Ammag protein a magnetic beads

Candidates were expressed in ExpiCHO-S™ cells. The VH and VL variable regions of antibody candidates were subcloned into expression vectors containing constant regions of IgG1 isotype. The candidates’ heavy chain and light chain expression vectors were co-transfected into ExpiCHO-S™ cells. After 8 days of growth, the supernatant was harvested and passed over AmMag™ Protein A Magnetic Beads(Genscript Biotech, Jiangsu, CN)according to the manufacturer’s instruction. Yields of purified antibody candidates were determined based on their UV absorbance at 280 nm by a Nanodrop instrument (Thermo Fisher Scientific, PA, USA).

Sequences from the unique variable regions were synthesized with adaptors for cloning at GenScript (China). Cloning was performed using in-house vectors for expression. Plasmids were transfected into human embryonic kidney 293T cells (ATCC) using PEI (Sigma, #24885) in a 24-well plate and incubated at 37°C, 5% CO₂ for 3 days before collecting supernatant for subsequent test. Positive clones in supernatant test were transfected into HEK 293F (Gibco, USA, R79007) or ExpiCHO cells (Thermo Fisher Scientific, A29127) for small-scale recombinant antibody production. Antibody was purified using Protein A (AmMag Protein A Magnetic Beads, Genscript, L00695) affinity chromotography, and purity was measured by both SDS-PAGE (SurePAGE, Bis-Tris, 10 × 8, 4% to 12%, 12 wells, Genscript, M00653) and SEC-HPLC (GE Healthcare).

Antibodies approved by the regulatory for clinical use include BRII-196/BRII-198, S309, REGN10933/REGN10987, COV2-2196/COV2-2130 and CB6 were selected for evaluation in the current study. All antibodies except BRII-196/BRII-198 were evaluated using parental IgG antibodies without Fc modification. Apart from BRII-196/BRII-198 derived from our own laboratory, the rest antibodies were synthesized according to the sequences released in Protein Data Bank (PDB) (52 (link), 54 (link)–56 (link)). Antibodies were produced by co-transfection of the heavy and light chain expression vectors into 293F cells using polyethyleneimine (PEI) (Polysciences). After 96h, antibodies secreted into the cell supernatant were captured by AmMag Protein A Magnetic Beads (Genscript L00695) and eluted by solution buffer Glycine pH 3.0. All antibodies were further purified by gel-filtration chromatography with Superdex 200 High-Performance column (GE Healthcare). The final protein concentrations were determined by nanodrop 2000 Spectrophotometer (Thermo Scientific).

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.

BDG-Ab mutants were purified by the AmMagTM ProteinA Magnetic Beads (GenScript) kit after the plasmids were transfected into 293T cells. Antibody potency was verified by the indirect ELISA method. The ELISA plates were coated with 20 ng/well of BDG, and BDG-Ab mutants were diluted at concentrations of 1,000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, and 3.90625 ng/mL, respectively. The plates were added to the dilutions separately, incubated at 37°C for 1 h, washed and placed in goat anti-rabbit-HRP antibody for 30 min at 37°C, and then washed and developed with TMB substrate for 15 min at 37°C. We defined the BDG-Ab potency as the reciprocal of the BDG-Ab concentration at OD = 0.