Bare fused silica capillary

Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is used for separation of denatured protein size variants under non-reduced or reduced conditions. For non-reduced CE-SDS (nrCE-SDS), drug product samples of ABP 501 and adalimumab RPs were denatured with sodium dodecyl sulfate (Beckman Coulter, Jersey City, NJ, USA) at 60 °C for 5 min. For reduced CE-SDS (rCE-SDS), β-mercaptoethanol (Thermo Scientific) was added to the protein denaturation step to reduce the disulfide bonds. After denaturation, both non-reduced and reduced samples were injected onto a bare, fused silica capillary (Beckman Coulter, Jersey City, NJ, USA) and separated based on hydrodynamic size resulting from an applied electric field in which migration of smaller-sized proteins is inversely related to overall size. Analytes were monitored by UV absorbance at 220 nm, and purity evaluated by determining the peak area of each species as a percentage of the total peak area.

High molecular weights were measured through the size exclusion high performance liquid chromatography (SE-HPLC). A sample was injected onto a TSK-GEL G3000SW_XL analytical column, which was connected to a Waters HPLC system; monitoring was done by ultraviolet (UV) detection (λ = 280 nm). A mobile phase consisting of 100 mM sodium phosphate and 200 mM sodium chloride, pH 6.8, was used. The flow rate was 0.5 mL/min, and monomers and impurities were detected at a UV wavelength of 280 nm. Data were acquired and processed by Empower™ software.
Purity and impurity results for %IgG and %2H1L were measured through capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). CE-SDS analysis was conducted with a high-performance capillary electrophoresis system (PA 800 plus Pharmaceutical Analysis System; Beckman Coulter). The sample was electrokinetically introduced onto a capillary (Beckman Coulter, bare fused-silica capillary, 50 µm/30.2 cm) by applying voltage at − 5 kV for 20 s and was separated in the capillary cartridge. Electrophoresis was performed at a constant voltage and monitored by UV detection (λ = 220 nm) through the capillary window and aperture. Data were acquired and processed by 32 Karat or Empower software with integration capabilities.