1. SYNTHESIS, PURIFICATION AND STABILITY STUDY OF ISLET NEOGENESIS-
ASSOCIATED PROTEIN- PEPTIDE AND ANALOGS
A Thesis Presented to
The Faculty of the Department of Chemistry
Northeastern Illinois University
In Partial Fulfillment
Of the Requirements of the Degree
Masters in Science
In Chemistry
By Andrew Apals
May, 2016
2. ii
Abstract of Thesis Presented to the Graduate School
Of Northeastern Illinois University
In Partial Fulfillment of the Requirements for the
Master’s Degree in Science
SYNTHESIS, PURIFICATION AND STABILITY STUDY OF ISLET NEOGENESIS-
ASSOCIATED PROTEIN- PEPTIDE AND ANALOGS
By
Andrew Apals
May 2016
Research Advisor: Dr. Jing Su
Master of Science: Chemistry
The pentadecapeptide fragment IGLHDPSHGTLPNGS of the islet neogenesis-
associated protein (INGAP-P) has displayed itself as a promising therapeutic agent for the
treatment of diabetes. Studies have shown this peptide to initiate islet neogenesis and to increase
β- cell mass in animal models, including human. In this study, the INGAP-P and its linear and
cyclic analog have been synthesized following the protocols of fluorenylmethyloxycarbonyl-
solid phase peptide synthesis (Fmoc-SPPS). Preliminary determination of the linear and cyclic
analogs in the final SPPS mixture has been determined by utilizing matrix- assisted laser
desorption ionization with time-of-flight mass spectrometry (MALDI-TOF-MS). Preliminary
identification of the INGAP-P in the SPPS mixture has been determined utilizing electrospray
ionization mass spectrometry (ESI-MS).
Also in this study, the parameters of reverse phase- high performance liquid
chromatography with UV detection have been studied as to separate and analyze the components
of the respective SPPS product mixtures. The linear range of quantitation has been determined
3. iii
for a purified INGAP-P analog standard. It was found to be in the 15 to 1000 µg/mL range. The
limit of quantitation (LOQ) was found to be 7µg/mL, with a working limit of detection (LOD)
found to be 3.65µg/mL. A percent coefficient of variance was found to be 0.298 for the injection
precision of a 500µg/mL concentration.
Further, a study into the degradation of the INGAP-P analog in its acidic solvent has also
been conducted, verifying that degradation at room temperature in the auto- sampler causes
relatively rapid degradation of the peptide. In a study to increase the resolution of the
synthesized INGAP-P from a co- eluate peptide of +101 Da in mass, it has been found that a
change in column type from an octyldecyl- stationary phase to that of a phenyl- stationary phase
is the parameter most effecting such an increase in resolution. Finally, a potential for using an
analytical HPLC instrument for the fractionalization and purification of a SPPS product mixture
has been displayed in this study.
4. iv
ACKNOWLEDGEMENTS
I am grateful to my research advisor, Dr. Jing Su, for her support and scientific guidance
and belief in my capabilities. I would like to thank Dr. Sargon Al- Bazi, Dean of the Department
of Chemistry and Master’s Program, for all his most valuable experience and knowledge that he
related through his HPLC courses. I would also like to thank Niroshi Meegoda, M. S.,
Laboratory Manager, for all of her help in acquiring the needed glassware, solvents and reagents,
and other apparatus needed for my project.
I would like to also thank the colleagues of Dr. Su at Northwestern University and the
Simpson Querrey Institute for their part in obtaining the mass spectrometry data of my peptide
product mixture samples with their respective instrumentation.
I am the most grateful to my wife Ella and daughter Nicole for supporting me all these
years as I obtained my Associate, Bachelor, and then Master Degrees. It most undoubtingly was
a sacrifice on their part, as I was missing in body and spirit the duration of these years as I
worked on my goal of becoming a chemist.
Lastly, I would like to thank my peers in the Master’s Program. Most notably, Sandra
Neri, Rafal Turek, Martin Shlaymoon and Matilda McFarland for our discussions on the HPLC
instrumentation, among other things.
5. v
TABLE OF CONTENTS
Page
ABSTRACT.................................................................................................................................... ii
ACKNOWLEDGEMENT............................................................................................................. iv
LIST OF FIGURES .........................................................................................................................x
LIST OF TABLES....................................................................................................................... xiii
ABBREVIATIONS ..................................................................................................................... xiv
CHAPTER 1 INTRODUCTION.....................................................................................................1
1.1 Background of Study .................................................................................................................1
1.1.1 Islet Neogenesis- Associated Protein and its Active Pentadecapeptide Fragment ...........1
1.1.2 Solid Phase Peptide Synthesis ..........................................................................................3
1.1.3 Mass Spectrometry Characterization of Peptides .............................................................6
1.1.3.1 Matrix- Assisted Desorption Ionization MS Analysis..........................................6
1.1.3.2 Electrospray Ionization MS Analysis ...................................................................8
1.1.4 Reverse Phase-High Performance Liquid Chromatography..............................................9
1.2 Objectives of Research ............................................................................................................10
1.2.1 Synthesize INGAP-P and Analogs ..................................................................................11
1.2.2 RP- HPLC Method Development ....................................................................................11
CHAPTER 2 SYNTHESIS OF INGAP PEPTIDE AND LINEAR AND CYCLIC ANALOGS
WITH PRELIMINARY IDENTIFICATION BY MS ..................................................................13
2.1 Introduction..............................................................................................................................13
2.1.1 Rink Amide Resin and Fmoc- Protection........................................................................13
2.1.2 Side- Chain Protecting Groups ........................................................................................13
6. vi
TABLE OF CONTENTS (Continued)
Page
2.1.3 Coupling Reagents...........................................................................................................15
2.1.4 Ninhydrin Test for Free Amino Groups............................................................................17
2.2 Experimental Section...............................................................................................................17
2.2.1 Chemicals and Reagents ..................................................................................................17
2.2.2 Instrumentation ................................................................................................................18
2.2.3 Synthesis Protocol............................................................................................................19
2.2.3.1 Synthesis of Linear Analog..................................................................................19
2.2.3.2 Synthesis of INGAP-P.........................................................................................21
2.2.3.3 Acetylation of Resin- Bound Peptides.................................................................21
2.2.3.4 Cyclization of INGAP Linear Analog .................................................................21
2.2.3.5 Cleavage of Peptides............................................................................................22
2.2.4 Mass Spectrometry Analysis.............................................................................................22
2.2.4.1 MALDI- TOF MS Analysis of the Linear and Cyclic Analogs of INGAP.........22
2.2.4.1.1 Analysis of the Linear INGAP Analog Product Mixture.....................22
2.2.4.1.2 Analysis of the Cyclic INGAP Analog Product Mixture.....................23
2.2.4.2 ESI MS Analysis of the INGAP-P Product Mixture ............................................24
2.3 Conclusion ...............................................................................................................................26
CHAPTER 3 HPLC METHOD DEVELOPMENT AND PARTIAL VALIDATION.................27
3.1 Introduction..............................................................................................................................27
3.1.1 Choice of Organic Modifier.............................................................................................27
3.1.2 Choice of Column and Gradient Mode............................................................................27
7. vii
TABLE OF CONTENTS (Continued)
Page
3.1.3 Use of TFA as an Ion- Pairing Reagent...........................................................................28
3.2 Experimental Section...............................................................................................................28
3.2.1 Chemicals and Reagents ..................................................................................................28
3.2.2 Instrumentation ................................................................................................................28
3.2.3 Method Development.......................................................................................................30
3.2.3.1 Peptide Standard Solubility...................................................................................30
3.2.3.2 UV Absorbance Selection.....................................................................................30
3.2.3.3 Sample Preparation...............................................................................................32
3.2.3.4 Mobile Phase Preparation .....................................................................................32
3.2.3.5 Column Testing.....................................................................................................32
3.2.3.6 Initial Gradient Parameters and Considerations ...................................................33
3.2.3.6.1 First Gradient Run to High Composition Organic Modifier..................33
3.2.3.6.2 Gradient Rate .........................................................................................34
3.2.3.6.3 Gradient Time Table..............................................................................34
3.2.3.7 Equilibration of Column .......................................................................................37
3.2.3.8 Injection Volume ..................................................................................................37
3.2.3.9 Flowrate ................................................................................................................38
3.2.4 Method Validation ...........................................................................................................38
3.2.4.1 HPLC Instrumentation and Parameters ................................................................39
3.2.4.2 Linearity................................................................................................................39
3.2.4.2.1 Preparation of Concentrations................................................................39
8. viii
TABLE OF CONTENTS (Continued)
Page
3.2.4.2.2 Linearity Determination.........................................................................40
3.2.4.3 LOQ/ LOD Determination....................................................................................41
3.2.4.4 Injection Precision ................................................................................................41
3.3 Conclusion ...............................................................................................................................42
CHAPTER 4 ROOM TEMPERATURE DEGRADATION STUDY
IN ACIDIC SOLVENT ................................................................................................................43
4.1 Room Temperature Degradation Experiment of 500 µg/mL Concentration...........................43
4.2 Examination of 100 µg/mL Concentration Degradation .........................................................45
4.3 Discussion and Conclusion......................................................................................................46
CHAPTER 5 RESOLUTION OF INGAP-P FRACTION ............................................................48
5.1 Effect of Decreasing %B/min on Resolution...........................................................................49
5.2 Effect of Increased %TFA in MP on Resolution.....................................................................49
5.3 Effect of Organic Modifier on Resolution...............................................................................49
5.3.1 IPA..................................................................................................................................50
5.3.2 Methanol .........................................................................................................................50
5.4 Effect of Column Type on Resolution.....................................................................................51
5.5 Conclusion ...............................................................................................................................53
CHAPTER 6 FRACTIALIZATION UTILIZING AN ANALYTICAL INSTRUMENT ............54
6.1 Scheme for Purifying SPPS Products ......................................................................................54
6.2 Conclusion ...............................................................................................................................57
CHAPTER 7 CONCLUSION AND FUTURE DIRECTIONS.....................................................59
9. ix
TABLE OF CONTENTS (Continued)
LIST OF REFFERENCES.............................................................................................................61
10. x
LIST OF FIGURES
Figure Page
1-1 Chemical structure of INGAP-P................................................................................................2
1-2 Typical SPPS vessel on shaker..................................................................................................3
1-3 Generalized SPPS scheme of a single residue coupling............................................................4
1-4 MADI- TOF...............................................................................................................................7
1-5 Electrospray ionization..............................................................................................................8
1-6 Diagram of HPLC instrument....................................................................................................9
1-7 INGAP-P and linear and cyclic analogs..................................................................................11
2-1 Rink amide resin and Fmoc group ..........................................................................................13
2-2 Fmoc removal by piperidine....................................................................................................14
2-3 Trt- and tBu- protecting group structures................................................................................14
2-4 Dde- and oDMb- protecting group structures..........................................................................15
2-5 Removal of Dde- and oDMb- protection by hydrazine...........................................................15
2-6 HBTU/HOBt coupling mechanism .........................................................................................16
2-7 PyBOP coupling mechanism...................................................................................................16
2-8 DIC/ HOAt coupling mechanism ............................................................................................17
2-9 Ninhydrin test: mechanism of Ruhemanns blue evolution from reaction with a free amine..18
2-10 Ninhydrin test for free amines...............................................................................................18
2-11 Abbreviated structures of INGAP-P and linear and cyclic analogs ......................................19
2-12 MALDI-TOF MS of linear INGAP-P analog........................................................................23
2-13 MALDI-TOF MS of cyclic INGAP-P analog.......................................................................24
2-14 Cyclization of linear analog...................................................................................................25
11. xi
LIST OF FIGURES (Continued)
Figure Page
2-15 ESI MS plot window report of extracted INGAP-P fraction.................................................25
3-1 UV spectrum of 1250 µg/mL LIP standard in H2O/0.1%TFA...............................................30
3-2 UV absorbance comparison at 220,215, and 232 nm..............................................................31
3-3 Column inefficiencies..............................................................................................................33
3-4 Phenomenex C-18, 3.2X 250mm, 5micron, 100 pore size......................................................33
3-5 Exemplification of the initial gradient.....................................................................................34
3-6 Change of gradient rate with increase in resolution 1000µg/mL LIP standard.......................35
3-7 LIP standard vs crude linear analog product mixture..............................................................36
3-8 No injection run- baseline extrapolation .................................................................................37
3-9 Linearity determination utilizing two concentration point scattered plot................................41
4-1 Overlay of degradation progression of 500µg/mL sample......................................................44
4-2 Degradation of 500µg/mL over time.......................................................................................44
4-3 Overlay of degradation progression of 100µg/mL sample......................................................45
4-4 Degradation of 100µg/mL over time.......................................................................................46
4-5 Formation of possible degradation product iso- aspartate analog...........................................47
5-1 A) Comparison of SPPS INGAP-P crude product mixture and lyophilized INGAP-P extract
B) Report image of lyophilized extract....................................................................................48
5-2 The effect of decreasing %B/min on resolution ......................................................................49
5-3 Effect of doubling TFA in mobile phase on resolution...........................................................50
5-4 Effect of IPA as an organic modifier on resolution.................................................................51
5-5 Effect of methanol as an organic modifier on resolution ........................................................51
12. xii
LIST OF Figures (Continued)
Figure Page
5-6 (A) Change in column type:3 Phenyl Column Gradient: 1%B/min, flow rate: 1mL/min
(B) Change in column type:3 Phenyl Column Gradient: 1%B/min, flow rate: 0.75mL/min ..52
6-1 Fractionalization of linear INGAP analog crude SPPS mixture
on an analytical instrument............................................................................................................55
6-2 Comparison of collected fraction to crude peptide mixture ....................................................56
6-3 Scheme for extracting purified INGAP-P from lyophilized extract by fractionalization
of an overloaded analytical column...............................................................................................57
13. xiii
LIST OF TABLES
TABLE Page
3-1 PREPARATION OF LIP- ST SAMPLES FOR HPLC...........................................................40
3-2 INJECTION PRECISION STATISTICAL VALUES FOR 500µg/mL SAMPLE
INJECTIONS.................................................................................................................................42
16. 1
CHAPTER 1 INTRODUCTION
1.1 Background of Study
1.1.1 Islet Neogenesis- Associated Protein and its Active Pentadecapeptide Fragment
The islet neogenesis- associated protein (INGAP) is a 16.8 kDa protein found expressed
in different subsectors of the pancreas, including islet, duct and exocrine cells (1). The protein is
a member of a regulatory protein subfamily of secreted C- Type lectins, subdivisions based on
the primary sequence, with INGAP belonging to the large Reg 3 subfamily (2), found in
predominantly the pancreas, stomach, and liver of rats, mice, hamsters, and humans (3).
Expression of INGAP has been implicated in the acute- phase response as its expression is
influenced by inflammatory cytokines and bacterial infection (4).
Promising in vivo results in different animal models makes it clear that INGAP plays a
role in generating newly, physiologically functional pancreatic islets. INGAP has been found to
be sufficient in increasing β- cell mass in normal animals and reverse diabetes in streptozotocin-
induced diabetic animals (5). Overexpression of INGAP in transgenic mice renders them
resistant to the induction of diabetes (4).
A pentadecapeptide fragment of INGAP (aa 104-118, chemical structure displayed in Figure 1-
1), called the INGAP peptide (INGAP-P, commercially known as Exsulin), has been found to be
as effective as INGAP in initiating new islet formation and reversing streptozotocin- induced
diabetes in hamsters and mice (6). INGAP-P has been studied extensively in both in vitro and in
vivo studies. The most compelling data displayed by INGAP-P is as follows: i) induces in vitro
regeneration of human islets from dedifferentiated islet- derived, duct- like structures ii) dose
17. 2
dependently stimulates β- cell mass in rodents, dogs, and cynomolgus monkeys iii) increases
insulin secretion and β-cell size and upregulates the expression of genes related to β- cell
function in rat neonatal islets in vitro (4).
These positive results have led to clinical trials investigating its efficacy and safety in
humans. In these trials, INGAP-P was found to improve glucose homeostasis in patients with
type-2 diabetes and to display a significant increase in C- peptide secretion a measure of
endogenous insulin secretion, in patients with type-1 diabetes (7,8).
The previously mentioned data points to INGAP-P as a promising treatment of diabetes
as it exhibits both islet- neogenesis and insulinotropic activities. Yet, it has a relatively short
plasma half- life; and the need for higher dose administration of INGAP-P calls for more
prioritized studies into its mechanism of action, which, like its mother protein INGAP, is still
unknown (7).
Such characterization studies into INGAP-P’s mechanism of activity would call for
quantities of INGAP-P to be readily at hand in the lab. Further, if analogs of INGAP-P are
needed in activity and structural studies, an in- house, synthesis method would be most
advantageous. Solid phase peptide synthesis offers such a method.
Figure 1-1 Chemical structure of INGAP-P
Commercially known as Exsulin
18. 3
1.1.2 Solid Phase Peptide Synthesis
In solid phase peptide synthesis (SPPS), a peptide is constructed from its C- terminal that
has been chemically bonded, or coupled through amide bond formation, to an insoluble support
of bead- like resin. A main advantage of SPPS is that separation of the growing peptide from
soluble reagents and solvents can be accomplished by simple washing and filtration using a glass
fritted vessel with stopcock (Figure 1-2). With this in mind, excess reagents can be employed to
drive coupling reactions to completion with a minimum of loss as the peptide remains bonded to
the support throughout the synthesis (9).
In a generalized scheme of SPPS (Figure 1-3), the C- terminal of the first amino acid is
coupled via amide bond formation to a free amino that is terminal on a linker of the resin solid
support. All reactive amino acid side- chains must have protecting groups that are not affected by
the reaction conditions of peptide assembly. By contrast, a temporary protecting group is
employed on the α- amino of the amino acid that is being coupled. After linkage of the first
amino acid to the resin is confirmed, the α- amino protecting group is removed under basic
Figure 1-2 Typical SPPS vessel on shaker
19. 4
conditions. Consequently, the next side- chain, α- amino protected amino acid of the sequence is
coupled to the first on- resin amino acid. A series of de-protection and coupling steps are carried
out until the peptide has been completed at the N- terminus, after which a cleavage cocktail is
employed to remove the peptide from resin as well as remove all side- chain protecting groups
(9).
The side chain- protecting groups of the amino acids can be termed as relatively
permanent groups, contrasting with the temporary groups that are protecting the α- amino. G.
Barany and R. B. Merrifield, pioneers of SPPS, would term these groups as being “orthogonal”,
which they designate as, “…classes of protecting groups which are removed by differing
chemical mechanisms. Therefore, they can be removed in any order and in the presence of the
other classes. Orthogonal protection schemes allow milder overall reaction conditions as well as
the synthesis of partially protected peptides (10)”. The Fmoc/tBu
Figure 1-3 Generalized SPPS scheme of a single residue coupling
For each coupling, the following steps are followed:
1)Fmoc removal from resin/last coupled amino acid
2)Coupling of next amino acid
20. 5
(Fluorenylmethyloxycarbonyl/tert- butyl) SPPS scheme can be called truly orthogonal, as the
Fmoc protection on the α- amino of the amino acids as well as starting resin are base labile. This
contrasts with the tBu, as well as trityl (triphenylmethyl, Trt), protection of the side chains,
which are acid labile (11). In practice, tBu as well as Trt protection and alkoxylbenzyl- based
linkers are used as they can be removed by trifluoroacetic acid (TFA). The advantage of using
TFA is that it is an excellent solvent for peptides, and can be used in standard laboratory
glassware, and since it is volatile is easily removed by evaporation. For these reasons, the
Fmoc/tBu scheme is a widely popular peptide synthesis route (9).
In most cases the product is a mixture of peptides. This includes the target peptide along
with other products of synthesis, including peptides with amino acid additions and omissions, as
well as peptides derived from side reactions. Additions may be caused by technician error where
de- protection and coupling steps are not adequately logged. Omissions may be caused by
incomplete de- protection, which has been demonstrated to be linked to slow or incomplete
couplings (12). Side reactions include the following: diketopiperazine formation (the de-
protected free amino of second coupled amino acid displaces the oxyl- group of the ester bond on
the linker, forming a cyclic product cleaved from resin), aspartimide formation (a nitrogen of the
amide backbone displaces the OtBu- protection on an adjacent aspartic acid forming aspartimide
with accompanying epimerization during the cyclic reopening with subsequent de-protection
steps yielding piperides), and guanylation (induced by use of uranium coupling reagents) (13,14)
Purity and complexity of peptide products can be examined preliminarily by mass spectrometry
(MS).
21. 6
1.1.3 Mass Spectrometry Characterization of Peptides
Mass spectrometry measures the intrinsic property of a molecule, that of its mass, with
very high sensitivity. The instrument can be divided into two parts: the ionization
instrumentation/ technique and the mass analyzer. Originally, MS required charged, gaseous
molecules for analysis. Biomolecules, because they are large and polar(peptides) and not easily
transferred into the gas phase and ionized, were subjected to limited analysis. MS analysis of
biomolecules came on a much larger scale in the 1990’s when new ionization techniques had
been developed. Most notably, matrix- assisted laser desorption ionization- time- of- flight
(MALDI-TOF) mass spectrometry as well as liquid chromatography- electrospray ionization
(HPLC-ESI) became highly utilized in the biosciences (15), and has been utilized in this study
for the preliminary determination of the target peptide in the SPPS product mixture.
1.1.3.1 Matrix- Assisted Desorption Ionization MS Analysis
Matrix- assisted desorption ionization (MALDI) is considered one of the soft ionization
techniques in mass spectrometry (Figure 1-4). This is because most ionization techniques in MS
fragment the molecular ion, the norm yielding a peak intensity that is a fraction of a more intense
peak of a fragment. By contrast, MALDI generally yields a peak for the molecular ion of the
greatest intensity in the scan. An advantage of this is that a sample of peptides can be introduced
and a single spectral scan can display all the molecular ion peaks of each peptide in the mixture.
The MALDI technique, in the gas phase, generates protonated molecules through the
following protocol: A large excess of matrix material is co- precipitated with the analyte
molecules by pipetting a sub microliter volume of the test mixture onto a metal substrate and
allowed to dry. The dried mixture is then irradiated by nanosecond laser pulses, usually from a
small nitrogen laser with a wavelength of 337nm. The matrix is typically a small organic
22. 7
molecule with absorbance at the wavelength of the laser employed. For biomolecules, α- cyano-
4- hydroxycinnnamic acid or 2,5- dihydrobenzoic acid is usually used as the matrix material. The
laser pulses then desorbs the matrix and analyte molecules, yielding protonated analyte
molecules that are then introduced into a mass analyzer, commonly a time of flight (TOF) mass
analyzer. This process yields a mass spectrum of protonated molecular ions + 1[(M+H)+
], which
can reflect components in a sample mixture (15).
Figure 1-4 MALDI- TOF
A) Matrix-Assisted Laser Desorption Ionization
B) Time- Of- Flight Mass Analyzer
23. 8
1.1.3.2 Electrospray Ionization Analysis
Electrospray ionization (Figure 1-5) is another soft ionization technique in MS that, for a
given molecule, produces a signal strength, or peak height, that increases linearly with analyte
concentration. In this ionization technique, a liquid that contains the analyte is pumped through a
hypodermic needle at low microliter- per- minute flow rates. This needle is at a high voltage, and
causes the eluting liquid to electrostatically disperse (electrospray) small, micrometer sized
droplets which rapidly evaporate, with a charge imparted upon the analyte molecules. This
technique takes place at atmospheric pressure and thus is a soft technique without fragmentation
of analyte ions in the gas phase. The analyte ions are then directed into mass spectrometer of
high efficiency, such as a quadrupole mass analyzer (15).
Because the ESI technique takes place at atmospheric pressure, originates from a flow of
mobile liquid, and yields peak intensities linearly related to analyte concentration, it is adaptable
as a detection technique for eluting components of high performance liquid chromatography
(HPLC), used routinely to separate components of a mixture. HPLC is an instrument that
separates components of a sample mixture with great efficiency. HPLC combined with UV/Vis
detection is the more commonly found instrument in laboratories. Consequently, developing a
Figure 1-5 Electrospray ionization
24. 9
method of separating and analyzing components of a SPPS mixture would be most valuable in
the peptide synthesis process. In addition, studying the parameters involved in a HPLC-UV
method of peptide mixture separation makes it possible to adjust the method to fit other studies
such as degradation and standard impurity studies.
1.1.4 Reverse Phase-High Performance Liquid Chromatography
HPLC has the capability to resolve and quantify components of a reaction mixture, as
well as that of a biological matrices utilizing UV detection (17). Figure 1-6 displays a diagram of
the instrument. Microliters of solvated sample are injected into a continuously flowing mobile
phase (MP) of solvent. The MP carries the sample components to the top of a column, packed
with particles that are coated with a chemically bonded stationary phase, this stationary phase
being hydrophobic. Upon entering the column, the sample components, at first traveling as a
Figure 1-6 Diagram of HPLC instrument
25. 10
single band, begin to differentiate in speed through the column based on their respective
hydrophobicity. The components with more hydrophobicity start to partition into the stationary
phase more than components of lesser hydrophobicity, and thus will spend more time on the
column and as such, the components elute out of the column at different times, now traveling as
differentiated bands to the UV detector flow cell. Here, absorbance at a particular wavelength is
registered while interface with a computer program yields a scan of UV absorbance vs time,
where ideally each component yields peak at a characteristic time, otherwise known as its
retention time. The heights or areas of the respective peaks can be correlated to the individual
component’s concentration, given that this concentration is in the analyte’s linear range of UV
absorbance (18). The resolution (separation efficiency) of a mixture of components such as
peptides is influenced by factors such as stationary phase, size of column, particle size, carbon
loading, mobile phase, buffer concentration, and flow rate. Most peptide separations are run
under a gradient change of organic solvent composition over time. The change in organic solvent
percentage over time can also be utilized in resolution of peaks (19). Reverse phase- high
performance liquid chromatography (RP-HPLC) is widely used in the separation and analysis of
proteins and peptides. It has the capability of separating peptides that differ by a single amino
acid residue (16).
1.2 Objectives of Research
The objectives of this research have been guided by the need to study the biological
properties of INGAP-P and analogs at Northeastern Illinois University. It has entailed the
synthesis of INGAP- P and two analogs, as well as the studying of parameters involved in RP-
HPLC method development for peptide separations using RP- HPLC with UV detection.
26. 11
1.2.1 Synthesize INGAP-P and Analogs
One of the objectives of this research was to synthesize INGAP-P, linear and a cyclic
analog (Figure 1-7) using the protocols of Fmoc SPPS. To preliminarily identify the target
peptide in the SPPS product, samples for MALDI- TOF and ESI evaluation were outsourced.
1.2.2 RP- HPLC Method Development
The development of a RP- HPLC method for the separation and analysis of peptides is a
first at Northeastern Illinois University. The primary objective of this study has been to learn the
Figure 1-7 INGAP-P and linear and cyclic analogs
27. 12
HPLC parameters involved in the evolution of a method, and how to adapt these parameters to fit
a fixed need, whether it be calibration of a standard, analysis of a reaction mixture or degradation
products, or increasing the resolution of peaks. The expectation in this work is to contribute a
data base of new information that will assist in future utilization of RP- HPLC at NEIU as a tool
for the analysis of peptides, whether it be quantitation of a target peptide in a SPPS reaction
mixture, in enzyme- degradation studies, or even in studies involved in cellular research.
28. 13
CHAPTER 2 SYNTHESIS OF INGAP PEPTIDE AND LINEAR AND CYCLIC
ANALOGS WITH PRELIMINARY IDENTIFICATION BY MS
2.1 Introduction
2.1.1 Rink Amide Resin and Fmoc- Protection
The resin used was the rink amide resin (Figure 2-1). This resin has a predetermined
density of amino groups, 0.5mmol/g (substitution of resin), and therefore maximum yield of a
target peptide synthesized on- resin can be predetermined. 2- amine groups of the amino acids
and starting resin, were N- Fmoc protected. The Fmoc group can be removed by the base
piperidine (Figure 2-2) (20).
2.1.2 Side- Chain Protecting Groups
In the synthesis of INGAP- P and its linear and cyclic analogs, the trityl group (Trt) was
used as side- chain protection for the coupled asparagine and histidine residues. The tert- butyl
groupl (tBu) was used to protect the residues with hydroxyl and carboxylic side- chains (Figure
2-3). Both of these groups are cleaved in the acidic conditions of the final TFA cocktail (9).
For the intermolecular cyclization of the linear INGAP analog, lysine and glutamic acid
residues containing the protecting groups - Dde and - ODmab, respectively (Figure 2-4), were
incorporated into the peptide. These protecting groups can be cleaved selectively while other side
chain protecting groups remain intact, with coupling of the internal lysine and glutamic acid
Figure 2-1 (A) Rink amide Resin (B) Fmoc group
29. 14
before the final acidic cleavage. Both Dde and ODmab are cleaved through hydrazinolysis that is
Figure 2-2 Fmoc removal by piperidine
Figure 2-3 Trt- and tBu- protecting group structures
Trt- protecting group for histidine residues
tBu- protection groups for acid and hydroxyl residues
30. 15
followed by elimination of the p- aminobenzyl group by a 1, 6 electron shift (Figure 2-5) (21).
2.1.3 Coupling Reagents
Three different coupling reagents were utilized at different points in the synthesis.
HBTU/ HOBt (Figure 2-6) coupling reagent pair was used to couple the first amino acid to the
resin for the linear/cyclic peptide. Consequently, all further amino acid couplings were
accomplished using PyBOP, which reduces the minimal time needed for each coupling (Figure
2-7). For the cyclization of the linear analog, the DIC/ HOAt coupling reagent pair was used
(Figure 2-8).
Figure 2-4 Dde- and oDMb- protecting group structures
Fmoc-Lys-Dde and Fmoc-Glu-ODmab
Utilized for internal cyclization as these can
Be selectively removed by hydrazine
Figure 2-5 Removal of ODmab- protection
from glutamic acid by hydrazine
Dde- is removed in similar fashion from lysine
31. 16
HOAt and HOBt are considered additives, also referred to as auxiliary nucleophiles.
These compounds are found to reduce racemization, and sometimes increase coupling efficiency.
PyBOP is also known to decrease racemization and increase coupling efficiency. In Figure 2.7,
the initial activation of the carboxylic acid evolves a molecule of (-) OBt, which in turn acts as
an auxiliary nucleophile (22,23,24).
Figure 2-6 HBTU/HOBt coupling mechanism
Figure 2-7 PyBOP coupling mechanism
32. 17
2.1.4 Ninhydrin Test for Free Amino Groups
The following steps were followed for each residue coupling starting with the Fmoc-
protected resin: [(1) De-protection (resin/ 2- amine of amino acids)→ (2) evaluation of free
amino group → (3) amide bond formation→ (4) evaluation of coupling]ⁿ, where “n” is the
number of residues in the peptide. The ninhydrin test is often used in SPPS to evaluate free
amino groups in solution. In the presence of free amino groups, the ninhydrin molecule reacts
with the amino group, yielding the fluorophore Ruhemanns Blue (Figure 2-9). As such, the
intense blue of a qualifying ninhydrin test positive is desired after each de- protection step. A test
negative/ no color change is alternatively the desired result after each coupling, indicative of
saturation of any free amino groups by the previous coupling step (Figure 2-10) (25).
2.2 Experimental Section
2.2.1 Chemicals and Reagents
All Fmoc, side- chain protected amino acids and coupling reagents were obtained from
the Anaspec and Millipore chemical companies. All other chemicals and reagents were obtained
from Sigma- Aldrich and Fisher, respectively.
Figure 2-8 DIC/ HOAt coupling mechanism
33. 18
2.2.2 Instrumentation
All MALDI- TOF mass spectra were acquired from Northwestern University (NU) via
my mentoring professor, Dr. Jing Su. The ESI spectra were received also from NU at the
Figure 2-9 Ninhydrin test: mechanism of Ruhemanns blue
evolution from reaction with a free amine
Figure 2-10 Ninhydrin test for free amines
TOP: Test Positive BOTTOM: Test Negative
34. 19
Simpson Querrey Institute, which utilizes the Agilent 6520 quadrupole- time- of- flight mass
spectrometer (Q-TOF) (26).
2.2.3 Synthesis Protocol
The abbreviated structures of INGAP-P and linear and cyclic analogs synthesized in this
study are displayed in Figure 2-11.
2.2.3.1 Synthesis of Linear Analog
300mg of Rink amide resin was used in order to obtain 0.15 mmols of desired product
peptide (amino- group density of resin 0.5mmol/g). The resin was added to a 25mL glass
sintered filter reaction vessel with stopcock, and washed with DMF twice and then allowed to
swell in 10mL DMF for 20 minutes. After draining, 10 mL of 20% piperidine/ DMF was then
added and the vessel shaken for 30 minutes in order to remove the Fmoc- protecting groups. The
solvent was drained and the sample washed four times with 10 mL of DMF, followed by a
Kaiser’s test (ninhydrin test) to confirm the exposure of the free amino groups. The test included
removal of a sample of beads to a test tube and two drops each of the test solutions added,
Figure 2-11 Abbreviated structures of INGAP-P and
linear and cyclic analogs
35. 20
followed by heating the mixture at approximately 100 ºC for about five minutes. A vivid dark
blue color indicates free amino- groups and thus adequate de- protection. The resin was then
coupled to the first amino acid Fmoc- serine(OtBu)-OH. This was accomplished with the
addition of three equivalents of amino acid(0.45mmol) and three equivalents(0.45mmol) of each
of the coupling reagents HBTU and HOBT to the reaction vessel with approximately 6 mL
DMF, with 261 µL (10 eq) DIPEA also added. This solution was shaken for three hours, the
vessel drained and washed with DMF three times, and a resin sample removed and tested with
the ninhydrin test protocol previously stated, with a resulting non- color change indicating a
complete reaction of all the resin amino- sites with the first amino acid.
After coupling of the first amino acid Fmoc- Ser(OtBu)- OH of the linear analog to the
resin, the coupling reagents were changed as to quicken the peptide synthesis. After the first
coupling of serine to the resin, the PyBOP coupling reagent was used in lieu of HBTU/ HOBT in
the rest of the coupling reactions. Three equivalents (0.45 mmol) each of Fmoc- amino acid,
PyBOP (234.18 mg, 0.45mmol) and 78.5 µL DIPEA(0.45mmol) in approximately 6 mL of DMF
(about 2-3 volume of resin) were added to the washed resin, and the vessel shaken for 1- 1.25
hours.
Starting at glycine (8th amino acid in sequence), the Kaiser ninhydrin test displayed
positive results after the first coupling (indicated incomplete coupling with the growing peptide
on the resin), and so from glycine to the last amino acid in the sequence, the coupling reaction
was conducted twice for each amino acid, with a ninhydrin test negative observed after each
double coupling.
36. 21
2.2.3.2 Synthesis of INGAP-P
The resin preparation and PyBOP coupling protocols cited above were also used to
synthesize 0.15 mmols of INGAP-P. Time for the latter half couplings (residues 7- 15) was
extended so as to improve coupling efficiencies. This was depicted in the first- coupling
ninhydrin tests for these latter amino acids, which displayed test negative results (double
couplings were avoided).
2.2.3.3 Acetylation of Resin- Bound Peptides
After coupling and de- protection of the last amino acid, the resin- bound peptides were
acetylated. In a solution of 6 mL DMF, 142.8 µL (10 equivalent) acetic anhydride and 391.8 µL
(15 equivalent) DIPEA, the resin was shaken for an hour and washed with DMF. A final
ninhydrin test indicated complete acetylation of amino- moieties.
2.2.3.4 Cyclization of INGAP Linear Analog
A 0.0375mmol sample of the on- resin, linear INGAP analog was also cyclized. This
was accomplished first by selective removal of the protecting groups on Lys- Dde (coupled
amino acid 3) and Glu- ODmab (last coupled amino acid 16 on the resin- bound peptide),
followed by on- resin lactam formation. This was accomplished by first selectively de- protecting
the Dde- and ODmab- amino acids. The resin was first swelled for four hours in DMF, after
which a solution of 2% v/v hydrazine monohydrate in DMF (twice resin bead) was added and
then agitated for five minutes, washed and then agitated again in the hydrazine solution for seven
minutes. The de- protection agents were removed by suction, and the resin washed with DMF,
followed by washing with 10% DIPEA in DMF. Next, the de- protected peptide was cyclized.
The washed resin was added to a solution of DIC (9.395 µL, 1.5 eq. = 0.05625 mmol) and 66.6
µL of 0.1 M HOAt in DMF (1.0eq, 0.0375 mmol) in DMF (0.6 mL), and then the vessel shaken
37. 22
for 18 hours. The cyclized on- resin peptide was then washed with DMF and DCM and dried
under vacuum for 2 hours prior to cleavage.
2.2.3.5 Cleavage of Peptides
The peptide- bound resin was washed with DCM followed by placement under a vacuum
for 6 hours. Next, the resin was stirred in a cleavage cocktail (TFA/phenol/water/TIPS- 88/5/5/2
by volume) for two hours. This solution was then filtered under a vacuum and collected in a 25
ml RBF. At 40º C, the RBF was attached to a rotovap and 80 % of the TFA removed. The
remainder to the solution was transferred to a 50 ml falcon tube, and five times this volume of
cold diethyl ether was slowly added along the walls of the tube, which precipitated a white solid.
The falcon tube was then centrifuged below 0º C. The ether was then decanted out to give the
crude peptide solid.
2.2.4 Mass Spectrometry Analysis
2.2.4.1 MALDI- TOF MS Analysis of the Linear and Cyclic Analogs of INGAP
Portions of the linear and cyclic analog were given to Dr. Su for MALDI- TOF analysis
at Northwestern University. The following are the results and discussion of each spectrum.
2.2.4.1.1 Analysis of the Linear INGAP Analog Product Mixture
The molecular mass of the linear INGAP analog calculates to 1700.84 Da (27). The
obtained MALDI- TOF mass spectrum is displayed in Figure 2-12. The two most prominent
peaks are at 1701.8 m/z and 1838.8 m/z. The peak at 1701.8 m/z is indicative of the protonated,
linear INGAP analog. The peak at 1838.9 m/z is just as prominent, and may be an amino acid
addition of histidine, as the mass of the histidine residue is 137 Da.
38. 23
2.2.4.1.2 Analysis of the Cyclic INGAP Analog Product Mixture
The molecular mass of the cyclic INGAP analog calculates to 1682.8 Da (mass of linear
analog minus mass of water). Its MALDI- TOF mass spectrum is displayed in Figure 2-13. Its
expected peak in the spectrum as a protonated species is minimal at 1683.8 m/z. The linear
analog nevertheless is displayed prominently at 1701.9 m/z. Also of moderate intensity is the
1838.8 m/z peak found in the linear spectrum.
The intensity of the cyclic analog peak is only a fraction that of the linear analog’s
intensity. It is also worth noting, that beside the fact that the cyclization reagents and reaction
conditions were not optimized, a successful peptide cyclization is, to a point, sequence
dependent. A minimum of cyclized product is not uncommon, due to the spatial orientation of
the peptide backbone and steric hindrance (Figure 2-14) (28).
Figure 2-12 MALDI-TOF MS of linear INGAP-P analog
(MM: 1700.8 amu)
39. 24
2.2.4.2 ESI MS Analysis of the INGAP-P Product Mixture
The full INGAP-P product mixture was taken by Dr. Su to the Simpson Querrey Institute,
Northwestern University, where it was lyophilized. The lyophilized product mixture was
fractionalized by the institute via preparatory HPLC, and the fractions analyzed on HPLC- Q-
TOF- ESI instrumentation. Figure 2-15 displays the received qualitative plot window report. The
Figure 2-13 MALDI-TOF MS of cyclic INGAP-P Analog (MM: 1682.8 amu)
Zoom of 1683.8 displayed at bottom
40. 25
scan displayed is of the fraction that eluted the INGAP-P peptide, whose mass calculates to
1541.7 Da (27).
The mass at 1541.7 m/z is most likely the protonated INGAP-P. The reason the peak is
not at an expected plus 1/ 1542.7 m/z may be to improper calibration of the instrument. The peak
Figure 2-14 Cyclization of linear analog
(rigid amide backbone may constrain a
cyclization coupling
Figure 2-15 ESI MS plot window report of extracted INGAP-P fraction
(MM: 1541.7 amu)
From preparative HPLC
41. 26
at 1642.76 m/z in each of the successive scans indicates that an unexpected product co- eluted
with INGAP-P, whose chromatogram will be seen in the HPLC section. The 1642.76 m/z peak is
101 Da more in mass than that of the INGAP-P peak. It may be due to an additional threonine in
the INGAP-P sequence.
2.3 Conclusion
The analysis of a SPPS product mixture by either MALDI- TOF or ESI has been found to
be adequate for the preliminary determination of the synthetic target in the product mixture.
Further, these analytical methods are advantageous in that they offer insight as to what other
major side- components may be in the mixture. Due to amino acid additions or omissions or
side- reactions during SPPS.
42. 27
CHAPTER 3 HPLC METHOD DEVELOPMENT AND PARTIAL VALIDATION
3.1 Introduction
RP- HPLC is extensively used in the separation and analysis of peptides and proteins
(17). For a method of separation for small peptides such as the INGAP-P and analogs, standard
initial parameters, obtained from reviews and articles (29,30,31), have been utilized. These
standard parameters include mobile phase selection, column type, use of gradient, and the use of
TFA as an ion-pairing reagent.
3.1.1 Choice of Organic Modifier
Acetonitrile(ACN) was used as an organic modifier solvent B, otherwise called a co-
solvent. It is volatile and easily removed from collected fractions. It has low viscosity,
minimalizing back pressure. Further, it has little UV absorbance at low wavelengths, with a
quantitative cutoff of 200nm (amide bond absorbance 205- 220nm, with 215 and 220nm having
been found suitable for detection wavelength throughout the study) (19).
3.1.2 Choice of Column and Gradient Mode
For peptides, a column with a octyldecyl (C-18) bonded stationary phase is typically
employed, and has been used throughout most of this study. Column dimensions are typically
250mm in length and 1- 4.6 mm inner diameter. Particle size is generally 5µm, with pore sizes
100- 150 Å.
It is theorized that the retention of larger peptides (>30 residues) and proteins is
adsorption at the top of the column, where they are retained but one time, with little or no
partitioning. For smaller peptides, it is believed that a combination of adsorption and partitioning
is the mechanism of retention. This theory suggests the use of a gradient mode in HPLC, as the
retention of a peptide is most sensitive to %B organic modifier. Too high %B would elute the
43. 28
component peptides too quickly and the component peptides would elute as one peak without
separation, whereas to low %B would yield peptide peak broadening (19).
3.1.3 Use of TFA as an Ion- Pairing Reagent
TFA is the most common ion- pairing reagent used in the RP- HPLC separation of
proteins and peptides. As a volatile molecule, it is more easily driven off in preparatory fractions.
It also protonates ionizable silanol groups on columns that can cause tailing and unideal peak
shapes. Further, it ion- pairs with the amino groups on peptides in order to increase retention of
otherwise un- retainable molecules. Under standard protocols, 0.1% TFA (v/v) is used in the
aqueous phase solvent A, while a slightly lesser percentage, 0.085%, is used in ACN so as to
decrease baseline drift that accompanies the change in dielectric constant of TFA in the organic
modifier as the percent B increases in gradient mode. These amounts of TFA yield a pH of
around 2 (19). As such, TFA has been employed in all mobile phases utilized in this study.
3.2 Experimental Section
3.2.1 Chemicals and Reagents
All solvents were obtained from Fisher Chemicals. These include HPLC- Grade water,
acetonitrile, isopropanol, and methanol. The LCMS- grade TFA was obtained from Optimal
Chemical.
3.2.2 Instrumentation
• Hitachi U-2910 Spectrophotometer
• 1100 Series HPLC System with MWD (UV/VIS Detector), Agilent Technologies
(HPLC/MWD).
G1311A Quaternary Pump
G1329A Auto sampler
44. 29
G1365B Multi-Wavelength UV/VIS Detector (MWD)
G1322A Degasser
G1330B Thermostat
G1316A Column Compartment
Chemstation data acquisition system for LC, Agilent Technologies
Dell computer with Microsoft XP operating system
• 1100 Series HPLC System with VWD (UV/VIS Detector), Agilent Technologies
(HPLC/VWD).
G1311A Quaternary Pump
G1322A Degasser,
G1329A ALS (Thermostat Autosampler),
G1316A VWD (Variable Wavelength Detector)
Chemstation data acquisition system for LC, Agilent Technologies
Dell computer with Microsoft XP operating system
• 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
45. 30
3.2.3 Method Development
In the testing of different columns for elution efficiency and integrity, a linear INGAP-P
analog standard (LIP-St)1679 m/z (replaced with peptide sequence Ac-IGLHHDPSHGTLPNGS-
NH2) was obtained from Dr. Su, previously synthesized in her lab. It was determined to be of
97% purity. When testing HPLC parameters, the LIP-St along with SPPS product were utilized.
3.2.3.1 Peptide Standard Solubility
The LIP-St has fairly high hydrophobicity and low solubility in water. 1 mL of aqueous
mobile phase (H2O/0.1% TFA) has been used throughout the study as a solvent for the HPLC
samples.
3.2.3.2 UV Absorbance Selection
A UV spectrum was taken of a 1250µg/mL solution of the LIP-St in H2O/0.1%TFA and
its spectrum is displayed in Figure 3-1. This is a typical UV spectrum of peptides not containing
the residues with aromatic side- chains (tyrosine, phenylalanine, and tryptophan) that absorb
light in the 250 to 290nm ultraviolet range. Typically, wavelength absorbance is observed
Figure 3-1 UV spectrum of 1250µg/mL LIP standard
in H2O/0.1%TFA
46. 31
between 205- 220nm, where the amide bond as well as histidine absorbs (17). For comparison,
Figure 3-2 displays the MWD scans at 215, 220, and 232nm for the detection of the LIP
Figure 3-2 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
47. 32
standard. The 215nm chromatogram yields the greatest response with reasonable baseline
fluctuations, and so this wavelength was chosen for detection.
3.2.3.3 Sample Preparation
All samples were weighed out and added to the appropriate volume of solvent (HPLC-
grade water/ 0.1% TFA). The resulting sample solution was vortexed for thorough dissolving,
and filtered through syringe with a 0.2µ pore syringe tip.
3.2.3.4 Mobile Phase Preparation
The aqueous solvent A was prepared in one liter volumes. One liter of water was
measured out using a round bottom volumetric flask. One milliliter was removed and replaced
with one milliliter of LCMS- grade TFA via micropipette, yielding a 0.1% TFA solution (v/v).
Solvent B was prepared by measuring out 0.5 liters of ACN in a volumetric flask and removing
0.425 milliliters of the ACN. This was replaced with 0.425 milliliters of TFA. These respective
solutions were then vacuum filtrated using a 0.2µ filter. These solvents were then degassed in the
sonicator for a minimum of 25 minutes.
3.2.3.5 Column Testing
A series of columns were tested for efficiency and integrity. The factors evaluated were
peak width (in chromatographic terms, N the number of theoretical plates) and peak symmetry
(in particular, peak tailing) of the LIP-St response. Nine different columns were tested. Figure 3-
3 displays the chromatograms for two of the columns tested. The Phenomenex C-18, 4.6 X
250mm, 5µm particle, 125 Å pore size (Phenomenex C-18/4.6mm) column displays “ghost
peaks”. This is indicative of a column that is poorly packed or deteriorated. The Agilent Eclipse
C-18, 4.6 X 250mm, 5µ particle, 100 Å pore size (Agilent C-18) column displays peak tailing,
which decreases theoretical plate number as well as resolution.
48. 33
Figure 3-4 displays the chromatogram of the LIP standard using the Phenomenex C-18,
3.2 X 250mm, 5µ particle, 100 Å pore size (Phenomenex C-18/3.2) column. This column
showed excellent plate number of 47K, along with a tailing factor of 1.3 within the acceptable
range (0.9-2). The Phenomenex C-18/3.2mm column as such displayed the required efficiency,
and so was used as the column of choice later in the study.
3.2.3.6 Initial Gradient Parameters and Considerations
3.2.3.6.1 First Gradient Run to High Composition Organic Modifier
Initially, a sample is run from a low %B to a high %B. In this way, a rough estimate can
be ascertained as to where the peptide(s) of interest elutes, and in this way initial and final %B
can be determined for the gradient time table. Further, it makes sure that no other components,
peptide or impurity, are eluting later after the main peptide(s) of interest, which may remain
retained on the column after an analytical run, taking up stationary phase space needed for the
Figure 3-3 column inefficiencies
LEFT: Tailing RIGHT: Ghost peaks
Figure 3-4 Phenomenex C-18, 3.2X
250mm, 5 µm, 100 Å Pore Size
49. 34
partitioning of the components of interest (in other words, the column should be clean of any
retained material before successive analytical runs). In addition, these late eluates may re- appear
as ghost peaks. Figure 3-5 displays a gradient from 5-60%B. It can be seen that no other
components are eluting at the latter half of the chromatogram.
3.2.3.6.2 Gradient Rate
The gradient rate, or slope, is the change in % B organic modifier over time. A standard
gradient rate used through most of the study is 1%B/ min. Improvement in resolution,
nevertheless, can be obtained by decreasing the gradient rate. This decrease is accompanied by
decreased peak height and increased peak width as well as increased retention times. Figure 3-6
exemplifies this. The sample is that of 1000µg/ mL of LIP standard that displays closely eluting
impurities. The top chromatogram has a gradient rate of 1%B/min. Seemingly two unresolved
peaks are observed at the front of the LIP peak. As the bottom chromatogram displays,
decreasing the gradient rate to 0.25%B/min improves resolution of the peak of interest from
these preceding peaks. The changing of gradient rate will be discussed more in Chapter Five.
3.2.3.6.3 Gradient Time Table
Elements of the gradient time table include the gradient rate, the initial and final %B
composition, starting isocratic hold time, and equilibration time.
Figure 3-5 Exemplification of an initial gradient to
Assure no other peaks or impurities are eluting at
High % ACN Gradient 5-60%B in 55 minutes
1250µg/mL LIP-St
50. 35
As stated in the previous section, the gradient rate is initially established at 1%B/min.
This rate exhibits sharply defined peaks with higher plate numbers, and peptides are eluted
faster, thus saving time.
The initial and final %B range can be determined from a preliminary run to higher %B
organic modifier content. These two parameters are set as to fit the peaks of interest into the
elution window. Figure 3-7 displays the chromatograms of the LIP-St (top) and that of the SPPS
crude linear INGAP-P analog mixture (bottom) synthesized in this study. The gradient is
1%B/min, 5 minute isocratic hold at initial 5%B, and then a 45 minute ramp to 50%B. The
vertical line in the LIP standard scan designates where approximately 20.5%B has reached the
detector. The retention time of the last notable peak in the SPPS crude mixture scan marks
approximately 29%B reaching the detector. The scan is clean of notable peaks after this point, so
it would be reasonable to change the final %B of the gradient to 30%B, resulting in valuable time
1% ACN/ min
Gradient Rate
0.25% ACN/ min
Gradient Rate
Figure 3-6 Change of gradient rate with
Increase in resolution
1000µg/mL LIP-St
Resolution of LIP-St with
preceding impurity: 0.550
Resolution of Lip-St with
Preceding impurity: 0.791
51. 36
saved. The peaks appearing at the beginning isocratic hold would co- elute if the gradient were to
be started immediately without an isocratic hold.
Considering the LIP-St scan at the top of Figure 3-7, it appears that no notable peaks
elute before the 15%B mark at minute 17.5. In order to save time, the gradient start time can be
changed to 15%B and the isocratic hold reduced. This scans does not display any peaks after the
25%B mark, and so for the validation experiments the gradient time table used was as follows:
15%B hold for 3 minutes, ramp to 30%B in 15 minutes, and hold for three minutes.
A gradient with baseline drift necessitates determination of a minimum baseline
equilibration time when considering the sequential running of samples in the auto- sampler.
Enough time is needed at the tail end of a run for the baseline to return to zero before the next
injection. Figure 3-8 displays a no injection gradient run (10-30%B). Note how the risen baseline
dips below the extrapolated zero baseline as the gradient is returned to the starting, isocratic
10%B. This baseline behavior is thought to be caused by TFA retention to the stationary phase in
high %A aqueous MP. As the gradient increases in %B organic modifier, the TFA is eluted, yet
Figure 3-7 LIP standard vs crude linear analog product mixture
Exemplification of the choice of initial and final %B
LIP Standard
Crude Linear Analog Mixture
52. 37
the sudden return to the more aqueous starting composition causes TFA in the aqueous solvent to
dilute as it is saturating the column (32). After a duration of time, the baseline is restored to zero,
and so this restoration time must be considered in the timetable.
3.2.3.7 Equilibration of Column
Before the start of experimental runs, the column was conditioned so as to become
acclimated to the gradient. Unlike isocratic equilibration, which requires a run at %B of a
method and a minimal of time for the baseline to remain constant, methods with gradient mode
require that you run a minimum of two no injection gradient runs (30). An equilibrated column is
identified by the baseline returning to the starting zeroed baseline (Figure 3-8).
3.2.3.8 Injection Volume
5 µL and 10 µL injection volumes were tested using 750 and 1000µg/mL samples. The 5
µL injection had displayed better plate number (40.0k vs 30.8) and tailing factor (1.123 vs 1.321)
for the test solutions, as well as a decreased peak width at half height. These values are very
important considerations as they represent the narrowness and symmetry of a peak which in turn
can relate to baseline resolution when considering a mixture of peptides with very similar
hydrophobicity and thus retention times. Because of this, 5µL was used as the injection volume
in method validation.
Figure 3-8 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
53. 38
3.2.3.9 Flowrate
Because of the dimensions of the Phenomenex column (3.1X250mm), a flowrate of
0.8mL/min was chosen as the back pressure hovered around 200psi. Dr. Al- Bazi suggested to
stay at this flow rate as a higher flowrate would increase back pressure to above 200psi,
detrimental to the well- being of the HPLC instrument.
3.2.4 Method Validation
Figure 3-7 displays a comparison of LIP-St and linear INGAP-P mixture. With a purified
standard of the peptide of interest, it is possible to identify and quantify the target peptide in a
mixture such as that of a SPPS product mixture utilizing RP- HPLC.
This was one of the preliminary objectives of this research at the start, to develop an
HPLC method for the separation and quantitation of the INGAP-P, cyclic and linear analogs in
their respective SPPS product mixtures.
Nevertheless, this preliminary objective was not possible because of a lack of purified
standard for the peptides synthesized. As such, this validation section was based on the purified
LIP standard received from Dr. Su (not the same peptide as the linear INGAP-P analog
synthesized in this study, and so the validation conducted is more of a practice of these
validations steps).
Developing such a method always requires the validation process that involves tests for
specificity, robustness, limit of quantitation/detection (LOQ, LOD) accuracy, and precision, just
to name a few (33).
This practice in validation had its limitations in the form of lack of standard, nevertheless.
Only a limited amount of standard was available (<25 mg throughout the study). Accuracy or
54. 39
adequacy in the validation steps cannot be stated, so the following was truly a practice in the
validation steps of linearity, LOQ/ LOD determination, and injection precision.
3.2.4.1 HPLC Instrumentation and Parameters
• Agilent 1260 Infinity (HPLC/DAD)
• Phenomenex C-18,3.2 X 250mm, 5 µm particle, 100 Å pore size
• Solvernt A: HPLC H2O/ 0.1%TFA Solvent B: HPLC CAN/ 0.085%TFA
• Gradient: 3 min hold at 15%B, ramp 15 minutes to 30%B, Hold 30%B 3 minutes
• UV detection at 215 nm, no reference
• 5 µL injection
3.2.4.2 Linearity
3.2.4.2.1 Preparation of Concentrations
Nine milligrams of lyophilized LIP standard were dissolved in 3mL of H2O/ 0.1%TFA,
vortexed, and filtered. Through serial dilution of the 2250 µg/mL solution was conducted to
prepare 500µL of HPLC samples of the following concentrations: 15, 25, 50, 100, 250, 500, 750,
1000, 1250, 1500, 2000, and 2250 µg/mL (Table 3-1), using the following formula:
mₐ (2250 µg/mL) Vₐ= mₓ (desired concentration) Vₓ(0.5mL)
where Vₐ is the volume of stock required, which is then diluted to 0.5mL. These microliter
volumes used in the preparation were extracted using 1000-100, 200-20, and 10- 0.10
micropipettes, respectively.
55. 40
TABLE 3-1 PREPARATION OF LIP- ST SAMPLES FOR HPLC
Concentration
of LIP
Standard
(µg/mL)
Volume of 2250
µg/mL Stock
Solution (µL)
HPLC Sample
Volume (µL)
15 3.3 500
25 5.5 500
50 11.1 500
100 22.2 500
250 55.6 500
500 111.1 500
750 166.7 500
1000 222.2 500
1250 277.8 500
1500 333.3 500
2000 444.4 500
2250 500 500
3.2.4.2.2 Linearity Determination
In a continual sequence, starting at the lowest concentration 15 µg/mL, each
concentration was run twice consecutively with progression to higher concentrations. Interspaced
in the sequence were blank runs after the 500 and 1250 µg/mL runs.
Linearity was determined between 15 and 1000 µg/mL (Figure 3-9), yielding a linearity
formula y= 6.8745x- 27.992, with a regression equation R squared value of 0.9998, meeting the
validation requirement for linearity (33).
56. 41
3.2.4.3 LOQ/ LOD Determination
Limits of quantitation (LOQ) and detection (LOD) were also determined. The 15 µg/mL
concentration from the linearity experiment was utilized in serial dilution for these
determinations. At first an analytical run was undertaken of the 15 µg/mL solution. It was found
by the linearity formula to have degraded to 14.6 µg/mL (degradation will be discussed in
Chapter Four). Serial dilution of this 14.6 µL/mL concentration was conducted to establish the
LOQ and LOD. Using the Signal-to-Noise (S/N) reporting of the OpenLab CDS program, the
LOQ was established at 7µg/mL with a S/N of 10/1. The LOD was established at 3.65µg/mL,
with a S/N of 4.65/1 (after dilution of this concentration, no signal was determined, and so the
working LOD is the more precise designation for this concentration).
3.2.4.4 Injection Precision
An injection precision experiment was also performed. It had utilized the 500 µg/mL
concentration from the initial linearity study. An analytical run established that this concentration
had also degraded (Chapter Four), yet it, nevertheless, can be used to test the precision of
Figure 3-9 Linearity determination utilizing two concentration point
Scattered plot
57. 42
repetitive injection. This sample was injected six times consecutively (statistical evaluation in
Table 3-2). The average, standard deviation, and coefficient of variance (CV,%) were calculated
using the Excel program. The coefficient of variance of 0.298% meets the precision requirements
of the Food and Drug Administration (FDA)(33).
TABLE 3-2 INJECTION PRECISION STATISTICAL VALUES FOR
500µg/mL SAMPLE INJECTIONS
Injection
Precision
500µg/mL
Sample
Area Average
Standard
Deviation
CV, %
3402.91
3416.64
3430.18 3420.89 10.191 0.298
3429.46
3425.15
3420.97
3.3 Conclusion
Validation cannot be separated from the development of a method. The validation
process affirms reproducibility and repeatability, as well as accuracy. It is integral to establishing
a method or assay for quantitation of a peptide in a SPPS product mixture. The lack of standard
was a detrimental issue of this study, and forced the use of practice at validation.
Nevertheless, a method for the quantitation and identification of a peptide in a mixture
seems very viable from the method development process undergone in this study.
58. 43
CHAPTER 4 ROOM TEMPERATURE DEGRADATION
STUDY IN ACIDIC SOLVENT
From the Method Validation section of the previous chapter, it was apparent that the
samples are prone to degradation as a loss in area response occurred with the 15 and 500 µg/mL
concentrations from as little as 16 hours at room temperature. A room temperature degradation
test was performed using the 500 µg/mL concentration from the linearity determination, which
was freshly prepared at the time. Further evaluated in the chapter is the degradation of the 100
µg/mL concentration from the linearity determination, though examination of this concentration
is not from a pre- determined experiment.
4.1 Room Temperature Degradation Experiment of 500 µg/mL Concentration
The HPLC parameters are the same as cited in Section 3.2.4.1
The 500 µg/mL concentration from the linearity experiment had initially been exposed to
room temperature in the auto- sampler for 16 hours. After the completion of the linearity
sequence, the sample was immediately wrapped in paraffin wax and placed in the freezer of the
lab refrigerator. It stayed in the freezer in this fashion for 22 days, before being thawed out. After
thawing and returning to room temperature (approximately 30 mins), the sample was vortexed
for five seconds, and an analytical run was conducted, marking time zero for this degradation
study. The sample was placed in a cabinet, where the temperature averaged 75 º F for the
duration of the experiment. The sample was removed and an analytical run conducted at hour 24,
48, and 84. The overlay of these four scans (including original linearity scan of freshly prepared
sample) is displayed in Figure 4-1. The first eluting peak of the overlay scan seems to be the
main degradation product. Not displayed is a peak that increases in area with time starting in the
59. 44
24 hour degradation analysis, eluting right after the solvent front. Figure 4-2 displays the percent
recovery values versus time and environmental conditions
Figure 4-2 Degradation of 500µg/mL LIP- St over time
Figure 4-1 Overlay of degradation progression of 500µg/mL sample
Impurity peak area does not change throughout experiment
Peak at far left is the major degradation product,
Increasing in area as degradation experiment progresses
208 Hr Room
Degradation
208 Hr Room
Degradation
60. 45
4.2 Examination of 100 µg/mL Concentration Degradation
The HPLC parameters are the same as cited in Section 3.2.4.1
The 100 µg/mL concentration from the linearity experiment had initially been exposed to
room temperature plus the temperature in the auto- sampler for 16 hours. After the completion of
the linearity sequence, the sample was immediately wrapped in paraffin wax and place in the
freezer of the lab refrigerator for three days. It was thawed and run through a series of repeated
injections while testing injection precision of this concentration. At this time, it had been
exposed to 24 hours in the auto- sampler, where it was removed, wrapped with paraffin wax and
returned to the freezer of 20 days before it was removed, thawed, vortexed, and then a
chromatogram received from an analytical run. The overlay of the chromatograms is displayed in
Figure 4-3. As in the degradation overlay of the 500µg/mL sample, the first eluting peak of the
scan seemingly is the main degradation product. Also, as in the 500µg/ml degradation
chromatograms, there is a peak that begins to appear after the solvent front. Figure 4-4 displays
the percent recovery values versus time/ environmental conditions.
Figure 4-3 Overlay of degradation progression of 100µg/mL sample
61. 46
4.3 Discussion and Conclusion
The LIP standard peptide has been shown to degrade relatively quickly in the acidic
H2O/TFA, pH 2 solvent. The INGAP-P has a highly conserved N- IGLHDP- motif (4), and the
LIP-St carries the D-P (aspartic acid- proline) sequential residues. Guidelines for the storage and
handling of synthetic peptides (34,35) suggest that the aspartic acid- proline sequence in acidic
conditions can form a cyclic imide intermediate (Figure 4-5), with hydrolysis either reverting to
the original aspartic acid residue, or the iso- aspartate analog (degradation product). Therefore, it
is a possibility that the degradation peak identified in the overlay scans is this analog of the
aspartic acid residue.
A more thorough study is needed on its stability. A less harsh solvent may be the answer
to a more stable sample solution. Such a study may involve increasing the organic
content of the solvent while at the same time decreasing the acidity/basicity when working with a
peptide of limited availability.
Figure 4-4 Degradation of 100µg/mL over time
63. 48
CHAPTER 5 RESOLUTION OF INGAP-P FRACTION
Figure 5-1 displays the analytical chromatograms of the extracted, lyophilized fraction
containing the INGAP-P based on the ESI-MS and the chromatogram of the crude INGAP-P
product mixture from SPPS. The original objective of this thesis was to use the extracted
lyophilized fraction from the Simpson Institute as the standard for method development for the
quantitation and identification of the target in the crude mixture. Nevertheless, this was not
feasible as the ESI MS identified an additional INGAP-P mass + 101 Da peptide co-eluting with
the INGAP-P.
The objective of this study was to increase resolution of these seemingly co- eluting
peptides. Different parameters have been cited as effecting the resolution of peptides. These
include a gradient rate (36), as well as concentration of TFA in the MP that can display a change
in α(selectivity) and in turn resolution (19). Other parameters include a change in organic
modifier (37), such as isopropanol (IPA) and methanol. A last parameter tested is a change in
column/ stationary phase (38). The following sections display the resulting effect on resolution
of the parameter changes listed above.
Figure 5-1 A) Comparison of SPPS INGAP-P crude product mixture
And lyophilized INGAP-P extract
B) Report image of lyophilized extract
(Co-elution with +101Da peptide)
1000ppm Crude
SPPS Mixture
250ppm Lyophilized
INGAP-P Fraction
64. 49
5.1 Effect of Decreasing %B/min on Resolution
Figure 5-2 compares the chromatograms with progressive reduction of %B/min.
Retention times increased as gradient rate decreased. The peaks decreased in height and
increased in width, yet the tops of two peaks can be identified with the 0.125%B/min gradient,
unlike the appearance of a preceding shoulder on a peak as displayed in the chromatogram with a
gradient of 1%B/min.
5.2 Effect of Increased %TFA in MP on Resolution
TFA was increased to double the original amounts (Solvent A: H2O/0.2%TFA, Solvent
B: ACN/0.17%TFA). The peaks begin to broaden, notably the fronting peak, with less
differentiation displayed (Figure 5-3).
5.3 Effect of Organic Modifier on Resolution
A change in organic modifier has the potential to increase resolution as the solvent has
different interactions with the components of the sample. It is even possible to reverse the elution
Figure 5-2 The effect of decreasing %B/min on Resolution
Solvent A: H20/0.1%TFA
Solvent B: ACN/0.085%TFA
Sample: 1250 µg/mL lyophilized fraction
300-mAU 50-mAU
65. 50
order by changing the organic modifier. In the following runs, IPA and methanol were
respectively tested.
5.3.1 IPA
The MP solvents used were solvent A: H2O/0.1%TFA and solvent B: IPA/0.1%TFA.
HPLC parameters are those cited in Section 3.2.4.1 (excepting MP composition and flow rate).
Because IPA has a higher viscosity than ACN, the flow rate was decreased to 0.5mL/min to keep
the back pressure at a reasonable level(<250psi). The chromatogram is featured in Figure 5-4.
IPA does not seem to increase resolution over ACN (32).
5.3.2 Methanol
Methanol was next tested as an organic modifier with solvent A: H2O/0.1%TFA and
solvent B: Methanol/0.1%TFA. HPLC parameters of Section 3.2.4.1 were used (excepting MP
composition, flow rate, and final %B). The final %B of the gradient was raised as methanol is a
weaker, less hydrophobic modifier. The flow rate was also reduced because of higher viscosity to
0.65ml/min with consideration of the back pressure. Figure 5-5 displays the chromatogram. Two
Figure 5-3 Effect of doubling TFA in mobile phases on resolution
Solvent A: H2O/ 0.2%TFA
Solvent B: ACN/ 0.17%TFA
Sample: 1250 µg/mL lyophilized fraction
200-mAU
Time
66. 51
sharply defined peaks appear at minute 30.2. It may be that methyl- esterification has taken place
with the solvent. Nevertheless, this was a one run examination, and further runs would need to be
conducted to validate repeatability.
5.4 Effect of Column Type on Resolution
The stationary phase/ column type was changed in order to increase resolution. This is
typically the last step taken in increasing resolution. The column utilized was the ACE 3-Phenyl
4.6 X 150mm column with a flowrate of 1mL/min and gradient rate of 1%B/min, sample 850
Figure 5-4 Effect of IPA as an organic modifier
Elution order may be transitioning
Solvent A: H20/0.1%TFA
Solvent B: IPA/0.1%TFA
Sample: 850 µg/mL lyophilized fraction
Figure 5-5 Effect of methanol organic modifier on resolution
Solvent A: H20/0.1%TFA
Solvent B: Methanol/0.1%TFA
Sample: 850 µg/mL lyophilized fraction
200-mAU
Time
Time
Resolution between identical peaks:
0.621
67. 52
µg/mL (Figure 5-6(a)). Figure 5-6(b) displays the same sample and gradient rate, except with a
flow rate change to 0.75mL/min. A reduction in flow rate can also improve resolution, and this
can be slightly identified by comparing the peaks of the two chromatograms.
The column type from a qualitative point of view increased resolution the greatest when
compared to the previous parameter changes. The phenyl stationary phase has strong π-π
interactions with the elution components (18), and this appears to be more discriminatory than
Figure 5-6(a) Change in column type:C3 Phenyl Column
Gradient: 1%B/min, flow rate: 1mL/min
Solvent A: H2O/0.1%TFA
Solvent B: ACN/0.085%TFA
Sample: 850 µg/mL lyophilized fraction
Resolution: 0.484
Time
mAU-215
Figure 5-6(b) Change in column type: 3 Phenyl Column
Gradient: 1%B/min, flow rate: 0.75mL/min
Solvent A: H2O/0.1%TFA
Solvent B: ACN/0.085%TFA
Sample: 850 µg/mL lyophilized fraction
Resolution: 0.674
Time
mAU-286
68. 53
the hydrophobic interactions with the C-18 column. Two clearly defined peaks are displayed in
Figure 5-6(a) and Figure 5-6(b), far removed from the initial analytical run of the extracted,
lyophilized fraction utilizing the C-18 column that appears almost as a single peak.
5.5 Conclusion
Changing the column’s stationary phase while at the same time decreasing the flow rate
appears to have improved resolution of the INGAP-P and INGAP-P mass +101Da peptide the
greatest when compared to decreasing the gradient rate, increasing MP TFA%, or changing the
organic modifier. Nevertheless, resolution is not quite at the adequate level of 2 (18) that
indicates complete baseline separation. It is possible that increasing the length of the 3 Phenyl
column (from 150 to 250mm) may further increase resolution as the number of theoretical plates
typically increases with column length. Further, other types of columns should also be tested
when searching for this optimal resolution.
Nevertheless, a minimum of a resolution of 1, baseline resolution, has not been
accomplished in this study. Further experiments changing mobile phase composition and
stationary phase need to be conducted in order to establish an optimized method for acquiring
acceptable resolution.
69. 54
CHAPTER 6 FRACTIALIZATION UTILIZING AN ANALYTICAL INSTRUMENT
Fractionalization is typically carried out on a preparatory HPLC instrument. Such a
preparatory HPLC instrument is not available at Northeastern Illinois University, though it
would be most valuable in SPPS as a target peptide such as the INGAP-P can be purified from
the synthetic mixture. Yet, it is possible to fractionalize limited amounts of sample using the
analytical HPLC instruments at the NEIU.
Such a technique would be invaluable in the SPPS synthesis of peptides as the target
peptide can be purified to an extent from the other components of the mixture. Most notably, the
lyophilized peptides can be resolved and the INGAP-P physically separated from the (+ 101) Da
peptide. The purity of pooled fractions than can be re- evaluated utilizing MALDI-TOF or ESI
evaluations. If purity of pooled fractions is adequate, and an acceptable amount can be extracted,
it may be possible to use this sample as a standard for the calibration of a linearity curve that can
be used to determine the amounts of target peptide in a SPPS reaction mixture, as well as using
such an extracted sample in the validation process. Further, the major product form the previous
room temperature degradation study could be purified with such a technique and analyzed by
MS.
The following steps and concepts of analytical fractionalization were taken from citation
(39) and personal correspondence with Rick Dauer, Engineer at Corden Pharma Colorado (40).
6.1 Scheme for Purifying SPPS Products
In order to add a larger amount of sample to the instrument, the injector was by-passed
through use of an unused channel whose tubing is detached from the degasser (this sample is
sonicated/de-gassed before introduction). A volume of the 2250 µg/mL (2mL) of the crude,
linear INGAP-P analog sample was then pumped into the system and followed by a volume of
70. 55
the starting composition solvent until it was approximated to have reached the top of the column.
Once at the top of the column, the gradient was run. From an analytical run, it can be estimated
where to start collection of the fractions. The analytical column has been overloaded in this
protocol. Figure 6-1 displays the analytical chromatogram of the crude linear INGAP-P analog
product mixture, followed by the consecutively collected 60-second fractions from the
overloading protocol. Figure 6-2 displays a zoom of one of the collected fractions against the
crude mixture scan.
Figure 6-3 outlines how this fractionalization procedure can extract purified peptides
from the lyophilized extract. Displayed is the analytical chromatogram overlaid with a
hypothetical scan of the overloaded -lyophilized extract (not to true intensity scale, the
absorbance could be thousands-fold). Fractional collection between 11.4 – 12.4min would yield
a highly purified peptide represented by the first eluting peak of the analytical scan. Fractional
collection between 13.35 and 14min would yield a purified peptide represented by the second
Figure 6-1 Fractionalization of linear INGAP analog crude SPPS mixture
on an analytical instrument
71. 56
eluting peak of the analytical scan. The intermediate fractional collection between 12.4-
13.35min would yield a mixture of the two peptides. If resolution was increased for the
lyophilized extract, then this intermediate period of peptide mixture would reduce with an
increase in extracted purified peptide yield, displaying the importance of optimizing resolution of
this extracted lyophilized fraction.
Figure 6-2 Comparison of collected fraction to crude peptide mixture
TOP: Crude peptide mixture
BOTTOM: 6th minute designation fraction
72. 57
6.2 Conclusion
If a systematic procedure of fractional collection of target peaks(peptides) from a SPPPS
product mixture utilizing the overloading of an analytical column can be developed, it seems
very advantageous and a natural step in the purification of peptides. The crude test conducted
shows that this may be possible. Pooled, purified fractions containing the peptide could be
Figure 6-3 Scheme for extracting INGAP-P from
Lyophilized extract through overloading
of an analytical column
(overload trace not to scale)
73. 58
extracted from the solvent and lyophilized, and then used as standards in HPLC method
development, or even in cellular studies.
74. 59
CHAPTER 7 CONCLUSION AND FUTURE DIRECTIONS
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 has been ascertained in the final product mixture. RP-
HPLC with UV detection is a natural accompaniment to the SPPS process, as it has high
efficiency at the separation of peptides, whether it be a synthetic or biological sample, and it is
routinely utilized for the quantitation of target peptides in such samples. Such quantitation
necessitates the need for a minimum of purified standard that can be used in the calibration of
linearity as well as in the validation of the developed method.
Originally, a portion of the INGAP-P product that was synthesized via Fmoc-SPPS was
meant to be fractionalized and purified on the preparatory HPLC instrument at the Simpson
Querrey Institute of Northwestern University, and this purified product used as a standard in
HPLC method development and validation. Yet, the facility could not separate a co- eluting
M +101 Da peptide from the INGAP-P fraction. In lieu of this standard, a purified INGAP-P
analog synthesized by Dr. Su’s group previously was utilized in studying the parameters
involved in method development, in the process knowledge was gained in the optimization of
parameters in the separation of peptides. Unfortunately, the quantity of such standard was not
enough for validation of the method. In addition, the acidic solvent used for sample preparation
degraded the standard peptide relatively quickly. Future HPLC method developments for the
separation of peptides should study the choice of sample solvent more extensively before moving
on in the method development process.
Nevertheless, the extracted INGAP-P fraction offered an excellent exercise in the
parameters that can affect the resolution of peptides, in the case of this study, two peptides that
75. 60
are in essence co- eluting. The choice of column type was determined as the parameter with the
greatest potential at separation of unresolved peaks, and as such, column type should be more
extensively studied in the beginning of the HPLC method development process.
With the co- eluting INGAP-P fraction, it is potentially possible to extract, with the
HPLC instrumentation at NEIU, an INGAP-P fraction of high purity that may yet be used as a
standard in SPPS product mixture quantitation. As shown with the SPPS crude product mixture
of the linear analog, it is possible, through the overloading of an analytical column, to extract
fractions of this SPPS product mixture.
Experiments should be furthered in the fractionalization of a sample on an analytical
instrument. An optimized method may make it 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.
Structural- activity relationship of INGAP-P and analogs (particularly the cyclic analog)
would shed light on design of new beta cell- promoting peptide drugs.
76. 61
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