platform process for antibody fragments using cellulose …...antibody fragments containing kappa...

Wild type PpL

PpL mutant(KanCapTM L ligand)

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

SpG agarose (B)

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

SpG agarose (A)

0

5

10

15

20

25

30

35

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

DBC

(mg/

mL-

gel)

KanCapTM G

Kaneka Eurogentec S.A.BioseparationRue du Bois Saint-Jean, 14 4102 Seraing, Belgiumbioseparation@eurogentec.com

KANEKA CORPORATIONPharma & Medical New Development Strategic Unit1-12-32, Akasaka, Minato-ku,Tokyo 107-6028, Japanbioproducts@kaneka.co.jp

Kaneka US Innovation CenterBiochromatography7979 Gateway Blvd., Suite 220, Newark, CA 94560 bioproducts@kaneka.com

Contact Information GeneFrontier CorporationTodai-Kashiwa Venture Plaza Rm#308 5-4-19, Kashiwa-no-Ha, Kashiwa, Chiba 277-0882, JAPAN contactus@genefrontier.com

This research is partially supported by the developing key technologies for discovering and manufacturing pharmaceuticals used for next-generation treatments and diagnoses both from the Ministry of Economy, Trade and Industry, Japan (METI) and from Japan Agency for Medical Research and Development (AMED).from Japan Agency for Medical Research

Enhanced binding capacity of KNK PpL Resin, KanCapTM L

Leakage in washing step

Elution Peak

Enhanced binding capacity of KNK SpG Resin, KanCapTM G

In vitro affinity maturation of SpG

Dynamic binding capacity (DBC) at 5 % breakthrough was measured at a residence time of 4 min.KanCapTM G : SpG mutant selected by affinity maturation was immobilized to cellulose base matrix. SpG agarose (A), (B) : Commercial SpG resin (agarose base matrix) Fab(A), Fab (B) from monoclonal IgG, Fab(C) from human serum IgGs

Figure 4. Chromatographic analysis of binding capacity of SpG resins

Figure 3. Association constant (KA) to Fabs of SpGmutants selected by in vitro affinity maturation

0

2

4

6

8

10

12

K A(×

106

M-1

)

Fab (a) Fab (b)

Rational design and engineering of PpL

PpL agarose

No Leakage

ElutionPeak

MkDa

976645

30

20.1

14.4

VH+CH1

VL+CL

Fab

ReducedNon-reduced

M. Marker1. Fab in E.coli lysate* (Fab from humanized IgG1 κ)2. Flow through 3. Wash 4. Elution *Mixture of purified monoclonal Fab (containing κ light chain) and E.coli lysate (cytoplasmic fraction)

Figure 9. SDS-PAGE analysis of Fab purification samples from E.coli. lysate using KanCapTM L

KanCapKanCapTMTMTM G or KanCapKanCapKanCapTMTMTM G or KanCapKanCapKanCapKanCapTM

G or G or TMTMTMTM L

(Capture)

Intermediate

purificationPolishing

Proposed antibody fragments purification process

SpG mutants DNA library

Figure 2. In vitro affinity maturation by using a ribosome display system, PUREfrex®RD

The association constant of selected SpG mutants increased from 30 to 80-fold compared to wild type SpG.

Fab was highly purified with high yield from E.coli lysate only by one-step chromatography using KanCapTM L.

Conclusions Acknowledgements

PpL mutants, KanCapTM L ligand, was rationally designed based on a structure modeling and sequence diversity of PpL, showed wide binding spectra.

KanCapTM L

Figure 8. Chromatographic analysis of binding capacity of PpL resins

Chromatogram of Fab (B) injection to PpL resins

Dynamic binding capacity (DBC) at 5 % breakthrough was measured at a residence time of 4 min.KanCapTM L: PpL mutant engineered by rational design was immobilized to cellulose base matrix. PpL agarose: Commercial PpL resin (agarose base matrix) Fab (A), Fab (B) from monoclonal IgG, diabody from Yeast Supernatant

KanCapTM G showed higher binding capacity for all types of human Fabsthan commercial SpG resins and no leakage in washing step.

KanCapTM L could capture wide range of antibody fragments containing kappa light chain than commercial PpL resin could hardly bind.

High recovery and purity in Fab purificationHigh recovery and specificity in Fab purification

2 3 41MkDa

66

45

30

20.1

M. Marker, 1. Fab in E.coli lysate*1 (left), Yeast Supernatant*2 (right), 2. Flow through , 3. Wash , 4. Elution*1 Mixture of purified polyclonal Fab (containing λ light chain) and E.coli lysate (cytoplasmic fraction)*2 Yeast Supernatant including expressed monoclonal Fab (containing κ light chain)

Non-reduced condition

Fab(κ type)

Recovery (%)(A) (B)

KanCapTM G 97 85

SpG agarose (A) 19 12

Light chain (LC) monomer

Figure 5. SDS-PAGE analysis of Fab purification samples using KanCapTM G

KanCapTM G could capture both types of Fabs, kappa and lambda type and eliminate byproduct (e.g. LC monomer, LC dimer) from Fab expressed in Yeast Sup.

Recovery (%)

KanCapTM L 94

KanCapTM G has high binding capacity and recovery for all types of human Fabs.

KanCapTM L has wide binding spectra and high binding capacity for the antibody fragments containing kappa light chain.

Kaneka proposes new purification platform process for antibody fragments using our novel Protein G and Protein L chromatography resin.

Load: 27 mg Load: 2 mg

modification

(y) (z)(w) (x)

(y) (z)(w) (x)

affinity

Diabody Fab(A) Fab(B)

PpL agarose

0

10

20

30

40

50

60

Diabody Fab(A) Fab(B)

DBC

(mg/

mL-

gel)

KanCapTM L

Objective：Increasing productivity of antibody fragment purification

Platform process for antibody fragmentsPlatform process for antibody fragmentsusing cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resinsKeiichi Karasugiusing cellulose based affinity chromatography resinsKeiichi KarasugiKeiichi Karasugi1*

using cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resins1*,

using cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resins1*, Fuminori

using cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resinsFuminoriFuminori Konoike

using cellulose based affinity chromatography resinsKonoikeKonoike1,2

using cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resins1,21,2, Dai Murata

using cellulose based affinity chromatography resins, Dai Murata, Dai Murata1,2

using cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resins1,21,2,

using cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resins1,2, Masakatsu

using cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resinsMasakatsuMasakatsu Nishihachijo

using cellulose based affinity chromatography resinsNishihachijoNishihachijo1,2

using cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resins1,21,2,

using cellulose based affinity chromatography resinsusing cellulose based affinity chromatography resins1,2, KazunobuKazunobu MinakuchiMinakuchi 1,2Keiichi Karasugi

(Keiichi Karasugi((1Keiichi KarasugiKeiichi KarasugiKeiichi KarasugiKeiichi Karasugi((11KANEKAKeiichi KarasugiKeiichi Karasugi , Keiichi Karasugi

KANEKAKANEKA Corporation, Corporation, Corporation, 2, Dai Murata, Dai Murata, Dai Murata , NishihachijoNishihachijoNishihachijo

22Manufacturing Technology Association for Biologics)

Fabκ type

VL

CLCH1

Protein LProtein G

Fabλ type

CH1

VL

CL

Figure 1. Characteristics of affinity ligands for antibody fragments

PDB : 1PGXPDB : 2KAC

Wild type Protein G (Streptococcal protein G : SpG)

Binding site CH1(+CL)Target Fab, etc

Weakness Low affinity to almost all of CH1

Wild type Protein L (Peptostreptococcus magnus Protein L : PpL)

Binding site VL (only κ types)Target Fab, scFv, diabody, etc

Weakness Low affinity to some of VL×

2 3 41MkDa

66

45

30

20.1

(B) Yeast Sup.(A) E.coli lysate

Figure 7. The difference of binding spectra between wild type and improved Protein L

E.coli lysate

z

180

clone 1 …… clone 9

Figure 6. Schematic representation of the PpL/Fab complex and surface analysis (generated by Pymol).

Fab

PpL

PDB: 1HEZ ……

0 20 40 60

Abs2

80

mL

leakage in washing step

Recovered Fab

Wild type (Load 5 mg)

KanCap G (Load 14 mg)

Chromatogram of Fab to SpG resins

scFv Bis-scFvFab Fab2

Target : antibody fragments containing CH1 Target : antibody fragments containing VL (κ)

diabody

2 41 2 3 413

Fab(λ type)

Wild type Protein G and Protein L need to modify their affinity as ligands for capture chromatography resin