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).
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).
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