plain 4 SYNTHESIS, PURIFICATION AND STABILITY STUDY OF ISLET
1. SYNTHESIS, PURIFICATION AND STABILITY
STUDY OF ISLET NEOGENESIS-
ASSOCIATED PROTEIN- PEPTIDE AND
ANALOGS
ANDREW APALS
Thesis defense presentation
April 28, 2016
Northeastern Illinois university
2. 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
3. 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)
4. • 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
6. 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
8. Reagents: Rink amide resin and Fmoc
A) Rink amide resin
Substitution density:
0.5mmol/g
B) The Fluorenylmethyloxycarbonyl
Group
Removed in de-protection of
2- amine in
20%piperidine/Dimethylformamide(DMF)• Starting resin and all amino
acids utilized N-Fmoc protected
9. The Intermittent Ninhydrin (Kaiser) Test
Coupling
De-
protection
Side chain
protection
Side chain
protection
NH-FMOC
NH2
Free amino
group
Carbamate
group
Ruhemanns Blue
10. 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
de- protection reactions
are washed away by
vacuum filtration
SPPS: A Series Of De-protection And Coupling Steps
11. • The first amino acid Fmoc-Ser(OtBu)-OH coupled using
HBTU/HOBt reagents
• Further amino acid couplings utilized PyBOP
as the minimum time required for each coupling
was reduced from 4 hours to 1 hour
• Coupling efficiencies are approximately equal
for these two reagents, yet kinetics quite
diffferent
HBTU/HOBt amide bond formation
In DMF with Diisopropylethylamide (DIPEA)
as base
PyBOP amide bond formation
In DMF w/ DIPEA
Synthesis of Linear INGAP-P Analog
14. 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
15. 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
16. Selective removal of Dde/ODmab de-
protects Lysine/Glutamic Acid
Coupling
Selective Deprotection And Cyclization Before Final Cleavage
18. On- resin linear INGAP-P analog
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
19. 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
20. 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
21. 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
22. 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
23. 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
25. 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 Da
Ac-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)
26. 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 Da
Ac-I-G-L-H-H-D-P-S-H-G-T-L-P-N-G-S-NH2
27. UV Spectrum of 1250µg/mL LIP Standard in
H2O/0.1%TFA
UV Detection Wavelength
Selection
28. 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 detections
BOTTOM: Plot of Area vs Wavelength
detection
UV Detection Wavelength Selection
220nm
215nm
232nm
29. 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
30. 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
31. 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
32. 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 injection
Gradient 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
33. Gradient Mode Considerations
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
34. Gradient Mode Considerations
No Injection Run- Baseline Extrapolation
As 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
35. RP-HPLC Parameters For Lip-St Peptide
Instrumentation: 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%TFA
Gradient rate: 1%B/min
Gradient time table:
Time
(minute)
% B
0 15
3 15
18 30
21 30
24 15
30 15
Flowrate: 0.8mL/min
36. Linearity Determination
• Two consecutive injections of each
concentration (µg/mL )
15, 25, 50, 100, 250
500,750,1000, 1250,
1500,2000, and 2250
• Blank runs between 500-750 and
1250-1500 µg/mL to assure cleanout
of previous runs
37. 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)
38. 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
39. Overlay of Degradation Progression
of 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
40. 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
41. 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
42. Extracted, Lyophilized Sample Containing INGAP-P
250µg/mL INGAP-P fraction
Solvent A: H2O/0.1% TFA,
Solvent B: ACN/0.085% TFA
Gradient: 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
43. 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
44. 300-mAU 50-mAU
Increasing Resolution of HPLC
Changing Capacity Factor By Decreasing Gradient Rate
The Effect of Decreasing %B/min on Resolution
Sample: 250 µg/mL lyophilized fraction
Solvent A: H20/0.1%TFA; Solvent B: ACN/0.085%TFA
45. Changinging α Selectivity To Increase Resolution
Effect of Doubling TFA in
Mobile Phases on Resolution
250µg/mL INGAP-P fraction
Solvent A: H2O/ 0.2%TFA
Solvent B: ACN/ 0.17%TFA
Gradient rate: 1%B/min
200-mAU
MP Composition- Increasing TFA
250µg/mL INGAP-P fraction
Solvent A: H2O/0.1% TFA,
Solvent B: ACN/0.085% TFA
Gradient: 1%B/min
Flowrate: 0.8mL/min
48. 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
49. Analytical parameters
2250 µg/mL linear INGAP-P analog crude mixture
Column: Phenomenex C-18 250x4.4mm 5µ 100 Å
Solvent A: H2O/0.1%TFA Solvent B: ACN/0.085%TFA
Flowrate: 1mL/min
Gradient rate: 1%B/min
10%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
50. 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
51. Scheme for extracting INGAP-P
from lyophilized extract through
overloading
of an analytical column
(overload trace not to scale)
Scheme For Purification
52. 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.
53. 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.
54. Acknowledgements
Jing Su, Ph.D.
Professor, Department of
Chemistry
Northeastern Illinois University
S. John Albazi, Ph.D.
Professor and Chair
Department of Chemistry
Northeastern 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
56. 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
On- resin linear INGAP-P analog
57. 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
58. Acetylation of resin-bound peptides
Resin-bound peptides acetylated with acetic
anhydride/ DIPEA in DMF
Acetylation
INGAP-P
Linear INGAP-P analog
59. 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)
60. 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
