Propac hic 10 column

Monospecific antibodies and κλ body distribution and integrity were assessed by electrophoresis, isoelectric focusing gel analysis (IEF), hydrophobic interaction high performance liquid chromatography (HIC-HPLC) and size exclusion high performance liquid chromatography SEC-HPLC. Purified IgGs were analyzed by electrophoresis in denaturing and reducing conditions. The Agilent 2100 Bioanalyzer was used with the Protein 80 kit as described by the manufacturer (Agilent Technologies, Santa Clara, CA, USA). The distribution of the different formats of IgG (monospecific lambda, kappa and bispecific antibody) was determined by isoelectric focusing (Cambrex pH 7–11 IsoGel agarose plates) and HIC-HPLC analysis using ProPac HIC-10 column (Dionex, Sunnyvale, CA, USA). A gradient of mobile phase A (0.01 M sodium phosphate dibasic buffer (Sigma-Aldrich, St Louis, MO, USA) + 1.5 M ammonium sulphate (Sigma-Aldrich, St Louis, MO, USA), pH 3.5) from 100 to 10% and a growing gradient of mobile phase B (0.01 M sodium phosphate dibasic buffer + 10% acetonitrile (Merck KGaA, Darmstadt, Germany), pH 3.5) from 0 to 90% were applied. A blank was performed with mobile phase A, pH 7.0. Aggregate and fragment levels were determined by SEC-HPLC with a Biosep-SEC-s3000 column (Phenomenex, Torrance, CA, USA) using a 200 mM sodium phosphate dibasic buffer, pH 7.0 mobile phase.