2.4.1. Nucleic acid removal by IEC

In IEC, Hitrap Q fast flow (FF) column (GE Healthcare, USA), was employed for HFn, HFn‐PAS and HFn‐PAS‐RGDK. pH 7, 8 and 9 were examined. Equilibration buffers and elution buffers at pH 7 and 8 were 20 mM PB without and with 1 M NaCl. 20 mM Tris‐HCl without and with 1 M NaCl buffers were pH 9 equilibration and elution buffer. Supernatants obtained after heat‐acid precipitation underwent Hitrap G25 desalting chromatography (GE Healthcare, USA) into corresponding equilibration buffer before being loaded onto Q FF column. Protein loading amount in each chromatography run was 10 mg. After sample loading and flow through (FT) peak, the column was eluted from 0 to 1 M NaCl linearly within five column volume (CV). Absorbance at 280 and 260 nm were recorded. Flow rate was 1 mL/min. Collected peaks underwent Bradford assay, nucleic acid concentration determination by Quan‐iT 1× dsDNA HS assay kit (Invitrogen, Thermo Fisher Scientific, USA) and 12% reducing SDS‐PAGE analysis. Purity was obtained from densitometry scan and recovery yield (step) was calculated as in Equation 1. In chromatography step, “the amount of target protein at purification stage n” means the amount of target protein in peak. “The amount of target protein at purification stage n − 1” means the target protein amount in loading sample. Nucleic acid removal was calculated as in Equation 2.

HIC screening was conducted for HFn and HFn‐PAS‐RGDK. The optimal condition was applied to the HFn‐PAS. Prior to HIC, the supernatants after heat‐acid precipitation were diluted five times with 100 mM PB, 1.2 M AS, incubated at 4°C for 0.5 h and then centrifuged at 12,000 rpm, 4°C, 15 min. During HIC, absorbance at 260 and 280 nm was recorded. Hitrap Octyl FF or Hitrap Butyl FF column (GE Healthcare, USA) was equilibrated with 100 mM PB, 1.0 M AS, pH 6.5 before sample loading. After FT peak finished, column was eluted with 0 to 100% elution buffer (20 mM PB, pH 6.5), one CV gradient. Flow rate was 1 mL/min. HIC peaks were collected and protein concentrations were determined using Bradford assay. Protein purities in peaks were analyzed using densitometry scan of SDS‐PAGE gel. Recovery yield (step) and nucleic acid removal were calculated using Equations 1 and 2. Recovery yield (overall), the recovery yield after heat‐acid precipitation and chromatography, was calculated as in Equation 3.