new ultra-high capacity toyopearl® protein a resin
DESCRIPTION
High capacity chromatography resin for the capture and purification of monoclonal antibodies Tosoh Bioscience, a provider of chromatographic solutions for the isolation and analysis of biomolecules, introduced a new Protein A chromatography resin designed for the purification of monoclonal antibodies (mAbs). It is well-suited for high capacity capturing of immunoglobulin out of high titer feedstock. TOYOPEARL AF-rProtein A HC-650F achieves 30% to 50% greater antibody adsorption than similar products. It exhibits dynamic binding capacities of greater than 70 g/L at residence times of 5 minutes and greater than 50 g/L at residence times of 2 minutes with feedstock titers from 1 g/L to more than 10 g/L. The recombinant ligand that is linked to the well proven methacrylic polymer backbone of TOYOPEARL media has been engineered to maintain capacity even after repeated exposure to alkaline solution. Its multipoint attachment to the TOYOPEARL matrix further enhances chemical and thermal stability. In practice this pays off for a low level of Protein A leaching and also for a high resistance to alkaline solutions applied in cleaning-in-place (CIP) procedures. Due to its rigid polymer matrix TOYOPEARL AF-rProtein A HC-650F also provides excellent pressure flow characteristics. Protein A resins constitute a substantial cost in state-of-the-art mAb purification processes. Factors such as operating cycles, capacity, and mAb titer can have an impact on total costs associated with mAb purification. The high capacity of the new TOYOPEARL AF-rProtein A HC-650F resin and its high alkaline resistance increase product throughput, reduce operating costs and increase manufacturing productivity.TRANSCRIPT
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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Tosoh Bioscience GmbH
Typical Antibody DSP Schemes
Capture Intermediate /Polishing
Protein A chromatography is the dominant capture step in mAb purification platforms.
Tosoh Bioscience GmbH
Protein A DBC - Operational Costs
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Tosoh Bioscience GmbH
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
Tosoh Bioscience GmbH
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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Tosoh Bioscience GmbH
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.
Tosoh Bioscience GmbH
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
Tosoh Bioscience GmbH
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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