New in Bio-separation by Developing High Capacity Sub-2 ... · Antibodix NP1.7 1.7 10,000 2-12 80...

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


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


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


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


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


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


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


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


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)


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


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)


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


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


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


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


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

New in Bio-separation by Developing High Capacity Sub-2 ... · Antibodix NP1.7 1.7 10,000 2-12 80...

Documents

Transcript of New in Bio-separation by Developing High Capacity Sub-2 ... · Antibodix NP1.7 1.7 10,000 2-12 80...