Captureselect ch1 xl pre packed column

The heavy and light chain genes of the obtained antibody were synthesized as eBlocks (Integrated DNA Technologies), and then cloned into human IgG1 and human kappa or lambda light chain expression vectors using Gibson assembly according to a previously described method [78 (link)]. The plasmids were transiently co-transfected into HEK293T cells at a mass ratio of 2:1 (HC:LC) using Lipofectamine 2000 (Thermo Fisher Scientific). On day 3 post-transfection, supernatant containing the IgG was collected for binding experiment. The expression of IgG was confirmed by SDS-PAGE gel electrophoresis and Coomassie Blue R-250 staining. Selected IgGs were purified using a CaptureSelect CH1-XL Pre-packed Column (Thermo Fisher Scientific).

Fab heavy and light chains were cloned into phCMV3. Heavy chain Y58F or F58Y mutants were constructed using the QuikChange XL Mutagenesis kit (Stratagene) according to the manufacturer’s instructions. The plasmids were transiently co-transfected into Expi293F cells at a ratio of 2:1 (HC:LC) using ExpiFectamine™ 293 Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The supernatant was collected at 7 days post-transfection. The Fab was purified with a CaptureSelect™ CH1-XL Pre-packed Column (Thermo Fisher Scientific).

The heavy and light chains of Fab were cloned into phCMV3 vector. Light chain mutants were generated using the QuikChange XL Mutagenesis kit (Agilent) following the manufacturer’s instructions. The plasmids were co-transfected into Expi293F cells at a 2:1 (HC:LC) mass ratio using ExpiFectamine 293 Reagent (Thermo Fisher Scientific). At 7 days post-transfection, the supernatant was collected, and the Fab was purified using a CaptureSelect CH1-XL Pre-packed Column (Thermo Fisher Scientific).

The heavy and light chains of Fabs were cloned into phCMV3 vector. PCR-based mutagenesis was performed to generate the allelic mutations. The plasmids were transiently co-transfected into ExpiCHO cells at a ratio of 2:1 (heavy chain to light chain) using ExpiFectamine CHO Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The supernatant was collected at 7 days post-transfection. The Fab was purified with a CaptureSelect CH1-XL Prepacked Column (Thermo Fisher Scientific), followed by a buffer exchange to Dulbecco’s Phosphate Buffered Saline (PBS, pH 7.4).

The anti-BACE1 affinity-matured 1A11 antibody (1A11AM) was used to generate bispecific antibodies. The engineering and expression of BBB00574 (1A11AM-BBB00515) and BBB00578 (1A11AM-BBB00533) were performed as previously described [16 (link),25 (link)]. Briefly, the DNA sequences encoding for the heavy chain composed of BBB00515 or BBB00533 fused to an Fc with knobs-into-holes (KiH) and ablated effector function mutations (human IgG1, L234A, L235A, P329G, T350V, T366L, K392L, T394W), the 1A11AM heavy chain with KiH and ablated effector function mutations (human IgG1, L234A, L235A, P329G, T350V, L351Y, F405A, Y407V), and the 1A11AM light chain (human kappa) were synthesized by Twist Bioscience and cloned in their pTwist CMV BetaGlobin WPRE Neo vector (Twist Bioscience). Expressions were performed using the Mirus CHOgro^® High Yield Expression System (Mirus Bio) according to the manufacturer instructions. Briefly, ExpiCHO-S™ cells (Mirus Bio) were transfected with the nanobody-human Fc fusions and the 1A11AM heavy chain and light chain with a transfection ratio of 2:1:3 with TransIT-PRO Transfection Reagent (Mirus Bio) [25 (link)]. After an incubation time of 14 days, the antibodies were purified, first with AmMag™ Protein A Magnetic Beads (Genscript) and then over a CaptureSelect™ CH1-XL Pre-packed Column (ThermoFischer Scientific) according to the manufacturer’s instructions.

The CR3022 Fab heavy (GenBank: DQ168569.1) and light (GenBank: DQ168570.1) chains were cloned into phCMV3. The plasmids were transiently co-transfected into Expi293F cells at a ratio of 2:1 (HC:LC) using ExpiFectamine 293 Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The supernatant was collected at 7 days post-transfection. The Fab was purified with a CaptureSelect CH1-XL Pre-packed Column (Thermo Fisher Scientific) followed by size exclusion chromatography. For crystallization, a VSRRLP variant of the elbow region was used to reduce the conformational flexibility between the Fab constant and variable domains [25 (link)].