Ion exchange chromatography of biotherapeutics: Fundamental principles and advanced approaches
Journal of Chromatography A,
Год журнала:
2025,
Номер
1742, С. 465672 - 465672
Опубликована: Янв. 9, 2025
Ion
exchange
chromatography
(IEX)
is
an
important
analytical
technique
for
the
characterization
of
biotechnology-derived
products,
such
as
monoclonal
antibodies
(mAbs)
and
more
recently,
cell
gene
therapy
products
messenger
ribonucleic
acid
(mRNA)
adeno-associated
viruses
(AAVs).
This
review
paper
first
outlines
basic
principles
separation
mechanisms
IEX
charge
variant
biotherapeutics,
examines
different
elution
modes
based
on
salt
or
pH
gradients.
It
then
highlights
several
recent
trends
when
applying
including:
i)
effective
use
gradients,
ii)
improvement
selectivity
by
using
organic
solvents
in
mobile
phase,
multi-step
combining
ion
pairing
exchange,
iii)
increase
throughput
ultra-short
columns
automated
screening
conditions.
The
also
discusses
incorporation
into
multidimensional
liquid
setups,
integrating
it
with
other
chromatographic
dimensions
analysis
complex
biotherapeutic
products.
covers
coupling
mass
spectrometry
(MS),
mobility
(IMS),
multi-angle
light
scattering
(MALS)
to
identify
various
species
contained
samples.
In
conclusion,
considered
today
essential
toolbox
quality
control
offers
a
unique
mechanism
can
be
coupled
highly
informative
detectors,
MS
MALS.
Язык: Английский
The Role of mD-LC–MS for Antibody Analysis in the Biopharmaceutical Industry
Deleted Journal,
Год журнала:
2025,
Номер
unknown, С. 4 - 10
Опубликована: Янв. 3, 2025
The
field
of
therapeutic
antibodies
has
witnessed
remarkable
growth
in
recent
years,
with
novel
biotherapeutics
formats
continually
emerging.
As
the
complexity
these
increases,
so
does
demand
for
robust
analytical
methods
to
characterize
them.
In
this
article,
we
explore
transformative
impact
multidimensional
liquid
chromatography–mass
spectrometry
(mD-LC–MS)
technology
on
antibody
characterization,
a
focus
specific
performance
enhancements.
This
article
explores
challenges
faced
by
traditional
chromatographic
and
discusses
how
mD-LC–MS
is
changing
way
evaluate
high
chromatography
(HPLC)
peaks
assess
performance.
Specifically,
highlights
enables
precise
peak
identification,
rapid
characterization
complex
antibodies,
early
detection
post-translational
modifications
improve
assessment
potential
critical
quality
attributes
(pCQAs)
optimize
overall
workflow.
advance
led
significant
time
savings
reduction
artificial
induced
consumption,
thereby
reducing
risk
sample
during
analysis.
Язык: Английский
Evaluation of ion pairing reversed-phase liquid chromatography for the separation of large RNA molecules
Journal of Chromatography A,
Год журнала:
2024,
Номер
1740, С. 465574 - 465574
Опубликована: Дек. 2, 2024
The
rapid
development
of
mRNA-based
therapeutics,
especially
post-COVID-19,
has
necessitated
the
precise
characterization
mRNA
quality
attributes,
including
sequence
integrity.
Ion-pairing
reversed-phase
liquid
chromatography
(IP-RPLC)
been
widely
accepted
as
a
reference
method
for
small
oligonucleotides.
Some
studies
have
already
investigated
use
IP-RPLC
RNA,
but
no
systematic
approach
developed
to
assess
impact
ion-pairing
agents
(IPAs)
on
separation
large
RNA
molecules.
This
study
addresses
this
gap
by
investigating
potential
and
molecules,
with
specific
focus
optimizing
IPAs
enhance
retention
selectivity.
Thirteen
different
IPAs,
varying
in
hydrophobicity,
were
systematically
tested
using
supermacroporous
polymeric
(divinylbenzene)
column
very
broad
pore
size
range
under
various
conditions,
temperatures,
pH,
IPA
concentrations.
results
demonstrate
that
moderately
hydrophobic
provide
superior
resolution
species
up
6000
nucleotides.
An
optimized
combination
100
mM
butylammonium
acetate
50
tripropylammonium
achieved
best
overall
separation,
significantly
improving
35%
compared
individual
IPAs.
also
identifies
optimal
conditions
mobile
phase
pH
7.0,
acetonitrile
organic
solvent,
temperature
65
°C.
In
second
step,
solution
increase
nucleotides
thereby
separate
nucleic
acids
ranging
from
1
allowing
characterize
IVT-mRNA
differing
length
their
integrity
fragmentation
or
monitor
presence
in-process
impurities
(nucleotides)
was
combining
two
LC
columns.
These
findings
analytical
toolbox
evaluating
critical
attributes
supporting
reliable
efficient
RNA-based
therapeutics.
Язык: Английский
Selected new approaches and future perspectives in liquid chromatography for the analysis of emerging modalities
European Journal of Pharmaceutical Sciences,
Год журнала:
2025,
Номер
unknown, С. 107101 - 107101
Опубликована: Апрель 1, 2025
Emerging
biopharmaceutical
modalities,
such
as
genetic
medicines
and
RNA
therapies,
offer
transformative
potential
for
treating
previously
intractable
diseases.
However,
these
complex
drugs
present
unique
analytical
challenges
due
to
their
intricate
structures,
sophisticated
manufacturing
processes,
modality-specific
product
quality
attributes.
Liquid
chromatography
(LC)
has
emerged
a
versatile
tool
addressing
challenges,
enabling
precise
characterization
control
strategies.
This
review
highlights
recent
advancements
in
LC
technologies,
including
low-adsorption
hardware,
ultra-wide
pore
size
exclusion
(SEC)
columns,
innovative
separation
modes
slalom
pressure-enhanced
liquid
(PELC).
These
developments
tackle
issues
non-specific
adsorption,
carryover,
inadequate
selectivity
while
improving
resolution
robustness
large
biomolecules
like
mRNA,
adeno-associated
viruses
(AAVs),
lipid
nanoparticles
(LNPs).
Novel
approaches,
tandem
SEC
systems,
gradient
dual
stationary
phase
gradients,
further
expand
the
scope
of
techniques
by
enhancing
separations
diverse
analyte
sizes
complexities.
Additionally,
practical
innovations
bracketed
injection
methods
new
enzymatic
tools
oligo-mapping
improve
reproducibility,
efficiency,
confidence
sequence
analysis.
not
only
address
current
limitations
but
also
pave
way
regulatory-compliant
which
will
support
broader
adoption
both
discovery
settings.
As
field
continues
evolve,
are
poised
play
pivotal
role
ensuring
safety,
efficacy,
consistency
next-generation
therapeutics.
Язык: Английский
Advanced two-dimensional liquid chromatography workflow for enhanced resolution of protein components in Indian Cobra (Naja naja) venom using hydrophobic interaction -reverse phase chromatography coupled with mass spectrometry
Journal of Chromatography Open,
Год журнала:
2025,
Номер
unknown, С. 100211 - 100211
Опубликована: Март 1, 2025
Язык: Английский
Establishing Multi-Dimensional LC-MS Systems for Versatile Workflows to Analyze Therapeutic Antibodies at Different Molecular Levels in Routine Operations
Pharmaceuticals,
Год журнала:
2025,
Номер
18(3), С. 401 - 401
Опубликована: Март 12, 2025
Background/Objectives:
Multi-dimensional
liquid
chromatography
coupled
with
mass
spectrometry
(mD-LC-MS)
has
emerged
as
a
powerful
technique
for
the
in-depth
characterization
of
biopharmaceuticals
by
assessing
chromatographically
resolved
product
variants
in
streamlined
and
semi-automated
manner.
The
study
aims
to
demystify
enhance
accessibility
this
but
inherently
complex
detailing
robust
user-friendly
instrument
platform,
allowing
analysts
switch
seamlessly
between
intact,
subunit,
peptide
mapping
workflows.
Methods:
Starting
from
commercially
available
Two-Dimensional
Liquid
Chromatography
(2D-LC)
system,
we
introduce
specific
hardware
software
extensions
leading
two
versatile
mD-LC-MS
setups,
slightly
different
configurations.
technique’s
efficacy
is
demonstrated
through
case
on
cation
exchange
method
charge
bispecific
antibody,
isolating
peak(s)
interest,
followed
online
sample
processing,
including
reduction
enzymatic
digestion,
subsequently
analysis.
Results:
accuracy
reproducibility
both
setups
proposed
were
successfully
tested.
Despite
peak
patterns
first
dimension,
systems
equally
effective
identifying
quantifying
underlying
species.
This
highlights
routine
usability
technology
(ultra)
high-performance
(UHPLC)
therapeutic
biomolecule.
Conclusions:
reliability
underscore
practicality
use
biopharmaceutical
Our
detailed
description
insights
simplify
access
advanced
broader
scientific
community,
regardless
expertise
level,
lower
entry
barrier
its
various
research
industrial
settings.
Язык: Английский
2D-CEX–FcRn–MS to Study Structure/Function Relation of mAb Charge Variants
Analytical Chemistry,
Год журнала:
2024,
Номер
96(45), С. 18122 - 18131
Опубликована: Окт. 29, 2024
The
automated
elucidation
of
the
interplay
between
monoclonal
antibody
(mAb)
structure
and
function
using
two-dimensional
liquid
chromatography-mass
spectrometry
(2D-LC-MS)
is
reported.
Charge
variants,
induced
through
forced
degradation,
are
resolved
by
first-dimension
(
Язык: Английский
Keeping up with a Quickly Diversifying Pharmaceutical Landscape
ACS Measurement Science Au,
Год журнала:
2024,
Номер
4(6), С. 615 - 619
Опубликована: Сен. 24, 2024
Small
molecules
and
antibodies
have
dominated
the
pharmaceutical
landscape
for
decades.
However,
limitations
associated
with
therapeutic
targets
deemed
"undruggable"
progress
in
biology
chemistry
led
to
blossoming
of
drug
modalities
approaches.
In
2023,
a
high
number
9
oligonucleotide
peptide
products
were
approved
by
Food
Drug
Administration
(FDA),
accounting
16%
all
drugs
approved.
Additionally,
first
time,
clustered
regularly
interspaced
short
palindromic
repeat
(CRISPR)-Cas9
gene
therapy
product
was
treatment
sickle
cell
disease.
New
possess
wide
range
physicochemical
properties
structures,
which
complicates
their
analytical
characterization.
Impurities
are
formed
at
each
step
solid
phase
synthesis
during
shelf
life.
Longer
chain
lengths
lead
higher
closely
related
impurities
that
become
increasingly
more
difficult
separate
from
full-length
product.
Chemical
modifications
such
as
phosphorothioates
(PS)
result
presence
diastereomers,
often
require
orthogonal
methods
profiling
strategies
prevent
interference
separation
achiral
impurities.
In-vitro
produced
mRNA
plasmid
DNA
also
present
variety
quality
attributes
need
be
determined,
polyA
tail
length
or
capping
efficiency.
Analytical
challenges
arise
modality
physiochemical
attributes,
fast
turnaround
times,
heightened
level
characterization
needed
enable
data-driven
decisions
early
development
process.
This
perspective
provides
author's
views
on
lessons
learned
employed
recent
years.
Язык: Английский