Post on 18-Mar-2020
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Small-Scale Physicochemical and Biophysical Characterization to Enable Peptide Delivery in Discovery
Candice Alleyne, Andrew Leithead, and Ellen Minnihan
Senior Scientists, Discovery Pharmaceutical Sciences
Merck & Co., Inc., Kenilworth, NJ, USA
Outline
• What makes peptides promising drug candidates?
• Challenges of peptide delivery
• Peptide characterization tool kit
• Case studies
– Solubility
– Aggregation
– Fibrillation
• Summary
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Why the Move to More Peptide Drugs?
• Involved in various physiological and pathological processes
– Play important roles in regulating cells process
• Low tissue accumulation
• High potency; high selectivity
• Potential for fewer side effects – lower toxicity and immunogenicity
• Broad range of targets
• May provide the best of small molecules and biologic approaches
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Peptide Protein,
mABs Small molecule
Craik DJ, et al. Chem Biol Drug Des. 2013;81:136-147.
General Challenges to Peptide Delivery
Challenging Peptide Properties
Molecular Weight • 500-5,000 Daltons (2-50 amino acid residues)
Solubility • Variable, dependent on amino acid residues, secondary
structure, pH, and pI (isoelectric point)
Permeability • Variable, but generally poor for linear peptides
Chemical Stability • Sensitive to hydrolysis, oxidation, deamidation
Physical Stability • Solution state or lyophilized solid stability required
• Adsorption to solid surfaces
Biophysical Stability • Risk of aggregation, fibrillation, denaturation
• Secondary and tertiary structure can affect activity
Metabolic Stability
• Sensitive to enzymatic degradation (eg, peptidases)
• Short plasma half-life
• Endotoxin and bioburden
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How can we assess these liabilities earlier in discovery with limited material: ie, <5 mg?
Peptide Characterization Tool Kit
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Purpose Characterization Technique Output
Solubility Reversed-Phase Ultra Performance
Liquid Chromatography (RP-UPLC)
Conventionally used to evaluate solubility in
biorelevant media and formulation vehicles
Secondary Structure
Changes
Circular Dichroism (CD) alpha-helix beta-sheet
Fourier Transform Infrared (FTIR) Fibrils and alpha-helix beta-sheet
Diffusion Ordered Spectroscopy
(DOSY-NMR)
Diffusion coefficient of various species in the
formulation
Fluorescence Assay used to monitor fibrillation in the
presence of dye
Particle Size
Size Exclusion Chromatography
(SEC)
Ideally suited to monitor irreversible
aggregation
Transmission Electron Microscopy
(Cryo-TEM) Particle morphology
Dynamic Light Scattering (DLS) Particle size distribution (PSD) in sub-micron
range
EPIC Critical Micelle Concentration CMC
Analytical Ultracentrifugation (AUC) Separation of sub-visible species
The Compound:
• Highly charged peptide (pI ~9)
• Very hydrophobic small molecule payload
• Biochemically conjugated via stable linker
• Total MW ~4k Da
The Challenge:
• Limited material: 1 mg of compound
available
• Requirements: 0.1 mg/mL solubility in a
very mild vehicle to enable in vivo
preclinical studies for immunology
• Peptide freely soluble in PBS,
pH 7.5 (>0.5 mg/mL) ≠ peptide conjugate
(<0.005 mg/mL)
Case Study 1: Small-scale Solubility Assessment of a Peptide/Small Molecule Conjugate
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The Strategy:
• Explore the effect of pH, buffer
composition, salt, and surfactant
on the solubility of the peptide
conjugate
• 18 conditions x 80 μL
sample/condition x 0.5 mg/mL
max target solubility 0.72 mg
total compound
The Results:
• Conjugate solubility strongly dependent on buffer composition and ionic strength
• pH sensitivity was less pronounced
• Addition of surfactant gave solubility boost
Solubility (mg/mL) Buffer Buffer +
NaCl Buffer +
Tw80 Water pH 2 ≥0.5 -- -- Water ACN ≥0.5 -- --
Citrate pH 4.4 <LOQ <LOQ <LOQ Acetate pH 4.9 ≥0.5 <LOQ ≥0.5
Succinate pH 5.1 <LOQ <LOQ <LOQ MES pH 5.6 0.1 <LOQ 0.18
Imidazole pH 6.2 ≥0.5 <LOQ ≥0.5 Hepes pH 6.6 0.4 <LOQ ≥0.5 RP-UPLC solubility measurement
Water pH 2 Water: ACN
Citrate pH 4.4 Acetate pH 4.9
Succinate pH 5.1 MES pH 5.6
Imidazole pH 6.2
HEPES pH 6.6
+ Tween 80
+ NaCl
• Ability to generate a uniformly disperse suspension of the peptide allowed for low-volume liquid handling
• Scouting broad formulation conditions revealed strong dependence on buffer composition and ionic strength
• Use of reverse-phase UPLC allowed for rapid, quantitative detection on sub-microgram peptide samples
Case Study 1: Small-scale Solubility Assessment of a Peptide/Small Molecule Conjugate (continued)
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• Stage: Discovery
– Objective: Monitor stability of a peptide in pH 7 buffer at 40°C for 4 weeks
• The Challenge:
– Limited amount of material available
– Can aggregates of the peptide be detected?
• The Strategy:
– Physical stability via SEC
• SEC allows for separation as a function of size using porous stationary phase and an isocratic mobile phase
• For 0.5 mg/mL sample concentration, 2 µL injection volume: only 1 µg/injection required
Case Study 2: Aggregation Assessment via SEC
Blank
3 weeks
2 weeks
1 week
Initial
SEC method
• Good qualitative first pass, material sparing approach to detect and quantify soluble molecular aggregates
• When coupled with MALS, can provide greater details on molecular weight; however, requires more material (50-100 µg/sample)
• Insoluble aggregates/fibrils not detected via this method due to column loading limitation
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Condition
Aggregation – SEC
Parent 4.3
% Area
Agg 1 3.8
% Area
Deg 1 4.4
% Area
Deg 2 4.7
% Area
Deg 3 5.0
% Area
Deg 4 5.2
% Area
40°C Initial 100.00
40°C 1 wk 94.92 2.90 1.97 0.21
40°C 2 wk 76.98 10.16 4.15 6.69 0.75 1.27
40°C 3 wk 30.13 4.75 4.48 10.31 4.39 45.51
40°C 4 wk - - - - - -
• Stage: Early Discovery
Objective: Develop method of detecting fibrillation on small scale with minimum material requirements
• The Challenge:
– Current method Thio T is quite sensitive and quantitative
– Requires 3-5 mL of sample/time point @ 3 mg/mL up to 15 mg
– Minimization of assay to plated version resulted in loss in sensitivity
• The Strategy:
– Develop 96-well plate based Congo Red assay utilizing UV absorbance
– Requires: 100 μL sample/time point @ 3 mg/mL 0.3 mg
– Medium to High Throughput
Case Study 3 – Fibrillation Assessment via UV and Fluorescence
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• Peptide Z was exposed to shear stress (stirring @ 300 rpm) at room temperature
• Shift in peak absorption wavelength is indicative of fibril formulation after 6 days
Biophysical Stability: Fibrillation (Congo Red Assay)
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
350 450 550 650 750 850A
bso
rban
ce
Wavelength (nm)
Initial
Day 1
Day 3
Day 6
0.00
10.00
20.00
30.00
40.00
50.00
60.00
0.0 5.0 10.0 15.0 20.0
Flu
ore
sce
nce
Res
po
nse
[A
U]
Fibril Content [μg/mL]
Fluorescence [AU] vs Fibril Concentration
Experiment 1 Experiment 2 Experiment 3 Standard Curve Fit
Lower 95% CL AU Upper 95% CL AU Lower 95% PL AU Upper 95% PL AU
AU = 1.0634 + 1.7789 * Conc, R^2 = 0.9746
Conditions AU1 AU2 AU3 Average
AU
Fibril Content
μg/mL
Initial 33.273 33.478 33.124 33.292 18.117
Wk 4 -80°C 33.274 33.092 33.220 33.195 18.063
Wk 4 -20°C 34.940 35.174 34.867 34.993 19.074
Wk 4 5°C 33.938 34.171 33.780 33.963 18.495
Wk 4 40°C 42.276 42.346 42.621 42.414 23.245
Slight increase in fibril
concentration under
temp stressed
conditions! AU=abritrary units.
• Congo Red assay
provides quick qualitative
read on conditions under
which fibrils form
• Can be used for
screening purposes
where peptide quantity
is limited
• Should be replaced with
more quantitative assay
like Thio T once peptide
availability no longer an
issue
Case Study 3 – Fibrillation Assessment via UV and Fluorescence (continued)
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Summary and Conclusion
• Peptide delivery comes with its own inherent liabilities
• Working with limited API to de-risk these liabilities at the early discovery stage adds another complication to the mix
• Miniaturization of analytical methods (ie, solubility, fibrillation) to cope with limited material is not trivial, but creative experimental design can frequently enable small-scale studies that may also be amenable to high throughput approaches
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Acknowledgements
Many thanks to my colleagues at Merck who provided data and guidance that made this presentation possible!
• Annette Bak
• Erika Bartholomew
• Andrew Leithead
• Dennis Leung
• Sachin Lohani
• Caroline McGregor
• Ellen Minnihan
• Grace Okoh
• Nathalie Toussaint
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Backup Slides
Routes of Delivery
Route Pros Cons
Oral Easy access, convenient to dose
Good patient compliance
High-dose delivery
Epithelial barrier
Chemical and enzymatic degradation
First pass gut and hepatic metabolism
Transdermal Easy access, convenient to dose
Good patient compliance
Large surface area for treatment
Low-dose delivery
Tough barrier to penetrate
Toxicity/irritation at site of application
Pulmonary/
inhalation
Large surface area for absorption
Thin epithelial barrier – moderate permeability
Very well perfused
Limited dose, dose volume
Reproducible deposition
Safety and lung function
Taste liability
Device development required
Intranasal Highly permeable epithelia
Convenient dosing
Commercially available devices
Limited dose, dose volume
Rapid clearance
Taste liability
Irritation potential
Ocular Easy access, convenient to dose (eye drops)
Good patient compliance (eye drops)
Limited dose, dose volume
Limited to local delivery
Irritation potential
Low permeability barrier
May require surgery (injections or implants)
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Source: Adapted from Mathias NR, et al. J Pharm Sci. 2010;99:1-20.
Why the Move to More Peptide Drugs?
Advantages
High potency
High selectivity
Broad range of targets
Potentially lower toxicity and immunogenicity
Low accumulation in tissues
High chemical and biological diversity
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• Peptide opportunities
may provide best of
small molecule and
biologics approaches
• Can we optimize
peptide design and
delivery to minimize
challenges with
peptides?
• How do we tailor
physicochemical and
formulation risk
assessment
approaches to
peptides?
Source: Adapted from Craik DJ, et al. Chem Biol Drug Des. 2013;81:136-147.
Fibrillation
• Peptides and proteins can assume a variety of possible conformations, the relative free energies of which are sensitive to various environmental conditions
– Concentration, shear, pH, ionic strength, and temperature
• Certain environmental conditions can lead to formation of aggregates/fibrils, which can lead to
– Loss of potency
– Manufacturing issues
– Increased immunogenicity
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Aggregate Native-state
Aggregation Folding
Energ
y
Aggregation vs Fibrillation
Aggregation • Process is an equilibrium between the
native and denatured states
• Irreversible changes to the denatured species can result in formations of fibrils
Fibrillation • A hydrophobic driven process where
nonpolar amino acid residues of peptides,
when exposed to water in unfolded state,
can lead to nucleation of fibrils
• Not bio-active; however, can induce
immune response
• Facilitated by air-water interface and
hydrophobic surfaces, eg, Teflon
Jorgensen L, et al. Expert Opin Drug Deliv. 2009;6(11):1219-1230. Jansen, et al. Biophys J. 2005;88:1344-1353.
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Techniques for Detecting Fibrils
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Bouchard M, et al. Protein Sci. 2000;9:1960-1967.
CD FTIR
Thioflavin T Assay for Detecting Fibrils
• Thio T assay
– Fibril-bound Thio T: rotation around is restricted, leading to
increase in fluorescence quantum yield
• This assay is low throughput using 3 mL cuvette
– Amendable to detect fibrils, monitor kinetics of fibril
formation, and investigate effect of excipients
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Courtesy: Sachin Lohani.
Congo Red Fibril Screening Assay
• Congo Red Assay – When CR binds to fibrils, a change in color from orange-red to rose
is induced that corresponds to a shift in the characteristic
absorbance spectrum of CR
• This assay is medium throughput and uses 96 well plate – Amendable to detect fibrils, monitor kinetics of fibril formation,
and investigate effect of excipients
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Spectra shift observed with CR and glucagon peptide
• Green line is non-stressed glucagon
• Red line is stressed glucagon
Courtesy: Nathalie Toussaint and Erika Bartholomew.
What Are Peptides?
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Peptide = chain of amino acids
polypeptidechain
Peptide Secondary Structures
Application 2010 2011 2012 2013 2014 2015 2016 2017 2018
CAGR%
2012-
2018
Parenteral 11,537 12,201 13,141 14,152 15,239 16,408 17,666 19,017 20,600 7.8
Oral 774 869 978 1,098 1,233 1,382 1,548 1,733 1,907 11.7
Pulmonary 375 427 483 546 617 695 782 880 1,017 13.2
Mucosal 383 428 512 607 715 836 974 1,130 1,144 13.5
Others
(intradermal
and nasal)
132 212 260 316 380 453 533 627 763 19.3
Total 13,200 14,138 15,374 16,719 18,183 19,775 21,504 23,386 25,432 8.8
A Business Case for Peptides: Projected Global Peptide Therapeutics Revenues
Source: Clinical trials.gov; Primary Interviews; Cancer Journals; TMR Analysis.
(Data in USD Million)
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