Sepax Proteomix® HIC Butyl NP5 Column

Heterogeneity of therapeutic monoclonal antibody are required to be studied to ensure the product safety, efficacy. Hydrophobic Interaction Chromatography (HIC) is an orthogonal separation tool to SEC, IEX, RP to analyze the variants resulted from post-translational modification and product degradation. Protein oxidation is one of the major variants to be monitored and analyzed.

 

Sepax Proteomix® HIC Butyl NP5 can successfully be used to analyze oxidized mAb variants and monitor the mAb oxidation process without further sample preparation, as illustrated below with methionine oxidized Rituximab. Further characterization can confirm the modifications.


 

Better mAb separation was achieved using Proteomix® HIC Butyl NP5 columns for the separation of Rituximab as illustrated below. A 4.6 x 100 mm longer column can further improve the resolution. 

 

Product Information:

 

Proteomix HIC Butyl NP5 4.6 X 100 mm (P/N: 431NP5-4610)

Proteomix HIC Butyl NP5 4.6 X 35 mm (P/N: 431NP5-4603)

 

Download: Application Note HICM1006
Column: Proteomix® HIC Butyl NP5, 5 µm, 4.6x 100 mm (PN: 431NP5-4610) Mobile phase: A: 100 mM sodium phosphate buffer, 2 M ammonium sulfate, pH 7.0, B:100 mM sodium phosphate buffer, pH7.0; Flow rate:0.8 mL/min, Pressure: 170 bar, System: UHPLC, Detector: UV 214 nm, Column temperature: 25 C, Injection: 10 µg mAb sample, 20 µg oxidized mAb, MAb oxidation: 10mg/mL was diluted to 5 mg/mL with water and 70% t-BHP was added to a final 5% concentration, incubate in dark and take time point, dilute to 2.5 mg/mL in 500 mM ammonium sulfate, 25 mM phosphate buffer
RituximAb Separation on HIC Columns
Column: Proteomix® HIC Butyl NP5, 5 µm, 4.6 x 35 mm, 4.6x 100 mm (PN: 431NP5-4603, PN: 431NP5-4610); Vendor T Butyl NP 2.5 4.6 X35 mm; Mobile phase: A: 100 mM sodium phosphate buffer, 2 M ammonium sulfate, pH 7.0, B: 100 mM sodium phosphate buffer, pH 7.0; Flow rate: 0.8 mL/min, System: UHPLC, Detector: UV 214 nm, Column temperature: 25 C Injection: 10 µg mAb for 4603, 20 mg for 4610, 2.5 mg/mL rituximab in 500 mM ammonium sulfate, 25 mM phosphate buffer
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