COMMENTARY

Strategies for the Assessment of Protein Aggregates

in Pharmaceutical Biotech Product Development
John den Engelsman & Patrick Garidel & Ronald Smulders & Hans Koll & Bryan Smith & Stefan Bassarab & Andreas Seidl & Otmar Hainzl &
Wim Jiskoot
Received: 4 August 2010 /Accepted: 28 September 2010 /Published online: 23 October 2010
# The Author(s) 2010. This article is published with open access at Springerlink.com
ABSTRACT Within the European Immunogenicity Plat-
form (EIP) (http://www.e-i-p.eu), the Protein Characteriza-
tion Subcommittee (EIP-PCS) has been established to
discuss and exchange experience of protein characterization
in relation to unwanted immunogenicity. In this commen-
tary, we, as representatives of EIP-PCS, review the current
state of methods for analysis of protein aggregates.
Moreover, we elaborate on why these methods should be
used during product development and make recommen-
dations to the biotech community with regard to strategies
for their application during the development of protein
therapeutics.
KEY WORDS aggregation
.
aggregates
.
analytical
characterization
.
biotech products
.
commentary
.
drug
development
.
particles
.
proteins
.
recommendation
INTRODUCTION
Despite the high quality of current therapeutic biotech
products and the resemblance of recombinant human
proteins and antibodies to endogenous human proteins,
protein immunogenicity remains an important concern.
Among the several factors playing a role in immunogenicity,
John den Engelsman and Patrick Garidel contributed equally in this article.
J. den Engelsman
:
R. Smulders
Biological and Sterile Product Development, MSD
Oss, The Netherlands
P. Garidel
:
S. Bassarab
Boehringer Ingelheim Pharma GmbH & Co. KG
Department of Process Science/Biopharmaceuticals
Biberach an der Riss, Germany
H. Koll
Biologics Research Protein Analytics
Roche Pharma Research and Early Development
Penzberg, Germany
B. Smith
UCB
Slough, Berkshire, UK
A. Seidl
:
O. Hainzl
Sandoz Biopharmaceuticals, Analytical Characterization, Hexal AG
Oberhaching, Germany
W. Jiskoot (*)
Division of Drug Delivery Technology
Leiden/Amsterdam Center for Drug Research
Leiden University
P.O. Box 9502, 2300 RA Leiden, The Netherlands
e-mail: w.jiskoot@lacdr.leidenuniv.nl
Pharm Res (2011) 28:920933
DOI 10.1007/s11095-010-0297-1
the presence of aggregates is considered an important
product-related factor that may increase the risk of an
immune response (1,2). Although little is known about which
aggregate species trigger the immune system, it is believed
that aggregates are more easily recognized by the immune
system than the native parent protein.
Aggregates (see Glossary, Table I) that may be present in
protein products can range from small (dimers) to large
assemblies (subvisible or even visible particles). They can be
formed during production, storage, shipment or delivery to
the patient. Numerous stresses (e.g., temperature fluctua-
tions, light, shaking, surfaces, pH adjustments, etc.) can
induce protein aggregation during each of these stages (3,4).
Aggregation can occur because of exposure to air-liquid or
liquid-solid interfaces, e.g., during mixing, during filling
and shipping, during reconstitution of lyophilized products,
or through contact with chromatography columns, pumps,
pipes, vessels, filters, etc. Also, solution contact with ice
during (adventitious or deliberate) freezing can cause
aggregation (5,6). Moreover, protein aggregates may in
some cases be induced by foreign particles, e.g., stainless
steel and other particles from filling pumps, rubber particles
from stoppers, salt crystals, glass particles generated during
heating of containers for depyrogenation, and silicone oil
droplets originating from siliconized syringes or stoppers
(710). Protein aggregation may also be followed or
induced by chemical degradations/modifications, e.g.,
oxidation (11,12).
The challenge in analyzing protein aggregates lies in the
unknown nature of the formed aggregates as well as the
wide size range of up to six orders of magnitude, from a few
nm to a few mm in diameter. Since no single one of the
currently available techniques is able to cover this size
range, a combination of several techniques is necessary.
However, each technique has its own strengths and
weaknesses. Moreover, the available methods differ in the
physical measuring principle and, consequently, in the
results and type of information obtained.
The aim of this commentary is to discuss the currently
available analytical methods to characterize protein
aggregates in relation to product quality and also the
interpretation of data resulting from these methods.
Moreover, we propose approaches to use these methods for
the characterization of protein therapeutics from early
product development through to commercialization.
This paper is a result of discussions among the co-authors
of this paper, who participate in the protein characterization
subcommittee (PCS) of the European Immunogenicity
Table I Glossary
Term Definition
Aggregates Assemblies of protein molecules other than the desired (e.g. monomeric)
species. Aggregates may differ in size (ranging from nm to m in diameter),
morphology (approximately spherical to fibrillar), protein structure
(native vs. denatured), type of intermolecular bonding
(covalent vs. non-covalent) and reversibility.
Extended characterization (EC) assays Assays that complement QC assays (see below), used throughout product
development to acquire a thorough physico-chemical characterization of
protein drug substance and drug product. Although EC assays should be scientifically
sound, potentially they cannot be validated in accordance with ICH guidelines
(ICH Q2(R1)). EC assays generally have a low(er) throughput and poor(er)
robustness, i.e. they may require advanced instruments and data interpretation
at an expert level.
Orthogonal methods Independent methods that fundamentally differ from each other in the physical
measuring principles that are used to investigate a certain aspect of a sample.
For example, aggregates may be detected by orthogonal microscopy, chromatography
or centrifugation methods.
Particles Undissolved species (other than gas bubbles or droplets) that are unintentionally
present in the product. Particles can be foreign (not intrinsic to drug substance)
or protein-related (i.e. large aggregates). Particles can be further categorized as
visible (>ca. 50 m) and subvisible (between ca. 0.150 m); submicron particles
(between ca. 0.11 m) are a subcategory of subvisible particles.
Quality control (QC) assays Assays that are used to release clinical batches throughout product development
and commercial batches after product launch (drug substance and drug product).
QC assays need to be validated in accordance with ICH guidelines (ICH Q2(R1)).
QC assays generally should have a high throughput and good robustness, i.e. they
require conventional instruments and data interpretation at a non-expert level.
QC assays are used for formal batch release testing and stability monitoring (GMP)
and may also be used for development support activities (next to EC assays).
Strategies for the Assessment of Protein Aggregates 921
Platform (EIP; see Table II). Within the EIP, the EIP-PCS
(Table II) was established to discuss product-related factors
associated with immunogenicity and methodologies for
protein characterization.
TECHNIQUES TO ANALYZE PROTEIN
AGGREGATES
Many techniques are available for the analysis of protein
aggregates or proteinaceous particles (and particles in
general). These techniques rely on different separation as
well as different detection principles and range from
relatively basic techniques such as visual or microscopic
inspection, through to high-tech methods such as analytical
ultracentrifugation (AUC) or mass spectrometry (4). In
order to obtain a broad view of the possible protein
aggregates that may be found in a protein sample, it is
recommended that complementary, orthogonal approaches
(see Table I) are used, especially in the development stage.
The various techniques available for the analysis of
protein aggregates or particles are summarized in Table III.
Furthermore, this table summarizes the basic principles,
describes types of analyses, e.g., qualitative (what can be
observed) and quantitative (size range, particle number),
and addresses some advantages and disadvantages of each
technique. Clearly, some of the analytical methods sum-
marized in Table III are focused directly on the primary
criterion of the protein aggregates, namely their size,
whereas other methods address other characteristics, e.g.
changes in secondary or tertiary structure. The conse-
quence of these distinct analytical features (e.g. different
separation, detection and characterization principles) is that
diverse aspects of protein aggregates are observed and
detected. Whereas this allows us to get a comprehensive
picture of a products aggregate profile, comparison of the
data obtained from different analytical methods may be
inappropriate because different techniques essentially
measure different characteristics, and establishing correla-
tions may be impossible in some cases. The analytical
methods listed in Table III differ from each other also in
performance aspects, like assay robustness and sensitivity,
impact of sample preparation, amount of sample required,
and ease of assay validation. These aspects, summarized in
Table IV, dictate the use of the analytical technique at
different stages of the development of a product. Based on
discussions among the members of the EIP-PCS, we
identified techniques which are commonly used in the
industry as analytical quality control (QC) and extended
characterization assays. These methods and their advan-
tages and disadvantages in protein aggregate analysis are
discussed in more detail in the following paragraphs.
Pharmacopeia Methods for Assessing Visible Particles
As set forth by the United States Pharmacopeia (USP) and
the European Pharmacopeia (EP), injectable solutions need
to be practically/essentially free of visible particles.
Manufacturers are expected to test every container of a
GMP batch for human use for visible particles and to reject
contaminated containers. Furthermore, the manufacturers
are recommended to have procedures in place that describe
the Acceptable Quality Levels (AQL) of the inspection
process and criteria for the maximum number of rejects per
batch. The wording practically/essentially free reflects
the limitations of the inspection process, i.e., the visual
inspection process is subjective and probabilistic and simply
cannot guarantee the total absence of visible particles from
all containers.
Originally, the compendial guidance was primarily
focused on particles originating from external sources such
as manufacturing materials and primary packaging compo-
Table II EIP-PCS
EIP EIP-PCS
The European Immunogenicity Platform (EIP) was created in 2007
by experts in the field of immunogenicity (http://www.e-i-p.eu).
The EIP represents companies, institutes and professionals involved in
research, development, testing, validation, application, production or
marketing of immunogenicity assessment tools, as well as those servicing
the biotechnology community. Its mission is to build know-how and
expertise in the field of immunogenicity, driven by a close interaction
between industry and scientific advisors. Its scope is to interact with
authorities regarding immunogenicity guidelines, formulate active
recommendations regarding immunogenicity, stimulate research
addressing the clinical and non-clinical effects of unwanted immunogenicity,
and boost collaborations between academia and pharmaceutical
companies. Through its working-group structure, the EIP can react in a
focused way on regulatory and scientific evolutions in the immunogenicity
field.
One of EIPs working groups, the Protein Characterization Subcommittee
(EIP-PCS) was established early 2008. The mission of the EIP-PCS is to
discuss and exchange experience with protein characterization in relation
with immunogenicity, in order to increase our fundamental understanding
of product-related causes of immunogenicity. One of the aims of the EIP-
PCS is to define common strategies and methodologies for protein
characterization in relation with immunogenicity, in the context of which
the current paper was established.
922 den Engelsman et al.
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Strategies for the Assessment of Protein Aggregates 925
nents. However, for protein products, visible particles may
also be product related. That is, protein products may
contain insoluble (visible) particles derived from the protein
product itself. In these cases, sterile filtration is often not
effective because insoluble protein particles may reform
over time. Nowadays, regulators expect that manufacturers
try to limit the presence of proteinaceous particles by
applying good formulation strategies and practices (13).
However, in specific cases, the presence of particles may be
accepted as an inherent product attribute, provided that
these particles do not pose a quality or clinical safety
concern. One example of this is insulin. In the EP it is
stated that insulin solutions may form insoluble particles
over time. Moreover, intermediate-acting and long-acting
insulin suspensions are particulate by definition. Neverthe-
less, insulin has proven to be a very safe product for many
years in a large number of patients. Also, for patients with a
compromised immune system, a product with non-optimal
appearance may be acceptable, provided that clinical data
do not reveal major safety issues.
Assay of visible particles is typically done by trained
operators under controlled conditions. Automated or semi-
automated instrumental methods may be applied, but
even then the human factor may be relevant, e.g. during
the qualification of the automatic inspection process
(i.e., manual testing to check if the AQL is met). The EP
describes a specific visual inspection method (the black and
white box). The threshold of visibility is believed to be ca.
50 m for a spherical particle. However, detection of visible
particles is a problematic process (14), and in daily practice
the detection limit very much depends on the individual
operator, the inspection device, the inspection time as well
as the morphology, number and refractive index of the
particles. Companies may use various additional, non-EP
methods for manual visual inspection to improve the
sensitivity or ergonomics of the process, such as the use of
aids such as magnifying glasses or differences in lighting
conditions or observation times and swirling procedures.
These operations may have a pronounced effect on reject
percentages.
Table IV Typical Use of Techniques in Industry with Respect to Aggregate Analysis
Method Validation Quantification Robustness
a
Sensitivity
a
Sample throughput
a,b
QC method
c
Visual inspection Yes No Medium Medium High Yes
Optical microscopy No Possible Medium N/A Low No
Fluorescence microscopy No No Low High Low No
Electron microscopy No No Low N/A Low No
Flow imaging No Yes Low N/A Medium No
Atomic force microscopy No No Medium N/A Low No
Turbidity Yes No High Medium Medium Yes
DLS No No Medium High High No
SEC-MALLS No No (MALLS part) Medium High High No
Light obscuration Yes Yes Medium Medium Medium Yes
Native mass spectrometry No No Low Medium Low No
Macro-IMS No No Low N/A Medium No
AUC No Yes Low Medium Low No
SEC Yes Yes High Medium High Yes
AF4 Yes Yes Medium Medium High No
SDS-PAGE Yes Possible Medium Medium High Yes
Native PAGE Yes Possible Medium Low Medium No
CE-SDS Yes Yes Medium Medium High Yes
UV-VIS spectroscopy No No Medium Medium High No
Infrared spectroscopy No No Medium N/A Low No
Raman spectroscopy No No Medium N/A Low No
Fluorescence spectroscopy No No Medium N/A High No
Circular dichroism spectroscopy No No Medium N/A Medium No
NMR spectroscopy No No Medium Medium Medium No
a
Scoring (low, medium, or high) was based on consensus of opinion of the authors; N/A = not available;
b
Low: <10; medium; 1025; high >25 per day and per
operator;
c
QC = quality control; all listed methods can be used for extended characterization; see Table I for definitions
926 den Engelsman et al.
Although visual inspection itself is pretty straightforward,
interpretation of final inspection results can be very
difficult. For instance, abnormal reject rates may be
observed after GMP batch manufacture in a large-scale
commercial facility, whereas no problems were noticed for
clinical batches produced in a small-scale facility. In such a
situation a root cause investigation can be very laborious
and time-consuming because the abnormal reject rate may
be caused by either a manufacturing failure, a change in
manufacturing conditions, a difference in visual inspection
conditions between the large- and small-scale facilities, or a
combination of these factors. In order to minimize particle
problems as much as possible, it is recommended to use
harmonized inspection procedures and acceptance criteria
throughout development (especially for release testing) and
to evaluate the performance of the visual inspection method
before manufacture of GMP batches.
Pharmacopoeia Methods for Assessing
Subvisible Particles
The pharmacopoeial guidance with regard to assessing
subvisible particles is applicable to all parenteral solutions or
lyophilizates, including those containing proteins. As to
analytical methodology, the US and EU pharmacopeias are
harmonized and basically describe two methods to count
particulate matter, namely light obscuration and micros-
copy. These methods are accompanied by acceptance
criteria defining the maximum numbers of subvisible particles
allowed per container, or per mL for large-volume injectable
solutions. Interestingly, these criteria are different between the
two methods, reflecting the differences in detection principle.
The measuring range of most light obscuration instruments
is ca. 2100 m diameter, with varying precision and
reproducibility. Potential pitfalls of the method lie in the
sample preparation, which has to ensure that no sample
contaminations or other artifacts occur. Air bubbles and
silicone oil droplets from the primary packaging may mistak-
enly be counted, non-spherical particles cannot adequately be
measured, and the technique is unable to discriminate
proteinaceous particles from unrelated, non-proteinaceous
solid particles. The microscopy method uses a suitable
binocular microscope. The sample is filtered, and the filter-
dried particles are then counted (manually or automatically) on
the filter. With this method, potential contaminations can
occur. Furthermore, the method is extremely labor intensive,
which limits its use for routine, large throughput applications.
The current compendial acceptance criteria for subvisible
particles are usually met for modern injectable solutions.
However, it should be realized that these criteria were
originally not set to control proteinaceous particles. Further-
more, criteria exist only for relatively large subvisible particles
(greater than 10 and 25 m diameter), whereas smaller
particles (between 0.1 and 10 m diameter) are not
considered. An animated discussion is currently ongoing
between regulators, industry and academia to determine
whether subvisible particles could pose a significant immuno-
genicity risk (15,16). The amount of proteinaceous subvisible
particles in terms of mass, however, is typically very low
(ng/ml-g/ml range), and it is unclear whether such an
amount could indeed trigger an immune response. Unfortu-
nately, the current compendial methods for subvisible particle
testing have several limitations regarding robustness, sensitiv-
ity and type of particles that can be quantified accurately.
Therefore, currently the industry is evaluating technologies
such as micro-flow imaging (17). However, whether these
new technologies really perform better and can be applied in
a QC environment remains to be established.
High Performance Size Exclusion Chromatography
The basic principle of high performance size exclusion
chromatography (SEC) is simple. Solute molecules are
passed through a column containing porous beads. Small
molecules penetrate the pores of the beads, while larger
molecules or aggregates do not; therefore, small molecules
take a longer path through the column and elute later.
Nominally, the ability to enter the pores is dictated by
molecular size, so if it is assumed that the analyte molecule
is spherical, the elution position (relative to molecular
weight standards) may be used to estimate molecular
weight. However, any such estimate is an approximation,
as most molecules tend to not be spherical, and actually the
hydrodynamic radius, rather than the molecular weight, is
measured (18). Assuming monomer and oligomers have the
same extinction coefficient, the absorbance at 280 nm or
lower (214 or 220 nm) may be used to quantify the extent
of aggregation of a sample. Analytical SEC typically
employs small columns, in which samples of a few to tens
of g of sample are sufficient to obtain a result.
Aqueous, non-denaturing buffers are compatible with
SEC. The basic method may be modified to gain insight
into the quality of aggregates, for instance by addition of
denaturant to the eluent (19). This can disperse non-
covalently associated aggregates. Alternatively, samples
may be treated prior to SEC, for instance by reduction
(and reaction of cysteinyl thiols with an alkylating agent), to
break disulfide bonds. The addition (to the eluent) of
fluorescent dyes that bind to denatured protein (and so
inducing an enhancement of the fluorescence) allows
detection of very low amount of denatured protein (20).
Quantification is not possible, however, since the fluores-
cence depends on both the type and extent of denaturation.
Finally, additional detectors may be added to give
complementary information, such as molecular weight
and stoichiometry of complexes from use of on-line light-
Strategies for the Assessment of Protein Aggregates 927
scattering, UV absorbance, and refractive index detectors
(21,22).
Various factors may affect the results of SEC, as
discussed recently by Carpenter et al. (23). For instance,
low solution ionic strength (e.g., 50 mM or less) may
encourage hydrophobic interactions of the eluting protein
with the column matrix, thereby slowing elution, affecting
resolution and peak shape. Addition of arginine to the
eluent may inhibit interaction between solute and column
matrix (24). Detergents in the sample (as opposed to the
eluent), though nominally of small molecular weight, can
behave as large molecules if they form micelles (above their
critical micelle concentration), appearing in the chromato-
gram as UV-absorbing peaks (25) and potentially also
giving rise to light scattering and fluorescence signals.
There is an upper limit to the size of aggregate
detectable by SEC, because larger aggregates can be
filtered out by frits in the system or by the column itself.
As a consequence, large material (large protein aggregates)
may disappear and be overlooked in the analysis. They also
build up on the top of the column and gradually degrade its
performance, seen as broadened peaks, poorer resolution
and decreased yields (smaller peaks). Another form of
aggregate that may be missed is that formed by very low
affinity intermolecular association, as these may dissociate
into monomers following a change in conditions from those
of the sample to those experienced during chromatography
(e.g., dilution or change in temperature) (26). For detection
of such low affinity aggregates other methods could be
used, such as AUC, or method conditions of SEC could
possibly be adjusted.
SDS-PAGE and Capillary Electrophoresis-SDS
SDS-PAGE is a very common, fairly robust method that is
easy to perform and can supply information on approximate
molecular weight and quantity, when using a suitable
method of quantitative staining and gel scanning. The
presence of SDS means that non-covalent aggregates are
disrupted, so the method only detects covalent aggregates. If
reducing conditions are used, SDS-PAGE can discriminate
between aggregates held together by disulfide bonds and
those held together by other (non-reducible) covalent bonds.
SDS-PAGE is becoming replaced by its capillary
electrophoresis counterpart, CE-SDS, as the latter is better
suited for robust quantification. With CE-SDS, one can
achieve similar results in a technically slightly different way,
with automation of running of samples and quantification
by UV absorption rather than dye-binding. For both
methods, low g amounts of sample are needed for the
analysis, throughput is medium to high, and turn-around
quick. A combination of SEC and SDS-PAGE and/or
CE-SDS is usually part of QC analytics.
Dynamic Light Scattering
Dynamic light scattering (DLS), also known as quasi elastic
light scattering (QELS) and photon correlation spectrosco-
py (PCS), is a technique used for the determination of
the size distribution of particles in the diameter range of
12 nm to 35 m (27). DLS is a non-destructive technique
for the characterization of colloidal systems like protein
solutions, allowing a re-use of the sample for further
characterization, possibly useful if only a small amount of
product is available, such as in early stage development.
Using modern equipment, the volume required for a DLS
analysis can be as low as a few l. The concentration ranges
between 0.150 mg/ml for protein solutions. Measure-
ments of highly concentrated solutions are becoming
feasible owing to the application of photon cross-correlation
approaches (28), for example. Although the sensitivity of
this technique for detection of large particles in particular is
unsurpassed, quantification is not possible. DLS yields
qualitative results, not quantitative results.
Analytical Ultracentrifugation
AUC, mostly used in sedimentation velocity mode (SV-AUC),
is a powerful technique to characterize the sedimentation
behavior of macromolecules and the presence of aggregates in
solution. Recently, Philo reviewed AUC as a method for
characterizing non-particulate, soluble protein aggregates,
including its strengths and weaknesses (29). One of the major
advantages of AUC is that protein therapeutics can often be
characterized without sample manipulation in relevant
solutions such as their formulation buffer, with some minor
exceptions (e.g. non-ideality may occur at high concentra-
tions). Quantification of aggregate species is possible, while
formation or disruption of aggregates due to sample
preparation, dilution or matrix effects is limited. However,
as noted by Philo (29), reproducibility is often relatively poor,
and assigning a limit of detection (LOD) or quantification
(LOQ) for aggregate detection using SV-AUC is difficult for
several reasons. Among the several practical and experimen-
tal parameters that influence the accuracy and precision of
AUC experiments, cell (mis)alignment and the quality of the
centerpieces especially seem to be major reasons for
variations in quantification of low amounts of aggregates
(3032). Therefore, regular calibration and intensive main-
tenance of the system and its accessories are required to
assure their proper functioning. Furthermore, it should be
recognized that the mathematical calculations behind AUC
analyses (i.e. selection and use of data analysis software such
as Sedfit or Ultrascan, and appropriate models to describe
the size distribution) may also have significant impact on the
absolute aggregation levels reported. For antibody therapeu-
tics, and in accordance with our own experience, the LOQ is
928 den Engelsman et al.
approximately 1% at best, and precision for samples
containing <3% aggregates is low (30). Nevertheless, because
of some strong advantages over SEC or AF4, like the
absence of interactions of the molecules of interest with
columns or membranes, AUC can be a valuable tool for a
qualitative cross-check or analysis of trend of data obtained
by SEC or AF4. AUC can be used to verify that no
aggregate type potentially present in a sample is missed by
SEC or AF4. For a (semi)quantitative cross-confirmation,
AUC is only suited to analysis of aggregate contents greater
than about 35%. Therefore, we suggest using AUC only as
a qualitative or relative method (comparison of relative
aggregation levels) but not as an absolute quantitative
orthogonal method, at least not until the technical issues of
equipment and component quality resulting in limited
precision and reproducibility are solved.
Asymmetrical Flow Field-Flow Fractionation
Asymmetrical flow field-flow fractionation (AF4) is an
analytical technology to separate proteins ranging from a
few nanometers to a few micrometers diameter (33).
Separation in AF4 occurs in a thin flow channel. Perpen-
dicular to the laminar channel flow, a cross-flow is
generated that leads the channel through a membrane.
This cross-flow directs the analyte proteins to the mem-
brane, but they cannot leave the channel because the
membrane is impermeable to them. Since small proteins
have a relatively large diffusion coefficient, they will return
more rapidly to the fast streamlines of the laminar channel
flow than large proteins. Consequently, small proteins elute
from the channel before large proteins.
Like SEC, AF4 separation can be combined with various
detectors such as UV, refractive index and multi-angle laser
light scattering (MALLS). In addition, AF4 does not require
much sample preparation, and even very large (insoluble)
protein aggregates may be detected. On the other hand, the
technique is less mature than conventional chromatography
and often requires in-depth method development (e.g. related
to channel dimensions, types of membranes and flow rate) to
obtain acceptable separation and robustness. For that reason,
application of AF4 as a QC test has been limited.
Quality Control Versus Extended
Characterization Assays
Obviously, a plethora of techniques is available and suitable
for aggregate detection, quantification or characterization,
each method having its own strengths and weaknesses (see
Table IV). However, not all techniques are suitable to be
applied at all stages of product development. Clearly, there
is a distinction between techniques that are routinely used
in a QC environment and techniques that are used for
extended characterization during product development (see
Table IV). To assess aggregation in therapeutic protein
products in the QC environment, relatively simple, robust
and quick assays of SEC and SDS-PAGE and/or CE-SDS
are used alongside the compendial methods for larger
(sub)visible particles (whether or not of proteinaceous
nature).
Techniques such as DLS, AUC, SEC with online
MALLS detection (SEC-MALLS) and AF4 can be applied
to support SEC, but are generally not used for QC analysis
because of one or several of the following reasons: a) some
of these extended characterization assay instruments are
very time consuming, costly to buy and maintain, and not
suited for high throughput analysis; b) these assays are
typically less accurate, precise and robust than conventional
chromatography; c) the operation of challenging techniques
requires skilled operators, and data obtained require expert
interpretation; d) in terms of ICH guidelines these assays
are difficult or impossible to validate. For instance, in the
case of DLS, it is impossible to perform quantification of
the species present. Therefore, determination of the limit of
detection for a certain protein (aggregate) is impossible
according to ICH guidelines. However, qualitatively, DLS
is unsurpassed in detecting trace amounts (<0.01% w/v) of
large aggregate species in the submicron-to-low m range.
SEC-MALLS is very useful throughout product devel-
opment but is not recommended as a routine QC method
(see Table III). A major disadvantage of light scattering in
general is its distinct sensitivity towards large-sized partic-
ulate contaminations, like dust, or particles arising from
pump abrasion or column erosion. This feature requires
attention to be paid to elution buffers (low particle content
advantageous), with inclusion of in-line filters between SEC
column and MALLS detector to remove particles (but
possibly also aggregates), and prolonged equilibration times
to obtain a stable baseline. As a consequence, more time-
consuming efforts are needed to run the method compared
to the classical SEC method, whenever an analysis cycle has
to be started or the elution buffer or the column has to be
changed. Data evaluation is also more time consuming.
Robustness of the method is therefore also impacted
significantly. Another disadvantage is that MALLS is a
qualitative method and cannot be used for quantification.
AUC is also not suited to be used as a QC test. Besides
the required operator expertise and the technical challenges
of the system itself and the advanced data analysis that is
required for performing and evaluating AUC experiments,
the technique suffers from poor reproducibility and
robustness when compared to a technique such as SEC.
The reproducibility of an AUC experiment is very
dependent on practical and technical aspects and the setup
of the actual experiment (34). This evidently makes
validation according to ICH guidelines problematic.
Strategies for the Assessment of Protein Aggregates 929
WHY, HOW, AND WHEN TO APPLY
EXTENDED CHARACTERIZATION METHODS
DURING PRODUCT DEVELOPMENT
Two general levels can be discerned in a proposed strategy:
a) initial analysis of the product with robust methods (such
as SEC, SDS-PAGE/CE-SDS, light obscuration); b)
further analysis on a case-by-case basis, using less robust,
lower throughput techniques (such as AUC, AF4, DLS,
microscopy methods, etc.).
Support of Formulation and Process Development
Applying extended characterization assays to assess aggre-
gate levels during formulation and process development can
be useful for product understanding. In the case of
formulation development, understanding the effects that
excipients have on the stability of the protein and
understanding the underlying mechanisms enable rational
development of the drug substance and drug product.
Furthermore, during the manufacturing process, the pro-
tein is often exposed to harsh conditions. Using in-process
sampling during development and examining the structural
integrity of the product enables determination of the impact
of the different process (and holding) steps on product
quality and adjustment of the process to minimize struc-
tural perturbations and aggregation.
Extended characterization should also be used to
investigate the compatibility of the product with materials
that may induce aggregation, e.g. primary package materi-
als (such as syringes containing silicone coatings) or
leachables from glass or tubing. Additionally, extended
characterization is very well suited for troubleshooting. This
might arise in situations where QC methods show atypical
results, such as in a case where SEC shows new peaks or
unexpected elution profiles.
Extended Characterization as an Integral Part of SEC
Method Development
Extended characterization assays, like AUC, DLS, SEC-
MALLS or AF4 should be used as supportive methods for
SEC method development, rather than for qualification or
cross-validation of SEC. Applying extended characterization
contributes to the characterization of the SEC profile and
could detect aggregates that were not detected by SEC. In
order to add greater confidence, a trending analysis could be
performed by looking at the relative increase in aggregate
content using extended characterization assays in concert with
SEC. If a progressive increase of aggregates is only seen using
the extended characterization techniques during real-time
stability, actions should be taken accordingly, i.e. a root cause
analysis.
By integrating the extended characterization assays early
in development, the need for these analyses in the
commercial production stage may be diminished and would
keep QC manageable in a production environment. The
classical SEC method should then be sufficient and well
suited for characterization and release of batches for market
products.
Extended Characterization During
Comparability Exercises
Comparability exercises are important throughout product
development. In the broadest sense, product comparability
exercises need to be performed during changes in cell line,
process, or scale. During these exercises, besides the
standard set of analytical tools such as SEC and SDS-
PAGE, extended characterization is required to determine
whether any change has affected product quality. Addi-
tionally, comparability exercises may be used to determine
quality differences between samples for toxicological studies
and clinical batches of different phases. Such analyses may
then help to justify specifications for aggregation products.
Characterization of the Type of Aggregates
Despite optimization of formulation, handling and processing
of protein pharmaceuticals, aggregation cannot be totally
avoided, and trace amounts remain present and/or are
formed during storage. Assessment of the risk of aggregates
in a product is difficult, however, because the relationship
between the nature of protein aggregates and their potential
immunogenicity is currently unknown (1,2). It is important to
realize that most (marketed) protein products induce
immune responses, which in many cases do not have
clinically relevant consequences. However, in some cases,
the consequences can be severe and potentially fatal.
Unfortunately, fully predictive in-silico, in-vitro and in-vivo
preclinical models to assess unwanted immunogenicity are
currently lacking. As a consequence, unwanted immunoge-
nicity of a protein pharmaceutical is typically addressed in
human trials, and its incidence and consequences may
become fully apparent only after the product has been
widely used as market product. Extended characterization
data, if available, may then help to establish a relationship
between the presence and nature of aggregates in a protein
pharmaceutical on the one hand and immunogenicity data
emerging from use of the product in humans on the other. It
may be difficult to establish a firm relationship between
immunogenicity and a certain quality parameter. However,
if such a relationship for a certain protein exists, the only way
to reveal this is to make sure that clinical batches are (or can
be) characterized in the best way possible. To this end, it is
necessary to have good understanding of the product quality
930 den Engelsman et al.
at the time of batch release and at the time of administration
to the patient. In order to find a good correlation between an
immune response (or the lack of it) and a product quality
attribute, well-established extended characterization methods
are crucial. In case of unexpected immune responses to
commercial batches, these methods can then also be applied.
Strategy for Application of Extended
Characterization Methods During Product
Development
Application of extended characterization methods through-
out the whole chemistry, manufacturing and control (CMC)
development process of protein products is now generally
recognized as being very important. In the end, this
characterization should firmly contribute to the establish-
ment of relevant specifications for aggregates, according to
which the quality of commercial products can be assessed
appropriately.
Strategy to Perform Extended Characterization
as Early as Possible
An extensive analytical program in early development can
pave the way for efficient late-stage development. A good
understanding of a products characteristics and factors that
affect its structural stability is key to developing a stable
product that can be produced in a consistent and
reproducible way. However, extensive use of extended
characterization tests can also be challenging in terms of
resources and timelines. That is, the extended character-
ization methods are typically low throughput, and this may
become a problem when their application is considered
during routine stability studies (e.g., of clinical batches),
process development studies, in-use testing and forced
degradation studies. One possible way to deal with this is
to use the extended characterization methods to support
the development of simpler but still high-quality methods
such as SEC. This may significantly reduce the need for
laborious assays in routine testing.
Strategy to Perform Extended Characterization
as Late as Possible
Should early characterization prove to be impossible, an
alternative approach that may be considered is retrospective
testing. In this approach, samples from development and
(clinical) stability programs are collected and stored frozen
(e.g. at 70C) rather than analyzed right away. A significant
advantage of such an approach is that relevant samples
collected throughout development can be analyzed side-by-
side using mature methods with well-known performance (i.e.,
analytical experience and technology usually evolve in parallel
with product development). Provided that it is demonstrated
that the freeze-thaw cycle does not influence the aggregation
status, this approach may result in a very comprehensive data
set (analytical variation is minimized) that can easily be used to
justify quality specifications. A drawback is that any learning
from analysis is not gained until late in the development
process.
Extended Characterization Tests as a Basis
for Future Risk Assessment
Decisions on the application of extended characterization
tests are driven by many parameters, including complexity
of the protein and the production process, availability of
resources, priority of the development program, as well as
the target product profile and (clinical) timelines. From a
patient safety perspective it may be more appropriate to
establish the characterization level based on a product risk
assessment. The greater the risk of safety issues, the more
extensive and more frequent should extended characteriza-
tion be. This approach would fit with general regulatory
expectations regarding risk-based product development.
Risk assessment is currently difficult, however. It is
complex and requires multidisciplinary interaction between
toxicologists, clinicians and product developers. However,
knowledge about product parameters that impact immu-
nogenicity, such as type of molecule (e.g. fully human vs.
chimeric) and its receptor (e.g. soluble versus cellular),
homology to endogenous counterpart, disease/indication,
dose regimen, route of administration, clearance rate and
patient immune status (e.g. suppressed versus activated), is
increasing rapidly. A key part of any risk assessment is
detailed knowledge of a products various characteristics,
and this is where extended characterization is important.
Thus, for example, a clinical development program (phase
13) may use several clinical preparations, and these
batches may display slight variations in quality (e.g. levels
of aggregation). There may be slightly different clinical
outcomes from these different batches. Comparison of
detailed product characterization with clinical outcome
may indicate particular product properties that affect its
immunogenicity and may provide data for inclusion in
future risk assessments.
CONCLUSIONS AND RECOMMENDATIONS
Aggregates are heterogeneous in size and in other physico-
chemical properties. Consequently, no single method covers
the analysis of all aspects of all aggregates. As each method
covers different aggregate characteristics, the results
obtained with a particular method are strictly linked to
that method. Accordingly, QC specifications depend on the
Strategies for the Assessment of Protein Aggregates 931
QC methods used and the aggregate class analyzed. In
addition to QC methods, extended characterization meth-
ods are indispensible during product development.
SEC and SDS-PAGE/CE-SDS are methods that are
robust enough for reproducible quantification of aggregates
and that allow routine use with sufficient sample through-
put. These methods use specific separation principles and
sample preparation methods which may impact the result
both qualitatively and/or quantitatively, so orthogonal
methods should be used to confirm that the applied
method, e.g. SEC, provides adequate results.
Below we list recommendations regarding the assessment
of protein aggregates in biotech product development:
& Employ robust, quantifiable methods for QC testing:
SEC (quantification of covalent and non-covalent
aggregates, but not low-affinity aggregates and larger
aggregates); SDS PAGE and/or CE-SDS (covalent
aggregates).
& Use extended characterization assays to support prod-
uct development, not to qualify or cross-validate.
& Use extended characterization assays to confirm the
performance of the QC analytical methods.
& Follow a risk-based approach, considering molecular
and safety aspects, in deciding on the application of
extensive as opposed to limited use of extended
characterization in product development.
& Archive samples for retrospective testing, and confirm
stability on storage.
ACKNOWLEDGMENTS
The authors thank Margit Jeschke and Markus Bluemel
from Novartis (Basel) for helpful discussions and Andrea
Herre and Werner Kliche from Boehringer Ingelheim
(Biberach an der Riss) for critically reading the manuscript.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License
which permits any noncommercial use, distribution, and
reproduction in any medium, provided the original author(s)
and source are credited.
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