Sepax Technologies www.sepax-tech.com
New in Bio-separation by Developing High Capacity Sub-2 Micron Non-Porous Ion-Exchange Resins
Ke Yang, Helen Gu*, Ryan Pringle and Xueying Huang
Sepax Technologies, Inc.5 Innovation Way, Newark, Delaware 19711
Sepax Technologies www.sepax-tech.com
AntibodixTM WCX Packingsfor Monoclonal Antibody Separation
AntibodixTM WCX resins are recently developed non-porous PS/DVB packing materials for ion-exchange chromatography, specifically for monoclonal antibody separation. Their particle sizes are controlled at 1.7, 3, 5 and 10 um. Those PS/DVB particles are surface modified with a proprietary technology that has negligible non-specific interactions with monoclonal antibodies. The smaller particle size offered higher resolving power for the variants of monoclonal antibodies.
Applications: Monoclonal antibodies (MAb) PEGylated Mab Peptide modified and other MAb derivatives
Sepax Technologies www.sepax-tech.com
Antibodix Phase – Chemical Structure
Structure FunctionHighly Cross-linked Non-porous PS/DVB
Rigid support to tolerate high pressure Minimize the diffusion
Hydrophilic Coating Eliminate non-specific interactionIon-exchange Layer Increased capacity
Controllable capacity Tunable hydrophobicity/hydrophilicity
Hydrophilic Layer
Ion-Exchange Group
ModifiedPS/DVB
Sepax Technologies www.sepax-tech.com
Sub-2m Non-Porous PS/DVB Beads
Narrow-dispersed Ion-exchange Particles
3.0 µm BeadsAntibodix NP3
5.0 m PS/DVB Non-Porous Particles
Sepax Technologies www.sepax-tech.com
AntibodixTM NP10 Columns - Lot-to-Lot ReproducibilityColumns: Antibodix-NP10 (10 m, 4.6x250 mm)Mobile phase: A, 10 mM phosphate, pH 6.0; B, A + 1.0 M NaClGradient: 0-100%B in 42 min; Flow rate: 1.0 mL/minSample: 1) Cytochrome C, 2) Lysozyme, 3) Ribonuclese A Injection: 5 L (1 mg/mL for each protein)Temperature: 25 oC; Detection: UV 214 nm
min0 2 4 6 8 10 12 14 16 18
12
3
Sepax Technologies www.sepax-tech.com
AntibodixTM NP10 Columns - Lot-to-Lot ReproducibilityColumn: Antibodix NP10 (10m, 4.6x250mm)Mobile phase: A, 10 mM Phosphate, pH 7.5; B, A + 100 mM NaClGradient: 15-55% B (30 min); Flow rate: 0.8 mL/minDetection: UV 214 nm; Temperature: AmbientSample: Monoclonal antibody (5 mg/mL); Injection: 5 µL
Min0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Lot 17122
Lot 17136
Lot 17136
Lot 17151
Lot 25102
Sepax Technologies www.sepax-tech.com
Comparison of WCX Columns for Antibody SeparationMobile phase: A, 10 mM Phosphate, pH 7.5; B, A + 100 mM NaClGradient: 15-55% B (30 min)Flow rate: 0.8 mL/min; Detection: UV 214 nmSample: Monoclonal antibody (5 mg/mL); Injection: 5 µLTemperature: Ambient
Min0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0
Vendor A WCX-10 (10µ, 4.0x250mm, Non-porous)Backpressure: 1490 psi
Antibodix NP10 (10µ, 4.6x250mm, Non-porous)Backpressure: 840 psi
Sepax Technologies www.sepax-tech.com
Antibodix NP1.7 Column for MAb SeparationColumn: Antibodix NP1.7 (1.7µm, 4.6x50mm)Mobile phase: A, 10 mM Phosphate, pH 7.5; B, A + 100 mM NaClGradient: 15-100% B (60 min)Flow rate: 0.3 mL/min; Detection: UV 214 nmSample: Monoclonal antibody (5 mg/mL); Injection: 5 µLTemperature: Ambient
AU
0.000.100.200.300.400.500.600.700.800.901.001.101.201.301.40
Min0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
N=15682
Sepax Technologies www.sepax-tech.com
Particle Size Impact on Antibody Separation
Min0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Antibodix NP3 (3 µm, 4.6x50 mm)Buffer: A) 10 mM PBS, pH 7.5
B) A + 100 mM NaClGradient: 15-55% (30 min)Flow rate: 0.5 mL/min (2180 psi)Injection: 5 µLSample: a monoclonal antibody
Antibodix NP1.7 (1.7 µm, 4.6x50 mm)Buffer: A) 10 mM PBS, pH 7.5
B) A + 100 mM NaClGradient: 15-100% (60 min)Flow rate: 0.3 mL/min (3425 psi)Injection: 5 µL (a monoclonal antibody)
(N=7260)
(N=15680)
Sepax Technologies www.sepax-tech.com
Column Dimension Impact on Antibody SeparationBuffer: A) 10 mM PBS, pH 7.5; B) A + 100 mM NaClGradient: 15-95% (60 min)Flow rate: 1.0 mL/minDetection: UV 214 nm; Temperature: Ambient Sample: a monoclonal antibody; Injection: 10 µL
Min0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Antibodix NP3(3 µm, 7.8x50 mm)
Antibodix NP3(3 µm, 7.8x100 mm)
N=6476
N=8659
Sepax Technologies www.sepax-tech.com
Mobile phase: A, 10 mM Phosphate, pH 7.5; B, A + 100 mM NaClGradient: 15-55% B (30 min); Flow rate: 0.8 mL/minDetection: UV 214 nm; Temperature: AmbientSample: Monoclonal antibody (5 mg/mL); Injection: 5 µL
Comparison of 5 & 10 µm Antibodix for MAb Separation
Min0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Antibodix NP10 (10m, 4.6x250mm)
Antibodix NP5 (5m, 4.6x250mm)
Sepax Technologies www.sepax-tech.com
Separation of a Monoclonal Antibody Sample
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
Min0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
N=12,170
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
Min0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
Min0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
N=12,170
Column: Antibodix NP5 (5µm, 4.6x250mm)Buffer: A, 10 mM Phosphate, pH 7.5; B, A + 100 mM NaClGradient: 15-55% B (30 min); Flow rate: 0.8 mL/minDetection: UV 214 nmSample: Monoclonal antibody (2.5 mg/mL); Injection: 10 µL
Sepax Technologies www.sepax-tech.com
Min0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
A
B
C
D
E
Separation of MAb-X22 with optimizing conditionsColumns: Antibodix-NP10 (10 m, 4.6x250 mm)Mobile phase: A, 10 mM phosphate, pH 7.5; B, A + 0.1M NaClFlow rate: 0.8 mL/minSample: MAb-X22; Injection: 10 L (1.5 mg/mL)Temperature: 25 oC; Detection: UV 214 nm
Gradient: A) 15-75%B in 30 minB) 15-65%B in 30 minC) 15-55%B in 30 minD) 15-47.5%B in 30 minE) 15-40%B in 30 min and 40%B after 30min
Sepax Technologies www.sepax-tech.com
Min0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
A
B
C
The Impact of initial salt on the separation of MAb-X22Columns: Antibodix-NP10 (10 m, 4.6x250 mm)Mobile phase: A, Phosphate buffer, pH 7.5; B, A + 0.1M NaClGradient: 15-65%B in 60 min; Flow rate: 0.8 mL/minSample: MAb-X22; Injection: 10 L (1.5 mg/mL)Temperature: 25 oC; Detection: UV 214 nm
Initial salt: A/B/C=20/10/5mM phosphate
Sepax Technologies www.sepax-tech.com
Min0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0
A
B
The impact of mobile phase pH on MAb-X22 separationColumns: Antibodix-NP10 (10 m, 4.6x250 mm)Mobile phase: A, 10 mM phosphate; B, A + 0.1M NaClGradient: 15-65%B in 60 min; Flow rate: 0.8 mL/minSample: MAb-X22; Injection: 10 L (1.5 mg/mL)Temperature: 25 oC; Detection: UV 214 nm
pH: A) pH 7.5B) pH 7.0
Sepax Technologies www.sepax-tech.com
Antibodix WCX-NP Columns - Summary
Products Particle Size(µm)
Pressure Limit(psi)
pH range Temperature limit (oC)
Antibodix NP1.7 1.7 10,000 2-12 80Antibodix NP3 3 8,000 2-12 80Antibodix NP5 5 6,000 2-12 80
Antibodix NP10 10 4,000 2-12 80
Based on uniform, non-porous PS/DVB based ion-exchange particles, Antibodix columns have high resolution and high efficiency with reasonable high capacity.
Particle size selection: 1.7, 3, 5 and 10 µm. Smaller particle size, higher separation efficiency
The chemistry are specifically designed for separation of monoclonal antibodies
The resolution and selectivity of monoclonal antibodies depend on the separation conditions
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