USD3103
KANEKA KanCapA◆ Affinity Sorbent
High Capacity, Highly NaOH-Resistant Protein A
Sorbent, for the Purification of Monoclonal Antibodies
KANEKA KanCapA chromatography sorbent is an industry-scalable Protein A affinity
sorbent that increases the overall productivity of monoclonal antibody (MAb) processes
through provision of high dynamic binding capacity and long service life. KANEKA
KanCapA sorbent is ideal for a MAb capture step and may be used in either a single
column batch operation or in a continuous process using a multicolumn chromatography
system.
Proprietary recombinant Protein A (rProtein A) ligand with improved selectivity
High binding capacity aligned to current MAb expression levels
Mild pH elution to protect MAb aggregation or denaturation
Good NaOH stability to prolong service time
Description
KANEKA KanCapA sorbent comprises a rigid cellulose matrix
to which the rProtein A ligand is covalently attached to ensure
stability over cycles, providing high flow performance at low
pressure so is compatible with the process requirements for
large scale MAb manufacture.
Figure 1
Schematic Representation of KANEKA’s New Protein A
Ligand Construction
KANEKA KanCapA sorbent is available in a variety of scouting
configurations including ScreenExpert RoboColumns◆ and PRC
prepacked columns designed for rapid method optimization or
small-scale preparative work. The sorbent is supplied in bulk as
a slurry in 20% (v/v) ethanol in several pack sizes ranging from
25 mL to 10 L.
Protein A Ligand
Native Protein A has a high affinity for the Fc region of
immunoglobulins, but also exhibits an undesired affinity for
the Fab region. In order to disrupt the Fab-Protein A interaction,
a low pH is often required that can lead to unwanted MAb
aggregation.
Figure 2
Pressure vs. Flow Rate for Columns from 4.4 cm to 45 cm I.D.
and 20 cm to 23 cm Bed Height
The Protein A ligand used in KANEKA KanCapA sorbent has
been modified and is a pentamer of the mutated C domain of
the native Protein A molecule. The C domain is known to exhibit
NaOH resistance and as a consequence, the KANEKA KanCapA
sorbent has good NaOH stability, requires mild elution conditions
and has no detectable Fab binding. The rProtein A is expressed
in bacteria and is free of animal content.
Main Properties
Base matrix
Average particle size
Ligand
Coupling chemistry
Dynamic binding capacity (DBC)1
Chemical stability
Working pH range
CIP condition
Operational flow rate
Residence time
1
Highly cross-linked cellulose
65~85 µm
Recombinant Protein A (alkaline-resistant)
Reductive amination
≥ 35 mg human polyclonal IgG/mL
packed resin
Stable in solutions commonly used in
affinity chromatography
pH 2~13
0.1 ~ 0.5 M sodium hydroxide
15 min contact time
Up to 500 cm/hr (bed height 20~25 cm)
≥ 3 min (4~6 min is recommended)
5% DBC determined by frontal analysis at 3 minute residence time
Operating Flow Rates
KANEKA KanCapA sorbent can be readily packed and
unpacked in laboratory, pilot and production-scale columns,
and supports high flow rates consistent with the requirements
of the latest production processes.
Figure 2 presents pressure versus flow rate data for KANEKA
KanCapA sorbent obtained in columns ranging from 4.4 cm to
45 cm I.D., with a bed height of approximately 20 cm, using
water. At 500 cm/hr, the pressure drop is 1.5 bar g (22 psi g)
which is consistent to process systems routinely used today.
Packing performance is reliable and scalable from laboratory to
large scale (60 cm I.D.) columns. Typical packing data is >4,000
plates per meter (1% acetone pulse at 60 cm/hr) and asymmetry
factors (AF) are of 1 – 1.2.
2
The pressures generated by packed beds are calculated by subtracting
the system pressure from total pressure.
Features and Benefits
High binding capacity for high titer processes
MAb expression levels have steadily increased over recent years
and titers >5 g/L are now common place. One key parameter
that affects overall productivity of MAbs is the dynamic binding
capacity (Figure 3). KANEKA KanCapA sorbent exhibits capacities
>50 g/L at 6 minute residence time which meets the performance expectations of a leading Protein A chromatography
sorbent. Dynamic binding capacites for two MAbs confirm
the good properties of the sorbent (Figure 4).
Figure 3
Residence Time vs. Dynamic Binding Capacity (Polyclonal
human IgG)
Figure 5
Life Cycle Study with Repeated Alkaline CIP of KANEKA
KanCapA Sorbent
A
Figure 4
Dynamic Binding Capacity of KANEKA KanCapA Sorbent for
Two MAb Molecules Expressed in CHO Cell Culture Supernatant
Excellent NaOH resistance leads to a long service life
KANEKA KanCapA sorbent can be cleaned with 0.1 or 0.5 N
NaOH. It can be effectively used up to at least 200 cycles with
periodic CIP using 0.1 M NaOH (15 minute contact time) with
no significant loss of capacity after 100 cycles (Figure 5). A small
loss of ~20% is observed after 300 cycles (data not shown). If
required, higher concentrations of NaOH of up to 0.5 M can be
used for periodic CIP of KANEKA KanCapA sorbent. Reusability
of a sorbent is especially important when considering continuous
processing using multicolumn chromatography, and the NaOH
resistance exhibited by KANEKA KanCapA sorbent positions it
well in this emerging area of bioprocessing. Leakage of Protein A
is <10 ppm and is below the limit of detection (1 ppm) after few
CIP cycles.
Milder elution pH
Protein A affinity sorbents are well-known to require an acidic pH
for the elution of bound MAbs. Unfortunately, exposure of the
MAb to an acidic pH often leads to denaturation and aggregation
of which affects the overall process yield. The modified rProtein A
ligand used in KANEKA KanCapA sorbent enables elution under
milder conditions of around pH 3.5 compared with a pH 3.0 or
lower required for the more traditional Protein A ligands. These
milder acidic solutions are effective with multiple MAbs with high
yield (Figure 6).
B
Remaining 5% DBC after repeated alkaline CIP with 0.1 M or 0.5 M
NaOH (A); yield and Protein A ligand leaching in the cycle use study with
0.1 M NaOH (B). Contact time to NaOH in all experiments: 15 minutes.
Figure 6
Elution pH Values of Five MAbs
Concentration of MAb loaded: 1 mg/mL sorbent. Elution pH determined
using a pH gradient study with 50 mM citrate from pH 6 to pH 2,
20 column volumes (CV).
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3
Applications
Regulatory
Benefits including post load washing steps
Protein A sorbents are well-known for their selectivity towards
MAbs. KANEKA KanCapA sorbent has been shown to further
improve the MAb purity by reducing residual HCP in the eluent.
As shown in Figure 7, the HCP content is very dependent on
the MAb feedstock. By including some additional wash steps
following the load to remove some residual HCP, it was possible
to improve the MAb purity during elution.
Figure 7
Comparison of Host-Cell Protein (HCP) Level in Elution Pool
Measured by HCP ELISA
A regulatory support file is available under a confidential
disclosure agreement. The entire production process is free
from animal derived components and is certified BSE-TSE-free.
Ordering Information
Part Number
Description
26080-026
26080-035
26080-058
26080-064
SR2KANCAPA
KANEKA KanCapA 25 mL
KANEKA KanCapA 500 mL
KANEKA KanCapA 5 L
KANEKA KanCapA 10 L
ScreenExpert RoboColumn KANEKA KanCapA
200 µL, row of 8
ScreenExpert RoboColumn KANEKA KanCapA
600 µL, row of 8
PRC Prepacked Column 5x50 KANEKA KanCapA,
1 mL
PRC Prepacked Column 8x100 KANEKA KanCapA,
5 mL
SR6KANCAPA
PRC05X050KANCAPA
PRC08X100KANCAPA
Washing protocols conducted
before elution. Elution buffer:
50 mM citrate buffer, pH 3.5.
Visit us on the Web at www.pall.com/biopharm
E-mail us at biopharm@pall.com
Corporate Headquarters
Port Washington, NY, USA
+1.800.717.7255 toll free (USA)
+1.516.484.5400 phone
biopharm@pall.com e-mail
European Headquarters
Fribourg, Switzerland
+41 (0)26 350 53 00 phone
LifeSciences.EU@pall.com e-mail
Asia-Pacific Headquarters
Singapore
+65 6389 6500 phone
sgcustomerservice@pall.com e-mail
International Offices
Pall Corporation has offices and plants throughout the world in: Argentina, Australia, Austria, Belgium, Brazil,
Canada, China, France, Germany, India, Indonesia, Ireland, Italy, Japan, Korea, Malaysia, New Zealand, Norway,
Philippines, Poland, Russia, Singapore, South Africa, Spain, Sweden, Switzerland, Taiwan, Thailand, United
Kingdom, and Vietnam. Distributors in all major industrial areas of the world. To locate the Pall office or distributor
nearest you, visit www.pall.com/contact.
The information provided in this literature was reviewed for accuracy at the time of publication. Product data
may be subject to change without notice. For current information consult your local Pall distributor or contact
Pall directly.
© 2015, Pall Corporation. Pall,
are trademarks of Pall Corporation. ® indicates a trademark registered
in the USA. Filtration.Separation.Solution.SM is a service mark of Pall Corporation. ◆KANEKA KanCapA is a
trademark of KANEKA Corporation. RoboColumn is a trademark of Atoll GmbH.
12/15, PDF, GN15.9840
USD3103