Protein l affinity chromatography

The PRLR-DbsAb was generated by BAPTS system, which was previously described in detail [27 (link),28 (link)]. CD3 antibody-fusion protein (Fragment A) was expressed in a stably transfected cell line CHO while PRLR antibody-fusion protein (Fragment B) was expressed in 293E cells using transient gene expression (TGE) technology. Both Fragment A and Fragment B were purified by Protein L affinity chromatography (GE Healthcare). The monoclonal antibodies used in this study including PRLR antibody (PRLR mAb), CD3 antibody (CD3 mAb) and PD-1 antibody (PD-1 mAb) were expressed by 293E cells and purified by Protein A affinity chromatography (GE Healthcare). The affinity of CD3 mAb and PD-1 mAb to their antigens was previously confirmed [27 (link),29 (link)]. The amino acid sequence used for the PRLR mAb is identical to that of the PRLR monoclonal antibody LFA102 [5 (link)]. All recombinant antibodies were dialyzed overnight to phosphate buffer saline (PBS) and sterilized by filtration using a 0.22 μm filter.

A simplified expression and purification schematic was developed in Fig. 3a. Briefly, Antibody fragments were purified by protein L affinity chromatography (GE Healthcare) from filtered cell culture supernatants referring to standard protocols. The supernatants were loaded and equilibrated with binding buffer (20 mM sodium phosphate, 500 mM NaCl, pH 7.4). Elution of antibodies was achieved at pH 2.8 (0.1 M sodium citrate) with a 20 CV linear gradient from 0% to 100%, followed by immediate neutralization of the sample. The samples were dialyzed overnight to splicing buffer (10 mM Tris-HCl, 0.5 M NaCl, pH 7.9), followed by splicing reaction of the antibody fragments catalyzed by the split intein. Imidazole were added into the splicing product at 30 mM final concentration, then the reaction mixture went into the Ni Sepharose affinity chromatography (GE Healthcare) and flowthrough containing the BsAb was collected. The flowthrough collect was further purified through protein A affinity chromatography (GE Healthcare). Antibody was eluted at pH 2.8 (0.1 M sodium citrate) with an elution of 20 CV linear gradient from 0% to 100%. The eluates were neutralized immediately, dialyzed overnight to PBS, and filter-sterilized over 0.22 μM dead-end filters. The protein concentration of the samples was measured by BCA protein assay kit.