Eluminol

HPLC-SEC was used to determine antibody concentration and purity, using a TSK_gel G3000SW_XL column (7.8 mm i.d. x 30 cm; Tosoh Bioscience). 100 µL of sample was injected for analysis, utilising a flow rate of 0.6 mL/min and a mobile phase which consisted of 0.2 M L-arginine, 0.05 M MES, 5 mM EDTA, 0.05% sodium azide (w/w), pH 6.5. The resultant concentrations were obtained by comparing the area under the peaks obtained at UV absorbance 280 nm with that of a calibration curve obtained using standard samples. The relative amount of HMW and LMW species was calculated based on the area of elution peaks before and after the monomeric peak, respectively. The area of the respective species obtained from HPLC-SEC was multiplied with the respective volume obtained from the AKTA system in order to perform mass balance analysis. Non-reducing SDS-PAGE gels (4–15% Criterion™ TGX Stain-Free™ Protein Gel, Bio-rad) were used according to manufacturer’s instructions, as a complementary approach to investigate the purity of the samples. Staining was performed with eLuminol™ (GeneCopoeia), with a total protein amount of 0.3 µg loaded per lane, as determined using Bradford assay (Thermo Fisher Scientific).

BsAb concentration and purity analysis were performed by size exclusion chromatography-HPLC (HPLC-SEC), using a TSKgel G3000SWXL column (7.8 mm i.d. × 30 cm; Tosoh Bioscience), flow rate: 0.6 mL/min; mobile phase: 200 mM L-arginine, 50 mM MES, 5 mM EDTA, 0.05% sodium azide (w/w), pH 6.5, UV 280 nm. The determination of bsAb concentrations was achieved by measuring the area under the main peak and referencing a calibration curve generated with known concentrations of antibody standards (Chen et al. 2022b (link)). In the HCCF, the main peak may potentially include other impurities, such as specific HCPs. To ensure accurate bsAb monomeric quantification, we applied a correction method by subtracting the integration of the main peak from the HCCF from that of the flowthrough obtained from PrismA chromatography. This effectively eliminated background interference from the calculation of bsAb concentration. The quantities of HMW and LMW components were determined by analysing the peak areas eluting before and after the main peak, respectively.
SDS-PAGE gels (Bio-Rad, non-reducing Gel: 4–15% Criterion TGX Stain-Free Protein Gel, reducing Gel: Any KD Criterion TGX Stain-Free Protein Gel) were used to visualize target proteins and impurities. 0.2 µg of monomeric bsAb was loaded per lane. The gels were stained using eLuminol™ (GeneCopoeia).