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Fcɣ Receptor immobilized Resin for Chromatogaphy

Judith Vajda, Toru Tanaka, Egbert Müller Tosoh Bioscience

BILS conference 2016, Berlin

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Outline

•  R&D of innovative chromatography resins

•  Fcγ Receptors

•  Fcγ-RIII immobilized on TSKgel resin for an analytical column

•  Application examples

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1. Protein engineering / Directed evolution techniques - Micro-DSC - CD spectra analyzer - BIAcore - Static light scattering analyzer (Optim2, AVACTA) 2. Directional controlled immobilization techniques - Unique immobilization system 3. Protein production facility - Large scale microbial fermentation system (over 1000L) - Large scale purification system

R & D of new Separation Media

TOYOPEARL or TSKgel

+

Proteinous ligand

=

Innovative separation media

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Fcγ-RI Fcγ-RII Fcγ-RIII Molecular Weight

30 kDa 20 kDa 20 kDa

Affinity for IgG High Low Low

Effect following binding to antibody

Induces phagocytosis

Regulation of Fcγ-R functionalities

Induces ADCC

Subclasses of Fcγ receptors

•  Important role in the immune response •  Unique recognition sites (Hinge region of Fc portion)

Fcγ-Receptors: Form and Functions

promising affinity chromatography ligand candidate

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•  Quality control of therapeutic antibodies is still very difficult.

Background and Target Application of Fcγ-R Resins

Lot to lot variation of Rituxan®/MabThera®

Source: Martin Schiestl et al. (Sandoz Biopharmaceuticals), Nature Biotechnology 29, 310-312 (2011)

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Quality attributes Influence Uncertainty Risk score Risk level

Aggregation 12 5 60 High

C-terminal lysine 2 2 4 Very Low

Deamidation 2 2 4 Very Low

Galactose (N-glycans) 16 3 48 High

Fucose (N-glycans) 20 3 60 High

Sialic acid (N-glycans) 12 5 60 High

High mannose (N-glycans) 16 5 80 Very High

Aglycosylation (N-glycans) 16 5 80 Very High

Oxidation 4 3 12 Low

DNA (impurity) 2 3 6 Very Low

MTX (impurity) 16 1 16 Low

HCP (impurity) 12 3 36 Middle

Protein Ａ (impurity) 16 1 16 Low

Source: N. Kawasaki et al., Pharm Tech Japan 28(12), 107, 2012.

Quality Attributes and Risk Level in MAb Production

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The binding affinity between Fcγ-RIII and a mAb is very important to destroy a cancer cell. The N-glycan structure of an antibody affects the affinity to the Fc receptor.

Mechanism of therapeutic Ab ADCC activity

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0

20

40

60

80

100

120

0 50 100 150 200

Sta

bilit

y (R

em

ainin

g ac

tivi

ty, %)

Incubation time (hours)

•  Wild-type Fcγ-RIII is unstable under acidic conditions

! Modification of Fcγ-RIII by directed evolution techniques

Ligand and resin development

•  FcR-immobilized resin/column using TSKgel resin packed into stainless steel hardware

Stability of wild-type (black) and modified FcR (red) at pH 3.0, 25 °C

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4 mAbs were obtained to test the FcR-column separation.

0 10 20

AB

S 28

0nm

Retention time (min) 0 10 20

AB

S 28

0nm

Retention time (min) 0 10 20

AB

S 28

0nm

Retention time (min) 0 10 20

AB

S 28

0nm

Retention time (min)

SEC analyses of mAbs on TSKgel G3000SWxl confirm purity. Low aggregate content.

Mabs for Evaluation

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0 20 40

AB

S 28

0nm

Retention time (min) 0 20 40

AB

S 28

0nm

Retention time (min)

0 20 40

AB

S 28

0nm

Retention time (min) 0 20 40

AB

S 28

0nm

Retention time (min)

FcR-column analyses of mAbs on FcR-column (proto-type)

•  4 mAbs were separated into three or four peaks •  All antibodies have unique separation profiles

Buffer A: 20 mM Na Acetate pH 5.0, 50 mM NaCl Buffer B: 10 mM Gly-HCl pH 3.0 Gradient: B 0% (0-2 min), B 0-100% (2-40 min), B 100% (40-50 min)

Fcγ-RIII Column Evaluation

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0 20 40

AB

S 28

0nm

Retention time (min) 0 20 40

AB

S 28

0nm

Retention time (min) 0 20 40

AB

S 28

0nm

Time (min) 0 20 40

AB

S 28

0nm

Time (min)

Rituxan® mAb A mAb B mAb C

FcR-column analyses of mAbs on FcR-column (proto-type)

Buffer A: 20 mM Na Acetate pH 5.0, 200 mM NaCl Buffer B: 10 mM Gly-HCl pH 3.0 Gradient: B 0% (0-2 min), B 0-100% (2-40 min), B 100% (40-50 min)

Fcγ-RIII Column Evaluation at optimized Conditions

•  Resolution improved by method optimization

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Buffer system Buffer A, 20 mM Sodium Acetate buffer pH 5.0, 50 mM NaCl Buffer B, 10 mM Gly-HCl buffer pH 3.0

Reproducibility

•  Separation profiles indicate well stability of Fcγ-RIII column

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-50

0

50

100

150

200

250

300

350

400

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

AB

S280

nm

Time (min)

Fr 1 Fr 2 Fr  3

20 25 30 35 40 45

AB

S280

nm

Time (min)

Fr 1

Fr 2 Fr 3

Broken line: Rituxan®

Preparative Rituxan® separation Component analysis of fractions

Preparative FcR Chromatography

•  Preparative FcR chromatography and component analysis

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ADCC activities of each fraction. Reaction time 6 hours; mAb conc. 4.1 ng/mL; Assay, ADCC reporter bioassay

(Promega G7015)

Retentive component

ADCC Activities of separated Fractions

0

50

100

150

200

250

Rituxan Fraction 1

Fraction 2

Fraction 3

AD

CC

act

ivity

(E

mis

sion

inte

nsity

, 10^

3)

®

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G0F

G1F

G1F

G2F

Rituxan® First Last Fraction

unknown

(%)

N-glycan Structure Analysis

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Lot-to-Lot deviations of antibodies can be examined by a comparison of peak areas.

•  Comparison of two Rituxan® Lots

Application Example: Quality Control

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Ligand Modified recombinant Fcγ-RIII (produced in E.coli, non-glycosylated) Binds Fc-containing Ig proteins, except IgG2

Base matrix Non-porous resin, 10 µm

Column size φ4.6 x 75 mm, 1.25 mL

Sample mass 5 mg of IgG (recommended)

Flow rate 0.3 mL/min (recommended)

Temperature 25 °C (recommended)

pH stability pH3 – 8

Recommended buffer system

A 20 mM Acetate pH 5.0, 50 mM NaCl B 10 mM Gly-HCl pH 3.0

Characteristics of Fcγ-RIII proto-type columns

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Conclusions

•  The Fcγ-RIII proto-type column can separate mAbs according to their glycan structure and ADCC activity

•  Non-glycosylated ligand produced in E.coli

•  Ligand stability at acidic conditions was achieved by directed evolution techniques