Tosoh Bioscience GmbH
TOYOPEARL AF-rProtein A HC-650F
Ultra-High Capacity Protein A Resin
Tosoh Bioscience GmbH
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.
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Protein A DBC - Operational Costs
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TOYOPEARL AF-rProtein A HC-650F
• 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
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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
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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.
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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
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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
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Leakage of rProtein A Ligand
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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
Tosoh Bioscience GmbH
TOYOPEARL AF-rProtein A HC-650F
• 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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