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org/ac Perspective

Selection of Analytical Technology and Development of Analytical

Procedures Using the Analytical Target Profile
Phil Borman,* Cristiana Campa, Ghislain Delpierre, Elliot Hook, Patrick Jackson, Wayne Kelley,
Michel Protz, and Olivier Vandeputte

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ABSTRACT: A structured approach to method development can help to ensure an

analytical procedure is robust across the lifecycle of its use. The analytical target profile
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(ATP), which describes the required quality of the reportable value to be produced by the
analytical procedure, enables the analytical scientist to select the best analytical technology on
which to develop their procedure(s). Once the technology has been identified, screening of
potentially fit for purpose analytical procedures should take place. Analytical procedures that
have been demonstrated to meet the ATP should be evaluated against business drivers (e.g.,
operational constraints) to determine the most suitable analytical procedure. Three case
studies are covered from across small molecules, vaccines, and biotherapeutics. The case
studies cover different aspects of the analytical procedure selection process, such as the use of
platform method development processes and procedures, the development of multiattribute
analytical procedures, and the use of analytical technologies to provide product
characterization knowledge in order to define or redefine the ATP. Challenges associated
with method selection are discussed such as where existing pharmacopoeial monographs link acceptance criteria to specific types of
analytical technology.

■ INTRODUCTION
Predefining what constitutes success for the measurement of a
quality attribute1 is a key input for a sound method
development strategy. In the pharmaceutical industry,
predefined acceptance criteria associated with the analytical
measurement can be summarized using the analytical target
profile (ATP).2−6
The ATP has been defined by USP ⟨1220⟩ as “a prospective
description of the desired performance of an analytical
procedure that is used to measure a quality attribute, and it
defines the required quality of the reportable value produced
by the procedure”.6 The ATP is informed by product/process
requirements (e.g., specification acceptance criterion for a
quality attribute) and is not linked to any specific analytical
technology. The ATP should enable the analyst to select the
best analytical technology for developing their procedure. This
will be driven by prior knowledge and/or experimental
verification relating to the performance of available analytical
technologies. Where more than one technology has been
demonstrated to meet the ATP, a review of business
requirements (e.g., throughput, automation, availability)
should be performed to aid selection. Once the technology is
selected, procedure specific performance indicators can be
defined which will support method development and ensure
the performance criteria in the ATP are achieved.
© XXXX The Authors. Published by
American Chemical Society
A Anal. Chem. XXXX, XXX, XXX−XXX
To enable the identification of optimal procedure con-
ditions, a broad initial screen of all plausible conditions is
recommended. This screen should be structured such that the
testing identifies potential sets of procedure conditions that are
likely to meet the ATP, which can subsequently be ranked, and
the most preferable ones checked to see whether there is a
likely robust region which will result in acceptable operation of
the procedure.
The output of a successful method development strategy
should lead to one or more sets of method conditions which
satisfies the ATP, ensures optimal method performance, and
identifies a region around the selected method conditions
which is likely to be robust over the analytical procedure
lifecycle. Other potential method conditions may also be
available for analysts to fall back on if unexpected failures arise
for the chosen procedure.
It is advisable to monitor the availability of new technologies
during product development and across the lifecycle, to ensure
the most up-to-date technology is being used. Following the
Received: September 6, 2021
Accepted: December 1, 2021
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Analytical Chemistry
pubs.acs.org/ac
above approach ensures a continued focus on product and
process needs and enables robust bridging between new and
existing analytical procedures, in case of change.
■ SCREENING AND SELECTION OF
FIT-FOR-PURPOSE ANALYTICAL TECHNOLOGIES
The process leading to selection of a fit-for-purpose analytical
procedure may start as soon as the performance expectations
(ATP) and operating environment are defined. The first step
of method development aims at identifying all available
technologies that can potentially be used to meet the ATP
requirements. This preliminary step is crucial to minimize the
risk of using routine or general-use analytical procedures that
are unable to meet the performance criteria. Technology
assessment also allows for the evaluation of any existing
platform analytical procedures or new technologies. Special
attention should also be given to orthogonal technologies
which can be used to analyze the same attribute but based on
different principles of measurement. Such technologies are
useful as back-up options in case of failure during development
of the selected technology. Orthogonal tests can also provide
additional product and analytical knowledge, thereby increas-
ing the confidence in the selected method.
After this first step, a roadmap leading to method selection
can be described as follows:
1. Scoring of the analytical technologies against both
required performance (ATP) and operational con-
straints (business drivers).
2. Experimental testing/screening of potentially fit-for-
purpose methods.
3. Final scoring and ranking leading to method(s) selection
and start of formal development. The approach
described for method screening and selection is
illustrated in Figure 1.
Figure 1. Method screening and selection approach.
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■
Perspective
METHOD DEVELOPMENT: GENERAL CONCEPTS
The application of quality by design (QbD) principles to
method development provides a systematic approach for
building in quality from the onset of development by
establishing method requirements (via the ATP) against
identified product quality attributes. The analytical QbD
framework2 allows decision making using a science- and risk-
based approach. These risk-based method development steps
involve (I) preliminary risk assessment, (II) risk management
if/where appropriate for high-risk analytical procedures. This
could involve improving method performance understanding
via modeling (e.g., multivariate experimentation) and (III) risk
confirmation, implementing method robustness and rugged-
ness. The ATP enables the analyst to select the most
appropriate analytical outputs to monitor during development.
The definition of surrogate performance criteria (also known
as analytical procedure attributes (APAs) linked to the ATP
acceptance criteria) underpins the method development
process. The APAs defined for a given method are technology
specific properties (for example, attributes for chromatography
measurements may include peak symmetry factor and
resolution). The expert analyst should identify the most
appropriate APAs used to study the performance during
development and to meet the ATP requirements. APAs are
also used to identify critical analytical procedure parameters
(CAPPs) through modeling studies, the output of which can
be used to define a method operable design region4,7 (MODR)
for the analytical procedure. CAPPs are analytical procedure
parameters whose variability have an impact on APAs and
consequently on the measured result.
Three examples of technology screening and method
development are covered below from across small molecules,
vaccines, and biotherapeutics. The above outlined concepts
will be covered for each example.
In the first example, HPLC specific method development
and screening principles are described using APAs linked to
the ATP acceptance criteria. The second example demon-
strates the method screening and selection process with an
evaluation of innovative approaches to address complex
biological CQAs during vaccine development. Example three
discusses how CQA knowledge gained during development
can drive revisions to the ATP and an evolving method
selection process in preparation for a commercial control
strategy.
■ EXAMPLE 1−SMALL MOLECULES: METHOD
DEVELOPMENT APPROACHES/PLATFORMS
Development of analytical procedures for the analysis of small
molecule pharmaceutical products or ingredients, following
quality by design principles, is well established.7−9
The structured analytical QbD method development
approach driven by the requirements of the ATP can be
applied to any analytical technology and has been applied to a
range of analytical techniques, such as HPLC,9 Karl Fischer,10
and colorimetry.11 Further technique specific method develop-
ment strategies have been developed and applied. For example,
in the case of developing an analytical procedure for the
determination of related organic impurities of an active
pharmaceutical ingredient, the ATP will direct the develop-
ment to an analytical technique (such as liquid chromatog-
raphy) that is capable of separating and quantifying the
impurities which are critical quality attributes (CQAs) that
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may impact the quality of the active pharmaceutical ingredient
(API). Once the technique is selected, a number of technique
specific method attributes together with their performance
criteria can be used to ensure the analytical procedure is likely
to meet the ATP, meet ICH Q2(R1)12 validation require-
ments, and to reliably be used across the entire method
lifecycle. The technique specific method attributes and their
performance criteria will be common to the majority of small
molecule HPLC impurity methods and so can be usually used
for all HPLC analytical procedures.
With the APAs defined, experimental scouting of analytical
procedure parameters can begin, typically starting for HPLC
with mobile phase pH, organic modifier, and column.9,13 See
Figure 2 for the number of mobile and stationary phases that
Figure 2. Mobile and stationary phases used in HPLC scouting.
are typically used. These are from a predetermined subset of
solvents/column, selected based on prior knowledge of
performance against the APAs, affording a degree of control
and commonality when this process is applied across multiple
assets.
This screening process is supported by data evaluation and
in-silico modeling to aid the initial method selection, before
undergoing iterative risk assessment and optimization to meet
the requirements of the ATP and APAs. As this process will be
common to the majority of small molecule HPLC methods, it
can be defined into a structured, technique specific method
development strategy (see Figure 3 for a simplified version of
what has previously been published by Vogt and Kord8) to
ensure consistent application of the QbD principles during
analytical procedure development.
Use of risk assessment is key throughout this strategy to
understand how the method is performing against the method
attributes and the ATP. Borman et al.14 demonstrates the
application of risk assessment and experimental design tools to
evaluate the final method robustness and identify method
controls as part of the method control strategy.
■
EXAMPLE 2−VACCINES: ANALYSIS OF
ADENOVIRUS VECTORS
Many physicochemical methods have been designed using the
QbD approach,15 but very few examples highlight how
biological methods can benefit from this iterative and
structured mindset. Using adenovirus vectors developed for
vaccination, this example will describe how QbD principles can
improve and foster the selection of fit-for-purpose methods,
the identification of multiattributes methods, and the deploy-
ment of innovative analytical methods addressing biological
attributes.
Virus-based vaccines and antigens produced using recombi-
nant DNA technology represent an important part of the
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Figure 3. HPLC method development strategy.
vaccines under development and available on the market.16 A
refinement of the recombinant DNA technology consists in
delivering the gene of interest directly into the host and in
using the host cell machinery to produce the corresponding
protein/antigen which, hence, induce the desired immune
response.16−18 Different virus vectors can be used to achieve
this strategy18−21 and include replication-defective adenovi-
rus.22−24
These adenovirus vectors are defective for viral replication,
thereby ensuring the patient’s safety but are fully capable of
hijacking the host cell machinery to produce the protein/
antigen of interest that will induce the desired immune
response.22−24 As for any other vaccines, several attributes are
part of the comprehensive understanding of the product. More
importantly, they are cornerstones of the control strategy since
they are key indicators of the efficacy, stability, and consistency
of the vaccine as well as of the process robustness. In this
example, three specific quality attributes will be discussed: two
related to the mode of action of replication-defective
adenovirus and one that is common to all vaccines.
To be efficacious, the replication-defective adenovirus vector
must enter the host cells where it can trigger the expression of
the gene of interest that will induce the desired immune
response.24 Therefore, two critical quality attributes of the
replication-defective adenovirus vectors that must be moni-
tored are the “infectivity” (i.e., capability to enter and to deliver
the gene of interest into the host cells) and the “transgene
expression” (i.e., control the expression of the gene of interest
in the recipient cells).24−27 Both attributes are key to
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Table 1. List of the Potential Fit-for-Purpose Technologies to Address the CQAs “Infectivity”, “Expression of the Gene of
Interest”, and “Identity”
demonstrate the functionality and the stability of the
nonreplicative adenovirus vector capsid components, the
efficient packaging of the full-length genomic DNA, and the
integrity of virus particles among other attributes.27−29 Being
produced through amplification in specific cell lines, any
change in process parameters can impact the functionality of
the replicative-deficient adenovirus vector since misfolded
proteins will result in nonrecognition by cell receptors,
misassembled capsid will result in unstable particles and/or
premature degradation, and incomplete genomic DNA will
result in decreased gene expression among other biological
misfunctions.30,31 As for any other vaccines, the identity of the
gene of interest must be verified to ensure that the right
antigen is used in the vaccine.32
After definition of the ATP for the identified CQAs, different
technologies were then evaluated with analytical subject matter
experts (SME). “Obvious” methods were first considered
before looking at multiattribute and innovative methods. The
identity of the antigen can typically be addressed by antibody-
based methods (e.g., ELISA or Western blot). Several methods
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were discussed to address the “transgene expression” attribute,
including nucleic-acid based technologies (NAT), antibody-
based methods, chromatography, electrophoresis, and spectro-
metric methods. Typically, cell-based methods (like CCID50,
TCID50, and IFU but also flow cytometry) were considered
for the infectivity attribute. Even though these methods meet
many analytical requirements (see Table 1) and could be used
as such to address the three CQAs, the technology evaluation
exercise with all SMEs led to a comparison of the “pros” and
“cons” of all technologies.
As shown in Table 1, some methods were identified as being
release-ready (such as electrophoresis, chromatographic
methods, antibody-based methods, and NAT), accepted by
the regulatory agencies (such as antibody-based methods and
NAT), ready for transfer to quality control (such as
electrophoresis, chromatographic methods, antibody-based
methods, and NAT), or specific and sensitive (e.g.,
spectrometric methods and NAT). Drawbacks were also
identified, such as the need for control materials (e.g., peptides
for mass spectrometry or characterized protein standards for
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Table 2. Summary of the Method Scoring for the Three Attributes (Infectivity, Identity, and Transgene Expression)a
a
For each ATP requirement, a score is given as follows: “1”, does not meet; “4”, partially meets; “7”, meets; and “10”, exceeds the requirements.
electrophoresis, chromatography, and antibody-based meth-
ods), the need for additional data (such as total protein
content for antibody-based methods), or the need for specific
reagents (i.e., monoclonal antibodies). A major outcome of the
method screening exercise was that all these methods were
identified as being able to address at least two attributes. More
particularly, the cell-based assays as well as the NAT methods
were identified as being able to address the three CQAs in a
single assay.
A second important step in the method selection is to ensure
that they meet the requirements defined in the ATP and the
associated business drivers. Table 2 shows how the method
screening was implemented to address the “infectivity” CQA
and how the performance of the different methods can be
ranked according to the expectations of the ATP. Although the
“infectivity” CQA is taken because of its criticality for vaccine
efficacy, it is important to note that a similar screening was
performed for the other CQAs considered in this case study.
The scoring/ranking is performed with all analytical SMEs to
foster brainstorming and innovation. The most promising
methods that will go into further practical assessment and
development were identified by comparing the capability of the
analytical methods to meet the ATP requirements defined for
all CQAs. Summing all the scores allows to provide a global
score for each method but also to discriminate between
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performance (ATP) versus operational (business drivers)
constraints. It is important to keep in mind that this scoring
is made to help in selecting what methods will go into further
development and is not, as explained later, a “score-based”
decision tool. Using this way of working also helps in capturing
the knowledge and rationales behind a go/no go choice.
Following the rationale shown in Table 2, three groups of
methods (i.e., electrophoresis/chromatography, spectrometry,
and antibody-based methods) out of five were not further
considered because their performance was not able to meet the
ATP requirements with regards to LOD/LOQ and combined
uncertainty.2 NAT-based and cellular-based assays were ranked
with the highest scores regarding performance and business
requirements. Besides, these groups of methods were also
identified as being able to target the three attributes at the
same time, which is an additional significant advantage for
downstream quality control. NAT-based assays were slightly
ahead in terms of industrialization because they were already
implemented in the quality control department. However,
since NAT methods target nucleic acids and not the protein/
antigen, the decision was made to move forward with the cell-
based assay even though flow cytometry was a new technology
for quality control. It is important to note that the main part of
the cellular-based assay method relies on cell-based technology
already implemented in quality control. As shown in Table 3,
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Table 3. Comparison between Flow Cytometry and NAT

Figure 4. All attributes in one single assay: (A) identity, (B) potency, and (C) transgene expression.

Table 4. Critical Analytical Procedure Parameters Evaluated

the need for training and budget to deploy the new technology during the method screening helped alleviate these risks and
in the final lab (i.e., quality control (QC)) were identified as allowed QC to anticipate the deployment of a new technology
the main risks associated to the use of a new cell-based assay. in a regulated environment, facilitating the deployment of the
Nevertheless, the priming and inclusion of the QC teams new assay in a short period of time in all involved laboratories.
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Notably, the screening process described here does not
necessarily lead to a clear-cut decision about the method to be
further developed. Instead, the screening is a tool which fosters
innovation, triggers the selection of multiattribute methods,
causing analytical experts and project leaders to leverage, in a
simple and visual way, the pros and cons of each considered
method and to make the best decision in a given context.
The next step in the development of the method consisted
of defining critical method parameters to establish an
optimized MODR. In a nutshell, the method consisted of
incubating permissive eukaryotic cells with drug substance,
drug product, or reconstituted vaccine samples to let the virus
grow. After 24 h of incubation, the cells were permeabilized
and a specific monoclonal antibody (labeled with a
fluorochrome) was used to detect and quantify the protein
expressed by the adenovirus-vectored transgene. As shown in
Figure 4, the three attributes (identity, potency, transgene
expression) were addressed in a single experiment. Indeed, the
presence of antibody-responsive cells and the specificity of the
antibody ensure that the identity of the product is verified. The
content of infective/functional virus present in the sample is
determined by the number of antibody-positive cells (each
infective/functional virus will infect one cell that will be
detected by flow cytometry). Finally, the transgene expression
level is obtained by comparing the median fluorescence
intensity to the one obtained with a reference lot, thereby
ensuring the consistency between batches.
The robustness of the cell-based flow cytometry multi-CQA
assay was evaluated through a QbD approach where the design
of experiments (DoE) with different CAPPs was analyzed. As
shown in Table 4, these parameters are grouped into three
categories: parameters related to the cell culture (such as
subpassages, percentage of confluence, and dilution of
inoculum), the composition of the growth medium (such as
fetal bovine serum (FBS), glutamine, and lots of reagents), and
the method of infection (such as operator and method of
dilution). As shown in Table 4, many of these parameters had
no impact on the performance of the method for determining
the potency and the transgene expression.
For the further development of the method, the DoE
approach allowed effort to be focused on the most critical
parameters of the assay, such as passage before culture, batch
of critical raw materials (e.g., glutamine, trypsin, and FBS), and
operator impact.
■
EXAMPLE 3−BIOTHERAPEUTICS: AN EVOLVING
ATP FOR MONOCLONAL ANTIBODY
AGGREGATION
Therapeutic monoclonal antibodies (mAbs) have become a
significant part of the pharmaceutical landscape, providing
powerful treatments for patients spanning a wide range of
diseases in the fields of oncology, cardiology, hematology,
neurological and autoimmune system related disorders, and
infectious disease.33−35 As with other therapeutic modalities,
biotherapeutics can benefit from the use of a systematic, risk-
based, and data driven approach for the development and
implementation of analytical methods. Understanding the
CQAs of a mAb is an important step in a QbD approach to
selecting the appropriate analytical technology and methods
used to establish a comprehensive product testing and control
strategy. Attributes that are identified as CQAs from the risk
assessment and attributes that need to be assessed further are
included in the method selection process establishing the
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overall testing strategy. Over the course of development,
information from analytical structure−function studies as well
as the clinical experience can lead to a reassessment of a CQA
and the identification of related attributes, which will in-turn
drive refinements of the initial ATP. This example for a mAb
will demonstrate how QbD principles can help to focus this
evolving testing strategy.
Therapeutic mAbs are complex proteins (immunoglobulins)
produced from a single clonal cell that have a high degree of
specificity for an antigen related to a disease (e.g., a tumor cell,
viruses, or bacteria) or an adverse immune response (e.g.,
asthma). Therapeutic mAbs work, in general, by binding to
antigens on the membrane or surface of a pathogen, such as a
cancer cell or virus, interfering with the disease pathway. This
is achieved by inhibiting absorption of a virus or replication of
a cancer cell. Additional mechanisms can include binding to
effector cells of the immune system, which will affect a
complement response or signal specific cells such as natural
killer cells or macrophages which mediate the destruction of
the pathogen.
Antibodies are categorized by five different isotypes.
Therapeutic mAbs are an immunoglobulin G isotope (IgG)
where the structure consists of four polypeptides composed of
two light chains and two heavy chains linked by disulfide
bridges and arranged in a Y shape configuration. The IgG
isotype is further defined by subtypes that differ by their
disulfide bond arrangement. IgG1, IgG2, and IgG4 are
commonly employed as therapeutic mAbs with the IgG4
typically used for immunotherapies.36 Therapeutic mAbs can
be described as having two characteristic domains: the variable
and the constant. The variable domain contains the
complementary determining regions (CDR) where the amino
acid sequence varies significantly from antibody to antibody
and is what makes the antibody highly specific for a particular
antigen. Therapeutic mAbs are further characterized by the
antigen binding fragment (Fab), involved in target binding,
and the crystallizable fragment (Fc) in the constant region.
The latter can play a role in the mechanism of action and
pharmacokinetics (PK) by binding to Fc receptors on innate
immune cells and activating the immune system. These
structural characteristics and some of the relevant physi-
ochemical and biological quality attributes to be considered in
a full product assessment are depicted in Figure 5.
The CQA assessment process follows the ICH Q11
guideline and involves the identification and prioritization of
the potential risks to patient safety or the product quality that
could impact the efficacy, consistent manufacture, or stability
of the product. The impact of each product quality attribute is
Figure 5. Structure of an mAb and its site-specific physiochemical and
biological attributes.
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typically assessed against biological activity, PK, immunoge-
nicity (specific to antidrug antibodies), and safety. Information
critical to the assessment includes the biology and mechanism
of action (MoA) of the molecule, therapeutic dose, process
and product-related variants, PK characteristics, biochemical
and biophysical characterization, and the stability and safety
profile. Additionally, in vitro (e.g., immunogenicity), non-
clinical, and safety studies are leveraged to guide the CQA
assessment and focus the scope of testing required.
Higher-order molecular weight species or aggregates are
typically assessed as a high risk CQA37 as they can impact
product quality and efficacy and pose a potential safety risk to
patients through enhanced immunogenicity.38 Aggregation is a
general term for the self-association of proteins outside of the
native quaternary structure.39 The formation of protein
aggregates is a common, yet challenging, problem for
biotherapeutics. During the downstream manufacturing steps,
the expressed protein is purified through various process
column steps (e.g., protein A chromatography, ion exchange
chromatography) and formulated using ultrafiltration/diafiltra-
tion. Most aggregates are removed during these processing
steps. However, aggregation occurs through a complex process
that can originate during the cell culture and downstream
process and product manufacturing, fill, and storage steps.
The formation of protein aggregates can be influenced by
post-translational modifications (PTMs), such as deamidation,
oxidation, iso-aspartic acid formation, disulfide bond scram-
bling, cross-linking, and by the presence of host-cell proteins
(residual proteins other than the intended product).40 Various
process manufacturing conditions can also influence the
formation of aggregation including pH, temperature, surface
area, and physical stress as well as extrinsic factors such as
storage conditions.41 The relationship between quality
attributes that can influence the amount and type of
aggregation and the impact of aggregation on other CQAs
related to safety and efficacy is depicted in Figure 6.
Figure 6. CQA relationships to aggregation.
While PTMs are monitored through characterization studies
and clearance of aggregates is monitored during the down-
stream process, the initial ATP would include the monitoring
of total aggregates and fragments in the drug substance, drug
product, and on stability. As development proceeds, this
selection process will be refined, linking related CQAs and
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establishing characterization and structure−function studies.
For example, during development, it will be critical to further
characterize the type of aggregation present because higher
order aggregates (e.g., greater than dimer) have an increased
potential to elicit adverse immunogenic responses in patients
and can, similar to the dimer, impact biological activity and
PK. 42 This approach will establish an evolving, but
comprehensive, ATP based on a clear assessment of the
structural features to monitor and the analytical controls
required for the commercial control strategy.
An example comparison of pros and cons for two techniques
considered for the initial method selection to detect aggregates,
and for the revised method selection when considering
additional characterization, is provided in Table 5. Addition-
ally, business drivers for the two techniques, such as how
quickly the method can be established in various laboratories,
the availability of technology, throughput, and speed of analysis
are provided in Table 6. While size exclusion chromatography
(SEC) is a common and readily available approach to detect
aggregates that has been reviewed elsewhere,43,44 it is
important to have a systematic evaluation using up-to-date
technical specifications and analysis requirements. The “pros”
and “cons” for particular technologies may be weighted
differently depending on the CQA details. Aggregation that
is suspected to be driven by different mechanisms may indicate
concerns about the stability of the aggregate for analysis and
lead to the selection of an orthogonal approach during
development of the process or even characterization of the
product.
In this example, the first goal for analysis is to establish a
method to quantitatively monitor the total % aggregate
throughout the various clearance steps and at release of the
product for clinical use. SEC with UV detection is most often
selected for process and quality control testing.45 The
separation and quantitation of aggregate species is achieved
based on the permeation of the analyte into and out of the
stationary phase pores (molecular sieving). There are benefits
with this as opposed to reverse phase and ion exchange
chromatography where analytes are retained by chemical
interactions with the stationary phase. SEC provides a robust,
rapid, quantitative approach to detecting aggregation with
good sample throughput. Equipment is readily available, low
cost, and already deployed in GMP QC laboratories. SEC is
relatively easy to train on and operate, meaning that
transferring the methodology can be a relatively uncomplicated
process. SEC can be coupled with fraction collection to assess
binding and bioactivity as part of critical structure−function
experiments and is compatible with a variety of detectors
including multiangle laser light scattering (MALS), expanding
SEC’s utility in providing characterization information for
aggregate species. Other separation techniques such as
hydrophilic interaction chromatography (HILIC) and RP-LC
may denature the protein. Many of these reasons also make
SEC ideal to deploy as in-process testing to monitor
manufacturing steps. For these reasons, SEC with UV
detection, as is the case here, is typically the appropriate
choice to fulfill the early phase development testing require-
ments. Figure 7 shows a chromatogram for a protein
therapeutic using a high-pressure, platform, SEC method that
provides faster analysis time with good resolution between the
monomer and higher molecular weight aggregate forms.
As development proceeds, it is critical to the commercial
control strategy to understand the impact of aggregation,
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Table 5. Pros and Cons of Methodology Selected to Detect and Characterize mAb Aggregation
particularly when significant levels of aggregate remain in the
final product or occur on stability. Understanding aggregation
involves not only separating and quantifying the total amount
of aggregates but also characterizing the aggregate forms
present and the potential impact on safety and efficacy. The
ATP was revised to select additional methods (complementary
and orthogonal to SEC-UV) that could confirm the accuracy
and range of higher order aggregate species and methods that
could collect samples of the observed peaks for character-
ization and binding studies.
MALS, in addition to UV absorbance, can be used as a
complementary detection scheme to determine the molecular
mass of the SEC fractions as seen in Figure 8. The molar mass
is calculated using light scattering intensity as a function of the
concentration at elution. While SEC coupled with MALS can
provide sensitive detection of higher order aggregates,46 SEC
has disadvantages that can challenge accurate characterization
of the full range of aggregate species in the product, and it is
generally accepted that additional techniques are needed for
the assessment of aggregates.47 Some of these disadvantages
include dilution effects where preparation of the sample can
induce the formation or dissociation of aggregates. Addition-
ally, adsorption of the protein to column or exclusion of
aggregates by the frit into the void can impact quantitation and
identification of higher order species.47 An orthogonal
pubs.acs.org/ac Perspective
approach to characterizing the aggregate species, particularly
confirming the accuracy and range of higher order aggregate
species, is sedimentation velocity analytical ultracentrifugation
(SV-AUC). SV-AUC measures the sedimentation coefficient of
the protein particles in a gravitational field. Radial scans of the
concentration profile are collected sequentially guided by UV
absorbance detection (typically 280 nm wavelength).48,49 The
distribution profile is used to provide an estimated molecular
weight and % abundance for each aggregate species. While SV-
AUC is sensitive to analyst control, has low throughput, and
can demonstrate relatively high measurement variability, SV-
AUC can also characterize insoluble or soluble aggregates. In
addition, SV-AUC avoids the issues with SEC previously
described that can impact quantitation and detection of the
range of higher order aggregate species.47,50 SV-AUC is an
orthogonal approach to confirm the SEC and SEC-MALS
observations.48,49
The ATP was revised using the additional requirements to
confirm the identification and range of higher order aggregates
in the product and the quantitative accuracy observed by SEC-
UV. Methods were screened against these requirements,
general method performance characteristics, and business
requirements. In addition to using SEC-UV, SEC-MALS and
SV-AUC were selected. Aggregate fractions from SEC were
collected and subjected to SEC-MALS demonstrating that
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Table 6. Evaluation of Business Considerations for Selected Aggregate Detection and Characterization Methodologya

a
Scoring = (++) high agreement with requirements, (+) meets requirements, (−) does not meet basic requirements or is not able to be evaluated.

Figure 7. Example SEC chromatogram (UHPLC). Inset showing the
zoom area.
aggregate species were predominately a dimer (Figure 8). SV-
AUC (not shown) confirmed that the drug product is
predominately a monomer, with noncovalently bound dimer
being the significant aggregate impurity.
Additionally, the aggregate fractions were tested in binding
studies using surface plasmon resonance (SPR). The SPR
J https://doi.org/10.1021/acs.analchem.1c03854
Figure 8. Example of SEC-MALS of collected aggregate fractions.
The dotted line indicates the molecular mass in Daltons as a function
of time.
studies revealed that binding to the antigen is impacted by
aggregates, reducing the specific binding activity of the
product. However, even larger percentages of dimer than
were observed (up to 5%) would have a negligible overall
effect on the potency of the product. The aggregate
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Analytical Chemistry
pubs.acs.org/ac
characterization work described here links directly to patient
safety. This, coupled with binding studies addressing efficacy,
could result in defining patient-centric specification limits and
avoiding limits derived solely by the process capability.37 The
result is further refinement of the ATP and a simpler
commercial analytical control strategy.
■
DISCUSSION
In small molecules, well-established pharmacopoeial mono-
graphs exist which link acceptance criteria to well-established
types of analytical technology. This makes method develop-
ment approaches for novel/different analytical technology
problematic. For example, Goodwin et al.51 describe the
situation for end product testing of tablet content, where the
traditional approach involves taking a composite sample of 10
tablets and performing a bulk assay by HPLC of 5 tablets in
duplicate. The mean of the two determinations is typically
reported with a limit of ±5% label claim in Europe and ±10%
in the U.S.
The application of at-line near-infrared (NIR) can be used
directly on intermediate materials during the manufacture of
final products without the need for any sample preparation and
has obvious benefits as discussed by Goodwin et al.51 For NIR
(and other spectroscopic analytical procedures), tablet content
is provided on individual tablets, and there is an obvious
potential for increasing the sample size well above 10 tablets
per batch for a more accurate determination of the batch mean.
Rather than rely on technology specific pharmacopoeial
guidance, an ATP can be used to ensure true technology
independent criteria can be established for the measurement of
quality attributes. Jackson et al.2 define an ATP for tablet
content where criteria for specificity, accuracy, and precision
are defined up front which can be adapted to any technology.
The vaccines example (Example 2: development of a cell-
based assay) demonstrated how a systematic approach for
technology evaluation and method screening enabled an
efficient analytical method which was aligned with regulatory
expectations to be developed which could analyze three very
different CQAs. This “multi-CQA” method could potentially
be converted into a platform analytical method (provided the
availability of specific monoclonal antibodies). Several backup
methods (including NAT assays) were also identified.
Unlike examples 1 and 2 where ATPs were already
established, the method development approach in example 3
for the aggregation of a monoclonal antibody in early
development was used to generate the structural understanding
required to establish a comprehensive analytical target profile.
A method was initially required to characterize and quantify
any resultant aggregation in the drug substance, drug product,
and on stability. As development proceeded, the method
selection process was refined linking related CQAs and
establishing characterization and structure−function study
goals. An ATP could only be developed once this character-
ization work was complete.
Fit-for-purpose analytical procedures were developed for
examples 1 and 2, which formed part of the overall control
strategy. Example 3 demonstrated how an ATP was revised
based on the knowledge gained from using a battery of
analytical technologies to better define the requirements for
measuring a CQA.
K https://doi.org/10.1021/acs.analchem.1c03854
■
Perspective
CONCLUSION
A well-constructed ATP enables all potential suitable analytical
technologies to be evaluated in an objective way. Consid-
eration of business drivers at the same time enables the
screening of methods to focus on the most preferred
technology that is likely to be robust and rugged. Multiple
ATPs can be considered together to identify a single
technology which can be used to develop a multiattribute
analytical procedure. In early development, for quality
attributes requiring assessment of impact on safety/efficacy,
analytical technologies can be used to develop structural
understanding which is required to establish an ATP in the
context of the overall control strategy.
■
AUTHOR INFORMATION
Corresponding Author
Phil Borman − Product Development and Supply, GSK,
Medicines Research Centre, Stevenage SG1 2NY, U.K.;
orcid.org/0000-0002-6742-5826;
Email: phil.j.borman@gsk.com
Authors
Cristiana Campa − Technical Research & Development,
Vaccines, GSK, 53100 Siena, Italy
Ghislain Delpierre − Analytical Research and Development,
GSK, 1330 Rixensart, Belgium
Elliot Hook − Global Pharma Analytical Science and
Technology, Pharma Supply Chain, GSK, Ware SG12 0DJ,
U.K.
Patrick Jackson − Product Development and Supply, GSK,
Medicines Research Centre, Stevenage SG1 2NY, U.K.;
orcid.org/0000-0002-0322-040X
Wayne Kelley − Product Development and Supply, GSK, King
of Prussia, Pennsylvania 19406, United States
Michel Protz − Analytical Research and Development, GSK,
1330 Rixensart, Belgium
Olivier Vandeputte − Analytical Research and Development,
GSK, 1330 Rixensart, Belgium
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.analchem.1c03854
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors wish to acknowledge Troy Adams, Marc Fiorucci,
Claudia Magagnoli, and Margarita Orticochea for their
contributions.
■
REFERENCES
(1) ICH Q8(R2) Pharmaceutical Development, 2009.
(2) Jackson, P.; Borman, P.; Campa, C.; Chatfield, M.; Godfrey, M.;
Hamilton, P.; Hoyer, W.; Norelli, F.; Orr, R.; Schofield, T. Anal.
Chem. 2019, 91 (4), 2577−2585.
(3) Barnett, K. L.; McGregor, P. L.; Martin, G. P.; Le Blond, D. J.;
Weitzel, M. L. J.; Ermer, J.; Walfish, S.; Nethercote, P.; Gratzl, G. S.;
Kovacs, E.; Pappa, H., Analytical target profile: Structure and
application throughout the analytical lifecycle. Pharmacopeial Forum
2016, 42 (5).
(4) Rignall, A.; Borman, P.; Hanna-Brown, M.; Grosche, O.;
Hamilton, P.; Gervais, A.; Katzenbach, S.; Wypych, J.; Hoffmann, J.;
Ermer, J.; McLaughlin, K.; Uhlich, T.; Finkler, C.; Liebelt, K. Pharm.
Technol. 2018, 42 (12), 18−23.
Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry pubs.acs.org/ac Perspective

(5) Elder, D. P.; Borman, P. Pharmaceutical Outsourcing 2013, 14 Winkel, J. G.J.; Aalberse, R. C.; Parren, P. W. H. I. Science 2007, 317
(4), 14−19. (5844), 1554−1557.
(6) USP ⟨1220⟩ Analytical Procedure Lifecycle Pharmacopeial (37) Ruesch, M. N.; Benetti, L.; Berkay, E.; Cirelli, D. J.; Frantz, N.;
Forum 2020, 46 (5). Gastens, M. H.; Kelley, W. P.; Kretsinger, J.; Lewis, M.; Novick, S.;
(7) Hanna-Brown, M.; Borman, P.; Bale, S.; Szucs, R.; Roberts, J.; Rellahan, B.; Pack, L.; Stroop, C. J. M.; Subashi, A.; Yin, P.; Zeng, M.;
Jones, J. Sep. Sci. 2010, 2, 12−20. Stults, J. J. Pharm. Sci. 2021, 110 (2), 771−784.
(8) Vogt, F. G.; Kord, A. S. J. Pharm. Sci. 2011, 100 (3), 797−812. (38) Den Engelsman, J.; Garidel, P.; Smulders, R.; Koll, H.; Smith,
(9) Li, Y.; Terfloth, G. J.; Kord, A. S. Am. Pharm. Rev. 2009, 12 (4), B.; Bassarab, S.; Seidl, A.; Hainzl, O.; Jiskoot, W. Pharm. Res. 2011, 28
571−583. (4), 920−933.
(10) Zhou, L.; Socha, J. M.; Vogt, F. G.; Chen, S.; Kord, A. S. Am. (39) Narhi, L. O.; Schmit, J.; Bechtold-Peters, K.; Sharma, D. J.
Pharm. Rev. 2010, 13 (1), 74−84. Pharm. Sci. 2012, 101 (2), 493−498.
(11) Zhou, L.; Vogt, F. G.; Overstreet, P. A.; Dougherty, J. T.; (40) Le Basle, Y.; Chennell, P.; Tokhadze, N.; Astier, A.; Sautou, V.
Clawson, J. S.; Kord, A. S. J. Pharm. Innov. 2011, 6 (4), 217−231. J. Pharm. Sci. 2020, 109 (1), 169−190.
(12) ICH Q2(R1) Validation of Analytical Procedures, 1994. (41) Philo, J. S.; Arakawa, T. Curr. Pharm. Biotechnol. 2009, 10 (4),
(13) Borman, P.; Roberts, J.; Jones, C.; Hanna-Brown, M.; Szucs, R.; 348−351.
Bale, S. Sep. Sci. 2010, 2, 2−8. (42) Ratanji, K. D.; Derrick, J. P.; Dearman, R. J.; Kimber, I. J.
(14) Borman, P.; Chatfield, M.; Jackson, P.; Laures, A.; Okafo, G. Immunotoxicol. 2014, 11 (2), 99−109.
Pharm. Technol. Eur. 2010, 4 (22), 38−46. (43) Brusotti, G.; Calleri, E.; Colombo, R.; Massolini, G.; Rinaldi, F.;
(15) Tome, T.; Ž igart, N.; Č asar, Z.; Obreza, A. Org. Process Res. Temporini, C. Chromatographia 2018, 81 (1), 3−23.
Dev. 2019, 23 (9), 1784−1802. (44) Hong, P.; Koza, S.; Bouvier, E. S. P. J. Liq. Chromatogr. Relat.
(16) Pollard, A.; Bijker, E. Nat. Rev. Immunol. 2021, 21, 83−100. Technol. 2012, 35 (20), 2923−2950.
(17) Brisse, M.; Vrba, S. M.; Kirk, N.; Liang, Y.; Ly, H. Front. (45) Philo, J. S. AAPS J. 2006, 8 (3), E564−E571.
Immunol. 2020, 11, 2578. (46) Ahrer, K.; Buchacher, A.; Iberer, G.; Josic, D.; Jungbauer, A. J.
(18) Sasso, E.; D’Alise, A. M.; Zambrano, N.; Scarselli, E.; Folgori, Chromatogr. A 2003, 1009 (1−2), 89−96.
A.; Nicosia, A. Semin. Immunol. 2020, 50, 101430. (47) Gandhi, A. V.; Pothecary, M. R.; Bain, D. L.; Carpenter, J. F. J.
(19) Humphreys, I. R.; Sebastian, S. Immunology 2018, 153 (1), 1− Pharm. Sci. 2017, 106 (8), 2178−2186.
9. (48) Gabrielson, J. P.; Arthur, K. K. Methods 2011, 54 (1), 83−91.
(20) Voellmy, R.; Bloom, D. C.; Vilaboa, N. Vaccines 2020, 8 (2), (49) Philo, J. S. Curr. Pharm. Biotechnol. 2009, 10 (4), 359−372.
230. (50) Arthur, K. K.; Kendrick, B. S.; Gabrielson, J. P. Methods
(21) Barry, M.; Rubin, J.; Lu, S. FEBS Lett. 2020, 594 (12), 1918− Enzymol. 2015, 562, 477−500.
1946. (51) Goodwin, D. J.; van den Ban, S.; Denham, M.; Barylski, I. Int. J.
(22) Afrough, S.; Rhodes, S.; Evans, T.; White, R.; Benest, J. Vaccines Pharm. 2018, 537 (1−2), 183−192.
2020, 8 (1), 131.
(23) Fedosyuk, S.; Merritt, T.; Peralta-Alvarez, M. P.; Morris, S. J.;
Lam, A.; Laroudie, N.; Kangokar, A.; Wright, D.; Warimwe, G. M.;
Angell-Manning, P.; Ritchie, A. J.; Gilbert, S. C.; Xenopoulos, A.;
Boumlic, A.; Douglas, A. D. Vaccine 2019, 37 (47), 6951−6961.
(24) Vitelli, A.; Folgori, A.; Scarselli, E.; Colloca, S.; Capone, S.;
Nicosia, A. Expert Rev. Vaccines 2017, 16 (12), 1241−1252.
(25) Kozlowski, D. A.; Bremer, E.; Redmond, D. E., Jr; George, D.;
Larson, B.; Bohn, M. C. Mol. Ther. 2001, 3 (2), 256−261.
(26) Suzuki, M.; Kondo, P.; Pei, Z.; Maekawa, A.; Saito, I.; Kanegae,
Y. Gene Ther. 2015, 22, 421−429.
(27) Takahashi, Y.; Vikman, E.; Nishikawa, M.; Ando, M.;
Watanabe, Y.; Takakura, Y. J. Gene Med. 2010, 12 (9), 739−746.
(28) Nambiar, B.; Cornell Sookdeo, C.; Berthelette, P.; Jackson, R.;
Piraino, S.; Burnham, B.; Nass, S.; Souza, D.; O’Riordan, C. R.;
Vincent, K. A.; Cheng, S. H.; Armentano, D.; Kyostio-Moore, S. Hum.
Gene Ther: Methods. 2017, 28 (1), 23−38.
(29) Wang, Z.; Zhao, J. Viruses 2019, 11 (8), 741.
(30) Brescia, M.; Janssen, J.; Liu, J.; Goncalves, M. Cells 2020, 9 (4),
869.
(31) Lee, C. S.; Bishop, E. S.; Zhang, R.; Yu, X.; Farina, E. M.; Yan,
S.; Zhao, C.; Zeng, Z.; Shu, Y.; Wu, X.; Lei, J.; Li, Y.; Zhang, W.;
Yang, C.; Wu, K.; Wu, Y.; Ho, S.; Athiviraham, A.; Lee, M. J.; Wolf, J.
M.; Reid, R. R.; He, T. C. Genes Dis. 2017, 4 (2), 43−63.
(32) FDA. Chemistry, Manufacturing, and Control (CMC) Information
for Human Gene Therapy Investigational New Drug Applications
(INDs), 2020.
(33) Kimiz-Gebologlu, I.; Gulce-Iz, S.; Biray-Avci, C. Mol. Biol. Rep.
2018, 45 (6), 2935−2940.
(34) Teo, E. C. Y.; Chew, Y.; Phipps, C. Crit. Rev. Oncol./Hematol.
2016, 97, 72−84.
(35) Mahmuda, A.; Bande, F.; Kadhim Al-Zihiry, K. J.;
Abdulhaleem, N.; Majid, R. A.; Hamat, R. A.; Abdullah, W. O.;
Unyah, Z. Trop. J. Pharm. Res. 2017, 16 (3), 713−722.
(36) van der Neut Kolfschoten, M.; Schuurman, J.; Losen, M.;
Bleeker, W. K.; Martinez-Martinez, P.; Vermeulen, E.; den Bleker, T.
H.; Wiegman, L.; Vink, T.; Aarden, L. A.; De Baets, M. H.; van de

L https://doi.org/10.1021/acs.analchem.1c03854
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