BioPharm International www.biopharminternational.com December 201 3 Electronically reprinted from December 2013 B io Pharm INTERNATIONAL The Scien ce & Busin ess of Bioph armace utica ls P h o t o C r e d i t : S C I E P R O / G e t t y I m a g e s mAb Purification W ith greater economic pressure on monoclo- nal antibody (mAb) production for thera- peutic and research uses, antibody titers in mammalian cell culture have increased dramatically over the past 20 years. As a consequence, down- stream processing must accept and handle higher titers of mAbs in har- vested cell-culture fluid (HCCF), and vendors of mAb purification technolo- gies must develop chromatography resins with high binding capacity to meet the demand. In addition, more cost-efficient cleaning procedures are necessary to extend the lifetime of chromatography resins and to reduce the cost for cleaning and validation. A team from Chugai Pharmaceutical (Japan) investigated the mAb purifi- cation performance of a new alkali- tolerant, prototype Protein A resin (Resin 3, MabSelect SuRe LX proto- type, GE Healthcare), which has the potential to address the demand for a more advanced, cost-effective mAb purification technology. The methodology for the produc- tion of monoclonal antibodies from a cell line by hybridization of mouse myeloma and mouse spleen cells from an immunized donor was first published in 1975 (1). As a technol- ogy that permits the generation of monoclonal antibodies against almost any target molecule, it immediately gained great interest as a source for potential drug candidates. Although it took some time for the first thera- peutic mAb to become commercially available in 1986 (2), the market for therapeutic mAbs has since grown rapidly. MAbs have proved to be suc- Comparing Protein A Resins for Monoclonal Antibody Purification Shohei Kobayashi and Yasufumi Ueda A prototype Protein A resin is evaluated for purification performance, reusability, and cost performance. Shohei Kobayashi and Yasufumi Ueda are in the API process development department, Pharmaceutical Technology Division, Chugai Pharmaceutical, Tokyo, Japan.
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7242019 Comparing Protein a Resins for MAb Purification
December 2013 wwwbiopharminternationalcom BioPharm International
Posted with permission from the December 2013 issue of BioPharm International reg wwwbiopharminternationalcom Copyright 2014 Advanstar Communications Inc All rights reservedFor more information on the use of this content contact Wrightrsquos Media at 877-652-5295
10749
mAb Purification
Resin 3 prototype resin The cal-
culations are based on an annual
mAb production amount of 500
kg and bioreactor size of 10000 L
for 1 gL titer or 5000 L for 3 gL
and 5 gL titers Column diam-eter was estimated by considering
a process time within 10 to 15 h
The total purification cost
per kilogram of produced anti-
body from 1 gL and 3 gL titers
including chemicals and water
using Resin 1 ($13000 and
$14000 respectively) can be
reduced by 29 and 46 respec-
tively by using Resin 2 instead as
shown in Figure 2
With Resin 3 in the case of atiter level of 3 gL the authors
found that the overall purifica-
tion cost can be even further
reduced by 26 With a lower
titer level of 1 gL however the
decrease was only 4
Under the selected conditions
Resin 1 was not suitable for puri-
fication of antibody from a 5 gL
titer The purification cost per
kilogram of produced antibody
from a 5 gL titer using Resin2 (7000 USD) can be reduced
by 32 by using Resin 3 (see
Figure 2)
These results show that the
use of Resin 3 in purification of
antibodies from high-titer feeds
significantly improves process
economy (see Figure 2)
SUMMARY
These data demonstrate that theResin 3 prototype has high capac-
ity and reusability with stable
step yield and impurity clear-
ance (eg DNA HCP) for more
than 100 cycles The engineered
Protein A ligand allows for the
use of rigorous and cost-effective
CIP and sanitization protocols
based on NaOH Furthermore
the ligand is protease stable
which leads to lower ligand leak-
age and the highly cross-linkedagarose matrix allows for high flow
velocities at production scale
In conclusion process economy
can be significantly improved by
the use of Resin 3 in purification of
monoclonal antibodies from high-
titer cell culture supernatants
ACKNOWLEDGEMENTSThe authors wish to thank
GE Healthcare Li fe Sciences(Uppsala Sweden) for providing
Resin 3 prototype resin
REFERENCES1 G Koumlhler and C Milstein Nature 256
(5517) 495-497 (1975)2 S Kozlowski and P Swann Adv Drug
Deliv Rev 58 (5-6) 707ndash 722 (2006)3 PA Scolnik mAbs 1 (2) 179-184 (2009)4 S Aggarwal Nat Biotechnol 29 (12)
1083-1089 (2011)5 B Kelley mAbs 1 (5) 443-452 (2009)6 S Lofdahl et al Proc Natl Acad Sci
USA 80 (3) 697-701 (1983)
7 D Colbert et al J Biol Response Mod 3(3) 255-259 (1984)
8 GE Healthcare ldquoDynamic bindingcapacity study on MabSelect SuReLX for capturing high-titer monoclonalantibodiesrdquo Application Note 28-9875-25 Edition AA
Rapid growth in mAb demand has triggeredindustry efforts to increase manufacturing
capacity with the consequence that theantibody titers in mammalian cell culture
have increased dramatically
7242019 Comparing Protein a Resins for MAb Purification
December 2013 wwwbiopharminternationalcom BioPharm International
Posted with permission from the December 2013 issue of BioPharm International reg wwwbiopharminternationalcom Copyright 2014 Advanstar Communications Inc All rights reservedFor more information on the use of this content contact Wrightrsquos Media at 877-652-5295
10749
mAb Purification
Resin 3 prototype resin The cal-
culations are based on an annual
mAb production amount of 500
kg and bioreactor size of 10000 L
for 1 gL titer or 5000 L for 3 gL
and 5 gL titers Column diam-eter was estimated by considering
a process time within 10 to 15 h
The total purification cost
per kilogram of produced anti-
body from 1 gL and 3 gL titers
including chemicals and water
using Resin 1 ($13000 and
$14000 respectively) can be
reduced by 29 and 46 respec-
tively by using Resin 2 instead as
shown in Figure 2
With Resin 3 in the case of atiter level of 3 gL the authors
found that the overall purifica-
tion cost can be even further
reduced by 26 With a lower
titer level of 1 gL however the
decrease was only 4
Under the selected conditions
Resin 1 was not suitable for puri-
fication of antibody from a 5 gL
titer The purification cost per
kilogram of produced antibody
from a 5 gL titer using Resin2 (7000 USD) can be reduced
by 32 by using Resin 3 (see
Figure 2)
These results show that the
use of Resin 3 in purification of
antibodies from high-titer feeds
significantly improves process
economy (see Figure 2)
SUMMARY
These data demonstrate that theResin 3 prototype has high capac-
ity and reusability with stable
step yield and impurity clear-
ance (eg DNA HCP) for more
than 100 cycles The engineered
Protein A ligand allows for the
use of rigorous and cost-effective
CIP and sanitization protocols
based on NaOH Furthermore
the ligand is protease stable
which leads to lower ligand leak-
age and the highly cross-linkedagarose matrix allows for high flow
velocities at production scale
In conclusion process economy
can be significantly improved by
the use of Resin 3 in purification of
monoclonal antibodies from high-
titer cell culture supernatants
ACKNOWLEDGEMENTSThe authors wish to thank
GE Healthcare Li fe Sciences(Uppsala Sweden) for providing
Resin 3 prototype resin
REFERENCES1 G Koumlhler and C Milstein Nature 256
(5517) 495-497 (1975)2 S Kozlowski and P Swann Adv Drug
Deliv Rev 58 (5-6) 707ndash 722 (2006)3 PA Scolnik mAbs 1 (2) 179-184 (2009)4 S Aggarwal Nat Biotechnol 29 (12)
1083-1089 (2011)5 B Kelley mAbs 1 (5) 443-452 (2009)6 S Lofdahl et al Proc Natl Acad Sci
USA 80 (3) 697-701 (1983)
7 D Colbert et al J Biol Response Mod 3(3) 255-259 (1984)
8 GE Healthcare ldquoDynamic bindingcapacity study on MabSelect SuReLX for capturing high-titer monoclonalantibodiesrdquo Application Note 28-9875-25 Edition AA
Rapid growth in mAb demand has triggeredindustry efforts to increase manufacturing
capacity with the consequence that theantibody titers in mammalian cell culture
have increased dramatically
7242019 Comparing Protein a Resins for MAb Purification
December 2013 wwwbiopharminternationalcom BioPharm International
Posted with permission from the December 2013 issue of BioPharm International reg wwwbiopharminternationalcom Copyright 2014 Advanstar Communications Inc All rights reservedFor more information on the use of this content contact Wrightrsquos Media at 877-652-5295
10749
mAb Purification
Resin 3 prototype resin The cal-
culations are based on an annual
mAb production amount of 500
kg and bioreactor size of 10000 L
for 1 gL titer or 5000 L for 3 gL
and 5 gL titers Column diam-eter was estimated by considering
a process time within 10 to 15 h
The total purification cost
per kilogram of produced anti-
body from 1 gL and 3 gL titers
including chemicals and water
using Resin 1 ($13000 and
$14000 respectively) can be
reduced by 29 and 46 respec-
tively by using Resin 2 instead as
shown in Figure 2
With Resin 3 in the case of atiter level of 3 gL the authors
found that the overall purifica-
tion cost can be even further
reduced by 26 With a lower
titer level of 1 gL however the
decrease was only 4
Under the selected conditions
Resin 1 was not suitable for puri-
fication of antibody from a 5 gL
titer The purification cost per
kilogram of produced antibody
from a 5 gL titer using Resin2 (7000 USD) can be reduced
by 32 by using Resin 3 (see
Figure 2)
These results show that the
use of Resin 3 in purification of
antibodies from high-titer feeds
significantly improves process
economy (see Figure 2)
SUMMARY
These data demonstrate that theResin 3 prototype has high capac-
ity and reusability with stable
step yield and impurity clear-
ance (eg DNA HCP) for more
than 100 cycles The engineered
Protein A ligand allows for the
use of rigorous and cost-effective
CIP and sanitization protocols
based on NaOH Furthermore
the ligand is protease stable
which leads to lower ligand leak-
age and the highly cross-linkedagarose matrix allows for high flow
velocities at production scale
In conclusion process economy
can be significantly improved by
the use of Resin 3 in purification of
monoclonal antibodies from high-
titer cell culture supernatants
ACKNOWLEDGEMENTSThe authors wish to thank
GE Healthcare Li fe Sciences(Uppsala Sweden) for providing
Resin 3 prototype resin
REFERENCES1 G Koumlhler and C Milstein Nature 256
(5517) 495-497 (1975)2 S Kozlowski and P Swann Adv Drug
Deliv Rev 58 (5-6) 707ndash 722 (2006)3 PA Scolnik mAbs 1 (2) 179-184 (2009)4 S Aggarwal Nat Biotechnol 29 (12)
1083-1089 (2011)5 B Kelley mAbs 1 (5) 443-452 (2009)6 S Lofdahl et al Proc Natl Acad Sci
USA 80 (3) 697-701 (1983)
7 D Colbert et al J Biol Response Mod 3(3) 255-259 (1984)
8 GE Healthcare ldquoDynamic bindingcapacity study on MabSelect SuReLX for capturing high-titer monoclonalantibodiesrdquo Application Note 28-9875-25 Edition AA
Rapid growth in mAb demand has triggeredindustry efforts to increase manufacturing
capacity with the consequence that theantibody titers in mammalian cell culture
have increased dramatically
7242019 Comparing Protein a Resins for MAb Purification
December 2013 wwwbiopharminternationalcom BioPharm International
Posted with permission from the December 2013 issue of BioPharm International reg wwwbiopharminternationalcom Copyright 2014 Advanstar Communications Inc All rights reservedFor more information on the use of this content contact Wrightrsquos Media at 877-652-5295
10749
mAb Purification
Resin 3 prototype resin The cal-
culations are based on an annual
mAb production amount of 500
kg and bioreactor size of 10000 L
for 1 gL titer or 5000 L for 3 gL
and 5 gL titers Column diam-eter was estimated by considering
a process time within 10 to 15 h
The total purification cost
per kilogram of produced anti-
body from 1 gL and 3 gL titers
including chemicals and water
using Resin 1 ($13000 and
$14000 respectively) can be
reduced by 29 and 46 respec-
tively by using Resin 2 instead as
shown in Figure 2
With Resin 3 in the case of atiter level of 3 gL the authors
found that the overall purifica-
tion cost can be even further
reduced by 26 With a lower
titer level of 1 gL however the
decrease was only 4
Under the selected conditions
Resin 1 was not suitable for puri-
fication of antibody from a 5 gL
titer The purification cost per
kilogram of produced antibody
from a 5 gL titer using Resin2 (7000 USD) can be reduced
by 32 by using Resin 3 (see
Figure 2)
These results show that the
use of Resin 3 in purification of
antibodies from high-titer feeds
significantly improves process
economy (see Figure 2)
SUMMARY
These data demonstrate that theResin 3 prototype has high capac-
ity and reusability with stable
step yield and impurity clear-
ance (eg DNA HCP) for more
than 100 cycles The engineered
Protein A ligand allows for the
use of rigorous and cost-effective
CIP and sanitization protocols
based on NaOH Furthermore
the ligand is protease stable
which leads to lower ligand leak-
age and the highly cross-linkedagarose matrix allows for high flow
velocities at production scale
In conclusion process economy
can be significantly improved by
the use of Resin 3 in purification of
monoclonal antibodies from high-
titer cell culture supernatants
ACKNOWLEDGEMENTSThe authors wish to thank
GE Healthcare Li fe Sciences(Uppsala Sweden) for providing
Resin 3 prototype resin
REFERENCES1 G Koumlhler and C Milstein Nature 256
(5517) 495-497 (1975)2 S Kozlowski and P Swann Adv Drug
Deliv Rev 58 (5-6) 707ndash 722 (2006)3 PA Scolnik mAbs 1 (2) 179-184 (2009)4 S Aggarwal Nat Biotechnol 29 (12)
1083-1089 (2011)5 B Kelley mAbs 1 (5) 443-452 (2009)6 S Lofdahl et al Proc Natl Acad Sci
USA 80 (3) 697-701 (1983)
7 D Colbert et al J Biol Response Mod 3(3) 255-259 (1984)
8 GE Healthcare ldquoDynamic bindingcapacity study on MabSelect SuReLX for capturing high-titer monoclonalantibodiesrdquo Application Note 28-9875-25 Edition AA
Rapid growth in mAb demand has triggeredindustry efforts to increase manufacturing
capacity with the consequence that theantibody titers in mammalian cell culture