SUPPLEMENTAL SECTION

Peptide Optimization at the Drug Discovery-

Development Interface: Tailoring of Physicochemical

Properties Toward Specific Formulation Requirements

Andreas Evers1,§,*, Stefania Pfeiffer-Marek2,§,*, Martin Bossart1, Christoph Heubel1, Ursula

Stock3, Garima Tiwari1, Birgit Gebauer1, Bettina Elshorst1, Anja Pfenninger4, Ulrike

Lukasczyk5, Gerhard Hessler1, Walter Kamm2, Michael Wagner1,*

Sanofi-Aventis Deutschland GmbH, Industriepark Hoechst, Frankfurt am Main, Germany,

1Integrated Drug Discovery, 2Tides Drug Product Pre-Development Sciences, 3Tides

Analytics, 4BioAnalytics, 5DMPK

§: Both authors contributed equally to this work.

*Correspondence to: Andreas Evers, phone: +49 305 12636, E-mail:

Andreas.Evers@sanofi.com ; Stefania Pfeiffer-Marek, phone: +49 305 26566, E-mail:

Stefania.Pfeiffer-Marek@sanofi.com ; Michael Wagner, phone: +49 305 46875, E-mail:

Michael.Wagner@sanofi.com

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SUPPLEMENTAL METHODS SECTION:

Peptide purity and confirmation with UHPLC-RP/UV-MS. Peptides were solubilized in

DMSO at a concentration of 1 mg/mL for 1 hour applying gentle agitation. Afterwards the

solutions were centrifuged for 20 minutes at 3000 RCF and a 3 µL aliquot of the supernatant was

injected on a UHPLC system consisting of an Agilent Infinity 1290 LC equipped with a diode-

array detector (DAD) hyphenated to an Agilent TOF 6230. Sample separation was accomplished

on a Waters Acquity column containing CSH C18 1.7 µm particles in a 2.1 x 150 mm column

dimension operated at 50 °C. The UV absorption was detected at 214 nm. Solvent A consisted of

water with 500 ppm TFA and solvent B composed of acetonitrile with 450 ppm TFA running

with a constant flow of 0.5 mL/min. After a column load phase the gradient increases the amount

of acetonitrile from 20% to 75% in a linear curve during 23 min followed by a wash phase.

Gradient details are shown in Table S1.

The mass spectrometer, equipped with a dual AJS electrospray source, was operated according to

the manufacturer’s instructions in the extended dynamic range modus. Spectra were recorded

with positive polarity in the range of 100 to 3000 m/z at a resolution of about 15000 and using

three reference masses (121, 922 and 2421) for improved mass accuracy. By analysing the MS-

spectra of the main peak the expected mass and isotopic distribution of each peptide batch was

confirmed. Peptide purity was calculated by the following equation after manual integration of

the UV chromatogram:

Purity [%]=( peak area)peptide

(total peak area)∗100 % (1)

where (peak area)peptide is the area of the chromatographic peak corresponding to the product and

total peak area refers to the sum of the area of all peaks present in the chromatogram.

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Peptide solubility with UHPLC-RP/UV. Solutions from solid samples were prepared in

specific buffer systems with a concentration of 10 mg/mL compound based on the previously

determined purity. Solid peptide was placed in a suitable glass vial and dissolved in 80%

volume of the buffering system while gently stirring for 1h under light protection at 200 rpm.

After re-adjustment of the pH value (if necessary) with hydrochloric acid or sodium

hydroxide, buffering solution was added up to the final volume. After 15 minutes of

homogenization at 200 rpm, the solution was centrifuged at 450 RCF. The supernatant was

filled in a glass vial for quantification of the dissolved amount of peptide. The following

primary buffer systems were used: a) 100 mM acetate buffer pH 4.5, b) 100 mM acetate

buffer pH 5.2, c) 100mM citrate buffer pH 6.5, and d) 100 mM phosphate buffer pH 7.4. In

addition two variations of each buffer system were tested: one with 2.7 mg/mL m-cresol and a

second with 5.0 mg/mL phenol added as preservatives.

After 15 minutes of centrifugation at 2500 RCF a 2 µL aliquot of the 1:10 diluted supernatant

was injected on the UHPLC system consisting of a Waters Acquity I-class UHPLC equipped

with a photodiode-array detector (PDA). Sample separation was accomplished with the same

method as described above (Peptide Purity). The sample concentration (Cpeptide) was determined

by relating the UV area of the main peak of the sample to a linear standard curve of a reference

peptide with known concentration (Cstandard). The different UV extinction coefficients (EC) of

sample and reference peptide were calculated based on the different amino acid sequences

according to reference1 and considered in the concentration calculation as described by the

following equation:

C Peptide=( peak area)peptide∗ECSample( peak area)standard∗ECPeptide

∗CStandard (2)

Chemical stability. Solutions from solid samples were prepared in specific buffer systems

with a concentration of 1 mg/mL compound based on the previously determined purity. Solid

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peptide was placed in a suitable glass vial and dissolved in 80% volume of the buffering

system while gently stirring for 1h under light protection at 200 rpm. After re-adjustment of

the pH value (if necessary) with HCl or NaOH, buffering solution was added up to the final

volume. After 15 min of homogenization at 200 rpm, the solution was filtered through 0.22

µm pore size under laminar flow conditions. The flowing buffer systems were used: a) 20 mM

acetate buffer pH 4.5, b) 25 mM acetate buffer pH 4.5, 2.7 mg/mL m-cresol, 15.3 mg/mL

glycerol, 3 mg/mL L-methionine, c) 20 mM phosphate buffer pH 7.4.

The obtained solutions were aliquoted into HPLC glass vials for storage at two different

temperatures for 28 days. One vial was stored at 2 °C as a reference and the second vial was

force degraded at 40 °C. Afterwards two different methods were applied to assess chemical

stability of the peptide by comparing the purity after storage at the two different temperatures.

Method A) Chemical degradation with UHPLC-RP/UV-MS. Prior to measurement the

vials were centrifuged for 15 minutes at 2500 RCF and a 2 µL aliquot of the supernatant was

injected on the UHPLC System consisting of a Waters Acquity I-class LC equipped with a

photodiode-array detector (PDA) that was hyphenated to a Waters LCT Premier TOF. Sample

separation was accomplished with the same method as described above (Peptide Purity). The

mass spectrometer, equipped with an ESI source, was operated according to the

manufacturer’s instructions in the W flight mode. Spectra were recorded with positive polarity

in the range of 300 to 3000 m/z at a resolution of about 10000. The MS-Spectra were created

and analyzed for each integrated UV-peak higher than approximately 0.2% of the total area

for the samples stressed for 28 days at 40 °C. Results were compared with that of the samples

stored at 2 °C in order to discriminate between impurities (also present at 2 °C) and

degradation products (only present after 40 °C stress). Overall, chemical stability was rated

through relative purity loss (degradation) calculated by the following equation:

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Relative Purity Loss [% ]=( Purity 2° C )−(Purity 4 0° C)

(Purity 2 °C)∗100 % (3)where the purity values were

calculated from the chromatograms at 214 nm after manual integration as described above.

In addition the mass balance is calculated as:

Mass Balance [% ]=(total peak area 40 °C )−(t otal peak area 2° C)

( t otal peak area2 °C )∗100 % (4)

where total peak area refers to the sum of the area of all peaks in the corresponding

chromatograms (214 nm). This value, that ideally equals 0%, is an indicator for physical

instability (e.g. precipitation would lead to a negative mass balance) and degradation

components eluting outside of the integration window (e.g. very unpolar components could elute

in the wash phase).

Method B) Increase of high molecular weight protein (HMWPs) with UHPLC-SEC/UV-

MS. Prior to measurement the vials were centrifuged for 15 minutes at 2500 RCF and a 2µL

aliquot of the supernatant was injected on the UHPLC System consisting of a Waters Acquity

H-class UHPLC equipped with a photodiode-array detector (PDA) that was hyphenated to an

Waters LCT Premier TOF. Sample separation was accomplished on an Agilent AdvanceBio

SEC column with 130 A 2.7 µm particles in a 4.6x150 mm column dimension operated at 30

°C. The UV absorption was detected at 214 nm. With an isocratic flow of a mixture of 60%

acetonitrile and 40% water containing 0.1% TFA at a flow rate of 0.2 mL/min the samples

were eluted within a 12 min run, followed by a 2 min wash time. Growth of HMWPs was

rated by relative HMWP increase calculated by the following equation:

Relative HMWP Increase [%]=(%Monomer 2° C)−(% Monomer 40 ° C)

(%Monomer 2° C)∗100 % (5)

with %Monomer being the percentage of the monomer peak relative to the total peak area.

In addition the mass balance is calculated as:

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Mass Balance [% ]=( total peak area 40°C )−(t otal peak area 2° C)

( t otal peak area2 °C )∗100 % (6)

The mass spectrometer, equipped with an ESI source, was operated according to the

manufacturer’s instructions in the W flight mode. Spectra were recorded with positive polarity in

the range of 300 to 3000 m/z at a resolution of about 10000. The MS-Spectra were created and

analyzed for the peptide monomer peak and the HMWP peaks to confirm the molecule size.

Capillary isoelectric focusing for isoelectric point (pI) determination. Solid peptide

samples (lyophilisate) were dissolved in de-ionized water. For injection in the iCE3 system

(ProteinSimple, California), the samples were further diluted with master mix solution

composed of 0.35% methylcellulose, 8% carrier ampholyte (pH 3-10) solution, 0.5% pI 4.22

marker and 0.5% pI 9.46 marker to obtain a 0.2 mg/mL peptide solution. Samples were

injected into a capillary cartridge with electrolytic tanks to fill the capillary column

completely. Peptides and pI markers were separated and focused within 6 minutes based on

their specific pI in the pH gradient that was created by applying 1500-3000 Volt. The UV

detector monitors the whole column which allows analyzing the peptides pI based on position

in the gradient calibrated by the specific pI markers.

Thioflavin T (ThT) binding. Investigations were carried out to determine fibrillation

tendencies under stress conditions by shaking at 37°C within Fluoroskan Ascent FL. For the

tests in Fluoroskan Ascent FL, 200 µL sample were placed into a 96 well mictrotiter plate PS

(Polystyrene), flat bottom, Greiner Fluotrac No. 655076. Plates were sealed with Scotch Tape

(Quiagen). Samples were stressed by continuous cycles of 30 s shaking at 960 rpm and 30 s

rest period at 37°C. Fibrillation kinetics was monitored by measuring fluorescence intensity

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every 20 minutes. Peptides were dissolved in a buffer system to a final concentration of 3

mg/ml. 20µL of a 10.1 mM aqueous ThT solution were added to 2 mL of peptide solution to

receive a final concentration of 100 µM ThT. Each sample solution was divided into eight

replicates. The following buffer systems were used: a) 100 mM acetate buffer pH 4.5, b) 100

mM acetate buffer pH 4.5, 2.7 mg/mL m-cresol.

Dynamic light scattering (DLS). Peptides were dissolved in an aqueous buffers to the final

concentration of 5 mg/mL using the following buffering systems: a) 15 mM acetate buffer pH

4.5, b) 15 mM phosphate buffer pH 7.4. In addition, each buffer system was tested with 2.7

mg/mL m-cresol added as preservative. Sample preparation included dissolution of freeze-

dryed peptide, pH adjustment, and a final filtration through 0.22µm pore size (Millipore,

Millex GV, PVDF membrane) into a clean vial. DLS measurements were performed on a

DynaPro Plate Reader II (Wyatt Technology, Santa Barbara, CA, US) using a low volume

non-treated black polystyrene 384 assay plate with clear bottom (Corning, NY, US).

Measurements were taken with a 830 nm laser light source at an angle of 158°. Samples were

measured in a 9 mm insert for 5 seconds (acquisition time) and 15 times (number of

acquisitions). Samples were tempered for 1 hour at +25°C and visually inspected.

Measurement was performed in triplicates. Therefore three times aliquots of 30 µL were

pipetted from the vial into a 384 assay plate and sealed. The plate was centrifuged for 2

minutes at 450 RCF. After removing the seal the samples were measured at 25°C. For every

peptide solution, DLS parameters were determined as an average over triplicates with non-

negatively constrained least squares (NNLS) methods using Dynals algorithms with

regularization fit.

Determination of DLS self-interaction parameter (kD).

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The DLS self-interaction parameter (kD) is a measure to describe inter-particle interactions,

where the particles are folded proteins or peptides.2,3 The parameter kD is derived from the

concentration dependence of the diffusion coefficient D, which is given by an expansion in

powers of the concentration c:

D (c )=D0(1+kD c+k iDc2+k jD c3+…) (7)

Neglecting the higher order terms, i.e. kiD = kjD = … = 0, the data can be fitted linearly

according to the equation:

D=D0(1+k D c) (8)

where D is the measured diffusion coefficient, D0 is the diffusion coefficient at a theoretic

sample concentration of zero, c is the sample concentration and kD is the DLS interaction

parameter. Experimental values of D were determined at peptide concentrations of 1 mg/mL,

2 mg/mL, 3.5 mg/mL, and 5 mg/mL and fitted to a straight line using equation (8).

The intercept of this graph is D0 and the slope of the line is kD/D0. kD can therefore be

calculated from the slope of the graph.

The parameter kD can be used to describe the interaction of protein or peptide molecules or

oligomers with oneself in solution and is theoretically related to the second virial coefficient

B22 as for example described by Harding and Johnson,

k D=2 B22 M −ks−ν (9)

where M is the molar mass, ks the first order concentration coefficient of sedimentation

velocity and 𝝂 the partial specific volume.4

Sample preparation included dissolution of freeze-dryed peptide, pH adjustment, and a final

filtration through 0.22µm pore size (Millipore, Millex GV, PVDF membrane) into a clean vial

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for the highest concentration. All lower concentrations were prepared by diluting the stock

solution using filtered matching placebo.

Hydrophobicity analysis and identification of aggregation hot spots. The following

amino acids were considered as hydrophobic: Val, Ile, Leu, Met, Phe, Trp, Cys, Ala, Tyr, Pro,

Gly, Aib, Lys(yE-yE-C16). All these amino acids were identified by colour coding (red) in

the peptides 3D structure. The most uninterrupted continuous patches were identified as

aggregation hot spots.

Charge, isoelectric point and electrostatic field calculation. For calculation of charge vs

pH titration curves, the pKa values of titratable main chain and side chain atoms of natural

and unnatural amino acids were computed using epik.5 The total peptide charge at different

pH values is the sum of the positive and negative charges of the individual amino acids that

were calculated according to the Henderson Hasselbalch equation.6 The isoelectric point was

calculated by identifying the pH value where the total peptide charge amounts to 0. For

electrostatic field calculation and visualization at different pH values, we used the Poisson-

Boltzmann (APBS) method7 and maestro.8

Molecular dynamics simulations. MD simulations were carried out using an explicit solvent

MD package, Desmond program (version 4.7, Desmond Molecular Dynamics System; D. E.

Shaw Research, New York, NY, USA and version 3.1, Maestro-Desmond Interoperability

Tools; Schrödinger) with inbuilt optimized potentials for liquid simulation (OPLS 2.1) force

field.9–11 The peptides were prepared for simulation by first checking their correctness using

the Protein Preparation wizard tool and Epik module was used for deriving the protonation

states of the peptides at two different pH values – 4.5 and 7.4. The system was prepared by S9

placing the peptides in a cubic box with periodic boundary conditions specifying the shape

and size of box as 10 Å × 10 Å × 10 Å distance. Predefined TIP3P water model was used as a

solvent and the systems were neutralized by adding appropriate number of ions. The solvated

systems were relaxed by implementing Steepest Descent and the limited-memory Broyden-

Fletcher-Goldfarb-Shanno algorithms in a hybrid manner. The simulation was performed

under NPT ensemble for 500ns implementing the Berendsen thermostat and barostat methods.

Nose-Hoover thermostat algorithm12,13 was used to maintain a constant temperature of 300 K

and MartynaTobias-Klein Barostat algorithm14 was employed for maintaining 1 atm of

pressure throughout the simulation, respectively. The short-range coulombic interactions were

analyzed using the short-range method with a cut-off value of 9.0 Å. The Particle Mesh Ewald

(PME) method15 was used for treating the long-range electrostatic interactions. All bonds

involving hydrogen atoms were constrained using the SHAKE algorithm.16

Mixed-solvent molecular dynamics simulations. The modeled peptide structures17 were

used as starting structures for mixed-solvent simulations which were carried out using a

mixture of water, acetate and m-cresol molecules. The program Packmol18 was used to place

the desired number of m-cresol and acetate molecules in the solvated systems. These solvated

systems were then neutralized using required number of Na+ ions (1 peptide, 4 Na+, 8 acetate,

10 m-cresol, 8580 T3P). The atom type definition and the associated force field parameters

were derived using Schrödinger suite version 2016-3. Relaxation and production MD

simulations were carried out as described above.

Analysis of molecular dynamics simulations. As an indicator of local backbone fluctuations

over the entire simulation length, standard deviation of Ψ and Φ backbone torsion angles

(Radians) was calculated from 100 snapshots that were generated at equal intervals from the

simulated trajectory. For comprehensive visual analysis of conformational peptide stability,

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these 100 snapshots were superimposed to the starting structures using the Cα atoms of a

sequence stretch with low backbone fluctuation (residues 21-27 in all simulations) as

reference. The same fitting parameters were furthermore used for the calculation of root mean

square fluctuations (rmsf) for each residue over the entire simulation.

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Supplemental Figures and Tables

Supplemental Figure S1. Standard Deviation of psi and phi backbone torsion angles (Radians) calculated in MD simulations of 500 ns as an indicator of local backbone fluctuations over the entire simulation length. The x-axis represents peptide sequences while the y-axis is radians.

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Supplemental Figure S2. Root-mean square fluctuation (rmsf) plot during molecular dynamic (MD) simulations. The figure shows Cα rmsf of peptides 1 and 2 at pH 4.5 in the absence and presence of cresol in MD simulations of 500 ns. The x-axis represents peptide sequences while the y-axis is average rmsf values.

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Supplemental Table S1. UHPLC gradient for peptide purity determination.

Time [min] Flow [mL/min] %A %B Curve

1 initial 0.5 80 20

2 3 0.5 80 20

3 23 0.5 25 75 linear

4 23.5 0.5 5 95

5 26.5 0.5 5 95 linear

6 27 0.5 80 20 linear

7 33 0.5 80 20 linear

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