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)

Rituximab Oxidation Analysis on Proteomix® HIC Butyl

RituximAb Separation on HIC Columns

Sepax vs. Competitor Brand and the Impact of Column Length

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