Pxc 17

Full-length human TPO ORF with a C-terminal 6× His tag, followed by a single stop codon sequence, was synthesized and cloned into pXC17.4 (Lonza Inc., Basel, Switzerland) with HindIII/EcoRI sites, expressed, and purified using the GS Xceed™ Gene Expression System (Lonza). The TPO ORF with a C-terminal 6× His tag in the pXC17.4 vector was transfected into CHOK1SV GS-KO cells by electroporation. For protein purification, TPO-expressing cells were cultured in CD-CHO medium (Thermo Fisher Scientific) for 5 days. The supernatant was collected and loaded onto a HisTrap excel column (Cytiva) equilibrated in 20 mM phosphate (pH 7.4) and 300 mM NaCl buffer. The bound protein was eluted with a phosphate buffer containing 300 mM imidazole. Fractions containing rhTPO-His, as verified by SDS-PAGE, were pooled and loaded onto a 10 mL HiLoad 26/600 Superdex 200 pg column (Cytiva) equilibrated in 2 × PBS. Fractions containing rhTPO-His were added to glycerol at a final concentration of 50% (v/v) and stored at −80 °C until use.

Based on publicly available sequence information on aducanumab [36 ] and gantenerumab [37 ], analogues were recombinantly produced. Gantenerumab was produced transiently in HEK293 cells using the Absolute Antibody HEXpress™ antibody expression platform and proprietary vectors (Absolute Antibody, Oxford, UK). Purification was done by affinity chromatography and size exclusion chromatography (SEC). The final buffer was PBS, pH 7.2 (Gibco, Cat. No. 20012–019). Aducanumab was produced transiently in CHOK1SV GS-KO cells using the single gene GS expression vectors pXC-184 and pXC-17.4 (Lonza Biologics, Cambridge, UK). Purification was done by protein A affinity chromatography and SEC and the final buffer was PBS, pH 7.2. The purity was estimated to be above 98% by SEC and by SDS-PAGE under denaturing conditions. Lecanemab was provided by Eisai Co., Ltd.