plain 4 SYNTHESIS, PURIFICATION AND STABILITY STUDY OF ISLET

SYNTHESIS, PURIFICATION AND STABILITY STUDY OF ISLET NEOGENESIS-

ASSOCIATED PROTEIN- PEPTIDE AND ANALOGS

ANDREW APALSThesis defense presentation

April 28, 2016Northeastern Illinois university

Outline Of Presentation

• Introduction• Solid phase peptide synthesis • RP-HPLC method development for

the separation and quantitation of target peptide in a synthetic mixture

• Room temperature degradation study of LIP-St in acidic solvent

• Resolution of INGAP-P fraction• Fractionalization using an analytical

instrument• Conclusion and future directions

A) 3-D structure of INGAP protein

B) Zoom on pentadeca-Bioactive site

The Islet Neogenesis- Associated Protein And Its Bioactive Pentadecapeptide Fragment (INGAP Peptide)

• Studies have consistently displayed the INGAP peptide as initiating pancreatic islet neogenesis and increasing Beta- cell mass

• The INGAP peptide has reversed streptozotocin- diabetes in animal models

• In clinical trials, INGAP peptide was found to improve glucose homeostasis in patients with Type II diabetes and to increase C- peptide secretion (a measure of endogenous insulin secretion) in patients with Type I diabetes

• Yet, INGAP peptide has a relatively short plasma half- life and the need for higher dose administration calls for prioritized studies into its mechanism of action, as this is still yet to be elucidated

• Petropavlovskaia,Daoud,Zhu,Moosavi,Ding,Makhlin,Assouline-Thomas,Rosenberg J Physiol Endocrinol Metab 2012 25 E917-E927

• Dungan,Buse,Ratner Diabetes Metab Res Rev 2009 25 558-565• Uwaifo,Ratner Endocrinol Metab Clin North Am 2005 35 155-197

The INGAP Peptide:A Promising Treatment For Diabetes

INGAP Peptide And Linear And Cyclic Analogs

Objectives Of Research

• Synthesize INGAP-P, linear and cyclic analogs utilizing the protocols of Fmoc SPPS

• Preliminarily identify target peptide in SPPS product mixture using MALDI-TOF and ESI MS analysis

• Study the parameters involved in developing a RP-HPLC method for the separation and quantitation of a target peptide in a SPPS product mixture

INCLUDED IN THE RP-HPLC STUDY

• Analyze degradation of a linear INGAP standard in the sample solvent

• Study the parameters involved in increasing resolution between unresolved peptides

• Demonstrate the potential for using an analytical RP-HPLC instrument in purifying a peptide in a crude product mixture

NH2 HOOC NH-FmocResin(Solid phase)

Amino acid 1

Side chain protection

Coupling[Activating reagents]

Resin NHOC Amino acid 1 NH-FmocResin NHOC Amino

acid 1NH-Fmoc

NH-FmocDe- protection[Basic reagent]

NH-H

NH-H HOOC Amino acid 2


NH-Fmoc Coupling[Activating reagents]

NHOC Amino acid 2 NH-Fmoc

The Fmoc group

Etc…………………

F-MOC Solid Phase Peptide Synthesis

Reagents: Rink amide resin and Fmoc

A) Rink amide resinSubstitution density:

0.5mmol/g

B) The FluorenylmethyloxycarbonylGroup

Removed in de-protection of2- amine in

20%piperidine/Dimethylformamide(DMF)• Starting resin and all aminoacids utilized N-Fmoc protected

The Intermittent Ninhydrin (Kaiser) Test

Coupling

De- protection



NH-FMOC

NH2

Free amino group

Carbamate group

Ruhemanns Blue

The flow of events for each coupling are:• [De-protection (resin/ 2- amine of amino

acids)→ • evaluation of free amino group → • amide bond formation→ • evaluation of coupling]ⁿ

The ease of solid phase peptide synthesis comes from the fact that the unreacted reagents of the respective coupling and deprotection reactions are washed away by vacuum filtration

SPPS: A Series Of De-protection And Coupling Steps

• The first amino acid Fmoc-Ser(OtBu)-OH coupled using HBTU/HOBt reagents

• Further amino acid couplings utilized PyBOPas the minimum time required for each coupling was reduced from 4 hours to 1 hour

• Coupling efficiencies are approximately equalfor these two reagents, yet kinetics quite

diffferent

HBTU/HOBt amide bond formationIn DMF with Diisopropylethylamide (DIPEA)

as basePyBOP amide bond formation

In DMF w/ DIPEA

Synthesis of Linear INGAP-P Analog

Ac-I-G-L-H-D-P-S-H-G-T-L-P-N-G-S-NH2

Ac-E-G-L-H-E-P-S-H-G-T-L-P-N-K-G-S-NH2

TFA cleavage cocktail cleaves peptides from resin/ side-chain protection

On- resin INGAP-P

On- resin linear INGAP-P analog

Amidated/ Acetylated product

TFA Cleavage Cocktail Cleaves Peptide From Resin And All Side- Chain Protection

Trt- group tBu- group

SPPS Product Mixture Of Peptides

Peptide mixture

Soft ionization [P1+][P2+]

[Px+]

Mass analyzer

m/z

Intensity

MS For Preliminary Identification Of Target Peptide In SPPS Product Mixture

• Mass spectrometry measure the intrinsic property of mass of an analyte

• Soft ionization techniques such as matrix-assisted laser desorption ionization (MALDI) and electrospray ionization yield molecular ion peaks for each peptide in a sample mixture with little/no fragmentation

• A single spectrum can be obtained of all peptide components

Reverse-phase High Performance Liquid Chromatography (RP-HPLC)

• Polar Mobile Phase/ Non polar Stationary Phase

• Non polar moves slower, polar moves faster

• Detected by UV-Vis detector

• Highly utilized in the separation and analysis of peptides and proteins

• Capable of resolving peptides differing by a single amino acid residue

Selective removal of Dde/ODmab de-protects Lysine/Glutamic Acid

Coupling

Selective Deprotection And Cyclization Before Final Cleavage

1-(4,4-dimethyl2,6-dioxocyclohexylidene)ethyl-

4-(N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino)benzyl

Removal of Dmab- protection from glutamic acid by hydrazine

Dde- is removed in similar fashion from lysine

Selective Removal of Dde and Dmab Side-Chain Protecting Groups

Dde

Dmab


Selective removal of Dmab/Dde

DIC/HOAt coupling

DIC/HOAt amide bond formation

Synthesis Of Cyclic INGAP-P Analog Through Select De-protection And Coupling Of A Sample Of Linear INGAP-P Analog

Cleavage And Extraction

• Resin-bound INGAP-P and linear and cyclic analogs were stirred in a cleavage cocktail of TFA/phenol/water/TIPS- 88/5/5/2 by volume for two hours. This cleaves the peptide from resin as well as removing the side-chain protecting groups

• The remaining solutions were filtered and had TFA driven off via rotovap, then peptides were precipitated via cold diethyl ether, centrifuged at 0ºC, and ether decanted to yield a crude product mixture of the INGAP-P, linear and cyclic analogs

Final Target Peptides

MALDI-TOF MS Of Linear INGAP-P Analog

Intensity

m/z

• Molecular Mass of linear INGAP-P analog: 1700.8 Da

• Its protonated molecular ion [M+1] can be distinguished at 1701.8 m/z in the spectrum

Ac-EGLHEPSHGTLPNKGS-NH2

MALDI-TOP MS Of Cyclic INGAP-P Analog

• Molecular mass of cyclic INGAP-P analog: 1682.8 Da

• Its protonated molecular ion [M+1] at 1683.8 m/z is barely discernable in the main spectrum at top, yet a zoom distinguishes a minor product yield

• Its intensity is12.5% that of the most intense peak at 1701.8 m/z, that of the linear analog

Ac-EGLHEPSHGTLPNKGS-NH2

Reasons For Low Product Yield Of Cyclic Analog

• Successful cyclization is sequence dependent with consideration of spatial orientation of the peptide backbone and steric hindrance

• Cyclization reagents and reaction conditions used not optimized

ESI-MS Qualitative Plot Window Report

• Molecular mass of INGAP-P: 1541.7 Da

• Peak at 1541.7 m/z represents the INGAP-P as instrument was not properly calibrated ([M+1] should be 1542.7 m/z)

• An additional peptide appears to co-eluent in this fraction with a INGAP-P mass + 101 Da represented by the peak at 1642.8 m/z

• This additional peak may be INGAP-P + threonine, caused by technician error Acquired from NU Simpson Querrey Institute

3100µg/mL Crude INGAP-P mixture

250µg/mL INGAP-P pooled fractions

1000µg/mL commercial INGAP-P

Why synthesize?

RP-HPLC Method Development for Analysis of INGAP-Peptides

• synthesized by Dr. Su’s group was utilized in method development.

• As lyophilized powder with ~ 97% purity.

LIP- St MM: 1679 DaAc-I-G-L-H-H-D-P-S-H-G-T-L-P-N-G-S-NH2

A linear INGAP-P peptide standard (LIP-St)

Solubility of LIP-St

The LIP-St has fairly high hydrophobicity and low solubility in water.

• Up to 6 mL of HPLC H2O were needed to dissolve 1 mg of LIP-St

• 1 mL of aqueous mobile phase (H2O/0.1% TFA) readily dissolved ample amounts of sample(> 4mg)

LIP- St MM: 1679 DaAc-I-G-L-H-H-D-P-S-H-G-T-L-P-N-G-S-NH2

UV Spectrum of 1250µg/mL LIP Standard in H2O/0.1%TFA

UV Detection Wavelength Selection

UV Absorbance Comparison at 220,215, and 232 nm for 1250 µg/mL LIP-St (Col: Phenomenex C-18, 3.1 X

250mm, 5 µm, 100 Å pore size. Gradient 10-30%B in 20 minutes,

flowrate 1mL/min)TOP: Comparison of chromatograms at

different wavelength detectionsBOTTOM: Plot of Area vs Wavelength

detection

UV Detection Wavelength Selection220nm

215nm

232nm

Mobile Phase Selection

Solvent A: H2O/0.1% Trifluoroacetic acid (TFA)Solvent B: ACN/ 0.085% TFA

Acetonitrile used as it has a low UV quantitative cutoff of 200nm and has a lower viscosity than ethanol or isopropanol, reducing column back pressure

TFA utilization 2-fold: 1)suppress ionization of acidic peptide residues as well as free silanols on column that can cause peak tailing 2) protonate and ion- pair with peptide basic residues. Overall effect of TFA is decrease peak tailing and to increase peptide retention of otherwise hydrophilic, unretainable peptides

15% less TFA used in ACN than water as TFA dielectric constant changes with increased composition of ACN, yielding a rising baseline in gradient mode.

Reduction of TFA in ACN helps to lesson this baseline drift

Injection Volume Selection

5µL injection LIP-St 10µL injection LIP-St

2600 µg/mL of linear INGAP-P analog crude mixture

• Lowering injection volume increases theoretical plate number while decreasing tailing factor

• This is particularly important when considering that a SPPS peptide mixture will have closely eluting peaks with similar hydrophobicities

Gradient Mode

• The most utilized mode in peptide separations

• yield column efficiency values N 10 plus fold over isocratic mode.

• Reducing gradient rate %B/min can increase resolution between peptide peaks

3min 10%B hold, 1%B/min to 30%B

3 min 20%B hold, 0.25%B/min to 25%B

Rs with preceding impurity: 0.550

Rs with preceding impurity: 0.791

1037.5 µg/mL LIP-St sample (Col: Phenomenex C-18 250x3.2 mm 5µm, 100 Å) ,5µL injection Solvent A: H2O/0.1%TFA; Solvent B: ACN/ 0.085%TFA

Gradient Mode Considerations

1250 µg/mL LIP-St sample (Col: Agilent Eclipse C-18 250x4.6 mm 5µm, 100 Å)Solvent A: H2O/0.1%TFA Solvent B: ACN/ 0.085%TFA 15µL injectionGradient 5-60%B in 55 minutes

Initially, a sample is run from a low %B to a high %B.

• a rough estimate can be ascertained as to where the peptide(s) of interest elutes

• initial and final %B can be determined for the gradient time table

• Determines that no other components, peptide or impurity, are eluting later after the main peptide(s) of interest

Initial Run To Assure Complete Peptide Elution


2600 µg/mL linear INGAP-P analog crude mixture (Col: Phenomenex C-18 250x3.2 mm 5µm, 100 Å)Solvent A: H2O/0.1%TFA Solvent B: ACN/ 0.085%TFA 15µL injection 5min 5%B hold, 1%B/min to 50%B

Isocratic hold time and Initial %B needs consideration of early eluent

Final %B needs consideration of late eluent, last peak elutes ≈31%B

Isocratic Hold, Initial/Final %B


No Injection Run- Baseline ExtrapolationAs gradient returns to initial solvent ratio, baseline dips below baseline as TFA re- saturates the C-18 stationary phase. For this reason, appropriate time is needed for re- equilibration between runs

Baseline Equilibration Consideration

RP-HPLC Parameters For Lip-St PeptideInstrumentation: Agilent 1260 Infinity

(HPLC/DAD)

G1311B Quaternary Pump

G1329B Auto sampler

G1315D Diode Array Detector

G1316A Column Compartment

OpenLab CDC ChemStation acquisition system for LC, Agilent Technologies

Column: Phenomenex C-18 250x3.2 mm 5µm, 100 Å

Sample solvent: H2O/ 0.1%TFA

Injection volume: 5µL

Wavelength detection: 215 nm, no reference

Mobile phase: Solvent A: H2O/ 0.1%TFA Solvent B: ACN/ 0.085%TFAGradient rate: 1%B/minGradient time table:

Time (minute)

% B

0 15

3 15

18 30

21 30

24 15

30 15

Flowrate: 0.8mL/min

Linearity Determination

• Two consecutive injections of each concentration (µg/mL )

15, 25, 50, 100, 250500,750,1000, 1250,1500,2000, and 2250

• Blank runs between 500-750 and 1250-1500 µg/mL to assure cleanout of previous runs

Limit Of Quantitation/Detection

Limit of Quantitation(USP): 7.3µg/mL with a signal to noise ratio 10.52/1

Limit of Detection(USP): 3.65µg/mL with a signal to noise ratio 4.47/1 (Limit of Detection defined at a signal to noise ratio of 3/1, so this is more of a working LOD)

Injection Precision

Area Average Standard Deviation CV, %

3402.91

3416.64

3430.18 3420.89 10.191 0.298

3429.46

3425.15

3420.97

Injection precision 500 µg/mL LIP-St

Overlay of Degradation Progressionof 500µg/mL Sample

500µg/mL LIP-St

Reduction of % peak area vs time at room temperature. 100%= freshly prepared

Degradation Study of INGAP peptides

500µg/mL Lip-St In H2O/0.1%TFA Ph2 At Room Temperature

Degradation Study of INGAP peptides

Overlay of Degradation Progression

of 100µg/mL Sample

Reduction of % peak area vs time at room temperature. 100%=

freshly prepared

100µg/mL Lip-St In H2O/0.1%TFA Ph2 At Room Temperature

Potential Degradation Product:Iso- Aspartate Analog

• Clark Int J Pept Protein Res 1987 30 808-821

Peptides with the ..D-P.. Sequence are prone to iso- aspartate isomerization

Extracted, Lyophilized Sample Containing INGAP-P

250µg/mL INGAP-P fractionSolvent A: H2O/0.1% TFA,Solvent B: ACN/0.085% TFAGradient: 1%B/min Flowrate: 0.8mL/min

• INGAP-P product mixture was partially purified by preparative HPLC• the INGAP-P containing fraction was lyophilized• Problem: This fraction also contained an unresolved component with M+101 Da

The Fundamental Resolution Equation In HPLC

N: Column efficiency, determined by column specifications (Column length, I.D., particle size, pore size, carbon load, etc.)

α: selectivity factor, determined by type of mobile and stationary phase utilized

k: capacity factor (retention times), determined by flow rate, gradient rate

300-mAU 50-mAU

Increasing Resolution of HPLC

Changing Capacity Factor By Decreasing Gradient Rate

The Effect of Decreasing %B/min on ResolutionSample: 250 µg/mL lyophilized fractionSolvent A: H20/0.1%TFA; Solvent B: ACN/0.085%TFA

Changinging α Selectivity To Increase Resolution

Effect of Doubling TFA in Mobile Phases on Resolution250µg/mL INGAP-P fractionSolvent A: H2O/ 0.2%TFASolvent B: ACN/ 0.17%TFAGradient rate: 1%B/min

200-mAU

MP Composition- Increasing TFA

250µg/mL INGAP-P fractionSolvent A: H2O/0.1% TFA,Solvent B: ACN/0.085% TFAGradient: 1%B/min Flowrate: 0.8mL/min

Solvent A: H20/0.1%TFA Solvent B: IPA/0.1%TFAFlowrate: 0.50mL/minSample: 850 µg/mL lyophilized fraction

200-mAU

Solvent A: H20/0.1%TFA Solvent B: Methanol/0.1%TFAFlowrate: 0.65mL/minSample: 850 µg/mL lyophilized fraction

Rs: 0.621

Changing Α Selectivity To Increase Resolution MP Composition- Changing Organic Modifier

Changinging α selectivity to increase resolution

3 Phenyl ColumnGradient: 1%B/min, Flow Rate: 1mL/min

Solvent A: H2O/0.1%TFA Solvent B: ACN/0.085%TFASample: 850 µg/mL lyophilized fraction

3 Phenyl ColumnGradient: 1%B/min

Flow Rate: 0.75mL/minSolvent A: H2O/0.1%TFA Solvent B: ACN/0.085%TFA

Sample: 850 µg/mL lyophilized fraction

Column: Ace 3-Phenyl 150x4.6mm, 3µm 100 Å

Decreasing flowrate with phenyl column increases resolution

Changing column type

mAU-215 mAU-286

Rs: 0.484 Rs: 0.674

Fractionalization Using An Analytical Instrument: A Valuable Technique To Add To Peptide Synthesis

• An optimized method can extract fractions of pure peptide

• Fractions can be pooled

• peptide extraction from solvent and lyophilization can yield a standard for HPLC method development and validation

Examples of utility• INGAP-P can be separated

from the INGAP-P mass + 101Da and used as a standard

• Degradation product can be extracted to access identity

Analytical parameters2250 µg/mL linear INGAP-P analog crude mixtureColumn: Phenomenex C-18 250x4.4mm 5µ 100 Å Solvent A: H2O/0.1%TFA Solvent B: ACN/0.085%TFAFlowrate: 1mL/minGradient rate: 1%B/min10%B Isocratic hold 3mins, ramp to 40%B in 30mins

• An unused channel is detached from degasser

• Pump turned on (1ml/min), drawing sample (2mL of 2250 µg/mL linear INGAP-P analog mixture)

• Pump is stopped and channel placed in starting MP composition 10%B

• Pump is turned on so as to carry sample to the top of the column 3mins

• Pump turned off, program set to start gradient 10%-40%B 30mins (1%B/min)

• Turn pump on, start collection of fractions here where gradient is detected (time from mixer to detector, wait 5mins, then start first 1-min fraction collections)

• Collect 1-min fractions for 12 mins (target peaks)

(Dauer, R. Engineer at Corden Pharma Colorado. Personal communication via LinkedIn messaging, 2015)

Fractionalization Using An Analytical Instrument

Analytical Runs Of Minute 6-9 FractionsAnalytical of 2250µg/mL sample

Minute-6 fraction

Minute-7 fraction

Minute-8 fraction

Minute-9 fraction

Target peaks

Collected fraction Minute-6 fraction

Scheme for extracting INGAP-P from lyophilized extract through overloading of an analytical column (overload trace not to scale)

Scheme For Purification

Conclusions

• Solid phase peptide synthesis has displayed itself as a straight forward and efficient method for the synthesis of target peptides such as INGAP-P and analogs

• With MALDI-TOF or ESI mass spectrometry, the target peptide can be ascertained in the final product mixture

• The study shows RP-HPLC can be a very viable technique in the separation and quantitation of a peptide in a mixture. Adjustment of parameters such as initial and final %B can control the elution window of a peptide mixture. Parameters such as gradient rate and column type can be changed as to increase resolution between unresolved peaks.

Future Directions

• Experiments should be furthered in the fractionalization of a sample on an analytical instrument. With an optimized method, it may be possible to home in on fractions of high purity of target peptide. Such purified samples can be pooled and the peptide extracted.

• The RP-HPLC method developed in this study will be used for studying stability of INGAP-P and analogs in vitro. Enzymatic degradation as well as chemical degradation under physiological conditions will be investigated.

• The synthesized peptides are currently being examined for their effects on stimulating islet beta cell growth.

• Structure-activity relationship of INGAP-P and analogs (particularly the cyclic analog) would shed light on design of new beta cell-promoting peptide drugs.

Acknowledgements

Jing Su, Ph.D.Professor, Department of ChemistryNortheastern Illinois University

S. John Albazi, Ph.D.Professor and ChairDepartment of ChemistryNortheastern Illinois University

Department of Chemistry at NEIU

My peers in the Master’s Program. Most notably, Sandra Neri, Rafal Turek, Martin Shlaymoon and

Matilda McFarland

Reagents: Coupling1. O-(Benzotriazol-1-yl)-N,N,N’,N’-

tetramethyluronium hexafluorophosphate/ N-Hydroxybenzotriazole (HBTU/HOBt)

2. benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP)

3. 1,3-Diiscaropropylcarbodiimide/ 1-Hydroxy-7-azabenzotriazole (DIC/HOAt)

Synthesis of Linear INGAP-P Analog Starting at glycine in the 8th position from the resin/C-terminal, the ninhydrin test

displayed a positive result after coupling,Indicative of incomplete amide bond formation

Because of this, coupling reaction was conducted twice,yielding a ninhydrin test positive

All further amino acid couplings until the last amino acid coupling followed the same pattern of requiring double coupling steps in order to achieve complete amide bond formation as indicated by the ninhydrin test


Synthesis of INGAP-P

PyBOP was used for all amide bond formation between amino acids of INGAP-P

For latter residue couplings (residue glycine in 7th position from resin to last amino acid isoleucine) coupling times were extended from 1 hour to 1.5 hours. This eliminated the need for double couplings of latter amino acids (as with the linear INGAP-P analog), as indicated by ninhydrin test negative after these couplings

On- resin INGAP- P

Acetylation of resin-bound peptidesResin-bound peptides acetylated with acetic anhydride/ DIPEA in DMF

Acetylation

INGAP-P

Linear INGAP-P analog

DIAGRAM OF MALDI- TOF(TIME-OF-FLIGHT MASS ANALYZER) INSTRUMENTATION

• Large excess of matrix material is co- precipitated with the analyte onto a metal substrate and allowed to dry

• Dried mixture is irradiated by nanosecond laser pulses, usually a nitrogen laser with wavelength 337nm

• Laser pulses desorb matrix and analyte molecules, yielding protonated analyte molecules (A) that are then introduced into a mass analyzer, typically the TOF mass analyzer (B)

DIAGRAM OF ESI PROCESS

• A liquid carrying the analyte is pumped through a hypodermic needed at µL/min

• Needle is at a high voltage, causing eluting liquid to electrostatically disperse small, µm droplets which rapidly evaporate, with a charge imparted on analyte molecules

• Because ESI takes place at atmospheric pressure, originates from a flow of liquid, and yields peak intensities linearly related to analyte concentration, it can be used as a detection method for high performance liquid chromatography

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plain 4 SYNTHESIS, PURIFICATION AND STABILITY STUDY OF ISLET