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High-Resolution U/HPLC Separation of Monoclonal Antibodies with the BIOshell™ IgG 1000 Å Diphenyl

High resolution U/HPLC separation of monoclonal antibodies (mAbs), mAb variants, and other large proteins with orthogonal selectivity to alkyl-bonded phases.

The BIOshell™ IgG 1000 Å Diphenyl column, based on Fused-Core® particles, is non-polar in nature.  Due to the aromatic nature of the phase, both methanol and acetonitrile organic solvents should be evaluated in the separation; as acetonitrile can act as a much stronger solvent when aromatic ligands are employed in the stationary phase in reversed-phase separations.  Typically, with mAb analysis workflows, column temperatures exceeding 60 °C are commonly employed; the BIOshell™ IgG 1000 Å Diphenyl column is compatible with such temperatures.

The BIOshell™ IgG 1000 Å Diphenyl column has shown unique advantages in resolving variants of antibody-drug conjugates (ADCs). This observation is most likely due to the interaction between the cytotoxic payload of the ADC with the diphenyl ligand.

 
BIOshell ™ IgG 1000 Å Diphenyl Specifications
Silica: Type B (High purity silica)
Particle Platform: Superficially porous particles (SPP)
Phase Chemistry: Diphenylmethyl
Particle Size: 2.7 µm
Pore Size: 1000 Å
Carbon Load: 1.0 %
Surface Area: 22 m²/g
pH Range: 2 - 9
Max Temperature at Low pH: 90 °C
Max Temperature at High pH: 40 °C
Endcapped: Yes
 

BIOshell™ IgG 1000 Å Diphenyl columns are perfectly suitable for the separation of monoclonal antibodies, antibody-drug conjugates, antibody fragments and large proteins with molecular weights ≤ 500 kDa with alternative selectivity to C4 and C18 column chemistries to ensure maximum resolution.

High resolution UHPLC separation of IgG2 mAbs

Figure 1. High resolution U/HPLC separation of IgG2 mAbs.

 
Chromatographic Conditions
Columns: BIOshell™ IgG 1000 Å C4, 15 cm x 2.1 mm, 2.7 µm
BIOshell™ IgG 1000 Å C18, 15 cm x 2.1 mm, 2.7 µm
BIOshell™ IgG 1000 Å Diphenyl, 15 cm x 2.1 mm, 2.7 µm
Mobile Phase: A: water/0.1% TFA, B: acetonitrile/0.1% TFA
Gradient: 32-40% B in 16 min
Flow Rate: 0.4 mL/min
Temperature: 80 °C
Detection: PDA at 280 nm
Injection Volume: 2 µL of 2 mg/mL trastuzumab in water/0.1% TFA
Sample: 2 mg/mL
 

BIOshell™ IgG 1000 Å Diphenyl is a high-speed, high-performance liquid chromatography column based on a superficially porous Fused-Core® particle design.  The Fused-Core® particle provides a thin porous shell of high-purity silica surrounding a solid silica core.  This particle design exhibits very high column efficiency due to the shallow diffusion paths.

Outstanding performance in U/HPLC. 2.7 µm SPP particle with 1000 Å pores. Ideal for the separation of large proteins.

2.7 µm SPP particle with 1000 Å pores

 

BIOshell™ IgG 1000 Å Diphenyl Guard Cartridges

BIOshell™ IgG 1000 Å Diphenyl Guard Cartridges

Finger-tight guard cartridge holder, Inlet
Provides easy replacement of guard cartrige.

Finger-tight guard cartridge holder, Outlet
Allows for replacement of cartridge without removing the guard holder from the flow path.

Titanium hybrid ferrule
Adds durability for multiple connections.

Auto-adjusting zero dead volume (ZDV) end fitting
Ensures optimum, ZDV connection to the analytical column.

Materials