactivationConstruct 1 time 0
Construct 4Construct 3
Construct 2Construct 1
Very slight decrease in structure
Slight decrease in structure
Red = time 0 Black = post vi
PROCESS STRESSES: Mock Viral Inactivation - Secondary Structure by CD
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PROCESS STRESSES: Mock Viral Inactivation (VI)
No change Minor change Moderate change Major change
Quality Attribute C-1 C-2 C-4 Comments
Aggregation
Purity
Charge Heterogeneity
Oxidation
Secondary Structure Possible ↓Construct 2
Turbidity
Melt Point *Quality attributes ranked in order of criticality
C3 was not considered as a candidate due to its obvious weak performance on most analytics and lack of in vitro activity
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PROCESS STRESSES: Shear/Mechanical Stress
Quality Attribute C-1 C-2 C-4 Comments
Aggregation
Purity
Charge Heterogeneity
Secondary Structure
Turbidity
Melt Point
No change Minor change Moderate change Major change *Quality attributes ranked in order of criticality
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PROCESS STRESSES: Freeze Thaw Study
Quality Attribute* C-1 C-2 C-4 Comments
Aggregation
Purity
Charge Heterogeneity
Secondary Structure
Turbidity
Melt Point
No change Minor change Moderate change Major change *Quality attributes ranked in order of criticality
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Slight increase in deamidation, except Construct 2
Significant increase in deamidation Least in Construct 2
Time (weeks)
0 1 2 3 4 5PE
NN
Y D
eam
idat
ion
(%)
0
5
10
15
20
25Construct 1 pH 7.2Construct 2 pH 7.2Construct 3 pH 7.2 Construct 4 pH 7.2
Time (weeks)
0 1 2 3 4 5
PEN
NY
Dea
mid
atio
n (%
)
0
5
10
15
20
25Construct 1 pH 5.0 Construct 2 pH 5.0 Construct 3 pH 5.0Construct 4 pH 5.0
FORCED DECOMPOSITION: pH Study - Deamidation of PENNY Peptide
pH 5.0 pH 8.0
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More clipping in Construct 1 Significant clipping in all constructs. Least in Construct 2, most Construct 1
Time (weeks)
0 1 2 3 4 5H
C C
lippi
ng (%
)
0
5
10
15
20
25Construct 1 pH 7.2Construct 2 pH 7.2Construct 3 pH 7.2 Construct 4 pH 7.2
Time (weeks)
0 1 2 3 4 5
HC
Clip
ping
(%)
0
5
10
15
20
25Construct 1 pH 5.0 Construct 2 pH 5.0 Construct 3 pH 5.0Construct 4 pH 5.0
FORCED DECOMPOSITION: pH Study - Heavy Chain Clipping
pH 5.0 pH 8.0
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FORCED DECOMPOSITION: Low pH (5.0) and Elevated Temperature
Quality Attribute C-1 C-2 C-4 Comments
Aggregation
Purity ↑Fragmentation
HC Clip
Isomerization
Deamidation
Charge Heterogeneity
Oxidation
Secondary Structure
Turbidity
Melt Point
No change Minor change Moderate change Major change *Quality attributes ranked in order of criticality
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FORCED DECOMPOSITION: High pH and Elevated Temperature
Quality Attribute* C-1 C-2 C-4 Comments
Aggregation
Purity ↑Fragmentation
HC Clip
Isomerization
Deamidation Significant ↑ in PENNY peptide
Charge Heterogeneity Significant ↑acidic forms
Oxidation
Secondary Structure
Turbidity Slight opalescent
Melt Point
No change Minor change Moderate change Major change *Quality attributes ranked in order of criticality
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Prob
abili
ty
Severity
Risk Analysis in Formulation
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Formulation Developability Ranking Construct 2>Construct 1>Construct 4>>Construct 3
Developability Ranking
Stress C-1 C-2 C-4
General
Freeze-thaw
Shear
Viral Inactivation
High temperature, pH 8.0
High temperature, pH 5.0
Favorable Neutral Weak Detrimental
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SUMMARY: Improving formulation strategies and outcomes by strengthening the Interface with discovery research
● Identify problematic candidates ● Identify analytical challenges ● Accumulate solution behavior data which will help guide development and
Quality by Design (QbD) ● Early information about the solution behavior of candidate proteins helps in
development strategies for robust dosage form development in a timely fashion.
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Acknowledgments
● Patrick Flanagan ● Rob Simler ● Nick Guziewicz ● Andrew Massetti ● Laura Geagan (AD) ● Lunds Anders (AD) ● Bill Brondyk (NPE) ● Bob Mattaliano
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