Prosep a media

IgG1 Fc (pEXPR-IgG1 Fc) was fused to either SpyTag (AHIVMVDAYKPTK) or KTag (ATHIKFSKRD) with additional GY-dipeptide at the C-terminus by using standard molecular cloning techniques. Native IgG1 glycosylation at position N297 was eliminated by introducing a N297A mutation by Quick Change Side-Directed Mutagenesis. Plasmids coding for chimeric cetuximab (C225, Erbitux^®) and trastuzumab (Herceptin^®) were kindly provided by Merck KGaA (Darmstadt). Cetuximab and trastuzumab variants containing SpyTag at the C-terminus of either the heavy chains, the light chains, or at both chains were prepared by standard molecular cloning techniques. A (GSG)₂-linker was introduced between the C-terminus of the antibody and SpyTag. IgG1 Fc and full-length antibodies were transiently expressed from HEK293F cells using the Expi293 Expression System (Life Technologies). Supernatants containing secreted proteins were conditioned and applied to spin columns with PROSEP-A Media (Montage, Merck Millipore). Columns were washed with 1.5 M Glycine/NaOH, 3 M NaCl, pH 9.0 and proteins eluted with 0.2 M Glycine/HCl pH 2.5 into 1 M Tris/HCl pH 9.0. Eluted proteins were dialyzed in 1 × DPBS (Life Technologies) using Amicon Ultra-15 (Merck Millipore, NMWL 10000 Da) and stored at 4 °C.

Isolated pYD plasmids of selected clones were used for subcloning of VH/VL regions into pTT5 vectors using the Expresso CMV-based system (Lucigen cloning) to allow full-length IgG expression in mammalian cells as described elsewhere [23 ]. In brief, cloned constructs were expressed in Expi293 cells after Expifectamin-mediated transient transfection according to the manufacturer´s instructions (Life Technologies). After 5 days, supernatants were harvested, and antibodies were purified via Antibody Purification Kit and Spin Columns with Prosep-A Media according to the manufacturers’ instructions (Merck KGaA).

VH regions as well as VL IGKV3-15*01 were cloned into pTT5 plasmids, which allow their expression as full-length IgG molecules in cell culture. Expi293 cells were transiently transfected with expression vectors following the instructions of the manufacturer (Thermo Fisher Scientific). Five days post transfection, antibody containing supernatants were harvested by centrifugation and purified by Antibody Purification Kit and Spin Columns with Prosep-A Media (Merck KGaA). After buffer exchange to PBS using Amicon Ultra-4 Centrifugal Filters (EMD Millipore) full length IgGs were analyzed by SDS-PAGE.
In addition aggregate formation was analyzed by analytical size exclusion chromatography. For this, a TSKgel SuperSW3000 column (4.6 × 300 mm, Tosoh Bioscience LLC) and an Agilent HPLC system was used. Differential scanning fluorometry on Prometheus NT.48 (Nanotemper Technologies) was applied to determine thermal stabilities of library candidates.