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

2

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

3

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

4

How can we assess these liabilities earlier in discovery with limited material: ie, <5 mg?

Peptide Characterization Tool Kit

5

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

8

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

9

• 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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13

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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

19

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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