Antibody-based therapeutics play a vital role in treating a wide range of diseases, but their manufacture can be complex, requiring efficient purification strategies to support product quality and process efficiency.
Traditional protein A affinity capture methods often struggle with engineered antibody formats, leading to process inefficiencies, reduced yields and higher production costs.
This whitepaper explores novel affinity capture technologies, which offer a powerful solution for purifying complex antibody formats with greater efficiency and higher yields.
Download this whitepaper to learn:
- How next-generation affinity capture methods simplify antibody purification
- The benefits of improved binding capacity, yield and impurity reduction
- Real-world case studies showcasing streamlined manufacturing processes
2 BioProcess International 21(5)e November 2023 Sponsored
Using affinity capture in
purification of antibodies
enables developers to reduce
the overall number of
chromatography steps in their
downstream processes. It can increase
product yield, reduce the time required
for bioprocess development, and
ultimately reduce time to market and
the overall cost of goods (CoG).
Because of its ability to bind to the Fc
region of monoclonal antibodies
(MAbs), protein A has been used as the
platform for affinity capture of IgGbased therapeutics for decades.
In addition to standard IgG MAbs,
many antibody formats are in
development: e.g., bispecifics, fusion
proteins based on the crystallizable
fraction (Fc) of antibodies, proteins
made up of just the antigen-binding
(Fab) fragments of antibodies, and
antibody–drug conjugates (ADCs).
Such engineered antibodies can pose a
challenge to protein A affinity capture if
they have altered or absent protein A
binding sites. These novel formats also
can be difficult to purify because some
tend to aggregate, form dimers, and
can come with elevated levels of high–
and low–molecular-weight (HMW, LMW)
species. Light-chain dimers can present
alongside some antibody fragments,
and overexpressed light chains can
complicate their downstream
processing further. In addition, some
engineered antibodies are sensitive to
low-pH/acidic conditions, which can
limit a developer’s choice of elution
buffer.
New Options for
Affinity Capture
Novel antibody modalities have driven
development of new options for
purification such as CaptureSelect
technology. CaptureSelect ligands offer
a unique affinity purification solution
based on camelid-derived single
domain (VHH) antibody fragments. The
small, 14-kD affinity ligands provide a
platform solution to many
biopharmaceutical purification
challenges and have been proven to
deliver increased yields and purities for
proteins of interest in many
applications. VHH affinity ligands are
produced by recombinant yeast culture
in a process that is free of animal-origin
materials. The molecules are highly
stable and screened for high specificity,
binding capacity, and desired elution
conditions for a target therapeutic of
interest. Thermo Fisher Scientific has
developed a number of unique
CaptureSelect affinity matrices to target
different binding sites on an array of
human therapeutic proteins (Figure 1).
CaptureSelect CH1-XL ligands bind
to the constant heavy domain (CH1) of
all human IgG subclasses and purify
100% of kappa and lambda Fabs,
providing an excellent platform for Fab
purification. Because of its high
selectivity, the CH1-XL ligand
precludes copurification of free light
chains and light-chain dimers. The
resin has a high dynamic binding
capacity (DBC) of ~19 mg/mL for a
polyclonal Fab with efficient elution at
relatively mild pH. Fab purity of 98%
has been achieved using
CaptureSelect CH1-XL resin, with a
nearly 90% yield.
CaptureSelect KappaXP resin was
designed for purification of Fab
fragments and bispecific antibodies.
The ligand provides 100% kappa
subtype coverage for all
immunoglobulins that contain a kappa
light chain. This resin has a high DBC
of 20–30 g/L for kappa Fabs and has a
DBC of 30-45 g/L IgG. Efficient elution
can be achieved at relatively mild
acidity (up to pH 6).
CaptureSelect LambdaXP ligands
offer 100% lambda-subtype coverage
and can be used to purify IgGs that
contain a lambda light chain, including
bispecifics and Fab fragments. The
resin has a high DBC of >35 g/L for
IgGs and can be eluted at pH 3.5–4.0.
CaptureSelect FcXP ligands bind
only the constant heavy domain CH3.
These were developed for Fc-fusion
proteins, chimeric antibodies, and
molecular formats in which the protein
SUPPLIER SIDE
Streamline Manufacturing of
Antibody-Based Therapeutics with
Novel Purification Approaches
Laurens Sierkstra
VH
VL
CL
CH1
CH2
CH3
VH
VL
CL
CH1
CH2
CH3
Example antibody subdomain structure
Sponsored November 2023 21(5)e BioProcess International 3
A binding site is either removed or
blocked. The resin also has a high
DBC — >40 g/L with 10% breakthrough
(BT) and 5-minute residence time —
and elutes efficiently at pH 4.0–4.5.
Case Study in
Streamlining Purification
This case study exemplifies rapid
implementation of CaptureSelect FcXL
resin, a predecessor of the FcXP resin
mentioned above, into a commercial
next-generation therapeutic
manufacturing process. A purification
process had been established for a
novel-format antibody based on an
affinity capture step followed by three
polishing steps. The process had a low
DBC because the antibody did not
bind well to protein A, and lower-pH
elution limited product stability. Other
problems included elevated fragment
levels and an environmentally
unfriendly regeneration solution. The
developer wanted to remove one
process step to enable a better fit
within a particular manufacturing
facility.
Several capture options were
screened for load density, yield, and
reduction of host-cell protein (HCP),
HMW, and LMW impurities.
CaptureSelect FcXL resin scored the
best on most attributes, including the
sum of LMW (Table 1). Following
implementation this resin for affinity
capture, the following results were
achieved:
• reduction of a four-step
chromatography process to three
steps, removing the need for one of
the polish steps
• consistent yields of ~80% in one
pilot and three good manufacturing
practice (GMP) runs
• increased binding capacity to
improve facility fit
• an improved impurity profile
• increased pool stability due to
elution at mild pH
• excellent scalability
• use of a more environmentally
friendly regeneration solution.
Case Study in
Improved Recovery
Using bind–elute polishing steps to
remove impurities such as HMW
components and dimers can be
cumbersome. In this case study, a
customer with a three-step purification
process step sought to improve product
recovery and process efficiency. The
original process used protein A capture
followed by anion-exchange polish in
flow-through (FT) mode and a mixedmode bind–elute to remove a high
percentage of aggregates (12%). Cation
exchange had been evaluated for
aggregate removal but was not efficient
for the MAb in this case. The existing
process provided only 90% monomer
recovery, with loading at 25 g/L and a
6-minute residence time.
The goal of our collaboration was to
replace the bind–elute mixed-mode
step with a FT operation to improve
recovery, aggregate clearance, and
efficiency. Mixed-mode
chromatography was replaced with
Poros Benzyl Ultra hydrophobicinteraction resin. Three attributes of
such resins make them extremely wellsuited for manufacturing scale: Product
resolution is maintained as linear flow
rate increases. High linear binding
capacity is possible over a large range
of flow rates. And scalability is linear
and predictable.
The final process cleared
aggregates to <1%, with 90% monomer
recovery after 25-g/L resin loading
Table 1: Comparing affinity-capture options for a novel-format antibody that had
suboptimal binding to protein A
(Existing)
Affinity A Affinity B Affinity C Nonaffinity
CaptureSelect
FcXL
Load density Medium Low Medium Very High High
HCP reduction High High Very High Low Medium
Yield High High Low Low High
HMW Medium Medium Medium Medium Medium
Main peak Medium Medium Medium High High
LMW High High High Medium Low
Figure 1: CaptureSelect ligands offer a range of highly specific affinity-capture options.
CaptureSelect
CH1-XL
CaptureSelect
KappaXP
LambdaXP
CaptureSelect
FcXP
Fab purification platform
Binding the CH1 domain
No binding of free light chains
Developed for Fab fragments and bispecifics
Binding CL-kappa domain
High dynamic binding capacity
Mild elution conditions
Developed for Fc-fusion proteins and IgGs that bind
to protein A poorly
Binding the CH3 domain (Fc)
Mild elution conditions
Fab F(ab')2
Fab
Bispecific IgG
containing κ or λ CL
IgG Fc-fusion
protein
4 BioProcess International 21(5)e November 2023 Sponsored
and a 6-minute residence time (Figure
2b). The FT process was coupled
directly to the upstream AEX process
with no need for buffer exchange,
which saved both time and money.
These results demonstrate the power
of HIC as an alternative strategy in
MAb-aggregate polishing.
Removing HCPs with
Affinity Polishing
Product- and expression-related HCPs
can be difficult to remove even by
multiple polishing options. HCPs that
coelute with proteins of interest are
particularly difficult to remove. If they
are not addressed sufficiently, a
product’s advancement from one
clinical phase to the next can be
delayed. Clusterin, cathepsin D,
galectin-3 binding protein, and
G-protein coupled receptor 56 are
among the many HCPs that can be
difficult to remove. To address such
challenges, Thermo Fisher Scientific
developed an approach called “affinity
for polish” that applies CaptureSelect
affinity ligands on POROS beads to
enable fast and efficient HCP removal
in FT applications. POROS
CaptureSelect ClusterinClear beads
were used to remove >95% of clusterin
as well as >65% of other top HCPs in
one customer evaluation.
New Solutions for New
Antibody Formats
Purifying the next generation of
antibody therapeutics requires a
diverse set of high-performance
chromatography options. The tools
described herein can deliver important
benefits including high purity and yield
in a single capture step, a reduction of
the number of necessary steps in a
downstream process, and seamless
upscaling for biomanufacturing as
products move through clinical phases
toward commercialization.
Laurens Sierkstra is senior director
of research and development for
bioproduction purification and analytics
at Thermo Fisher Scientific in Leiden,
Netherlands; laurens.sierkstra@
thermofisher.com.
POROS resin is a pharmaceutical grade
reagent for manufacturing and
laboratory use only. CaptureSelect
ligands and affinity matrix are for
research use or further manufacturing,
not for diagnostic use or direct
administration in humans or animals.
Figure 2: Results of the new process — final verification
Load Purified Antibody
HMW Dimer Monomer LMW
Eective reduction of dimer and HMW aggregates
in low-conductivity solutions, with high recovery
A successful MAb polish step for highly ecient aggregate
removal and near-complete monomer recovery
Mixed-Mode
MAb-A Process BE HIC FT
Load density (g/L resin) 25 80
Monomer purity FT (%) 99 >99
Monomer recovery FT (%) 90 98
Host-cell proteins (ppm) <LLoQ <LLoQ
Residence time (minutes) 6.0 1.2
0.1% 0.1%
85.5%
9.5% 0.5% 5.0% 0.1%
99.3%
Final Verification