New Ultra-High Capacity TOYOPEARL® Protein A Resin

Tosoh Bioscience GmbH

TOYOPEARL AF-rProtein A HC-650F

Ultra-High Capacity Protein A Resin


Antibody Biotherapeutics Increasing

Source: BIOPHARMA: Biopharmaceutical Products in the U.S. and European Markets (Rader,

RA; Published by BioPlan Associates, Inc.).

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Typical Antibody DSP Schemes

Capture Intermediate /Polishing

Protein A chromatography is the dominant capture step in mAb purification platforms.


Protein A DBC - Operational Costs

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• High capacity Protein A resin for mAb capturing

• Highest DBC of commercial Protein A resins.

• Binding capacity:

• 70 g/L DBC for IgG at 5 min residence time

• 50 g/L DBC for IgG at 2 min residence time

• Ideally suited for IgG titers up to 10 g/L and even higher

• High alkaline and chemical stability

• CIP over 200 times with 0.1 mol/L NaOH

• Easy cleaning with 0.1 mol/L to 0.5 mol/L NaOH, ethanol, urea, etc.

• Rigid TOYOPEARL base particle – easy packing

• Stable at room temperature at production site

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Specification

Particle size: 30 - 60 micron (> 80 %)

Ligand: recombinant Protein A (E. coli)

Ligand coupling: multi-point attachment

IgG capacity (static) > 68 g/L

(DBC; > 65g/L at 5 min residence time)

Bioburden:

• Bacterial count < 100 colonies/mL

• Endotoxin < 10 EU/mL

Foreign substances < 6 particles/100 mL

No animal source used

Shipping solvent* 20 % ethanol

Protein A ELISA kit available

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Recombinant Ligand Structure

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Comparison of Binding Capacities

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MabSelect SuReTM LX & MabSelect SuReTM are trademarks of GE Healthcare,

DBC values from brochure 28-9875-25 AA

Column: TOYOPEARL AF-rProtein A HC-650F (5 mm ID x 5 cm)

IgG solution: 1 g/L in 0.02 mol/L sodium phosphate (pH 7.4) + 0.15 mol/L NaCl

Residence time: 2, 3.5, 5 min Detection: UV @ 280 nm


DBC – Residence Time & IgG Load

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Resin: TOYOPEARL AF-rProtein A HC-650F

Column size: 5 mm ID ×5 cm

Mobile phase: 0.02 mol/L sodium phosphate, 0.15 mol/L NaCl, pH 7.4

Residence time: 2, 3.5, 5 min Detection: UV @ 280 nm

Sample: human IgG @ 1, 5, 10 g/L in mobile phase

DBC measured at 10 % breakthrough


Lot-to-Lot Variation of SBC

Static binding capacity for polyclonal hIgG of 4 production lots

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Purification of mAb from Harvest

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Product Purity

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Resin Rigidity

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Workflow

1. Sample loading with neutral pH buffer

2. Washing with neutral pH buffer

3. Elution of mAb with acidic pH (3.5-3.0) buffer

4. Washing with neutral pH buffer

5. Washing with 0.1 N NaOH

* After elution with acidic pH buffer, some aggregates and hydrophobic impurities

might still be adsorbed on the resin. Impurities are eluted with alkaline cleaning.

However, when acidic eluent changes directly to NaOH solution, those impurities may

be precipitated during NaOH washing and elution. In such cases, neutral buffer washing

prior to NaOH washing is recommended without precipitation of the impurities.


CIP Study with 0.1 N NaOH

Column size: 5 mm ID × 5 cm

Wash procedure:

A: 20 mmol/L Na2HPO4, 0.15 mol/L NaCl, pH 7.4 (10 CV washing buffer)

B: 0.1 mol/L citrate, pH 3.0 (5 CV elution buffer)

C: 20 mmol/L Na2HPO4, 0.15 mol/L NaCl, pH 7.4 (7 CV washing buffer)

D: 0.1 mol/L NaOH (3 CV – 15 min contact time)

E: 20 mmol/L Na2HPO4, 0.15 mol/L NaCl, pH 7.4 (5 CV washing buffer)

Capacity: DBC was determined at 10% breakthrough after every 20 cycles

>95 % of initial DBC remaining after 200 CIP cycles with 0.1 M NaOH


CIP Study with 0.5 N NaOH

80 % of initial DBC remaining after 40 CIP cycles with 0.5 M NaOH

Column size: 5 mm ID × 5 cm

Wash procedure:

A: 20 mmol/L Na2HPO4, 0.15 mol/L NaCl, pH 7.4 (10 CV washing buffer)

B: 0.1 mol/L citrate, pH 3.0 (5 CV elution buffer)

C: 20 mmol/L Na2HPO4, 0.15 mol/L NaCl, pH 7.4 (7 CV washing buffer)

D: 0.5 mol/L NaOH (3 CV – 15 min contact time)

E: 20 mmol/L Na2HPO4, 0.15 mol/L NaCl, pH 7.4 (5 CV washing buffer)

Capacity: DBC was determined at 10% breakthrough after every 5 cycles


Leakage of rProtein A Ligand


Application Data for Therapeutic mAb

First results obtained for a specific monoclonal

IgG indicate:

• Column packing in lab-scale (0.7 mL to 20 mL; Omnifit and XK

columns) as straight-forward and easy as for other

TOYOPEARL resins.

• Capacity is constant over a wide range of feed concentrations

(up to 15 mg/mL)

• DBC for short residence times (45 sec) remains close to 50

mg/mL

• Yields greater than 90 % could be obtained with pH 4.0 elution

buffer

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DBC at Various Loads & Velocities

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DB

C @

10%

BT

[m

g m

Ab

X/m

L]

Resin: TOYOPEARL AF-rProtein A HC-650F

Column size: 6.6 mm ID ×2 cm

Mobile phase: 100 mmol/L sodium phosphate pH 6.5

Detection: UV @ 280 nm

Sample: monoclonal antibody mAb X @ 1, 5, 10 g/L in mobile phase

Obtained for a monoclonal human IgG in our application lab



• Superior dynamic binding capacity for human IgGs

70 g/L at 5 min residence time

50 g/L at 2 min residence time

• Capacity increases with feedstock titers up to 10 g/L

• Alkaline resistance through engineered recombinant

ligand and multipoint attachment

• Minimized leaching through multi-point attachment

• High rigidity simplifies large column operation

• Attractive price

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New Ultra-High Capacity TOYOPEARL® Protein A Resin

Technology

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