Article

pubs.acs.org/OPRD

Applying QbD Principles To Develop a Generic UHPLC Method

Which Facilitates Continual Improvement and Innovation
Throughout the Product Lifecycle for a Commercial API
Jacky Musters,* Leendert van den Bos, and Edwin Kellenbach
MSD Oss, GTO API, Molenstraat 110, 5342 CC Oss, The Netherlands
ABSTRACT: The application of quality-by-design (QbD) principles for new chemical entities (NCEs) is highlighted in several
publications. QbD, however, is rarely applied to marketed APIs. Lifecycles of existing APIs are extending. With this extension the
focus on drug safety and compliance requires up-to-date knowledge of the product and its production process as over the years
expectations in this respect have shifted. We applied QbD principles in order to set up an improved control strategy for the nal
ve steps in the production route of a steroidal contraceptive, which has been produced for over 20 years within our facilities. A
generic UHPLC method was developed for the quantitative analysis of the available intermediate batches which have all resulted
in compliant API at full commercial scale. On the basis of the batch results, (statistically supported) specications are proposed
to create a range of proven acceptance criteria. This approach allows the application of the historical knowledge of the drug
substance and its manufacturing process gained over the years to future production by exploiting the process capability of
eliminating impurities and dealing with variability in the quality of the intermediates. Furthermore, it improves the process of
specication setting. Generic UHPLC methods enable rapid quantitative monitoring of quality. The use of a generic UHPLC
method in combination with the setup of a side-product ow scheme improves process understanding and supports chemical
entity design space development. Furthermore, the use of a generic UHPLC method for multiple intermediates also oers the
possibility to perform side-product tracing on the basis of retention times. A generic UHPLC method was developed according
to QbD principles to create a range of proven acceptance criteria for the assay and side-product determination for the nal ve
steps in the production route of a contraceptive API.

INTRODUCTION
Quality-by-Design is the concept of building quality into the
product,1,2 or, as quoted in ICH harmonized tripartite guideline
Q8,3 The goal of manufacturing process development for the
drug substance is to establish a commercial manufacturing
process capable of consistently producing drug substance of the
intended quality. QbD principles can also be implemented
within the process of developing an adequate control strategy,
especially when a lot of process knowledge has been gained
over the years or, as highlighted in ICH harmonized tripartite
guideline Q11,4 The control strategy might be developed
through several iterations as the level of process understanding
increases during the product lifecycle. The analytical method
development is a major contribution to the control strategy. On
the basis of articles that have been published regarding the
implementation of QbD principles in analytical method
development,58 we dened QbD in analytical method
development as a systematic approach, beginning with predened objectives, to obtain an increased scientic understanding and improved condence in the nal method
developed, followed by continuous verication and improvement throughout the method lifecycle.
Several examples have been published in the literature that
highlight the application of QbD principles for new chemical
entities (NCE).911 By exploiting techniques such as design of
experiments, process analytical technology (PAT) using
sophisticated probes (FT-IR, Raman, etc.) and state-of-the-art
scaled lab reactors, a thorough understanding of the
manufacturing process is gained. Few examples of applications
2012 American Chemical Society

of QbD to marketed products have been published. In our

opinion there are two reasons for this. The rst consists of the
regulatory obstacles related to changing process parameters and
updating analytical controls. The second is related to the
investments required to perform these investigations.
The lifecycles of existing APIs, however, are extending due to
decreased approvals of NCEs12 and the use of existing APIs in
novel formulations. The increasing focus on drug safety and
compliance requires improved knowledge of the various
impurities and their variances over the years. By applying
QbD principles, an increased knowledge and understanding of
the product and its production process is gained, which also
improves the process of specication setting. Finally, a greater
understanding of the product and its production process can
create a basis for a more exible regulatory approach.3,13
For commercial products a lot of knowledge has been gained
over the years related to impurities, (process) chemistry, safety,
etc. We applied this information to realize the implementation
of QbD in order to develop an improved control strategy for
the nal ve steps in the production route of a steroidal
contraceptive. The steroidal compound has been produced for
over 20 years within our facilities, and it takes >10 chemical
conversions to produce this steroid from commercially available
starting material of plant origin.
Currently, the intermediates are released by thin layer
chromatography (TLC). The TLC test performs a qualitative
Received: October 17, 2012
Published: December 18, 2012
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potential side-product ranges measured by the method to be

developed. We based the ATP on criteria for the API itself,
currently released by HPLC.
The objectives of the ATP are simply What do you want the
method to measure? and How well does it have to measure
it?. In this case the analytical method should be suitable for the
identication and accurate quantitative determination of the
assay of the intermediates in the range of 80120%, relative to
the API concentration. The combined accuracy and precision of
the method must be such that assay measurements fall within
100% 1%. The method should be able to separate all
intermediates and known side products. Furthermore, the
method should be able to perform an accurate quantitative
determination of the content of the potential side products in
the range of 0.051%, relative to the concentration of the
active compound. The combined accuracy and precision of the
method must be such that side-product content measurements
fall within 100% 15% for levels 0.1% and 100% 10% for
levels >0.1%.
In the second step expressed as Risk Assessment hazards are
identied, and risks associated with exposure to those hazards
are evaluated and eliminated.15 Examples of hazards are the
availability of facilities, equipment, utilities, and analyst
experiences. In preparation for the method development, also
API characteristics (such as structures, volatility, solubility, UV
sensitivity, etc.) were collected. The Risk Assessment step is
mentioned after the ATP in Figure 1, since it is an
indispensable step before selecting an analytical technique.
However, a more precise view would be that Risk Assessment is
applied to all steps in Figure 1 and will return throughout the
method lifecycle to identify factors that can contribute to
method performance and improvement.
On the basis of the ATP, the analytical method should be
suitable for the identication and accurate quantitative
determination of assays and side-product contents. Chromatography is the preferred technique for method development, since
compounds should rst be separated before they can be
detected and quantied. On the basis of previous experiences
we knew that the intermediates are not thermally stable. GC
was therefore not preferred. Since the intermediates and most
of the side products are uncharged apolar molecules of a similar
size (330 Da) the preferred technique to start with was
reversed phase liquid chromatography. The intermediates and
most of the side products contain chromophore groups. On the
basis of these structure properties, diode array was selected as
the detection technique. It was preferred to develop a generic
state-of-the-art analytical method which can be introduced as a
robust, reliable, and quantitative method for release testing
within the QC laboratories. The analysis runtime should be at
least equivalent in comparison with the current TLC methods,
and health, safety, and environmental considerations should be
taken into account. Furthermore, it is preferable to develop a
method which is MS-compatible and oers the possibility to
perform structure proposal or structure conrmation analysis
on side products obtained. The liquid chromatography
technique which can comply with all these requirements is
UHPLC.
A systematic approach was followed to eciently develop a
generic method for the steroidal intermediates. This approach
involves the use of a computer-assisted commercially available
chromatographic method development tool (ChromSword),
which is capable of performing fully automated method
development. Since our method development setup (see Figure

comparison of the samples against the reference standard. TLC

is a simple and quick, low-cost, analytical technique which
enables the analysis of multiple batches simultaneously. The
interpretation of TLC may be dicult and analyst dependent
(subjective). TLC has poorer separation eciency in
comparison, for example, to HPLC,14 and due to the qualitative
manner it is not possible to perform trending. The main
drawback, however, is the reference standard. This standard
does not represent all acceptable impurity ranges because
identity and levels of side products may vary batch-to-batch and
are not covered by the reference standard. This may result in
the rejection of intermediate batches which may have resulted
in compliant API.
The use of a quantitative and selective state-of-the-art
analytical technique together with (statistically supported)
specication setting should signicantly increase the range of
proven acceptance criteria for the intermediates. In addition,
critical quality attributes (CQAs) of intermediates can be
modied as product knowledge and process understanding
increases. This contribution presents an improved analytical
control strategy based on QbD principles.

RESULTS AND DISCUSSION

In the introduction we explained why it is crucial to investigate
the possibilities of applying QbD principles within the
manufacturing process of marketed APIs. In this section the
analytical method development for the ve nal processes in
the route towards the contraceptive API will be discussed. The
aim of QbD in analytical method development is to design a
robust and well-understood method that consistently delivers
an adequate performance. The overall QbD process ow for
analytical method development is depicted in Figure 1.

Figure 1. QbD process ow for analytical method development.

First we set up an analytical target prole (ATP). The ATP is

based on the intended quality of the API, represented by the
quality target product prole (QTPP). Critical quality
attributes (CQAs) and critical process parameters (CPPs) for
a given product and process, respectively, are used to achieve
this QTPP.3,4 For our research we focused on assay and
potential side-product CQAs. Other CQAs, such as, for
example, appearance, morphic form, or particle size distribution, are not within the scope of this study since these CQAs
are not controlled by the currently used TLC methods and also
cannot be controlled by the developed ultrahigh-performance
liquid chromatography (UHPLC) method. On the basis of
extensive product knowledge gained over the years, it is known
that the fate of potential side products is to be purged by the
process. The available released intermediate batches which were
used for method development and specication setting have all
resulted in high-purity API with acceptable assays. Thus far,
intermediates have been released by nonquantitative TLC
methods. The assay and potential side-product CQAs for the
intermediates will be expressed on the basis of the assay and
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Figure 2. Schematic diagram of the method development setup (adapted from ref 18).

2) contains an HPLC and not a UHPLC method, rst an

HPLC method was developed which was subsequently
converted to a UHPLC method. By using ChromSword in
combination with two external selection valves (one for the
selection of eluents and one for the selection of columns) along
with the prior knowledge and experience of the analytical
scientists, the (U)HPLC method development time can be
signicantly shortened, and the probability of achieving
optimum separation conditions can be enhanced.1618 See
Figure 2 for a schematic diagram of the method development
setup.
The development of a generic method for the intermediates
was started by making a selection of columns and mobile
phases for screening on the basis of the structural properties of
the steroidal compounds and the potential side products. The
selected HPLC technique to be used was reversed phase. Since
neither of the compounds contains basic groups with a pKa
value > 1.9 or acidic groups with a pKa value < 12.9, the mobile
phase does not necessarily need to be buered. The most
common mobile phase compositions, combinations of
acetonitrile/water and methanol/water, were tested. Since it
was required to convert the developed HPLC method to a
UHPLC method in a later stage, the column selection was
limited to columns for which also a 2-sub-m particle variant is
available. Since the column selection was not limited to a pH
range, both columns with a hybrid backbone as well as columns
with a silica backbone were selected.19 Typical alkyl-bonded
reversed phase columns (e.g., C18) do not always oer the
required selectivity. Therefore, also a phenyl phase was added
to the column set for screening.20 Phenyl phases can provide
alternative retention characteristics, due to a type of electron
donorelectron acceptor interaction between the electrons of
the phenyl ring of the bonded phases and the electrons of an
aromatic or polyunsaturated compound. Stationary phases
containing shielded groups (polar embedded groups),21 which
use an internal amide or carbamate group as the polar
functionality, are particularly suitable for the separation of basic

compounds. The polar groups embedded in the bonded silane

shield the free surface silanols from interaction with strong
bases. Although the API and the intermediates do not have any
basic properties, a stationary phase with shielding groups is
generally part of the column set for screening since these types
of stationary phases still might show an alternative selectivity.
After suitable sample preparation, the combinations of
columns and mobile phases were screened on HPLC using
the isocratic ne optimization mode within the automated
method development software ChromSword,22 see Table 1.
Table 1. Columns and mobile phases selected for the
screening experiment
HPLC columns
Xbridge C18, length 100 mm, diameter 3 mm, particle size 3.5 m
Xbridge Shield RP18, length 150 mm, diameter 3 mm, particle size 3.5 m
Xbridge Phenyl, length 150 mm, diameter 3 mm, particle size 3.5 m
Atlantis T3, length 150 mm, diameter 3 mm, particle size 3.5 m
mobile phases
water
methanol
acetonitrile

The sample mixture was injected onto each column, and per
column the isocratic ne optimization mode was run with both
organic modiers. The isocratic ne optimization mode is a
method development mode in which mobile phase compositions are automatically selected and run by the software. The
mobile phase composition starts at a manually selected level of
the percentage of the organic modier at which all components
will elute. In this case the required level of organic modier was
not determined during earlier experiments; therefore, a level of
95% was selected (if the level is known, this will certainly
reduce the screening time of the isocratic ne optimization).
After the rst run ChromSword calculates the number of peaks,
peak resolutions, and retention factors. On the basis of these
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Figure 3. Overview of the optimal isocratic method conditions.

Figure 4. Chromatogram of a mixture of API and the potential side products. Labels: 1 = side product A; 2 = side product B; 3 = side product C; 4 =
side product D; 5 = API.

converted to UHPLC conditions, and further optimization

was done on the UHPLC system using columns with 2-sub-m
particles. The small-particle technology23,24 provides not only
increased eciency but also the ability to work at increased
linear velocity without a loss of eciency, providing both
resolution and speed.25,26 The narrower the peaks are, the
easier they are to separate from each other. Also, peak height is
inversely proportional to the peak width; thus, as the particle
size decreases, an increase in sensitivity is obtained, since
narrower peaks are taller peaks.27 The intermediates III and IV
are semi-pure variants of the API, which dier only by the
presence of side products.
A gradient was introduced for the elution of the apolar
components to speed up the analyses and to improve the
sensitivity. The optimal ow was determined by increasing the
ow to a level at which the system pressure does not exceed the
critical point of 1000 bar for the most viscous mobile phase
composition (in practice, the maximum pressure was set
around 850 bar). The optimal injection volumes and sample
concentrations were determined in relation to the desired limit
of quantitation for the potential side products.
The nal method conditions after optimization are shown in
Table 2. A chromatogram of a sample of a mixture of API, the
intermediates I and II (all at an equal level of 0.25 mg/mL),
and potential side products (at a 1% level) is shown in Figure 5.
For ne-tuning the method conditions and to verify the
robustness of the generic method, a full factorial design of
experiments (DoE) was used in which the parameters, column

results, the mobile phase composition is automatically adopted

by the ChromSword software. After each run, the results of the
actual and prior runs are automatically compared, and the
software decides to continue optimizing or concludes that the
optimal mobile phase composition is obtained. The goal of the
isocratic ne optimization mode is to develop a method with a
minimal peak resolution of 2 for all components and a
maximum retention factor of 10 for the latest-eluting
component. The selection of the best column and mobile
phase composition is made by the analyst. The selection is
subjective, but straightforward. The best conditions are chosen
on the basis of the number of detected or separated peaks, peak
shape, and total run time.
On the basis of the results of the ChromSword screening, the
highest resolution within the shortest time was obtained using
an Xbridge Shield RP18 column in combination with an
acetonitrile/water mobile phase. A chromatogram obtained at
these isocratic conditions (see Figure 3) is shown in Figure 4.
The intermediates I and II have more apolar structural
properties and will not elute within an acceptable analysis time
using a water/acetonitrile 55:45 v/v % mobile phase. A gradient
should be introduced for the determination of these apolar
intermediates and potential side products. To suciently
separate the API, the intermediates, and the potential side
products within a generic HPLC method, run times have to be
increased signicantly, and the sensitivity for late-eluting
components will be decreased as a result of peak broadening
eects. Therefore, the HPLC method conditions were
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Table 2. Final method conditions after optimization on

UHPLC
column

Table 3. Varied parameters in full factorial DoE

Acquity BEH Shield RP18, 100 mm

2.1 mm; i.d. 1.7 m
40 C
95:5 v/v% water/acetonitrile
95:5 v/v% acetonitrile/water

column temp.
eluent A
eluent B
Mobile Phase
time (min)
eluent A (%)
0
75
1
75
11.5
5
12.5
5
12.6
75
17
75
ow (mL/min)
0.8
detection (UV, nm)
244
injection vol (L)
1

eluent B (%)
25
25
95
95
25
25

factor 1

factor 2

factor 3

run

column temp. in C

ow in mL/min

% acetonitrile in eluent B

1
2
3
4
5
6
7
8
9
10
11

40
50
50
30
30
40
30
30
50
50
40

0.8
0.9
0.7
0.9
0.7
0.8
0.9
0.7
0.7
0.9
0.8

95
92
92
92
92
95
98
98
98
98
95

acetonitrile in eluent B the resolution of the API decreases

faster when decreasing the column temperature compared to
the upper level of acetonitrile in eluent B, which is shown by
two nonparallel lines in the interaction plot which will nally
intersect.
When varying the ow in combination with the percentage of
acetonitrile in eluent B, there was no or hardly any interaction
with regard to the resolution factors. When varying the column
temperature in combination with the ow or the percentage of
acetonitrile in eluent B, interactions with regard to the
resolution factors were obtained for several peaks. However,
under all tested conditions the resolution factors are acceptable.
Figure 7 represents interaction plots for the API.
On the basis of the results from the full factorial DoE, the
method is considered to be robust; method parameters can be
varied in a sucient range. The method conditions are robust
and close to the optimum of the DoE. The optimal method
conditions based on the DoE results would be the conditions as
described in Table 2 in combination with a column temperature
of 30 C. However, at a temperature of 30 C, the increase of
the column pressure is unacceptable. With the conditions as
mentioned in Table 2, intermediate I, intermediate II, the API,
and the potential side products are baseline separated. In
addition, 30 C represents the extreme of the robustness range,
and it would be more adequate to select conditions inside this
range to ensure that small variations fall inside the studied

temperature, mobile phase composition, and ow, were varied

(Table 3).a
The API and its four intermediates were analyzed at the
dierent design conditions. Retention times and resolution
factors of the API, the intermediates, and the potential side
products were compared. No or hardly any interaction was
obtained between the factors tested with regard to the retention
time. To illustrate this, Figure 6 represents interaction plots for
the API.
A full factorial design contains all possible combinations of
low/high levels for all the factors. In an interaction plot twofactor interactions were confounded with one another. For
example, in the rst plot of Figure 6, the eect of interactions of
the factors (A) column temperature and (B) ow on the
retention time of the API is shown. Both at the upper and lower
ow levels tested, the retention time of the API decreases, when
the column temperature is increased. There is no interaction
between ow and column temperature regarding the retention
time of the API, which is shown by two parallel lines in the
interaction plot which do not intersect. In the second plot of
Figure 7 the eect of the interactions of the factors of (A)
column temperature and (C) percent of acetonitrile in eluent B
on the resolution of the API is shown. At the lower level of

Figure 5. Chromatogram of a mixture of the API, the intermediates I and II, and the potential side products at UV 244 nm. Labels: 1 = side product
E*; 2 = side product A; 3 = side product B; 4 = side product C; 5 = side product D; 6 = API; 7 = intermediate I; 8 = intermediate II. *Side product E
is a side product which is present in intermediate I.
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Figure 6. Interaction plots for the retention time of the API.

As shown in Figure 8 the strength of the method lies in its

generic character, which oers the possibility to perform side
product tracing based on retention times. The chromatograms
of subsequent intermediates along the route show at a glance
the formation, fate, and purge of potential side products
through every step of the manufacturing process. However, side
products can also convert. Therefore, it is advisable to use
additional techniques such as, for example, mass spectrometry
and multidimensional, heteronuclear NMR to perform full sideproduct tracing.
As mentioned in the introduction, drawbacks of the currently
used TLC methods are the subjective interpretation and the
qualitative determination of the purity against a reference
standard which does not represent all acceptable impurity
ranges. This may result in the rejection of intermediate batches
which would have resulted in compliant API. In contrast, using
a set of representative intermediate batches (which have all
resulted in compliant API) to set specications on side
products by using an objective, quantitative technique will
create a range of proven acceptance criteria encompassing all
the qualities of batches used in specication setting. This
approach thus allows applying the process and (historic)
product knowledge gained over the last 20 years for future
production by exploiting the process capability to eliminate
impurities and deal with variability in the quality of the
intermediates.
For the specication setting of each individual intermediate, a
set of representative batches (which have all resulted in
compliant API at full commercial scale) was analyzed. An

robustness intervals. Therefore, the column temperature of 40

C was unaltered.
Outside the design,column robustness, linearity, and
sensitivity were also veried. The method is robust using
dierent column batches of the same type (same vendor, serial
number, and dimensions). The API, the intermediates, and the
potential side products are baseline separated on each of the
tested columns, and the variations in retention times are minor
(<1%). Linearity was proven in both the side-product range
(0.051%) as well as the assay range (80120%). All the
potential side products at a level of 0.05% or higher can be
quantied. The accuracy and precision will be tested during the
validation. Before the start of the validation product
specications should be set. The process of specication setting
will be described in the next paragraph.b
For each intermediate approximately 10 batches have been
analyzed, using the generic UHPLC method. The identity,
content of side products, total side products, and purity (for
intermediates) or assay (for dierent API qualities) were
determined. The identity and assay were determined against an
API or, respectively, an intermediate standard at 100% level.
The assay is determined by weight %. The side products were
determined against a 1% dilution of the 100% standard and are
reported in area %. The reporting limit for the side products
was 0.05%. In Figure 8 an overview is shown of the
chromatograms of representative batches of the four
intermediates at UV 244 nm.
The chromatogram of intermediate IV and the nal API are
comparable, regarding the side product prole.
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Figure 7. Interaction plots for the resolution factor of the API.

Figure 8. Overview of the chromatograms of the intermediates. Labels: 1 = intermediate I; 2 = intermediate II; 3 = API; 4 = side product E; 5 = side
product A; 6 = side product D.

overview of the content ranges of side products in the API and

the dierent intermediates at UV 244 nm is shown in Table 4.
The batch results over approximately 10 batches were
summarized, and the lowest, highest (as shown in Table 4), and

average levels were determined for each individual side product,

the total of side products, and the assay. For the proposal of an
upper level specication the average results plus 3 times the
standard deviation were calculated. For the assay results, a
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Table 4. Content ranges of side products, based on batch analyses for the last ve steps in the synthetic route of the APIa
average side product content in area %
RRT
0.46
0.50
0.57
0.63
0.66
0.75
0.91
1.00
1.31
1.33
1.38
1.75
1.82
1.83

side product
D
A
E
I
F
B
C
API
K
G
J
int. I
H
int. II

assay (%) minmax

a

int. I minmax
1.222.71
b
0.230.25
b
0.080.12
b
0.054.26
b
b
0.080.10
b
n.a.
0.060.10
b

int. II minmax

int. III minmax

0.050.09
1.282.67
b
0.150.23
b
0.140.18
b
0.272.19
b
b
0.050.06
3.046.39
b
n.a.

0.080.30
b
b
b
b
1.213.35
n.a.
0.050.13
b
b
b
b
b

9298

8593

int. = intermediate. b<0.05%.

int. IV/API minmax

b
b
b

9699

b
b
b
n.a.
b

b
b
100

CONCLUSION
For commercial APIs a thorough understanding of the
manufacturing process and extensive product knowledge is
gained over the years. Series of representative intermediate
batches which have all given commercial API batches within the
specications can be used to create a range of proven
acceptance criteria, encompassing all the qualities of batches
used in specication setting.
Drawbacks of the currently used TLC methods are the
subjective interpretation and the qualitative determination of
the purity against a reference standard which does not
represent all acceptable impurity ranges. This could result in
the rejection of intermediate batches which would have resulted
in compliant API at full commercial scale. A generic UHPLC
method was developed according to QbD principles for the
identity, assay, and side-product determination for the nal ve
steps in the synthetic route of the API. UHPLC is an objective,
quantitative technique which visualizes and allows specication
setting by applying the process and product knowledge gained
over the years for future production, by exploiting the process
capability to eliminate impurities and deal with variability in
quality of the intermediates. Specication setting was
performed using intermediate batches, which have all resulted
in compliant API at full commercial scale. Therefore,
specications levels are representative for the process, and
there are no scalability issues since it involves full commercialscale batches.
Generic methods allow realizing the full potential of UHPLC
in analytical eciency and also process understanding in
support of QbD; the introduction of a generic UHPLC method
has several advantages. Since analytical run times become
shorter by up to a factor of 4, it is possible to replace current
TLC release methods by quantitative UHPLC methods without
an increase in analysis time. Moreover, the generic character of
the UHPLC method enables an optimal eciency in process
control; columns and mobile phases should not be changed and
conditioned between analyses. Also system suitability should
only be proven once. Therefore, not only the analysis time but
also the lead time is reduced signicantly. In addition, analysts
will collect a lot of method knowledge and become experienced

proposal was determined additionally for the lower-level

specication by calculating the average assay minus 3 times
the standard deviation. Most of the individual side products
reported in Table 4 are distributed in a normal distribution
around the average value. The highest level obtained in one or
several batches diers 0.05% from the proposed specication
level (average plus 3 times the standard deviation). Since
specication setting was performed using intermediate batches,
which have all resulted in compliant API at full commercial
scale, these specication levels are representative of the process,
and there are no scalability issues since it involves full
commercial-scale batches. For side products C and D in
intermediate I and side product A in intermediate II we
observed an increased side-product content level in the oldest
batch analyzed, which has a major inuence on the average
values and therefore also on the average plus 3 times the
standard deviation values. For these side products we just
proposed the highest level obtained during batch analyses to
become the specication level (i.e., without adding 3 times the
standard deviation), since the process has only demonstrated
that the presence of these side products at the obtained levels
resulted in compliant API. Also the assays of intermediates I
and II are inuenced by the increased content of these side
products in one of the batches. Therefore, the min/max levels
reported in Table 4 are proposed as specication levels for the
assay of intermediates I and II. On the basis of the analytical
results it is shown that the use of intermediates close to the
proposed specication criteria results in a compliant API.c
By using mass spectrometry a structural proposal was made
for the relevant side products. Some of the side products were
isolated, and full structure elucidation by multidimensional,
heteronuclear-NMR was performed. A side-product ow
scheme helps to make the reaction process transparent and
improves process understanding. It shows at a glance the
formation, fate, and purge of potential side products through
every step of the manufacturing process. In combination with
full structure elucidation it additionally oers the possibility to
give a direct overview of the conversion of potential side
products.
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analytical columns were adapted, no new or coeluting

impurities were obtained. Second, peak purity for the main
peak of each intermediate was determined using diode array
detection. The obtained results gave us good condence in the
selectivity of the method.
c
Upon being asked by a reviewer what happens if the level of
one or all impurities achieves or even increases the specication
limit, and did we ever try to demonstrate that the resulting API
is within specications, we respond: First, a spike/purge
experiment on lab scale might be performed in the near future
using isolated side products to demonstrate the capability of the
process to eliminate impurities. Second, on the basis of all our
experiences with this process build over the years and taking
into account the process improvement steps which will be
introduced in addition to this new control strategy, we do not
expect the side products to be increased.

with the method within in a short period of time. Another

strength of generic UHPLC methods lies in the possibility to
perform side-product tracing on the basis of retention times
when using the same method for multiple intermediates.
Minimal application of QbD principles, restricted to the
development of an improved control strategy, has resulted in
increased scientic knowledge for several steps of the
manufacturing process. The use of a generic UHPLC method
in combination with the setup of a side-product ow scheme
improves process understanding and supports chemical entity
design space development.
The use of a quantitative and selective state-of-the-art
analytical technique together with a range of proven acceptance
criteria signicantly increases the range of intermediate
qualities, which is reected in a robust and reliable production
of a high-quality API.

AUTHOR INFORMATION

Corresponding Author

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*E-mail: jacky.musters@merck.com
Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS
We express our special thanks to R. Stol whose help,
stimulating suggestions, knowledge, and experience helped us
during the development of this new control strategy. In
addition we express our thanks to R. Hofstraat, W. Foppen, and
all the colleagues of the GTO section of MSD for their
generous support. Finally, it is our pleasure to acknowledge the
reviewers for their suggestions.

ADDITIONAL NOTES
A reviewer commented that only some factors are included in
the DoE and others that may have an impact are missing. The
reviewer also suggested measuring the inuence of varying the
detection wavelength and noted that we addressed robustness
but ruggedness is not discussed. Our response is: The factors
included in the DoE were carefully selected. The sample
preparation is straightforward, and no sample dilution or
extraction steps are applied. An extensive study has been
performed to determine the optimal injection volume and
injection mode. Optimal wavelengths were selected prior to the
UHPLC method development. The inuence of small
variations in wavelength will be tested during the validation
since this is part of determining the relative response factor for
the available side products. On the basis of our experiences with
performing this analysis and verifying the method, we have no
doubt about the ruggedness of the method. Varying the analyst,
equipment, and number of days will be tested in the precision
study during validation. On the basis of the method verication
experiments, we do not expect any problems there.
b
Upon being asked by a reviewer how are we sure that we
separated all components and how do we conrm that we do
not have impurities underneath the main peak(s) or impurities
coeluting, we respond: First, we have to clarify the term all
components. The term all components is used to mention
the components (API, intermediate I, intermediate II, and the
identied side products AD) added to the mixture used for
the automated method development. These components are all
separate. On the basis of the robustness testing of the UHPLC
method, in which combinations of method parameters and
a

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