Discovery Pharmaceutics Approaches and Impact on Optimizing Peptide Molecules within the context of Lead Optimization
Erika R. Bartholomew; Annette Bak, Ellen Minnihan, Nathalie Toussaint, James Ormes, Discovery Pharmaceutical Sciences
Merck Research Labs Kenilworth, NJ & Boston, MA
AAPS Annual Meeting October 28, 2015 Orlando, FL
Presentation Outline
• Peptides and Biologics
– Pharmaceutical landscape
– Introduction to peptides
• Structures, liabilities, and characterization techniques
• Formulation development and risk assessment in Discovery
• Case Studies: Peptides in the Lead Optimization Space
1. Case Study 1: Solubility and stability
2. Case Study 2: Biophysical stability
3. Case Study 3: Maximizing analysis with limited material
Presentation Outline
• Peptides and Biologics
– Pharmaceutical landscape
– Introduction to peptides
• Structures, liabilities, and characterization techniques
• Formulation development and risk assessment in Discovery
• Case Studies: Peptides in the Lead Optimization Space
1. Case Study 1: Solubility and stability
2. Case Study 2: Biophysical stability
3. Case Study 3: Maximizing analysis with limited material
4
C16H15F6N5O
MW: 407.314 g/mol C267H404N72O78S6
MW: 6063 g/mol
C6638H10160N1720O2108S44
MW: ~149,000 g/mol
Top selling pharmaceuticals in the US: 2013
Source: Njarðarson Group, ASU
http://cbc.arizona.edu/njardarson/group/top-pharmaceuticals-poster Slide courtesy Ellen Minnihan
Octreotide/Sandostatin: 1988;
MW 1000 g/mol
Cyclosporin: 1983; MW 1200
g/mol
Insulin: 1982; MW: ~5000 g/mol
Liraglutide: Victoza; 2008; MW 3751
g/mol
Peptides of Yesterday and Today
Recently Approved Products
C&EN Volume 89 Issue 22, pp 13-20
Year # Peptides /
Year
1980 5
1990 10
2010 17
140 Peptide-drugs candidates in clinical development!
Why Peptides?
“They understand that if you can unlock certain fundamental limitations of peptide therapeutics you might have the largest superclass of drugs that the industry has ever generated” – Joseph A. Yanchick III CEO Aileron Therapeutics
C&EN Volume 89 Issue 22, pp 13-20
Fosgerau, Hoffman, Drug Discovery Today Volume 20, 1, 2015
S Strengths
• Efficacy, Safety, Tolerability • Selectivity • Predictable Metabolism • Shorter time to market • Lower attrition rates • Standard synthetic protocols
O Opportunities • Discovery of new peptides • Focused libraries and optimized
design sequences • Formulation development • Alternative ROAs • Multi-functional peptides and
conjugates
W Weaknesses
• Chem/phys instable • Prone to hydrolysis and oxidation • Tendency for aggregation • Short half-life and fast elimination • Low membrane permeability
T Threats
• Immunogenicity • New advancements in genomics,
proteomics and personalized medicine • Significant number of patent expiries • Price and reimbursement environment • Increasing safety/efficacy requirements
for novel drugs
Structural Diversity Among Peptides & Biologics
Peptides
Proteins
Antibodies
Source: RCSB PDB Slide adapted from Ellen Minnihan
AAPS J. 2015,17(1):144-55
Common Stability Risks and Mitigation Strategies
Stability risk Formulation mitigation strategy Stability risk Formulation mitigation strategy
Solubility
pH modification and salt formation
Aggregation
Lower concentration
Optimization of ionic strength pH modification and salt formation
Addition of solubilizing excipients (i.e.,
surfactants, co-solvents) Addition of buffer excipients
Hydrolysis
Evaluation of stability across pH 3–10 range Optimization of ionic strength
Addition of buffer excipients to control pH Addition of solubilizing excipients (i.e.,
surfactants, co-solvents)
Low-temperature storage
Adsorption
Addition of surfactant and polymer
excipients
Oxidation
Addition of antioxidants Addition of albumin
Addition of chelating agents Appropriate container selection or surface
modification
Maintenance of pH <7
Denaturation
Addition of salts or metal ions
Anaerobic processing Appropriate pH
Protection from light Low-temperature storage
Low-temperature storage Microbial
contamination Addition of preservative excipients
Stability risk Formulation mitigation strategy Stability risk Formulation mitigation strategy
Solubility
pH modification and salt formation
Aggregation
Lower concentration
Optimization of ionic strength pH modification and salt formation
Addition of solubilizing excipients (i.e.,
surfactants, co-solvents) Addition of buffer excipients
Hydrolysis
Evaluation of stability across pH 3–10 range Optimization of ionic strength
Addition of buffer excipients to control pH Addition of solubilizing excipients (i.e.,
surfactants, co-solvents)
Low-temperature storage
Adsorption
Addition of surfactant and polymer
excipients
Oxidation
Addition of antioxidants Addition of albumin
Addition of chelating agents Appropriate container selection or surface
modification
Maintenance of pH <7
Denaturation
Addition of salts or metal ions
Anaerobic processing Appropriate pH
Protection from light Low-temperature storage
Low-temperature storage Microbial
contamination Addition of preservative excipients
Slide courtesy Ellen Minnihan
“Right-Sizing” Formulation Development and Risk Assessment in Discovery
Traditional: major preformulation investment on all projects with “plan
for success approach” in Discovery/Preclinical
Undesired State: insufficient preformulation investment to proactively
meet project issues; “gaps exist” in toxicology and early clinical studies
Desired State: balanced preformulation investment appropriate to meet
project needs in a stage-dependent fashion
Problems/limitations Material limitations; translatability; developability criteria less well-defined
Question How much and when should investment in formulation and developability
assessment occur?
Small Scale Stability / Formulation Workflow
Stage 1: Stability
assessment
Evaluate physical and chemical stability: Particle Size, Aggregation, Secondary/Tertiary Structure
Analyze by SEC-and RP-UPLC and DLS
Stage 2:
pH evaluation
Prep stock solutions at various pH levels to identify ideal pH for maximal
solubility/stability
Evaluate physical and chemical stability
Analyze by SEC-and RP-UPLC
Stage 3: Excipient evaluation
Stability Liabilities Identified – what excipients
can help?
Evaluate physical and chemical stability
Analyze by SEC- and RP-UPLC
Stage 4: Vetting lead formulation
After first 3 stages, choose best formulation
Perform in-depth stability studies
MALS and DSC
Fibrillation Assays
Engage downstream
partners for further developability assessment
Slide adapted from Ellen Minnihan
Presentation Outline
• Peptides and Biologics
– Pharmaceutical landscape
– Introduction to peptides
• Structures, liabilities, and characterization techniques
• Formulation development and risk assessment in Discovery
• Case Studies: Peptides in the Lead Optimization Space
1. Case Study 1: Solubility and stability
2. Case Study 2: Biophysical stability
3. Case Study 3: Maximizing analysis with limited material
Case Study I: Compound A
Intrinsic Properties
Salt Form Acetate
Physical Form Amorphous
Isoelectric Point (pI) 8.7
# Amino Acids 25
Molecular Weight 46,000
Solubility > 300 mg/mL
Hygroscopicity Very
hygroscopic
Subcutaneous bolus is main
method of administration for peptides.
The addition of a PEG-linker
increases half life, improves stability,
and reduces the potential for
aggregation.
200.00
250.00
300.00
350.00
400.00
450.00
500.00
0 1 2 3 4 5 6
Co
nce
ntr
ati
on
(m
g/m
L)
% EtOH (v/v)
MFC Compound A vs % EtOH (v/v)
% EtOH
(v/v) Buffer
MFC
(mg/mL)
0 6mM Acetate (pH 5) 240
0 5% Mannitol, 6mM Acetate (pH 5) 240
1 5% Mannitol, 6mM Acetate (pH 5) 280
2.5 5% Mannitol, 6mM Acetate (pH 5) 340
5 5% Mannitol, 6mM Acetate (pH 5) 440
Formulation Strategy
Formulate at the most stable pH
of the molecule (pH 5). Improve
aqueous solubility by maintaining a
pH of at least one unit above or
below the pI of the molecule (calc.
pI = 8.74).
MFC can be increased by EtOH
addition. The increase is proportional
to % EtOH (v/v)
Presentation Outline
• Peptides and Biologics
– Pharmaceutical landscape
– Introduction to peptides
• Structures, liabilities, and characterization techniques
• Formulation development and risk assessment in Discovery
• Case Studies: Peptides in the Lead Optimization Space
1. Considerations: Form, dose, handling, route of administration
2. Case Study 1: Solubility and stability
3. Case Study 2: Biophysical stability
4. Case Study 3: Maximizing analysis with limited material
Compound B: Biophysical Stability
• Problem Statement: We need to choose a candidate with developable physicochemical properties.
– Fibrillation is a common stability concern for peptides, especially for a liquid drug product.
– In the Discovery space, one strategy is to stress several lead peptides, in various formulations and compare them to the fibrillation of a similar marketed compound for reference
Physical Instability in Peptide Formulations
Irreversible Aggregation Reversible Aggregation
• Loss of colloidal stability, system conditions thermodynamically favorable self-association
• Where possible, formulate to prevent formation, to simplify characterization and release.
• De-risk effects on PK and immunogenicity
• Typically proceeds through change of conformation, loss of alpha helix and aggregation of beta sheet
• Results in loss of PK, immunogenicity Risk
• Formation during processing and shelf life unacceptable
Fibrils, precipitates
>500 nm
Monomer
~1nm
Oligomer
1-5 nm
Higher order
structure 10-200 nm
Slide courtesy Suzanne D’Addio
Thioflavin T Assay for Detecting Fibrils
Courtesy: Sachin Lohani.
• Thio T assay
– Fibril-bound Thio T: rotation around is restricted, leading to increase in
fluorescence quantum yield
– Amendable to detect fibrils, monitor kinetics of fibril formation, and
investigate effect of excipients
Compound B: 4-Week Biophysical Stability
• Glucagon is known to be highly unstable and form fibrils quickly
• Compound B is stable at 5°C and 40°C for 4 weeks under the conditions
tested
Marketed Compound
@ 40°C
Compound B
Slide courtesy Nathalie Toussaint
Glucagon: 100% Fibril
Formation @ 3h
Presentation Outline
• Peptides and Biologics
– Pharmaceutical landscape
– Introduction to peptides
• Structures, liabilities, and characterization techniques
• Formulation development and risk assessment in Discovery
• Case Studies: Peptides in the Lead Optimization Space
1. Considerations: Form, dose, handling, route of administration
2. Case Study 1: Solubility and stability
3. Case Study 2: Biophysical stability
4. Case Study 3: Maximizing analysis with limited material
Case Studies: Maximizing Data with Limited Material
Problem Statement: Compound C is going in a safety study, for a head-
to-head comparison with our pre-clinical candidates
• Need at least 6 hours chemical/physical stability for dosing in this safety
study, however…
• Safety group doesn’t have enough material to prepare fresh before each dose
• 2 mg available to conduct array of stability studies
Concentration
(mg/mL) Temp (deg C) Time Point (h) DLS (uL)
UPLC
(uL) Thio T
(uL) Total
(uL) Amount of
Drug (mg)
0.2
5
0 70 150 50 270 0.054
3 70 150 50 270 0.054
6 70 150 50 270 0.054
24 70 150 50 270 0.054
25
0 70 150 50 270 0.054
3 70 150 50 270 0.054
6 70 150 50 270 0.054
24 70 150 50 270 0.054
0.432
Less than 0.5 mg material necessary!
Case Studies: Compound C – UPLC Results
t=initial
t=3h
t=24h
Condition Time (h) Concentration
(mg/mL) % Claim
5 C
initial 0.2 100
3 0.199 99.5
24 0.188 94
RT
initial 0.2 100
3 0.204 102
24 0.183 91.5
Case Studies: Compound B: Thio T and DLS Results
Did not observe increase in
fluorescence, or trend indicating
fibril growth!
DLS does not indicate
significant change in initial
particle size, or aggregation
Conclusions
• Peptides as drug candidates offer many potential benefits due to potency, specificity and low toxicity, however there are a few unique challenges in formulation and development.
• Due to the potential upside in peptide drug development, there are ~4X more peptides entering the clinic each year.
• There are a few unique challenges in the characterization and development of peptides, including aggregation, biophysical stability, as well as well-known amino acid-induced chemical instability.
• Often, material limitations can make the necessary, in-depth formulation and characterization challenging, but with the development of more miniaturized and plate-based assays, it is possible.
Acknowledgments
• AAPS
• Annette Bak
• Ellen Minnihan
• Nathalie Toussaint
• Grace Okoh
• Candice Alleyne
• James Ormes
• Jenna Terebetski
• Nicole Buist
• Suzanne D’Addio
• Caroline McGregor
• Pete Wuefling
This presentation would not have been possible
without the excellent contributions from colleagues
at Merck & Co., Inc.
Back Up Slides
Complementary analytical methods
• Reverse-phase liquid chromatography (RP-HPLC) – allows for analysis of chemical purity of a protein sample by
chromatographic separation on a hydrophobic stationary phase; may be used to evaluate chemical stability during
forced degradation assays
• Q-TOF mass spectrometer – provides intact mass measurements; sample workup under varying conditions allows for
analysis of amino acid modifications, truncation and glycosylation patterns, and other post-translational modifications
• Capillary isoelectric focusing (cIEF) – a pH gradient generated by inducing an electrical field in a solution of carrier
ampholytes allows for determination of a protein isoelectric point (pI)
• Caliper LabChip GXII – a microfluidic electrophoresis instrument that allows for protein separation in a 96-well format on
the minute timescale
• Biacore – a surface plasmon resonance technique that allows for measurement of the binding affinity between a ligand
and its receptor; may be used to evaluate how formulation/storage/stressors may impact affinity of a biologic for its
target
• Differential Scanning Fluorimetry (DSF) – a rapid, fluorescence-based assay for screening thermal stability; demands
less sample and provides higher-throughput relative to DSC
• Asymmetric-Flow Field-Flow-Fractionation (AF4) – provides a gentle, stationary phase-free separation and molecular
weight determination of both soluble and colloidal species including antibodies, aggregates, liposomes, nanoparticles,
etc. over a significantly wider size range than is accessible by SEC
