# A Multidisciplinary Investigation to Determine the Structure and Source of Dimeric Impurities in AMG 517 Drug Substance

Authors: Maria Victoria Silva Elipe; Zhixin Jessica Tan; Michael Ronk; Tracy Bostick
Journal: International Journal of Analytical Chemistry
 (2009)

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## Abstract

In the initial scale-up batches of the experimental drug substance AMG 517, a pair of unexpected impurities was observed by HPLC.  Analysis of data from initial LC-MS experiments indicated the presence of two dimer-like molecules.  One impurity had an additional sulfur atom incorporated into its structure relative to the other impurity.  Isolation of the impurities was performed, and further structural elucidation experiments were conducted with high-resolution LC-MS and 2D NMR.  The dimeric structures were confirmed, with one of the impurities having an unexpected C-S-C linkage.  Based on the synthetic route of AMG 517, it was unlikely that these impurities were generated during the last two steps of the process.  Stress studies on the enriched impurities were carried out to further confirm the existence of the C-S-C linkage in the benzothiazole portion of AMG 517.  Further investigation revealed that these two dimeric impurities originated from existing impurities in the AMG 517 starting material, N-acetyl benzothiazole. The characterization of these two dimeric impurities allowed for better quality control of new batches of the N-acetyl benzothiazole starting material.  As a result, subsequent batches of AMG 517 contained no reportable levels of these two impurities


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## Body

## 1. Introduction

In the  early stages of new  drug development, understanding the impurity
profiles of the drug substance is critical when interpreting the data from
toxicology and clinical studies.  There
is a body of regulatory requirements with regard to identification and control of impurities.  A commonly used framework used in the
pharmaceutical industry is Q3A(R2), the International Conference on
Harmonization (ICH) guidance for controlling impurities in new drug substance
[1].  Although this guidance is intended only
for products approaching application for final market registration, many
companies consider similar elements when evaluating impurities in new chemical
entities during the clinical phases of development.Impurities in drug substances are classified into several categories in
the ICH guideline Q3A(R2): organic impurities, inorganic impurities, and
residual solvents.  The organic
impurities are of major concern for a new drug substance produced by chemical
synthesis because the potential toxicity of most of these impurities is unknown.  These impurities can originate from starting
materials, by-products, intermediates, degradation products, reagents, ligands,
and catalysts [1].  Knowledge of impurity
structures can provide important insight into the chemical reactions responsible
for forming these impurities as well as understanding potential degradation
pathways [2].  Such information is
essential in establishing critical control points in the drug substance synthetic
process and eventually ensuring its overall quality and safety.HPLC with UV detection is the most common analytical methodology used in
the pharmaceutical industry to monitor organic impurities in new drug
substances [2, 3].  These HPLC-UV methods
are frequently used to track impurity profiles across various batches of drug
substance which are often produced by different synthetic routes and at
different scales.  This is especially
important in the earlier phases of clinical development when, due to resources
and time constraints, the synthetic process is dynamic and not completely characterized,
and the source/quality of starting materials has not been thoroughly evaluated [4].
When a new impurity is detected above a particular threshold (e.g., >0.10%
according to ICH Q3A(R2) for commercial products), structural elucidation of
that impurity is typically initiated.  LC-MS
systems are widely available these days and are routinely used in initial
impurity identification efforts during early drug development phases [5].  The sensitivity of LC-MS allows for the
analysis of the impurities without isolation, which is often time consuming.  Coupled with knowledge of the sample’s
history (e.g., synthetic scheme, purification process, storage conditions,
stress conditions, etc.), it is often possible to propose the chemical
structure of the impurity solely based on LC-MS data [6, 7]. However, the LC-MS
data alone may not provide sufficient information to derive a chemical
structure.  In such cases, NMR
spectroscopy (1D and/or 2D) is often employed to gather further structural
information for impurity identification [8, 9].
Although online LC-NMR has gained some popularity in recent years
[10, 11], isolation or enrichment of impurity component for offline NMR
studies is still one of the most common approaches [12, 13].   Frequently, publications detailing the
identification of pharmaceutical impurities will focus on the application of a
selected technique and will document the proposed formation reaction for the
impurity.  Rarely does the publication
involve multiple analytical disciplines used to both identify the impurity and
to trace back to its ultimate source through a complex synthetic scheme [14].Preparation for the first kilogram-scale production of one of Amgen’s
investigational anti-inflammatory drugs, AMG 517, provides a case in which a
multidisciplinary investigation involving HPLC-UV, LC-MS, NMR, preparative HPLC,
and forced degradation was required for unequivocal impurity identification.  Two unexpected late eluting impurities were
detected by an HPLC-UV method during release testing of this first scale-up
batch of AMG 517 (see Figure1).  This first
kilogram-scale batch of AMG 517 was manufactured with a process that was not
well characterized (see Figure 2), using starting materials from outside vendors
with which we had  very little prior experience. Such situation is not uncommon in early
clinical drug development.  As the new
batch was slated for use in first-in-human clinical trials, characterization of
these impurities was required to enable process development which would lead to
better process control.  As a result of LC-MS
and NMR analyses, the structures of these impurities were proposed as a simple
dimer of AMG 517 and a thioether-linked dimer.
A typical impurity investigation may end here with proposal of impurity
structures.  However, the formation of
these impurities could not be explained by the synthesis scheme shown in Figure
2.  A forced degradation study of the
dimeric impurities provided a degree of certainty to the proposed structure for
the thioether impurity.  The desire to
understand the origin of these impurities in the drug substance led to
investigation of starting materials using HPLC-UV and LC-MS.  Information compiled from these studies
allowed us to work back through the synthetic scheme for AMG 517 to determine
the source of the dimeric impurities.  Knowing the origin of these impurities
ultimately allowed for better quality control of the AMG 517 drug substance.(a) HPLC-UV chromatogram
of a standard mixture and (b) a representative AMG 517 sample containing the
unknown impurities.  Chromatographic
conditions are in the experimental section and Table1.
(a)
Standard injection(b)
Sample injectionFigure 2
Synthetic pathway of AMG 517 during the
early stages of clinical development.


## 2. Experimental

### 2.1. Materials and Reagents

HPLC grade acetonitrile (ACN, Burdick and Jackson, Muskegon, Mich, USA),
trifluoroacetic acid (TFA, J. T. Baker, Phillipsburg, NJ, and Pierce, Rockford,
Ill, USA), and purified water from a Milli-Q unit (Millipore, Molsheim, France)
were used in the preparation of various mobile phases and diluents in
chromatographic analysis. Dimethyl-d6 sulfoxide (DMSO-d6) “100%” (D, 99.96%), used for NMR analysis, was from Cambridge Isotope Laboratories (Andover, Mass, USA).Samples of AMG 517 drug substance, N-(4-hydroxy- benzo[d]thiazol-2-yl)acetamide
(N-acetyl benzothiazole), and the enriched impurity fraction were provided by
the Chemical Process Research and Development Department of Amgen inc.,
(Thousand Oaks, Calif, USA).


### 2.2. HPLC

Analytical-scale chromatographic analyses were performed on an Agilent (Wilmington, Del,
USA) 1100
series HPLC system.  Mobile phase A was
0.1% TFA in water; mobile phase B was 0.1% TFA in ACN.  A Phenomenex (Torrance, Calif, USA) Luna C18(2) HPLC column (5μm,
150×4.6mm, at 30°C) was used for the separation and quantitation
of the AMG 517 impurities.  Two different
gradients with different flow rates were employed for the separation of AMG 517
and N-acetyl benzothiazole (see Table 1). A UV detection wavelength of 254 nm
and an injection volume of 30μL were used in the analysis of both
compounds.Table 1
Gradient
conditions used for the HPLC-UV and LC-MS analyses of AMG 517 and N-acetyl benzothiazole.
CompoundAMG 517N-acetyl Benzothiazole(HPLC-UV and LC-MS)(HPLC-UV)(LC-MS)Gradient
programTime (min)%BTime (min)%BTime (min)%B05050515651030103020701550155027982075207530982595Flow rate1.0 mL/min1.5 mL/min1.0 mL/minSample diluent50% ACN/50% water10% ACN/90% water


### 2.3. LC-MS

LC-MS experiments with accurate mass determination via high resolution
mass spectrometry were performed using an Agilent 1100 HPLC (configured with a
diode array UV detector) interfaced with a Waters (Milford, Mass, USA)
Micromass Q-Tof Ultima API quadrupole time-of-flight mass spectrometer.  The mass spectrometer was
configured with a lockspray electrospray
ionization (ESI) source to allow for the introduction of an internal mass
calibration solution, which provides for a 5 ppm mass error specification when
used in conjunction with tune settings producing ~20 000 mass resolution on
the instrument.LC-MS analyses of the enriched impurities, and of their hydrolysates,
were accomplished using a Phenomenex Luna C18(2) HPLC column (3μ, 100 Å, 2.0×150mm) and mobile phase
consisting of 0.1% aqueous TFA (mobile phase A) and 0.1% TFA in ACN (mobile
phase B). A flow rate of 0.2 mL/minute was used, and a column temperature of 30°C was maintained throughout each HPLC run. Gradient
conditions listed in Table 1 for the HPLC-UV analysis of AMG 517 were also used
for the LC-MS analysis of AMG 517 and its impurities.LC-MS analysis of the AMG 517 starting material, N-acetyl benzothiazole,
was accomplished using a Phenomenex Luna C18(2) HPLC column (3μ, 100 Å, 4.6×150mm). The same mobile
phase system described above was used at a flow rate of 1.0 mL/minute.  Column temperature was also maintained at 30°C.  The gradient conditions used for the LC-MS
analysis of N-acetyl benzothiazole are listed in Table 1.


### 2.4. NMR

Spectra were acquired at25°C and 27°C on Bruker DPX 400 and Bruker AVANCE
600 NMR instruments (Bruker BioSpin Corporation, Billerica,
Mass, USA)
equipped with 5 mm and 2.5 mm multinuclear inverse z-gradient probes,
respectively. H1  NMR
experiments were carried out at 400.13 and 600.13 MHz, respectively, and C13
NMR experiments were carried out at 100.61 and 150.90 MHz, respectively.  The data processing was performed on the
spectrometers.  Chemical shifts are
reported in the δ scale (ppm) by assigning the residual solvent
peak at 2.50 and 39.51 ppm to DMSO for H1 and C13,
respectively.  The 1D H1 and C13
NMR spectra were determined using a 30° flip angle with 1 second and 2 seconds
equilibrium delays, respectively.  The
90° pulses used  were 7.7 and 4.5 microseconds
for H1, and 22.0 and 12.50 microseconds for C13 in
experiments carried out on the 400 and 600 MHz spectrometers, respectively.  The H1, H1-2D correlation
spectroscopy (COSY) spectra were acquired into 2K data points in the f2
dimension with 128 increments in the f1 dimension, using a spectral width of
4789.3 Hz on the 400 MHz spectrometer and 7788.2 Hz on the 600 MHz instrument. The
nuclear Overhauser effect spectroscopy (NOESY) experiments were determined with
an 800 milliseconds mixing time, and with the same spectral width for f2
dimensions as COSY experiments, but with 256 increments in f1 dimension.  The delays between successive pulses were 1.5
and 2 seconds for 2D COSY and NOESY, respectively.  Both the H1, C13-2D
heteronuclear single-quantum correlation (HSQC) and H1, C13-2D
heteronuclear multiple bond correlation (HMBC) spectra were determined using
gradient pulses for coherence selection.
The H1, C13-2D heteronuclear multiple-quantum
correlation (HMQC) and the HSQC spectra were determined with decoupling during
acquisition.  The 2D HMQC and 2D HMBC
experimental data were acquired on the 400 MHz spectrometer with spectral
widths of 4789.3 Hz for H1 and 20123.9 Hz for C13, into
1K data points in the f2 dimension with 128 increments in the f1 dimension.  The 2D HSQC and 2D HMBC experimental data
carried out on the 600 MHz spectrometer were acquired with spectral widths of
6009.6 and 7788.2 Hz for H1 for HSQC and HMBC, respectively, and
27162.5 Hz for C13 dimension.  The data were acquired into 1K and 4K data
points in the f2 dimension for HSQC and HMBC, respectively, and with 256 and
128 increments in the f1 dimension for HSQC and HMBC, respectively.  Delays corresponding to one bond C13–H1
coupling (ca. 145 Hz) for the low-pass filter and to two-to-three bond C13–H1
long range coupling (7.7 Hz) were used for the HMBC experiments.  All 2D NMR data were processed using sine and
qsine weighting window functions with some line broadening.


## 2.1. Materials and Reagents

HPLC grade acetonitrile (ACN, Burdick and Jackson, Muskegon, Mich, USA),
trifluoroacetic acid (TFA, J. T. Baker, Phillipsburg, NJ, and Pierce, Rockford,
Ill, USA), and purified water from a Milli-Q unit (Millipore, Molsheim, France)
were used in the preparation of various mobile phases and diluents in
chromatographic analysis. Dimethyl-d6 sulfoxide (DMSO-d6) “100%” (D, 99.96%), used for NMR analysis, was from Cambridge Isotope Laboratories (Andover, Mass, USA).Samples of AMG 517 drug substance, N-(4-hydroxy- benzo[d]thiazol-2-yl)acetamide
(N-acetyl benzothiazole), and the enriched impurity fraction were provided by
the Chemical Process Research and Development Department of Amgen inc.,
(Thousand Oaks, Calif, USA).


## 2.2. HPLC

Analytical-scale chromatographic analyses were performed on an Agilent (Wilmington, Del,
USA) 1100
series HPLC system.  Mobile phase A was
0.1% TFA in water; mobile phase B was 0.1% TFA in ACN.  A Phenomenex (Torrance, Calif, USA) Luna C18(2) HPLC column (5μm,
150×4.6mm, at 30°C) was used for the separation and quantitation
of the AMG 517 impurities.  Two different
gradients with different flow rates were employed for the separation of AMG 517
and N-acetyl benzothiazole (see Table 1). A UV detection wavelength of 254 nm
and an injection volume of 30μL were used in the analysis of both
compounds.Table 1
Gradient
conditions used for the HPLC-UV and LC-MS analyses of AMG 517 and N-acetyl benzothiazole.
CompoundAMG 517N-acetyl Benzothiazole(HPLC-UV and LC-MS)(HPLC-UV)(LC-MS)Gradient
programTime (min)%BTime (min)%BTime (min)%B05050515651030103020701550155027982075207530982595Flow rate1.0 mL/min1.5 mL/min1.0 mL/minSample diluent50% ACN/50% water10% ACN/90% water


## 2.3. LC-MS

LC-MS experiments with accurate mass determination via high resolution
mass spectrometry were performed using an Agilent 1100 HPLC (configured with a
diode array UV detector) interfaced with a Waters (Milford, Mass, USA)
Micromass Q-Tof Ultima API quadrupole time-of-flight mass spectrometer.  The mass spectrometer was
configured with a lockspray electrospray
ionization (ESI) source to allow for the introduction of an internal mass
calibration solution, which provides for a 5 ppm mass error specification when
used in conjunction with tune settings producing ~20 000 mass resolution on
the instrument.LC-MS analyses of the enriched impurities, and of their hydrolysates,
were accomplished using a Phenomenex Luna C18(2) HPLC column (3μ, 100 Å, 2.0×150mm) and mobile phase
consisting of 0.1% aqueous TFA (mobile phase A) and 0.1% TFA in ACN (mobile
phase B). A flow rate of 0.2 mL/minute was used, and a column temperature of 30°C was maintained throughout each HPLC run. Gradient
conditions listed in Table 1 for the HPLC-UV analysis of AMG 517 were also used
for the LC-MS analysis of AMG 517 and its impurities.LC-MS analysis of the AMG 517 starting material, N-acetyl benzothiazole,
was accomplished using a Phenomenex Luna C18(2) HPLC column (3μ, 100 Å, 4.6×150mm). The same mobile
phase system described above was used at a flow rate of 1.0 mL/minute.  Column temperature was also maintained at 30°C.  The gradient conditions used for the LC-MS
analysis of N-acetyl benzothiazole are listed in Table 1.


## 2.4. NMR

Spectra were acquired at25°C and 27°C on Bruker DPX 400 and Bruker AVANCE
600 NMR instruments (Bruker BioSpin Corporation, Billerica,
Mass, USA)
equipped with 5 mm and 2.5 mm multinuclear inverse z-gradient probes,
respectively. H1  NMR
experiments were carried out at 400.13 and 600.13 MHz, respectively, and C13
NMR experiments were carried out at 100.61 and 150.90 MHz, respectively.  The data processing was performed on the
spectrometers.  Chemical shifts are
reported in the δ scale (ppm) by assigning the residual solvent
peak at 2.50 and 39.51 ppm to DMSO for H1 and C13,
respectively.  The 1D H1 and C13
NMR spectra were determined using a 30° flip angle with 1 second and 2 seconds
equilibrium delays, respectively.  The
90° pulses used  were 7.7 and 4.5 microseconds
for H1, and 22.0 and 12.50 microseconds for C13 in
experiments carried out on the 400 and 600 MHz spectrometers, respectively.  The H1, H1-2D correlation
spectroscopy (COSY) spectra were acquired into 2K data points in the f2
dimension with 128 increments in the f1 dimension, using a spectral width of
4789.3 Hz on the 400 MHz spectrometer and 7788.2 Hz on the 600 MHz instrument. The
nuclear Overhauser effect spectroscopy (NOESY) experiments were determined with
an 800 milliseconds mixing time, and with the same spectral width for f2
dimensions as COSY experiments, but with 256 increments in f1 dimension.  The delays between successive pulses were 1.5
and 2 seconds for 2D COSY and NOESY, respectively.  Both the H1, C13-2D
heteronuclear single-quantum correlation (HSQC) and H1, C13-2D
heteronuclear multiple bond correlation (HMBC) spectra were determined using
gradient pulses for coherence selection.
The H1, C13-2D heteronuclear multiple-quantum
correlation (HMQC) and the HSQC spectra were determined with decoupling during
acquisition.  The 2D HMQC and 2D HMBC
experimental data were acquired on the 400 MHz spectrometer with spectral
widths of 4789.3 Hz for H1 and 20123.9 Hz for C13, into
1K data points in the f2 dimension with 128 increments in the f1 dimension.  The 2D HSQC and 2D HMBC experimental data
carried out on the 600 MHz spectrometer were acquired with spectral widths of
6009.6 and 7788.2 Hz for H1 for HSQC and HMBC, respectively, and
27162.5 Hz for C13 dimension.  The data were acquired into 1K and 4K data
points in the f2 dimension for HSQC and HMBC, respectively, and with 256 and
128 increments in the f1 dimension for HSQC and HMBC, respectively.  Delays corresponding to one bond C13–H1
coupling (ca. 145 Hz) for the low-pass filter and to two-to-three bond C13–H1
long range coupling (7.7 Hz) were used for the HMBC experiments.  All 2D NMR data were processed using sine and
qsine weighting window functions with some line broadening.


## 3. Results and Discussion

### 3.1. Impurity Profiles in Kilogram-Scale Batches of AMG 517 Drug Substance

A stability
indicating HPLC-UV method was developed to separate and quantify AMG 517 along
with its potential impurities and possible degradants.
Figure1(a) represents a typical separation of
a standard mixture of AMG 517 in the presence of its known impurities and
degradants.  This method was used to
analyze the first six drug substance batches of AMG 517 during release and
stability testing of these lots.  A pair
of unexpected late-eluting unknown impurities was observed in all six batches
of AMG 517.  The area percent levels of impurity
Unknown 1 ranged from 0.15% to 0.44%, while Unknown 2 ranged from 0.06% to
0.21%. A representative chromatogram of an
AMG 517 drug substance lot containing these impurities is shown in Figure 1(b). These two
impurities were not detected at a reportable level in the
previous small-scale batches of AMG 517. Since these two unknown impurities eluted near
the retention time of the by-product in step 1 of the AMG 517 synthetic
reaction, it was concluded that these new impurities were highly hydrophobic and
may have structural features similar to the by-product (see Figure 2).Preliminary low-resolution LC-MS analysis on the drug substance provided
molecular mass and tandem mass spectrometry (MS/MS) fragment ion information
for these two impurities (data not shown).
The observed mass for the protonated Unknown 1 and Unknown 2 was 859 Da and 891 Da, respectively.  Since
the exact mass for AMG 517 is 430.0711 Da, an observed mass of 859 Da for Unknown 1 suggested that it
could be some sort of dimeric structure related to  AMG 517.  MS/MS data also suggested dimeric structures for
both impurities. Fragment ions that corresponded to the neutral loss of
multiple acetyl and hydrofluoric functional groups were observed in MS/MS
experiments performed on the protonated ions of both Unknown 1 and Unknown 2.  The mass difference between the two unknowns was
32 Da, which could be
attributed to either one additional sulfur atom or two additional oxygen atoms
in Unknown 2 relative to Unknown 1.  However,
the preliminary LC-MS analysis alone could not conclusively identify the structures
of these impurities due to the possible existence of multiple isomeric structures
consistent with the mass data.  To aid in
the structural elucidation efforts, an enriched fraction of these two
impurities was isolated via preparative-scale HPLC.  The isolated fraction contained about 35% of Unknown
1 and 62% of Unknown 2 based on UV detection at 254 nm.  LC-MS and NMR experiments were performed to characterize
this enriched fraction.


### 3.2. Accurate Mass Determination for Unknowns 1 and 2 in the Enriched Fraction

An accurate mass of 859.1342 Da was determined for Unknown 1 in the
enriched fraction.  Elemental composition
analysis was performed for this protonated mass. Instrument performance, the
synthetic pathway for AMG 517, and information gained from the preliminary
LC-MS analysis of the impurities were taken into account in setting parameters
for this analysis.  Based upon the
performance of  the mass spectrometer, the error between the observed
and
calculated masses
was limited to 5 ppm or less.  MS/MS
analysis indicated the presence of two trifluoromethyl groups, so the number of
atoms of F required was set to six.
MS/MS analysis also indicated the presence of two acetyl groups, so the
minimum number of atoms of O required was set to two.  Consideration of the synthetic pathway for
AMG 517  suggested
that a
molecule containing less than four atoms of N was unlikely.  All elemental composition analyses performed
as part of this investigation utilized a similar strategy to logically identify
the most likely elemental formula for an observed mass.The elemental composition analysis for the observed mass of Unknown 1
determined that the elemental formulaC40H24N8O4F6S2 was the best fit for the impurity.  This
elemental composition was consistent with a dimer of AMG 517 minus two hydrogens
(elemental formula C20H13N4O2F3S).
The mass error between the observed mass
for Unknown 1 and the calculated mass for a dimer of AMG 517 was 0.3 ppm.An accurate mass of 891.1058 Da
was determined for Unknown 2 in the enriched fraction.  Elemental composition analysis using this
protonated mass determined that the elemental formulaC40H24N8O4F6S3 was the best fit for the impurity.  This
elemental composition was consistent with a dimer of AMG 517 with the addition of
a sulfur atom [dimer+S].  The mass error
between the observed mass for Unknown 2 and the calculated mass for [dimer+S] was
0.8 ppm.Another possible elemental
formula for Unknown 2 isC40H24N8O6F6S2 which corresponded to an AMG 517 dimer with two additional oxygen atoms [dimer+2O].
The mass error between the observed mass
for Unknown 2 and the calculated mass for the [dimer+2O] was 20.7 ppm.  Based on the accurate mass data, it was
concluded that [dimer+S] was a more likely structure for Unknown 2.The MS data was consistent with
dimeric structures for both Unknown 1 and Unknown 2 but provided no definitive
structural linkage information.  The
structure of AMG 517 itself and the synthetic scheme shown in
Figure2 did not
provide any obvious possible point of linkage.
Therefore, NMR analyses were performed on the enriched fraction to help
elucidating the structures of these impurities.


### 3.3. NMR

AMG 517 and its enriched impurity fraction containing
Unknowns 1 and 2 were first analyzed byH1 and C13 NMR to further investigate the connectivity.  Proton assignments were made based on
chemical shifts, proton-proton coupling  constants, and COSY and NOESY spectra (see
Tables 2 and 3). Carbon assignments were based on chemical shifts,
carbon-fluorine coupling constants, and HMQC, HSQC, and HMBC spectra (see Tables
2 and 3).  All assignments referring to
the structures of AMG 517 and impurities are depicted in these two tables.Table 2
H1 and C13 chemical shifts (δ/ppm) of AMG 517 standard in DMSO-d6 (400 MHz).
PositionH1
(δ/ppm, J/Hz)(a)C13
(δ/ppm)(a)1130.9 (q,JC–F = 31.9 Hz)2, 67.92 (d, 2H, J = 8.2 Hz)(b)125.9 (q,JC–F = 3.7 Hz)3, 58.44 (d, 2H, J = 8.2 Hz)128.04139.77163.498.79 (s, 1H)158.6(e)11170.3(d)127.97 (s, 1H)104.213124.0 (q,JC–F = 272.2 Hz)15143.5167.35 (m, 1H)(c)119.1177.39 (t, 1H, J = 7.7 Hz)(c)124.2187.93 (m, 1H)(b)119.619133.620141.422158.4(e)2412.42 (s, 1H)25169.5(d)262.13 (s, 3H)22.6(a)Signal splitting patterns: s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet; (b), (c)overlapping; (d), (e)interchangeable assignment.Table 3
H1
and C13 chemical shifts (δ/ppm) of the enriched impurity fraction
in DMSO-d6.
H1
(δ/ppm,
J/Hz)(a)C13
(δ/ppm)(a)PositionUnknown 2Unknown 1Unknown 2Unknown 11131.0 (q,J2C–F =
31.7 Hz)131.0 (q,J2C–F =
31.7 Hz)2, 67.92 (d,
2H, J = 8.3 Hz)7.95 (d,
2H, J = 8.3 Hz)125.9125.93, 58.45 (d,
2H, J = 8.3 Hz)8.50 (d,
2H, J = 8.3 Hz)128.0128.04139.6139.67163.6163.598.81 (s,
1H)8.89 (s,
1H)158.5158.6(b)11170.1170.2128.00 (s,
1H)8.11 (s,
1H)104.2104.413124.0 (q,J1C–F =
273.2 Hz)124.0 (q,J1C–F =
273.2 Hz)15143.7143.5167.45 (d,
1H, J = 8.2 Hz)7.59 (d,
1H, J = 8.0 Hz)120.4119.9177.49 (d,
1H, J = 8.2 Hz)7.68 (d,
1H, J = 8.0 Hz)128.1123.918122.6131.219136.6132.820141.6141.922158.5158.4(b)2412.54 (s, 1H)12.55 (s, 1H)25169.7169.7262.12 (s,
3H)2.13 (s,
3H)22.522.6(a)Signal splitting pattern: s = singlet, d = doublet, q = quartet; (b)interchangeable assignments.NMR analysis was also conducted on AMG 517 for
comparison (see Table2).  The H1
NMR spectrum showed the presence of all the protons of the molecule including
the exchangeable NH proton. The H1 NMR spectrum showed the presence
of three aromatic systems, an AA’BB’ spin system (δ 7.92 and 8.44 ppm) for a p-disubstituted benzene ring, two singlets (δ 7.97 and 8.79 ppm) for another aromatic ring, and an ABX spin system (δ 7.35, 7.39, and
7.93 ppm) for a 1,2,3-trisubstituted benzene ring.  The downfield chemical shift of the singlet
at 8.79 ppm together with the singlet at 7.97 ppm suggested a 4,6-disubstituted
pyrimidine as one of the aromatic rings in the molecule.  The C13 NMR spectrum showed the
presence of all the carbons of the molecule. Three of these carbons were coupled
to F19; C-13 as a quartet through one C–F bond (δ 124.0, J1[C13, F19] = 272.2 Hz),
C-1 as a quartet through one C–C and one C–F bonds (δ 130.9, J2[C13, F19] = 31.9 Hz),
and C-2, 6 as a quartet  through two C–C
and one C–F bonds (δ 125.9, J3[C13, F19] = 3.7 Hz)
(see Table 2).TheH1 NMR spectrum of the enriched fraction containing the
impurities indicated that the sample was a mixture of two components
structurally related to AMG 517, present at a ratio of 1:1.94 based on the
areas of their related aromatic signals.
Based on the HPLC-UV data from the enriched fraction, the major
component present corresponded to Unknown 2, and the minor component to Unknown
1.  The H1 NMR spectrum of the
impurities contained signals corresponding to the same substitution patterns
observed for AMG 517 (see Figure 3). H1
NMR and H1,
H1-2D NOESY spectra indicated the presence of
a p-disubstituted benzene ring, a
4,6-disubstituted pyrimidine, a 2,4,7-trisubstituted benzothiazole ring, and an
N-acetyl group.  The only difference
between AMG 517 and these two related compounds is the substitution pattern of
the benzothiazole.   The H1 NMR spectrum showed more
distinct chemical shift differences for the protons H-16 and H-17 from these
two AMG 517-related compounds (see Figure 3 and
Table 4).  The signals from Unknown 1 were shifted
downfield compared to Unknown 2.  The elemental molecular formulae for Unknowns 1 and 2 were indicative of dimer structures.  Only one set of resonances was observed for
each of the two unknowns. This indicated that the unknowns were symmetrical
dimers.  The
monomers were connected through carbon C-18 based on the presence of an AB
system, their chemical shifts, and the coupling constants for the benzothiazole
ring.  The H1, C13-2D
HSQC spectrum supported the H1 NMR data showing only two aromatic
C–H (C-16 and 17) on the benzothiazole ring of the impurities. The absence of a
C–H signal for C-18, as was observed in AMG 517, was noted in the NMR spectra
in both of the unknowns (see Figure 4). C13 NMR, H1, C13-2D
HSQC, and H1, C13-2D HMBC spectra of the impurities showed
more distinct chemical shift differences for the carbons C-16, C-17, C-18, and
C-19.  This indicated that the difference
between these two impurities was in the linkage through C-18, either directly
or through a heteroatom (see Table 4).  The
possibility of having a sulfur atom connecting the two AMG 517 monomers for Unknown
2 was considered very plausible based on the MS data and the chemical shift
data (see Table 4).Table 4
PartialH1 and C13
chemical shifts (δ /ppm) of the benzothiazole ring for AMG 517 and the enriched impurity
fraction in DMSO-d6.
H1  (δ/ppm,  J/Hz)C13
(δ/ppm)PositionAMG 517(a)Unknown 2(b)Unknown 1(b)AMG  517(a)Unknown 2(b)Unknown 1(b)167.35 (m, 1H)(c)7.45 (d, 1H, J = 8.2 Hz)7.59 (d, 1H, J = 8.0 Hz)119.1120.4119.9177.39 (t, 1H, J = 7.7 Hz)(c)7.49 (d, 1H, J = 8.2 Hz)7.68 (d, 1H, J = 8.0 Hz)124.2128.1123.9187.93 (m, 1H)119.6122.6131.219133.6136.6132.8(a)Data
from 400 MHz NMR instrument; (b)data from 600 MHz instrument;
(c)overlapping signals.Aromatic region of theH1
NMR spectra of the enriched impurity fraction ((a) 600 MHz) and AMG 517 ((b)
400 MHz) in DMSO-d6. In (a), numbers designated as prime (e.g., 3′)
represent signals of Unknown 1, with all others representing signals of Unknown
2.
(a)(b)Aromatic region of theH1, C13-2D
HSQC spectrum of the enriched impurity fraction ((a) 600 MHz) and the H1, C13-2D
HMQC spectrum of AMG 517 ((b) 400 MHz) in DMSO-d6.  In (a), numbers
designated as prime (e.g., 3′) represent signals of Unknown 1, with all
others representing signals of Unknown 2.
(a)(b)


### 3.4. LC-MS Analysis of the Hydrolysate of the Enriched Impurities

The MS data for the two impurities strongly supported
a thioether-linked dimer of AMG 517 as the structure for Unknown 2. TheH1
and C13 NMR data provided
indirect evidence of such thioether linkage but could not afford direct
measurement of the heteroatom. However, the formation of this impurity in the
synthesis of
AMG 517 (see Figure 2) did not seem
as plausible as  the oxidation
of a heteroatom from a reaction mechanistic standpoint.  There was a significant difference between the
calculated mass values for the two potential structures for Unknown 2, however,
the relatively high mass of the impurity resulted in a large number of
potential elemental formulae.  To simplify
the elemental composition analysis, a chemical degradation experiment was
performed.  The enriched fraction was
treated with 0.5 equivalent of aqueous HCl in DMSO-d6 and heated overnight at 70°C.  This
experiment  furnished
low-molecular-weight fragments
of the impurity that could not be generated via MS/MS. These low-mass fragments
resulted in a small number of potential elemental formulae for each observed
mass.Multiple hydrolysis fragments
were observed in LC-MS after forced degradation of the enriched fraction with
hydrochloric acid (see Figure5).  Accurate
mass data collected in the LC-MS analysis of the acid treated enriched impurity
fraction was used to identify peaks corresponding to the expected hydrolysis
fragments (see Figure 5).  The scheme in Figure
6 shows the expected acid hydrolysis fragments from Unknown 2, with Unknown 2
and its fragments  presented using both the thioether and bis-sulfoxide structures
being considered for the impurity. A number of deacetylation products were also
observed.  This LC-MS analysis
demonstrated that all of the expected fragments for Unknown 2 (and some for
Unknown 1) were formed during the forced degradation.Figure 5
UV chromatogram from the LC-MS analysis of
the acid hydrolyzed impurities.  Labeled
peaks correspond to hydrolysis fragments of Unknown 1 (U1) and Unknown 2 (U2).Figure 6
Potential fragments produced
by acid hydrolysis of Unknown 2. Structures on the left represent fragments
expected to be generated from the thioether, those on the right from the bis-sulfoxide.  Fragment 241 would be common to both
structures.Table5 shows the accurate mass assignments for the acid
hydrolysis products of Unknown 2 as well as the calculated exact mass for each product
that was expected to arise from both the proposed thioether and bis-sulfoxide
structures. The mass error (observed mass versus calculated mass of the
hydrolysis fragments) is shown for each proposed structure for Unknown 2. The
mass error range for hydrolysis products arising from the thioether structure was
0 to 3.5 ppm; the mass error range for the corresponding bis-sulfoxide was 17.5
to 40.7 ppm. Thus, the accurate mass data collected for the acid hydrolysis fragments
allowed for elimination of the bis-sulfoxide as a potential structure for
Unknown 2.Table 5
Mass
error analysis of the observed accurate mass for fragments generated by the
acid hydrolysis of the enriched impurity fraction. The analysis is conducted
for both the thioether and bis-sulfoxide structures proposed for Unknown 2.
(ND: not detected; N/A: not applicable.)
Obs.
mass
(M
+ H)+
(Da)Calc.
mass
thioether
(M
+ H)+
(Da)Calc.
mass
bis-sulfoxide
(M
+ H)+
(Da)Mass
error
thioether   (ppm)Mass
error
bis-
sulfoxide
(ppm)Unknown 2891.1081891.1060891.12372.417.5Mono-deacetyl849.0967849.0954849.11321.519.4Bis-deacetyl807.0874807.0848807.10263.218.8U2-669669.0674669.0655669.08322.823.6Mono-deacetyl627.0552627.0549627.07270.527.9627.05581.427.0Bis-deacetyl585.0458585.0443585.06212.627.9U2-447447.0250447.0250447.04280.039.8Mono-deacetyl405.0158405.0144405.03223.540.5405.01573.240.7Bis-deacetylND363.0039363.0216N/AN/A241241.0579241.0583241.05831.71.7These mass error results
strongly supported an elemental formula ofC40H24F6N8O4S3 (thioether) for Unknown 2, and essentially ruled out an elemental formula of C40H24F6N8O6S2
(bis-sulfoxide) for the impurity.


### 3.5. LC-MS Analysis of N-Acetyl Benzothiazole Starting Material

Although the MS and NMR data
provided great confidence in the proposed dimeric structures for these two
late-eluting impurities, the chemical reactions described in
Figure2 were not
likely to generate such impurities.
Since the dimeric linkages are in the benzothiazole portion of AMG 517,
it was possible that these two impurities were originated from existing
impurities in the AMG 517 starting material, N-acetyl benzothiazole, which was
prepared via multistep synthesis from 2-methoxybenzenamine by a contract
manufacturer.  To determine if N-acetyl
benzothiazole was a potential source for generating Unknowns 1 and 2, additional experiments
were performed to evaluate the impurity profiles of N-acetyl benzothiazole.A different HPLC method was developed
for the analysis of N-acetyl benzothiazole (see
Table1).  Although the supplier’s Certificate of
Analysis indicated an HPLC purity of >99% area for various batches of N-acetyl
benzothiazole, retrospective analysis by Amgen’s HPLC method resulted in
purities ranging from 96.2 to 98.2% area.
LC-MS analysis was performed on lot A, the starting material used in the
production of the six kilogram-scale AMG 517 batches.  Analysis of this lot indicated that there were
multiple impurities, some  of which had the potential to generate Unknowns 1 and
2 (see Figure 7).  These impurities were
designated by their nominal mass values as determined by the LC-MS analysis (e.g., MS447 corresponds to a compound
with an observed mass of 447 Da).
Table 6 provides a summary of the proposed structures for the observed impurities of N-acetyl
benzothiazole.Table 6
Summary of proposed structures of impurities
observed in the LC-MS analysis of N-acetyl benzothiazole.Figure 7
Expanded view of the UV chromatogram from the
LC-MS analysis of N-acetyl benzothiazole lot A (ca. 0.1 mg/mL in 10% ACN,3μg injected on column).An accurate mass of 447.0247 Da
was determined for the protonated ion of impurity MS447 in N-acetyl
benzothiazole.  Elemental composition
analysis using the observed mass determined that the elemental formulaC18H14N4O4S3 was the best fit for this impurity. This elemental formula, along with MS/MS
analysis  of MS447 (see Figure 8), supported a thioether linked dimer of benzothiazole
as the structure for MS447 (see Table 6).
This symmetrical thioether compound could participate in the same
reaction as AMG 517 step 2 to generate Unknown 2 (see
Figure 9).(a) MS/MS analysis (with accurate mass
determination) of the protonated ion of MS447.
(b) Schematic of the MS/MS fragmentation interpretation of MS447.
(a)(b)Figure 9
Proposed structures and
formation pathway for the two unknown impurities in AMG 517.An accurate mass of 405.0144 Da
was determined for the protonated ion of impurity MS405.  Elemental composition analysis using the observed
mass determined that the elemental formulaC16H12N4O3S3 was the best fit for the impurity.  MS405
was proposed to be the mono-deacetylated
form of MS447.Accurate mass determination for each of the
three peaks designated MS415 led to the assignment of exact mass values that were
in close agreement with each other (415.0522 Da, 415.0540 Da, and 415.0536 Da,
in order of elution), and elemental composition analysis using these observed
mass values points to the same elemental composition (C18H14N4O4S2)
as the most likely formula for each.  These three MS415 impurities in N-acetyl
benzothiazole could be positional isomers to each other.  One of these isomers, a symmetrical ortho
dimer (see Table 6), was a plausible precursor for the proposed structure of Unknown
1 (see Figure 9).Accurate mass determination for each of the
two peaks designated MS287 led to the assignment of exact mass values that are
in close agreement with each other (286.9486 Da and 286.9475 Da, in order of
elution), and elemental composition analysis using these observed mass values
points to the same elemental composition (C9H7N2O2SBr)
as the most likely formula for each.  Structures
consistent with these elemental formulae are shown in
Table 6.  The presence of these molecules in the benzothiazole
synthetic process could lead to the formation of impurities MS415 and MS447.


## 3.1. Impurity Profiles in Kilogram-Scale Batches of AMG 517 Drug Substance

A stability
indicating HPLC-UV method was developed to separate and quantify AMG 517 along
with its potential impurities and possible degradants.
Figure1(a) represents a typical separation of
a standard mixture of AMG 517 in the presence of its known impurities and
degradants.  This method was used to
analyze the first six drug substance batches of AMG 517 during release and
stability testing of these lots.  A pair
of unexpected late-eluting unknown impurities was observed in all six batches
of AMG 517.  The area percent levels of impurity
Unknown 1 ranged from 0.15% to 0.44%, while Unknown 2 ranged from 0.06% to
0.21%. A representative chromatogram of an
AMG 517 drug substance lot containing these impurities is shown in Figure 1(b). These two
impurities were not detected at a reportable level in the
previous small-scale batches of AMG 517. Since these two unknown impurities eluted near
the retention time of the by-product in step 1 of the AMG 517 synthetic
reaction, it was concluded that these new impurities were highly hydrophobic and
may have structural features similar to the by-product (see Figure 2).Preliminary low-resolution LC-MS analysis on the drug substance provided
molecular mass and tandem mass spectrometry (MS/MS) fragment ion information
for these two impurities (data not shown).
The observed mass for the protonated Unknown 1 and Unknown 2 was 859 Da and 891 Da, respectively.  Since
the exact mass for AMG 517 is 430.0711 Da, an observed mass of 859 Da for Unknown 1 suggested that it
could be some sort of dimeric structure related to  AMG 517.  MS/MS data also suggested dimeric structures for
both impurities. Fragment ions that corresponded to the neutral loss of
multiple acetyl and hydrofluoric functional groups were observed in MS/MS
experiments performed on the protonated ions of both Unknown 1 and Unknown 2.  The mass difference between the two unknowns was
32 Da, which could be
attributed to either one additional sulfur atom or two additional oxygen atoms
in Unknown 2 relative to Unknown 1.  However,
the preliminary LC-MS analysis alone could not conclusively identify the structures
of these impurities due to the possible existence of multiple isomeric structures
consistent with the mass data.  To aid in
the structural elucidation efforts, an enriched fraction of these two
impurities was isolated via preparative-scale HPLC.  The isolated fraction contained about 35% of Unknown
1 and 62% of Unknown 2 based on UV detection at 254 nm.  LC-MS and NMR experiments were performed to characterize
this enriched fraction.


## 3.2. Accurate Mass Determination for Unknowns 1 and 2 in the Enriched Fraction

An accurate mass of 859.1342 Da was determined for Unknown 1 in the
enriched fraction.  Elemental composition
analysis was performed for this protonated mass. Instrument performance, the
synthetic pathway for AMG 517, and information gained from the preliminary
LC-MS analysis of the impurities were taken into account in setting parameters
for this analysis.  Based upon the
performance of  the mass spectrometer, the error between the observed
and
calculated masses
was limited to 5 ppm or less.  MS/MS
analysis indicated the presence of two trifluoromethyl groups, so the number of
atoms of F required was set to six.
MS/MS analysis also indicated the presence of two acetyl groups, so the
minimum number of atoms of O required was set to two.  Consideration of the synthetic pathway for
AMG 517  suggested
that a
molecule containing less than four atoms of N was unlikely.  All elemental composition analyses performed
as part of this investigation utilized a similar strategy to logically identify
the most likely elemental formula for an observed mass.The elemental composition analysis for the observed mass of Unknown 1
determined that the elemental formulaC40H24N8O4F6S2 was the best fit for the impurity.  This
elemental composition was consistent with a dimer of AMG 517 minus two hydrogens
(elemental formula C20H13N4O2F3S).
The mass error between the observed mass
for Unknown 1 and the calculated mass for a dimer of AMG 517 was 0.3 ppm.An accurate mass of 891.1058 Da
was determined for Unknown 2 in the enriched fraction.  Elemental composition analysis using this
protonated mass determined that the elemental formulaC40H24N8O4F6S3 was the best fit for the impurity.  This
elemental composition was consistent with a dimer of AMG 517 with the addition of
a sulfur atom [dimer+S].  The mass error
between the observed mass for Unknown 2 and the calculated mass for [dimer+S] was
0.8 ppm.Another possible elemental
formula for Unknown 2 isC40H24N8O6F6S2 which corresponded to an AMG 517 dimer with two additional oxygen atoms [dimer+2O].
The mass error between the observed mass
for Unknown 2 and the calculated mass for the [dimer+2O] was 20.7 ppm.  Based on the accurate mass data, it was
concluded that [dimer+S] was a more likely structure for Unknown 2.The MS data was consistent with
dimeric structures for both Unknown 1 and Unknown 2 but provided no definitive
structural linkage information.  The
structure of AMG 517 itself and the synthetic scheme shown in
Figure2 did not
provide any obvious possible point of linkage.
Therefore, NMR analyses were performed on the enriched fraction to help
elucidating the structures of these impurities.


## 3.3. NMR

AMG 517 and its enriched impurity fraction containing
Unknowns 1 and 2 were first analyzed byH1 and C13 NMR to further investigate the connectivity.  Proton assignments were made based on
chemical shifts, proton-proton coupling  constants, and COSY and NOESY spectra (see
Tables 2 and 3). Carbon assignments were based on chemical shifts,
carbon-fluorine coupling constants, and HMQC, HSQC, and HMBC spectra (see Tables
2 and 3).  All assignments referring to
the structures of AMG 517 and impurities are depicted in these two tables.Table 2
H1 and C13 chemical shifts (δ/ppm) of AMG 517 standard in DMSO-d6 (400 MHz).
PositionH1
(δ/ppm, J/Hz)(a)C13
(δ/ppm)(a)1130.9 (q,JC–F = 31.9 Hz)2, 67.92 (d, 2H, J = 8.2 Hz)(b)125.9 (q,JC–F = 3.7 Hz)3, 58.44 (d, 2H, J = 8.2 Hz)128.04139.77163.498.79 (s, 1H)158.6(e)11170.3(d)127.97 (s, 1H)104.213124.0 (q,JC–F = 272.2 Hz)15143.5167.35 (m, 1H)(c)119.1177.39 (t, 1H, J = 7.7 Hz)(c)124.2187.93 (m, 1H)(b)119.619133.620141.422158.4(e)2412.42 (s, 1H)25169.5(d)262.13 (s, 3H)22.6(a)Signal splitting patterns: s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet; (b), (c)overlapping; (d), (e)interchangeable assignment.Table 3
H1
and C13 chemical shifts (δ/ppm) of the enriched impurity fraction
in DMSO-d6.
H1
(δ/ppm,
J/Hz)(a)C13
(δ/ppm)(a)PositionUnknown 2Unknown 1Unknown 2Unknown 11131.0 (q,J2C–F =
31.7 Hz)131.0 (q,J2C–F =
31.7 Hz)2, 67.92 (d,
2H, J = 8.3 Hz)7.95 (d,
2H, J = 8.3 Hz)125.9125.93, 58.45 (d,
2H, J = 8.3 Hz)8.50 (d,
2H, J = 8.3 Hz)128.0128.04139.6139.67163.6163.598.81 (s,
1H)8.89 (s,
1H)158.5158.6(b)11170.1170.2128.00 (s,
1H)8.11 (s,
1H)104.2104.413124.0 (q,J1C–F =
273.2 Hz)124.0 (q,J1C–F =
273.2 Hz)15143.7143.5167.45 (d,
1H, J = 8.2 Hz)7.59 (d,
1H, J = 8.0 Hz)120.4119.9177.49 (d,
1H, J = 8.2 Hz)7.68 (d,
1H, J = 8.0 Hz)128.1123.918122.6131.219136.6132.820141.6141.922158.5158.4(b)2412.54 (s, 1H)12.55 (s, 1H)25169.7169.7262.12 (s,
3H)2.13 (s,
3H)22.522.6(a)Signal splitting pattern: s = singlet, d = doublet, q = quartet; (b)interchangeable assignments.NMR analysis was also conducted on AMG 517 for
comparison (see Table2).  The H1
NMR spectrum showed the presence of all the protons of the molecule including
the exchangeable NH proton. The H1 NMR spectrum showed the presence
of three aromatic systems, an AA’BB’ spin system (δ 7.92 and 8.44 ppm) for a p-disubstituted benzene ring, two singlets (δ 7.97 and 8.79 ppm) for another aromatic ring, and an ABX spin system (δ 7.35, 7.39, and
7.93 ppm) for a 1,2,3-trisubstituted benzene ring.  The downfield chemical shift of the singlet
at 8.79 ppm together with the singlet at 7.97 ppm suggested a 4,6-disubstituted
pyrimidine as one of the aromatic rings in the molecule.  The C13 NMR spectrum showed the
presence of all the carbons of the molecule. Three of these carbons were coupled
to F19; C-13 as a quartet through one C–F bond (δ 124.0, J1[C13, F19] = 272.2 Hz),
C-1 as a quartet through one C–C and one C–F bonds (δ 130.9, J2[C13, F19] = 31.9 Hz),
and C-2, 6 as a quartet  through two C–C
and one C–F bonds (δ 125.9, J3[C13, F19] = 3.7 Hz)
(see Table 2).TheH1 NMR spectrum of the enriched fraction containing the
impurities indicated that the sample was a mixture of two components
structurally related to AMG 517, present at a ratio of 1:1.94 based on the
areas of their related aromatic signals.
Based on the HPLC-UV data from the enriched fraction, the major
component present corresponded to Unknown 2, and the minor component to Unknown
1.  The H1 NMR spectrum of the
impurities contained signals corresponding to the same substitution patterns
observed for AMG 517 (see Figure 3). H1
NMR and H1,
H1-2D NOESY spectra indicated the presence of
a p-disubstituted benzene ring, a
4,6-disubstituted pyrimidine, a 2,4,7-trisubstituted benzothiazole ring, and an
N-acetyl group.  The only difference
between AMG 517 and these two related compounds is the substitution pattern of
the benzothiazole.   The H1 NMR spectrum showed more
distinct chemical shift differences for the protons H-16 and H-17 from these
two AMG 517-related compounds (see Figure 3 and
Table 4).  The signals from Unknown 1 were shifted
downfield compared to Unknown 2.  The elemental molecular formulae for Unknowns 1 and 2 were indicative of dimer structures.  Only one set of resonances was observed for
each of the two unknowns. This indicated that the unknowns were symmetrical
dimers.  The
monomers were connected through carbon C-18 based on the presence of an AB
system, their chemical shifts, and the coupling constants for the benzothiazole
ring.  The H1, C13-2D
HSQC spectrum supported the H1 NMR data showing only two aromatic
C–H (C-16 and 17) on the benzothiazole ring of the impurities. The absence of a
C–H signal for C-18, as was observed in AMG 517, was noted in the NMR spectra
in both of the unknowns (see Figure 4). C13 NMR, H1, C13-2D
HSQC, and H1, C13-2D HMBC spectra of the impurities showed
more distinct chemical shift differences for the carbons C-16, C-17, C-18, and
C-19.  This indicated that the difference
between these two impurities was in the linkage through C-18, either directly
or through a heteroatom (see Table 4).  The
possibility of having a sulfur atom connecting the two AMG 517 monomers for Unknown
2 was considered very plausible based on the MS data and the chemical shift
data (see Table 4).Table 4
PartialH1 and C13
chemical shifts (δ /ppm) of the benzothiazole ring for AMG 517 and the enriched impurity
fraction in DMSO-d6.
H1  (δ/ppm,  J/Hz)C13
(δ/ppm)PositionAMG 517(a)Unknown 2(b)Unknown 1(b)AMG  517(a)Unknown 2(b)Unknown 1(b)167.35 (m, 1H)(c)7.45 (d, 1H, J = 8.2 Hz)7.59 (d, 1H, J = 8.0 Hz)119.1120.4119.9177.39 (t, 1H, J = 7.7 Hz)(c)7.49 (d, 1H, J = 8.2 Hz)7.68 (d, 1H, J = 8.0 Hz)124.2128.1123.9187.93 (m, 1H)119.6122.6131.219133.6136.6132.8(a)Data
from 400 MHz NMR instrument; (b)data from 600 MHz instrument;
(c)overlapping signals.Aromatic region of theH1
NMR spectra of the enriched impurity fraction ((a) 600 MHz) and AMG 517 ((b)
400 MHz) in DMSO-d6. In (a), numbers designated as prime (e.g., 3′)
represent signals of Unknown 1, with all others representing signals of Unknown
2.
(a)(b)Aromatic region of theH1, C13-2D
HSQC spectrum of the enriched impurity fraction ((a) 600 MHz) and the H1, C13-2D
HMQC spectrum of AMG 517 ((b) 400 MHz) in DMSO-d6.  In (a), numbers
designated as prime (e.g., 3′) represent signals of Unknown 1, with all
others representing signals of Unknown 2.
(a)(b)


## 3.4. LC-MS Analysis of the Hydrolysate of the Enriched Impurities

The MS data for the two impurities strongly supported
a thioether-linked dimer of AMG 517 as the structure for Unknown 2. TheH1
and C13 NMR data provided
indirect evidence of such thioether linkage but could not afford direct
measurement of the heteroatom. However, the formation of this impurity in the
synthesis of
AMG 517 (see Figure 2) did not seem
as plausible as  the oxidation
of a heteroatom from a reaction mechanistic standpoint.  There was a significant difference between the
calculated mass values for the two potential structures for Unknown 2, however,
the relatively high mass of the impurity resulted in a large number of
potential elemental formulae.  To simplify
the elemental composition analysis, a chemical degradation experiment was
performed.  The enriched fraction was
treated with 0.5 equivalent of aqueous HCl in DMSO-d6 and heated overnight at 70°C.  This
experiment  furnished
low-molecular-weight fragments
of the impurity that could not be generated via MS/MS. These low-mass fragments
resulted in a small number of potential elemental formulae for each observed
mass.Multiple hydrolysis fragments
were observed in LC-MS after forced degradation of the enriched fraction with
hydrochloric acid (see Figure5).  Accurate
mass data collected in the LC-MS analysis of the acid treated enriched impurity
fraction was used to identify peaks corresponding to the expected hydrolysis
fragments (see Figure 5).  The scheme in Figure
6 shows the expected acid hydrolysis fragments from Unknown 2, with Unknown 2
and its fragments  presented using both the thioether and bis-sulfoxide structures
being considered for the impurity. A number of deacetylation products were also
observed.  This LC-MS analysis
demonstrated that all of the expected fragments for Unknown 2 (and some for
Unknown 1) were formed during the forced degradation.Figure 5
UV chromatogram from the LC-MS analysis of
the acid hydrolyzed impurities.  Labeled
peaks correspond to hydrolysis fragments of Unknown 1 (U1) and Unknown 2 (U2).Figure 6
Potential fragments produced
by acid hydrolysis of Unknown 2. Structures on the left represent fragments
expected to be generated from the thioether, those on the right from the bis-sulfoxide.  Fragment 241 would be common to both
structures.Table5 shows the accurate mass assignments for the acid
hydrolysis products of Unknown 2 as well as the calculated exact mass for each product
that was expected to arise from both the proposed thioether and bis-sulfoxide
structures. The mass error (observed mass versus calculated mass of the
hydrolysis fragments) is shown for each proposed structure for Unknown 2. The
mass error range for hydrolysis products arising from the thioether structure was
0 to 3.5 ppm; the mass error range for the corresponding bis-sulfoxide was 17.5
to 40.7 ppm. Thus, the accurate mass data collected for the acid hydrolysis fragments
allowed for elimination of the bis-sulfoxide as a potential structure for
Unknown 2.Table 5
Mass
error analysis of the observed accurate mass for fragments generated by the
acid hydrolysis of the enriched impurity fraction. The analysis is conducted
for both the thioether and bis-sulfoxide structures proposed for Unknown 2.
(ND: not detected; N/A: not applicable.)
Obs.
mass
(M
+ H)+
(Da)Calc.
mass
thioether
(M
+ H)+
(Da)Calc.
mass
bis-sulfoxide
(M
+ H)+
(Da)Mass
error
thioether   (ppm)Mass
error
bis-
sulfoxide
(ppm)Unknown 2891.1081891.1060891.12372.417.5Mono-deacetyl849.0967849.0954849.11321.519.4Bis-deacetyl807.0874807.0848807.10263.218.8U2-669669.0674669.0655669.08322.823.6Mono-deacetyl627.0552627.0549627.07270.527.9627.05581.427.0Bis-deacetyl585.0458585.0443585.06212.627.9U2-447447.0250447.0250447.04280.039.8Mono-deacetyl405.0158405.0144405.03223.540.5405.01573.240.7Bis-deacetylND363.0039363.0216N/AN/A241241.0579241.0583241.05831.71.7These mass error results
strongly supported an elemental formula ofC40H24F6N8O4S3 (thioether) for Unknown 2, and essentially ruled out an elemental formula of C40H24F6N8O6S2
(bis-sulfoxide) for the impurity.


## 3.5. LC-MS Analysis of N-Acetyl Benzothiazole Starting Material

Although the MS and NMR data
provided great confidence in the proposed dimeric structures for these two
late-eluting impurities, the chemical reactions described in
Figure2 were not
likely to generate such impurities.
Since the dimeric linkages are in the benzothiazole portion of AMG 517,
it was possible that these two impurities were originated from existing
impurities in the AMG 517 starting material, N-acetyl benzothiazole, which was
prepared via multistep synthesis from 2-methoxybenzenamine by a contract
manufacturer.  To determine if N-acetyl
benzothiazole was a potential source for generating Unknowns 1 and 2, additional experiments
were performed to evaluate the impurity profiles of N-acetyl benzothiazole.A different HPLC method was developed
for the analysis of N-acetyl benzothiazole (see
Table1).  Although the supplier’s Certificate of
Analysis indicated an HPLC purity of >99% area for various batches of N-acetyl
benzothiazole, retrospective analysis by Amgen’s HPLC method resulted in
purities ranging from 96.2 to 98.2% area.
LC-MS analysis was performed on lot A, the starting material used in the
production of the six kilogram-scale AMG 517 batches.  Analysis of this lot indicated that there were
multiple impurities, some  of which had the potential to generate Unknowns 1 and
2 (see Figure 7).  These impurities were
designated by their nominal mass values as determined by the LC-MS analysis (e.g., MS447 corresponds to a compound
with an observed mass of 447 Da).
Table 6 provides a summary of the proposed structures for the observed impurities of N-acetyl
benzothiazole.Table 6
Summary of proposed structures of impurities
observed in the LC-MS analysis of N-acetyl benzothiazole.Figure 7
Expanded view of the UV chromatogram from the
LC-MS analysis of N-acetyl benzothiazole lot A (ca. 0.1 mg/mL in 10% ACN,3μg injected on column).An accurate mass of 447.0247 Da
was determined for the protonated ion of impurity MS447 in N-acetyl
benzothiazole.  Elemental composition
analysis using the observed mass determined that the elemental formulaC18H14N4O4S3 was the best fit for this impurity. This elemental formula, along with MS/MS
analysis  of MS447 (see Figure 8), supported a thioether linked dimer of benzothiazole
as the structure for MS447 (see Table 6).
This symmetrical thioether compound could participate in the same
reaction as AMG 517 step 2 to generate Unknown 2 (see
Figure 9).(a) MS/MS analysis (with accurate mass
determination) of the protonated ion of MS447.
(b) Schematic of the MS/MS fragmentation interpretation of MS447.
(a)(b)Figure 9
Proposed structures and
formation pathway for the two unknown impurities in AMG 517.An accurate mass of 405.0144 Da
was determined for the protonated ion of impurity MS405.  Elemental composition analysis using the observed
mass determined that the elemental formulaC16H12N4O3S3 was the best fit for the impurity.  MS405
was proposed to be the mono-deacetylated
form of MS447.Accurate mass determination for each of the
three peaks designated MS415 led to the assignment of exact mass values that were
in close agreement with each other (415.0522 Da, 415.0540 Da, and 415.0536 Da,
in order of elution), and elemental composition analysis using these observed
mass values points to the same elemental composition (C18H14N4O4S2)
as the most likely formula for each.  These three MS415 impurities in N-acetyl
benzothiazole could be positional isomers to each other.  One of these isomers, a symmetrical ortho
dimer (see Table 6), was a plausible precursor for the proposed structure of Unknown
1 (see Figure 9).Accurate mass determination for each of the
two peaks designated MS287 led to the assignment of exact mass values that are
in close agreement with each other (286.9486 Da and 286.9475 Da, in order of
elution), and elemental composition analysis using these observed mass values
points to the same elemental composition (C9H7N2O2SBr)
as the most likely formula for each.  Structures
consistent with these elemental formulae are shown in
Table 6.  The presence of these molecules in the benzothiazole
synthetic process could lead to the formation of impurities MS415 and MS447.


## 4. Conclusion

An extensive investigation successfully utilized multiple analytical
disciplines to elucidate structures for two complex impurities in AMG 517 drug
substance and to trace the source of the impurities to a starting material used
in the manufacture of AMG 517.The structures of two unknown impurities in AMG 517 drug substance were
identified through extensive HPLC, LC-MS, high resolution MS, MS/MS, and 1D and
2D NMR studies.  The existence of an unexpected
C-S-C linkage in one of the impurities was confirmed.  Further investigation revealed that these
impurities originated from existing impurities in the N-acetyl benzothiazole
starting material used in AMG 517 synthesis.
This information was shared with the supplier of this starting material,
and the process for N-acetyl benzothiazole preparation was re-evaluated.  Better synthetic process controls and tighter
specifications were established resulting in higher quality N-acetyl
benzothiazole batches.  These two dimeric
impurities were not observed in subsequent larger-scale AMG 517 production runs.



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*Source: 768743-2008-12-17.xml*