Comparing Protein a Resins for MAb Purification

7242019 Comparing Protein a Resins for MAb Purification

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BioPharm International wwwbiopharminternationalcom December 2013

Electronically reprinted from December 2013

BioPharmINTERNATIONAL

The Scien ce amp 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

With 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 cellsfrom 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 PurificationShohei Kobayashi andYasufumi Ueda

A prototypeProtein A resin

is evaluatedfor purificationperformance

reusabilityand cost

performance

Shohei Kobayashi and

Yasufumi Ueda are in the

API process development department

Pharmaceutical Technology Division

Chugai Pharmaceutical Tokyo Japan



December 2013 wwwbiopharminternationalcom BioPharm International

mAb Purification

A L L F I G U R E S A R E

C O U R T E S Y

O F T H E A U T H O R S

cessful as targeted therapeutics

for a variety of diseases includ-

ing several forms of cancer

multiple sclerosis and immuno-

logical disorders such as rheu-

matoid arthritis and psoriasis In

2007 mAbs accounted for almosthalf of the top-20 best-selling

biotechnology drugs in the US

alone establishing them as an

important group of molecules (3)

Today mAbs constitute the single

largest class of biological drugs

and accounts for about 36 of

the total biologics market with

an annual sales growth rate of

approximately 10 (4)

COMMERCIAL983085SCALEPRODUCTION CHALLENGESThe rapid growth in mAb demand

has triggered industry efforts to

increase manufacturing capacity

with the consequence that the

antibody titers in mammalian

cell culture have increased dra-

matically Today a typical process

accumulates titers of 1ndash5 gL but

expression levels as high as 10ndash13

gL have been reported (5) The

increase in upstream productivity

creates a subsequent demand on

downstream processing to address

high-titer HCCF

Commercial-scale purification

of mAbs usually contains two

or three chromatographic steps

Protein A is the affinity chro-matography ligand of choice for

the first antibody capture step

because its high selectivity gives

excellent purity (typically gt 99)

and high yields Furthermore

Protein A-based resins form the

basis of almost all mAb-purifica-

tion platforms as they are easy

to use at both small and large

scale with generic experimental

protocols

Increased antibody titers cre-ate a potential purification chal-

lenge because of the limited

capacity of current Protein A res-

ins To handle the high titers

new resins with significantly

greater capacity are needed In

addition Protein A resins with

the ability to withstand repeated

cleaning-in-place (CIP) with low-

cost sodium hydroxide (NaOH)

considerably improves process

economics

PROTEIN A RESINSProtein A is a bacterial protein

from Staphylococcus aureus

with the capacity to bind mam-

malian antibodies of class immu-

noglobulin G (IgG) with high

affinity The gene for Protein Ahas been cloned and expressed

in Escherichia coli (6 7) allow-

ing for the production of large

q u a n t i t i e s o f r e c om b i n a n t

Protein A

Although recombinant Protein

A is widely used as an affinity

ligand for the capture and puri-

fication of antibodies its sen-

sitivity to alkaline conditions

prevents the use of rigorous and

cost-effective CIP and sanitiza-

tion protocols based on NaOH

Compared to conventional

Protein A resins one of the affin-

ity chromatography resins inves-

tigated Resin 2 (MabSelect SuRe

GE Healthcare) is based on a

modified alkali-tolerant Protein

A ligand Through protein engi-

neering the amino acids in one

of the IgG-binding domains par-

ticularly sensitive to alkali were

identified and substituted with

Table I Properties of Protein A resins degree of alkali resistance is indicated by +-

Resin Ligand Average particle size (microm) Alkali resistance Matrix Binding capacity

Resin 1 (rProtein ASepharose 4 Fast Flow)

rProtein A 90 +- Agarose ~27 gL resin

Resin 2 (MabSelect SuRe) Alkali-tolerant Protein A 85 +++ Agarose ~35 gL resinResin 3 (MabSelect SuReLX prototype)

Alkali-tolerant Protein A 85 +++(+) Agarose ~60 gL resin

Typical dynamic binding capacities according to resin manufacturer data

Table II Study outline column height = 100 mm column inner diameter = 10 mm run time = 5 h CV is column volume

Step Solution Volume Flow rate

Equilibrium20 mM citrate-phosp hate buff er pH 75 1 molL sodiumchloride (NaCl)

5 CV 300 cmh

Load Harvested cell-culture fluid 50 gL resin (residence time 6 min) 100 cmh

Wash 1 20 mM citrate-phosphate buffer pH 75 1 molL NaCl 5 CV 300 cmh

Wash 2 10 mM citrate-phosphate buffer pH 77 5 CV 300 cmhElution 50 mM acetic acid 6 CV 300 cmh

Regeneration 01 mol sodium hydroxide 3 CV 120 cmh

Storage (per 4 cycles) 2 benzyl alcohol 50 mM sodium acetate pH 50 5 CV 120 cmh




mAb Purification

more stable ones

A nove l p rototype Res in

3 offers an increased dynamic

binding capacity (DBC) at a

slightly longer residence t ime

EVALUATINGCAPACITY AND REUSABILITYTable I shows the Protein A res-

ins that were compared for per-

formance and cost-efficiency

According to the vendor Resin 3

exhibits higher DBC than Resin

2 at longer residence times (8)

A residence time of 6 min is

expected to give a DBC (at 10

breakthrough) of approximately

60 g antibody per liter resin

In this study the authors were

able to confirm this behav-

ior and for further studies a

residence time of 6 min and

a loading of 50 g antibody per liter

resin corresponding to approxi-

mately 80 of the DBC (at 10

breakthrough) were selected

Table II presents the outline of

the lifetime study An amount

of cell-culture supernatant cor-

responding to 50-g antibody per

liter resin was applied to the col-

umn at 6 min residence time

This was followed by a two-step

washing procedure to remove

unbound particles Bound anti-

body was eluted with 50 mMacetic acid and the column resin

was cleaned in place with five

column volumes (CV) of 01 M

NaOH

The results show that the step

yield was consistently over 95

and high log-reduction factors

of host-cell proteins (HCP) andDNA were achieved (see Figure 1)

In this study carryover was eval-

uated each 28th cycle and was

found to be less than 01 (ie

after cycle 28 56 84 and 112)

The lifetime study with mAb-con-

taining feedstock demonstrates

that the product quality DBC

and yield with Resin 3 were stable

for more than 100 purification

cycles No increase in pressure

was observed during the study

COST PERFORMANCECost performance is dependent

on product amount produced per

year batch size column size and

acceptable process time In this

study the cost of Protein A-based

production was calculated titer

by titer by the use of conven-

tional resins (Resin 1 which is GE

Healthcarersquos rProtein A Sepharose

4 Fast Flow and Resin 2) and

The engineered

Protein A ligand allows

clean-in-place and

sanitization protocols

based on sodium

hydroxideFigure 2 Cost-performance of Resin 3 prototype compared to conventional resins

(Resin 1 and Resin 2) Product amount is 500 kg fermenter size is 10000 L (for 1 gL)

or 5000 L (for 35 gL) column size is 20 cm bed height column lifetime is 120 cycles

for Resin 1 and 200 cycles for Resin 2 and 3 process time is 10-15 h

Figure 1 Resin lifetime study using harvested cell-culture fluid (HCCF) Step yield

is gt95 elution volume is 22 +- 07 column volumes (CV) and carry-over is lt11000

all show no trend Impurities are as follows DNA 32 log HCP 31 log Protein A

approximately 10 ppm (using data from GE Healthcare obtained from commercially

available resin) all show no trend




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




mAb Purification

A L L F I G U R E S A R E

C O U R T E S Y

O F T H E A U T H O R S

cessful as targeted therapeutics

for a variety of diseases includ-

ing several forms of cancer

multiple sclerosis and immuno-

logical disorders such as rheu-

matoid arthritis and psoriasis In

2007 mAbs accounted for almosthalf of the top-20 best-selling

biotechnology drugs in the US

alone establishing them as an

important group of molecules (3)

Today mAbs constitute the single

largest class of biological drugs

and accounts for about 36 of

the total biologics market with

an annual sales growth rate of

approximately 10 (4)

COMMERCIAL983085SCALEPRODUCTION CHALLENGESThe rapid growth in mAb demand

has triggered industry efforts to

increase manufacturing capacity

with the consequence that the

antibody titers in mammalian

cell culture have increased dra-

matically Today a typical process

accumulates titers of 1ndash5 gL but

expression levels as high as 10ndash13

gL have been reported (5) The

increase in upstream productivity

creates a subsequent demand on

downstream processing to address

high-titer HCCF

Commercial-scale purification

of mAbs usually contains two

or three chromatographic steps

Protein A is the affinity chro-matography ligand of choice for

the first antibody capture step

because its high selectivity gives

excellent purity (typically gt 99)

and high yields Furthermore

Protein A-based resins form the

basis of almost all mAb-purifica-

tion platforms as they are easy

to use at both small and large

scale with generic experimental

protocols

Increased antibody titers cre-ate a potential purification chal-

lenge because of the limited

capacity of current Protein A res-

ins To handle the high titers

new resins with significantly

greater capacity are needed In

addition Protein A resins with

the ability to withstand repeated

cleaning-in-place (CIP) with low-

cost sodium hydroxide (NaOH)

considerably improves process

economics

PROTEIN A RESINSProtein A is a bacterial protein

from Staphylococcus aureus

with the capacity to bind mam-

malian antibodies of class immu-

noglobulin G (IgG) with high

affinity The gene for Protein Ahas been cloned and expressed

in Escherichia coli (6 7) allow-

ing for the production of large

q u a n t i t i e s o f r e c om b i n a n t

Protein A

Although recombinant Protein

A is widely used as an affinity

ligand for the capture and puri-

fication of antibodies its sen-

sitivity to alkaline conditions

prevents the use of rigorous and

cost-effective CIP and sanitiza-

tion protocols based on NaOH

Compared to conventional

Protein A resins one of the affin-

ity chromatography resins inves-

tigated Resin 2 (MabSelect SuRe

GE Healthcare) is based on a

modified alkali-tolerant Protein

A ligand Through protein engi-

neering the amino acids in one

of the IgG-binding domains par-

ticularly sensitive to alkali were

identified and substituted with

Table I Properties of Protein A resins degree of alkali resistance is indicated by +-

Resin Ligand Average particle size (microm) Alkali resistance Matrix Binding capacity

Resin 1 (rProtein ASepharose 4 Fast Flow)

rProtein A 90 +- Agarose ~27 gL resin

Resin 2 (MabSelect SuRe) Alkali-tolerant Protein A 85 +++ Agarose ~35 gL resinResin 3 (MabSelect SuReLX prototype)

Alkali-tolerant Protein A 85 +++(+) Agarose ~60 gL resin

Typical dynamic binding capacities according to resin manufacturer data

Table II Study outline column height = 100 mm column inner diameter = 10 mm run time = 5 h CV is column volume

Step Solution Volume Flow rate

Equilibrium20 mM citrate-phosp hate buff er pH 75 1 molL sodiumchloride (NaCl)

5 CV 300 cmh

Load Harvested cell-culture fluid 50 gL resin (residence time 6 min) 100 cmh

Wash 1 20 mM citrate-phosphate buffer pH 75 1 molL NaCl 5 CV 300 cmh

Wash 2 10 mM citrate-phosphate buffer pH 77 5 CV 300 cmhElution 50 mM acetic acid 6 CV 300 cmh

Regeneration 01 mol sodium hydroxide 3 CV 120 cmh

Storage (per 4 cycles) 2 benzyl alcohol 50 mM sodium acetate pH 50 5 CV 120 cmh




mAb Purification

more stable ones



































NaOH


























The engineered


clean-in-place and


based on sodium














10749

mAb Purification
















shown in Figure 2






decrease was only 4








Figure 2)






SUMMARY


























USA 80 (3) 697-701 (1983)









mAb Purification

more stable ones



































NaOH


























The engineered


clean-in-place and


based on sodium














10749

mAb Purification
















shown in Figure 2






decrease was only 4








Figure 2)






SUMMARY


























USA 80 (3) 697-701 (1983)










10749

mAb Purification
















shown in Figure 2






decrease was only 4








Figure 2)






SUMMARY


























USA 80 (3) 697-701 (1983)






Documents

Comparing Protein a Resins for MAb Purification