Affinity Chromatography Resins - KANEKA KanCap™ · Affinity chromatography resins for full length...

Affinity Chromatography Resins

CelluloseBase Matrix

KANEKA KanCap™ resins use a highly cross-

linked cellulose-based matrix with an average

particle of size 65 – 85 µm and reduced non-

specifi c binding properties when compared to

other base matrices such as polymers or glass.

The cellulose-based matrix is biocompatible and

has been successfully used for many years in

KANEKA’s plasmapheresis systems.

The combination of highly crosslinked cellulose-

based matrix with the

in-house engineered

ligands makes

KANEKA KanCap™

series an excellent

tool to effi ciently

capture and purify

high titer of antibodies

and derivatives. n

KANEKA offers two protein A affi nity

resins for industrial–scale purifi cation

of full length monoclonal antibodies:

KANEKA KanCapA™

& KANEKA KanCapA™ 3G.

Both resins are manufactured under

a validated GMP process to ensure

a stable and reliable supply chain.

The entire GMP production process is

free of animal derived components and

is BSE-TSE Free certifi ed. Regulatory

Support Files are available under a

Confi dential Disclosure Agreement (CDA).

Affi nity chromatography resins for full length antibody purifi cationKANEKA KanCap™ chromatography resins are effi cient cellulose-based

affi nity sorbents intended for purifi cation of all antibody formats. With

their innovative proprietary ligands, KANEKA KanCap™ series resins

demonstrate increased antibody affi nity and high binding capacity.

With these new resins Kaneka offers a comprehensive solution for

purifying all antibody formats from classical full-length antibodies

to Fc-fusion proteins, bispecifi c and fragmented formats:

Cellulose-based matrix microscope image (x20k).

series

1. KANEKA KanCapA™ 3G

is a Protein A resin with

enhanced performance.

2. KANEKA KanCapA™

is a Protein A resin

intended for industrial

scale purifi cation of mAb’s.

3. KANEKA KanCap™ L

is a Protein L resin

dedicated for purifi cation

of a wide range of antibody

fragments and derivatives

containing the κ VL

domain.

4. KANEKA KANCap™ G

is a Protein G resin

recommended for purifying

antibody fragments and

derivatives containing the

CH1 domain.

100µm

KANEKA KanCapA™ 3G’s ligand is a multimer of a modifi ed

C domain designed to improve binding capacity, elution profi le,

impurity removal ability and alkaline stability. n

KANEKA KanCapATM3G a resin with enhanced performance

1.

> Uniqueimpurity removal properties

> Excellent elution profi le

> Enhanced binding capacity

#2

Affinity chromatography resins for full length antibody purification

Performance

Enhanced binding capacity

Dynamic binding capacity (DBC)

is closely linked to productivity

and production costs. It is a

key parameter to consider

when selecting an efficient

chromatography resin.

KANEKA KanCapA™ 3G shows

enhanced binding capacity

which makes it suitable

for monoclonal antibody

purification from high titer

feedstocks. n DBC DEPENDENCY on the residence time (Polyclonal Human IgG).

ELUTION pH COMPARISON of mAbs and Fc fusion derivatives

3.4

3.6

A B C D E F G H

3.8

4.0

4.2

Elu

tio

n p

H

0

20

40

60

80

0 2 4 6 8 10

DB

C a

t 5

% b

reak

thro

ug

h (m

g lg

G /

mL

re

sin

)

Residence time (min.)

100

80

60

40

20

0

0 100 200 300

Cycle Number

Re

mai

nin

g B

ind

ing

Cap

acit

y [%

]

0.1 M NaOH

0.5 M NaOH

100

80

60

40

20

0

0 100 200 300

Cycle Number

Yie

ld [

%]

Yield

50

40

30

20

10

0

0 100 200 300

Cycle Number

Lig

and

Le

ach

ing

[p

pm

of

lgG

]

Ligand Leaching

800

700

600

500

400

300

200

100

0

0.00 0.05 0.10 0.15

Pressure [MPa]

Lin

ear

Flo

w R

ate

[cm

/h]

0.20 0.25 0.30

30 cm l.D. x 20 cm height




4.4 cm l.D. x 20 cm height


0

5

10

15

20

25

30

35

Bin

din

g C

ap

acit

y (m

g/m

L-g

el)

Fab(A) Fab(B) Fab(C)

Competitor A Competitor B


KANEKA KanCapTM G


3.4

3.6

A B C D E F G H

3.8

4.0

4.2

Elu

tio

n p

H

0

20

40

60

80

0 2 4 6 8 10

DB

C a

t 5

% b

reak

thro

ug

h (m

g lg

G /

mL

re

sin

)


100

80

60

40

20

0

0 100 200 300

Cycle Number

Re

mai

nin

g B

ind

ing

Cap

acit

y [%

]

0.1 M NaOH

0.5 M NaOH

100

80

60

40

20

0

0 100 200 300

Cycle Number

Yie

ld [

%]

Yield

50

40

30

20

10

0

0 100 200 300

Cycle Number

Lig

and

Le

ach

ing

[p

pm

of

lgG

]

Ligand Leaching

800

700

600

500

400

300

200

100

0

0.00 0.05 0.10 0.15

Pressure [MPa]

Lin

ear

Flo

w R

ate

[cm

/h]

0.20 0.25 0.30







0

5

10

15

20

25

30

35

Bin

din

g C

ap

acit

y (m

g/m

L-g

el)




KANEKA KanCapTM G


DB

C a

t 5

% b

reak

thro

ug

h


mA

b m

on

om

er

con

cen

tra

tio

n (

SE

C a

rea

)

mA

b m

on

om

er

con

cen

tra

tio

n (

SE

C a

rea

)

Re

lati

ve d

istr

ibu

tio

n o

f H

CP

(/%

)

Re

lati

ve d

istr

ibu

tio

n o

f a

gg

reg

ate

(%

)

Elu

tio

n p

H

80 4.2

4.0

3.8

3.6

3.4

60

40

20

2 4 6 8 10

0

16.000

KANEKA KanCapATM 3G KANEKA KanCapATM 3GCompetitor A Competitor A16.000

Fraction # Fraction #

12.000 12.000

8.000 8.000

4.000 4.000

0 0

0 A B C D E F G H

Excellent Elution pHKANEKA KanCapA™ 3G’s ligand

was designed to allow elution of

monoclonal antibodies and their

Fc fusion derivatives under very

mild acidic conditions, between

pH 3.7 and 4.2. n

Unique impurity removal properties

KANEKA KanCapA™ 3G is a new

generation of Protein A affinity

chromatography resins carrying a

recombinant protein A ligand with

enhanced properties. The resin

has a better potential for impurity

removal compared to current

commercially available resins. n

0

4,000

8,000

12,000

16,000

1 2 3 4 5 6 7 8 9 10 11

pH 3.8

0

20

40

60

80

1 2 3 4 5 6 7 8 9 10 11

pH 3.7

Aggregate removalKANEKA KanCapA™ 3G Competitor A

Fraction #

mA

b m

on

om

er

con

cen

trat

ion

(S

EC

are

a)

Re

lati

ve d

istr

ibu

tio

n o

f ag

gre

gat

e (

/%)

0

4,000

8,000

12,000

16,000

1 2 3 4 5 6 7 8 9 10 11

mA

b m

on

om

er

con

cen

trat

ion

(S

EC

are

a) pH 3.8

0

20

40

60

80

1 2 3 4 5 6 7 8 9 10 11

pH 3.7

Fraction #

Host cell protein removalKANEKA KanCapA™ 3G Competitor A

Re

lati

ve d

istr

ibu

tio

n o

f H

CP

(/%

)

HOST CELL PROTEIN (left)

AND AGGREGATES (right)

separation during mAb elution by linear

pH gradient.

Monomer Peak

Host cell protein (HCP)

Aggregate

Molecules: IgG1 (CCCF),

Load: 5 mg/mL resin.

Elution: 50 mM Citrate buffer,

(gradient pH from 4.5 to 3, 10 CV).

KANEKA KanCapATM 3G

Competitor A

KANEKA KanCapATM 3G Competitor A KANEKA KanCapATM

#3

Molecules: A,G, H = Humanized IgG1 B, F= Human IgG2 C = Fc-fusion D, E = Chimeric IgG

Elution: 50 mM citrate buffer gradient pH from 6 to 3.

1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 111 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11

0 0

20 20

40 40

60 60

80 80

Affinity Chromatography Resins

KANEKA KanCapA™’s ligand is a multimer of a modified C domain

containing amino acid mutations to improve the resins’ alkaline

stability and antibodies elution profile. n

KANEKA KanCapATM 2.

> Track record of successful mAb GMP manufacturing for clinical trial materials

> Alkaline stability and long lifetime

> High flow rate operation and easy to scale up from pilot to process

Performance

Alkaline stability and long lifetime

KANEKA KanCapA™ can be sanitized

with 0.1 or 0.5 M NaOH. It can be

safely used up to 300 cycles with a

limited loss of Binding Capacity after

alkaline CIP with 0.1 M NaOH for

15 minutes contact time.

Over the CIP cycles, the elution yields

and the levels of the leached Protein A

remain stable. n

3.4

3.6

A B C D E F G H

3.8

4.0

4.2

Elu

tio

n p

H

0

20

40

60

80

0 2 4 6 8 10

DB

C a

t 5

% b

reak

thro

ug

h (m

g lg

G /

mL

re

sin

)


100

80

60

40

20

0

0 100 200 300

Cycle Number

Re

mai

nin

g B

ind

ing

Cap

acit

y [%

]

0.1 M NaOH

0.5 M NaOH

100

80

60

40

20

0

0 100 200 300

Cycle Number

Yie

ld [

%]

Yield

50

40

30

20

10

0

0 100 200 300

Cycle Number

Lig

and

Le

ach

ing

[p

pm

of

lgG

]

Ligand Leaching

800

700

600

500

400

300

200

100

0

0.00 0.05 0.10 0.15

Pressure [MPa]

Lin

ear

Flo

w R

ate

[cm

/h]

0.20 0.25 0.30







0

5

10

15

20

25

30

35

Bin

din

g C

ap

acit

y (m

g/m

L-g

el)




KANEKA KanCapTM G


3.4

3.6

A B C D E F G H

3.8

4.0

4.2

Elu

tio

n p

H

0

20

40

60

80

0 2 4 6 8 10

DB

C a

t 5

% b

reak

thro

ug

h (m

g lg

G /

mL

re

sin

)


100

80

60

40

20

0

0 100 200 300

Cycle Number

Re

mai

nin

g B

ind

ing

Cap

acit

y [%

]

0.1 M NaOH

0.5 M NaOH

100

80

60

40

20

0

0 100 200 300

Cycle Number

Yie

ld [

%]

Yield

50

40

30

20

10

0

0 100 200 300

Cycle Number

Lig

and

Le

ach

ing

[p

pm

of

lgG

]

Ligand Leaching

800

700

600

500

400

300

200

100

0

0.00 0.05 0.10 0.15

Pressure [MPa]

Lin

ear

Flo

w R

ate

[cm

/h]

0.20 0.25 0.30







0

5

10

15

20

25

30

35

Bin

din

g C

ap

acit

y (m

g/m

L-g

el)




KANEKA KanCapTM G


3.4

3.6

A B C D E F G H

3.8

4.0

4.2

Elu

tio

n p

H

0

20

40

60

80

0 2 4 6 8 10

DB

C a

t 5

% b

reak

thro

ug

h (m

g lg

G /

mL

re

sin

)


100

80

60

40

20

0

0 100 200 300

Cycle NumberR

em

ain

ing

Bin

din

g C

apac

ity

[%]

0.1 M NaOH

0.5 M NaOH

100

80

60

40

20

0

0 100 200 300

Cycle Number

Yie

ld [

%]

Yield

50

40

30

20

10

0

0 100 200 300

Cycle Number

Lig

and

Le

ach

ing

[p

pm

of

lgG

]

Ligand Leaching

800

700

600

500

400

300

200

100

0

0.00 0.05 0.10 0.15

Pressure [MPa]

Lin

ear

Flo

w R

ate

[cm

/h]

0.20 0.25 0.30







0

5

10

15

20

25

30

35

Bin

din

g C

ap

acit

y (m

g/m

L-g

el)




KANEKA KanCapTM G


RESIN LIFETIME EVALUATION after alkaline CIP with 0.1M NaOH for 15 min contact time. Remaining dynamic binding capacity at 5 % breakthrough (left); Yield (middle) and Protein A leaching (right). Ligand leaching: values less than the detection limit (1 ppm) are plotted as 1 ppm.

#4

100 100 100

80 80 80

60 60 60

40 40 40

20 20 20

0 0 0

Re

mai

nin

g B

ind

ing

Cap

acit

y (%

)

Cycle Number Cycle Number Cycle Number

Yie

ld (

%)

Lig

and

Le

ach

ing

(p

pm

of

IgG

)

0 100 200 300 0 100 200 300 0 100 200 300

0.1 M NaOH 0.5 M NaOH Yield Ligand Leaching

Mild acidic elution conditions for all mAbs

One of the most critical factors during monoclonal antibody

purifi cation is the pH used for their elution. In the case of

VH3-encoded mAbs, a low pH elution buffer is needed due

to their undesired binding to Protein A through the Fab region.

However it is well known that low pH elution leads to higher

aggregate formation.

To overcome this issue KANEKA KanCapA™ ligand has been

designed to remove the binding ability to Fabs. This allows milder

elution conditions. n

Operation at high fl ow rate and scalability from pilot to process

Thanks to its innovative highly crosslinked cellulose-based

matrix KANEKA KanCapA™ is perfectly suited for pilot and

large scale purifi cations. n

PRESSURE VS FLOW for packed columns of different diameters. The pressure generated by packed beds was calculated by substracting the pressure of the system from total pressure.

#5

ELUTION PROFILE of VH3-encoded mAb compared to other commercially available Protein A resins.Load: 5 g IgG /L resin; Strip solution: 1 M Acetic acid

pH 3.5pH 3.0

3.4

3.6

A B C D E F G H

3.8

4.0

4.2

Elu

tio

n p

H

0

20

40

60

80

0 2 4 6 8 10

DB

C a

t 5

% b

reak

thro

ug

h (m

g lg

G /

mL

re

sin

)


100

80

60

40

20

0

0 100 200 300

Cycle Number

Re

mai

nin

g B

ind

ing

Cap

acit

y [%

]

0.1 M NaOH

0.5 M NaOH

100

80

60

40

20

0

0 100 200 300

Cycle Number

Yie

ld [

%]

Yield

50

40

30

20

10

0

0 100 200 300

Cycle Number

Lig

and

Le

ach

ing

[p

pm

of

lgG

]

Ligand Leaching

800

700

600

500

400

300

200

100

0

0.00 0.05 0.10 0.15

Pressure [MPa]

Lin

ear

Flo

w R

ate

[cm

/h]

0.20 0.25 0.30







0

5

10

15

20

25

30

35

Bin

din

g C

ap

acit

y (m

g/m

L-g

el)




KANEKA KanCapTM G


800

700

600

500

400

300

200

100

0

0.00 0.05 0.10 0.15 0.20 0.25 0.30

Pressure (MPa)

Lin

ear

Flo

w R

ate

(cm

/h)

KanCapAAgarosePolymerGlass

A280

VolumeELUTION STRIP

A280

ELUTION STRIP

A280

VolumeELUTION STRIP

A280

ELUTION STRIP

A280

Volume

Volume Volume Volume

ELUTION STRIP

A280

ELUTION STRIP

A2

80


A280

VolumeELUTION STRIP

A280

ELUTION STRIP

A280

VolumeELUTION STRIP

A280

ELUTION STRIP

A280

Volume


ELUTION STRIP

A280

ELUTION STRIP

A2

80


A280

VolumeELUTION STRIP

A280

ELUTION STRIP

A280

VolumeELUTION STRIP

A280

ELUTION STRIP

A280

Volume


ELUTION STRIP

A280

ELUTION STRIP

KanCapATM

AgarosePolymerGlass

3.4

3.6

A B C D E F G H

3.8

4.0

4.2

Elu

tio

n p

H

0

20

40

60

80

0 2 4 6 8 10

DB

C a

t 5

% b

reak

thro

ug

h (m

g lg

G /

mL

re

sin

)


100

80

60

40

20

0

0 100 200 300

Cycle Number

Re

ma

inin

g B

ind

ing

Ca

pac

ity

[%]

0.1 M NaOH

0.5 M NaOH

100

80

60

40

20

0

0 100 200 300

Cycle Number

Yie

ld [

%]

Yield

50

40

30

20

10

0

0 100 200 300

Cycle Number

Lig

an

d L

ea

chin

g [

pp

m o

f lg

G]

Ligand Leaching

800

700

600

500

400

300

200

100

0

0.00 0.05 0.10 0.15

Pressure [MPa]

Lin

ear

Flo

w R

ate

[cm

/h]

0.20 0.25 0.30







0

5

10

15

20

25

30

35

Bin

din

g C

ap

acit

y (m

g/m

L-g

el)




KANEKA KanCapTM G


3.4

3.6

A B C D E F G H

3.8

4.0

4.2

Elu

tio

n p

H

0

20

40

60

80

0 2 4 6 8 10

DB

C a

t 5

% b

reak

thro

ug

h (m

g lg

G /

mL

re

sin

)


100

80

60

40

20

0

0 100 200 300

Cycle Number

Re

ma

inin

g B

ind

ing

Cap

acit

y [%

]

0.1 M NaOH

0.5 M NaOH

100

80

60

40

20

0

0 100 200 300

Cycle Number

Yie

ld [

%]

Yield

50

40

30

20

10

0

0 100 200 300

Cycle Number

Lig

an

d L

ea

chin

g [

pp

m o

f lg

G]

Ligand Leaching

800

700

600

500

400

300

200

100

0

0.00 0.05 0.10 0.15

Pressure [MPa]

Lin

ear

Flo

w R

ate

[cm

/h]

0.20 0.25 0.30







0

5

10

15

20

25

30

35

Bin

din

g C

ap

acit

y (m

g/m

L-g

el)




KANEKA KanCapTM G


4.4 cm l.D. x 20cm height

7 cm l.D. x 20cm height





SCHEMATIC REPRESENTATION of KANEKA KanCapTM L’s ligand affi nity to different kappa light chain of different antibody fragments in comparison to wild type protein L.

Affi nity

Fab Variants

Affi nity

Fab Variants

Wild type Protein L KANEKA KanCapTM L Protein L

KANEKA offers also TWO solutions

for purifying antibody fragments

and derivatives that lack the Fc region:

KANEKA KanCap™ L and

KANEKA KanCap™ G.

Affi nity chromatography resins for antibody fragments and derivatives purifi cation

KANEKA KanCap™ L is a newly developed cellulose-based Protein L

resin designed for capture and purifi cation of a wide range of antibody

fragment formats such as scFv, Fab and F(ab’)2. KANEKA KanCapTM L

ligand was designed to bind a wide spectra of molecules and to exibit

high affi nity to the VL domain of the antibody kappa light chain. It

is also an alternative tool for purifying full length antibody formats

containing a kappa light chain which poorly bind to Protein A resins. n

KANEKA KanCapTM L 3.

> High binding capacity for antibodies containing a κ light chain

> Wide binding range for antibody molecules containing a κ light chain FULL LENGTH ANTIBODY

Fc

Fab

Heavy Chain

Constant Region

Variable Region

FULL LENGTH ANTIBODY

Heavy Chain

#6

Affi nity Chromatography Resins

DBCSBC

DBCSBC

Fab (A) from humanized IgG1 kappa, Fab (B) from chimeric IgG1 kappa

Bin

ding

Cap

acit

y (m

g/m

L)

0

10

20

30

40

50

60

70

CompetitorKANEKA KanCapTM L

DBCSBC

DBCSBC


Bin

ding

Cap

acit

y (m

g/m

L)

0

10

20

30

40

50

60

70

CompetitorKANEKA KanCapTM L

1 min. 4 min.

Fab A Fab B Fab A Fab B Fab A Fab B Fab A Fab B

4 min. 4 min. 4 min.1 min. 1 min. 1 min. 1 min. 4 min. 4 min. 4 min. 4 min.1 min. 1 min. 1 min.


KANEKA KanCapTM L Competitor

DYNAMIC AND STATIC binding capacity evaluation.Fab (A) from humanized IgG1 kappa; Fab (B) from chimeric IgG1 kappa

70 70

60 60

50 50

40 40

30 30

RT 1 min. RT 4 min. SBC RT 1 min. RT 4 min. SBC

20 20

10 10

0 0

Bin

din

g C

ap

aci

ty (

mg

/mL

)

Fab A Fab B

Fab A Fab B

VH

CH1

CL

KANEKAKanCapTM G KANEKA

KanCapTM L

VL

KANEKA KanCap

VL

G

Light Chain

n KANEKA and the KANEKA logo are trademarks of

KANEKA.n KANEKA KanCapA™ is a

trademark of KANEKA.n KanCap™ is a trademark

of KANEKA.n RoboColumn® is a

registered trademark of Repligen GmbH.

n KANEKA products may not be resold or

transferred, modifi ed for resale or transfer, or used

to manufacture commercial products for these purposes

without written approval from KANEKA.

n The information in this brochure were obtained by

KANEKA CORPORATION.n Disclaimers: All

experimental data are provided “as is”, without

any warranty of accuracy or completeness.

©2018 KANEKA CORPORATION, JAPAN

All rights reserved.

Affi nity chromatography resins for antibody fragments and derivatives purifi cation

KANEKA KanCapTM G is an innovative cellulose-based affi nity

resin designed for effi cient capture and purifi cation of antibody

fragment formats containing the human CH1 domain such as Fab

and F(ab’)2. KANEKA KanCapTM G ligand exhibits an increased

binding affi nity for the CH1 domain. It can also be used to purify full

length antibody formats. n

KANEKA KanCapTM G 4.

> High binding capacity for antibody molecules containing CH1 or Fc region

series

#7

CH1

VHVL

CL

CH1

VHVL

CL

Low affi nity

High affi nity

KANEKA KanCapTM G Protein G

Wild type Protein G

SCHEMATIC REPRESENTATION of KANEKA KanCapTM G’s ligand affi nity to CH1 domain

SDS-PAGE analysis of non-reduced Fab samples purifi ed from yeast supernatant M. Marker; 1.Supernatant; 2.Flow-through; 3.Wash; 4.Eluate

DYNAMIC BINDING capacity evaluation at 4 min. residence time.Fab (A) from monoclonal chimeric IgG1

Fab (B) from polyclonal human IgG (containing κ and λ types)

Fab (C) from monoclonal humanized IgG1

3.4

3.6

A B C D E F G H

3.8

4.0

4.2

Elu

tio

n p

H

0

20

40

60

80

0 2 4 6 8 10

DB

C a

t 5

% b

rea

kth

rou

gh

(mg

lgG

/ m

L r

esi

n)


100

80

60

40

20

0

0 100 200 300

Cycle Number

Re

mai

nin

g B

ind

ing

Cap

acit

y [%

]

0.1 M NaOH

0.5 M NaOH

100

80

60

40

20

0

0 100 200 300

Cycle Number

Yie

ld [

%]

Yield

50

40

30

20

10

0

0 100 200 300

Cycle Number

Lig

and

Le

ach

ing

[p

pm

of

lgG

]

Ligand Leaching

800

700

600

500

400

300

200

100

0

0.00 0.05 0.10 0.15

Pressure [MPa]

Lin

ear

Flo

w R

ate

[cm

/h]

0.20 0.25 0.30







0

5

10

15

20

25

30

35

Bin

din

g C

ap

acit

y (m

g/m

L-g

el)




KANEKA KanCapTM G


KANEKA KanCapTM G Competitor A Competitor B

0

5

10

15

20

25

30

35

Fab(A) Fab(A) Fab(A)Fab(B) Fab(B) Fab(B)Fab(C) Fab(C) Fab(C)

Bin

din

g C

apac

ity

(mg

/mL

-ge

l)

JAPAN Kaneka Corporation1-12-32, Akasaka, Minato-ku,Tokyo 107-6028, [email protected]

USAKaneka US Innovation Center7979 Gateway Blvd., Suite 220Newark, CA [email protected]

EUROPEKaneka Eurogentec S.A.Rue du bois Saint-Jean, 144102 Seraing, [email protected]

www.bioseparation.kaneka.com

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