Overview of Analytical Methods for Characterizing the Charge Heterogeneity of Antibody-Drug Conjugates
International Journal of Advanced Pharmaceutical Sciences and Research,
Год журнала:
2024,
Номер
4(5), С. 16 - 22
Опубликована: Авг. 26, 2024
Antibody-drug
conjugates
(ADCs)
are
ideal
candidates
for
selective
therapy.
ADCs
display
considerable
heterogeneity
resulting
from
various
modifications.
One
of
the
molecular
characteristics
is
charge
heterogeneity.
Several
analytical
methods
could
be
used
to
characterize
ADC
including
ion
exchange
chromatography
(IEC),
isoelectric
focusing
(IEF),
and
capillary
electrophoresis
(CE).
This
work
aimed
give
insights
into
different
techniques
determine
pI
distribution
profile
ADCs.
Язык: Английский
Exploring imaged capillary isoelectric focusing parameters for enhanced charge variants quality control
Frontiers in Chemistry,
Год журнала:
2025,
Номер
13
Опубликована: Фев. 20, 2025
Biopharmaceuticals
are
increasingly
utilised
in
the
treatment
of
oncological,
inflammatory,
and
autoimmune
diseases,
largely
due
to
their
exceptional
specificity
targeting
antigens.
However,
structural
complexity,
heterogeneity,
sensitivity
pose
crucial
challenges
production,
purification,
delivery.
Charge
heterogeneity
analysis,
a
Critical
Quality
Attribute
these
biomolecules
used
Control,
is
often
performed
using
separative
analytical
techniques
such
as
imaged
capillary
Isoelectric
Focusing
(icIEF).
Recognized
gold
standard
by
industry,
icIEF
leverages
pH
gradient
provide
high-resolution
profiling
charge
variants
biotherapeutics.
In
this
review,
critical
experimental
parameters
for
method
development
context
biotherapeutic
drug
QC
will
be
discussed.
Key
aspects,
including
sample
preparation,
properties,
carrier
ampholytes,
stabilizers,
detection
examined,
supported
recent
literature.
Advances
technology
its
expanding
applications
underline
robustness,
reproducibility,
compliance
with
regulatory
standards,
affirming
pivotal
role
ensuring
identity
consistency
biological
products.
Язык: Английский
Analysis of therapeutic monoclonal antibodies by imaged capillary isoelectric focusing (icIEF)
Analytical Methods,
Год журнала:
2024,
Номер
16(31), С. 5450 - 5458
Опубликована: Янв. 1, 2024
Imaged
capillary
isoelectric
focusing
(icIEF)
is
a
preferred
analytical
method
for
determining
points
(pIs)
and
charge
heterogeneity
profiles
in
biotherapeutic
proteins.
In
this
study,
we
optimized
the
icIEF
an
in-house
IgG1κ
monoclonal
antibody
(mAb-1)
assessed
its
reproducibility,
robustness,
autosampler
stability.
The
was
used
to
evaluate
batch-to-batch
consistency
pIs
multiple
lots
of
mAb-1
determine
relative
percentages
variants.
We
also
tested
method's
performance
using
another
IgG1
mAb,
commercially
available
as
Herceptin
(trastuzumab).
Additionally,
designed
native
denaturing
platform
methods
11
other
marketed
mAbs,
with
ranging
from
6.0
(eculizumab)
9.22
(tocilizumab).
Язык: Английский
A new paradigm for optimized experimental design in cIEF platforms aimed at an accurate robust and reliable mAbs charge-variant assessment
Scientific Reports,
Год журнала:
2024,
Номер
14(1)
Опубликована: Ноя. 15, 2024
The
success
of
therapeutic
monoclonal
antibodies
(mAbs)
and
their
biosimilars,
highlights
the
challenge
to
control
purity,
identity,
stability.
For
this
purpose,
among
several
orthogonal
methodologies,
imaged
capillary
Iso
Electric
Focusing
(icIEF)
is
one
leading
techniques.
Despite
isoelectric
point
(pI)
being
a
univocal
parameter
relying
on
protein's
primary
sequence
its
post-translational
modifications
(PTMs),
current
Charge
Variants
Profile
Assessment
(CVPA)
carried
out
by
cIEF
relies
relative
comparisons
with
corresponding
reference
standards,
because
inconsistent
outputs
for
same
sample
across
different
instruments
preclude
uniqueness
measured
parameters.
We
demonstrate
that
refining
calibration
approach
in
iciEF
method
allows
obtaining
more
reliable
objective
pIs,
deeper
understanding
pH
gradients
along
capillaries.
are
confident
our
advancements
will
enhance
CVPA,
exploring
concept
charge
identity.
This
crucial
constructing
biobanks
developing
algorithms
quickly
identify
divergences
from
originators,
thus
ensuring
drug
quality,
efficacy,
safety.
Moreover,
an
experimental
design
optimized
minimize
bias
("Unbiased"
Experimental
Design—UED)
study
resolution
as
multivariate
function
input
variables.
endeavor
aims
develop
optimal
methods
tailored
specific
ranges.
Язык: Английский
Development of a novel, high-throughput imaged capillary isoelectric focusing-Western method to characterize charge heterogeneity of monoclonal antibody heavy and light chains
Gangadhar Dhulipala,
Alanna Broszeit,
Kun Lu
и другие.
mAbs,
Год журнала:
2024,
Номер
16(1)
Опубликована: Ноя. 15, 2024
Charge
heterogeneity
is
one
of
the
commonly
monitored
quality
attributes
in
biotherapeutics.
It
can
impact
stability,
efficacy,
and
safety
products,
but
it
also
affect
pharmacokinetics,
binding
affinity,
overall
biological
activity
molecules.
Given
substantial
size
complexity
antibodies,
subtle
variations
or
specific
modifications
that
result
charge
might
be
concealed
when
mAbs
are
analyzed
under
native
conditions.
Two-dimensional
gel
electrophoresis
has
traditionally
been
used
to
characterize
antibody
heavy
chain
(HC)
light
(LC)
variants.
The
procedures,
however,
laborious,
method
only
qualitative.
ChromiCE
was
developed
as
an
alternative
approach
provide
quantitative
analysis,
labor
intensive,
requiring
separation
HC
LC
by
chromatography
before
imaged
capillary
isoelectric
focusing
(iCIEF)
analysis.
We
thus
a
novel,
automated
high-throughput
iCIEF-Western
directly
quantify
variants
with
high
sensitivity
denatured
reduced
selectively
characterized
using
detection
antibodies
LC.
In
addition,
reduced,
allows
for
analysis
up
96
samples
overnight,
offering
good
precision
throughput
minimal
analyst
hands-on
time.
Further,
applied
different
aspects
drug
development,
such
comparability,
release
stability
testing
given
its
ability
identity,
well
qualitative
comparative
Язык: Английский
An Imaged Capillary Isoelectric Focusing Separation of the Linear and Cyclic Variants of a Mimotope of the Cancer‐Related CD20 Antigen–Validation and Statistical Evaluation
Journal of Separation Science,
Год журнала:
2024,
Номер
48(1)
Опубликована: Дек. 30, 2024
Imaged
capillary
isoelectric
focusing
was
successfully
applied
for
separating
an
in-house
synthesized
closely
related
peptide
pair,
that
is,
a
linear
12-mer
(Rp5-L)
and
its
cyclic
15-mer
variant
(Rp5-C).
Rp5-L
represents
mimotope,
epitope
mimicking
peptide,
of
the
CD20
antigen,
which
is
over-expressed
in
B-cell-related
tumors.
Peptide
identity-including
successful
disulfide
bond
formation
Rp5-C-was
confirmed
with
matrix-assisted
laser
desorption
ionization-time
flight
mass
spectrometry.
The
purity
products
determined
by
reversed-phase
high-performance
liquid
chromatographic
method
ultraviolet
detection.
apparent
point
(pI)
Rp5-C
5.99
6.47,
respectively.
An
appropriate
combination
carrier
ampholytes
allowed
their
baseline
separation
analysis
time
<20
min.
Method
validation
done
peptides
three
flanking
pI
markers
covering,
example,
repeatability
intermediate
precision.
Calibrations
on
different
days
resulted
identical
slopes
Rp5-C,
respectively,
as
statistically
Welch's
t-test
pooled
over
8
days.
calibration
data
mimotopes
were
evaluated
outliers,
normality,
homoscedasticity,
autocorrelation
complementary
statistical
procedures,
identified
otherwise
unnoticed
outlier
marker.
linearity
Rp5-L,
tested
Mandel's
fitting
test
lack-of-fit
test.
For
calculated
limits
detection
quantification
≤0.31
≤0.96
µmol/L,
Язык: Английский