Gene cloning → Expression vector construction → Host cell transfection → Selection of stable clones → Scale-up culture → Expression & Harvest → Purification → Quality control.

CHO cells: Glycosylation profile similar to humans, suitable for full-length antibodies, with high expression yield.

HEK293 cells: High transfection efficiency, suitable for rapid small-scale expression.

E. coli: Low cost, short cycle, suitable for antibody fragments (e.g., scFv, Fab).

SDS-PAGE, CE-SDS (Capillary Electrophoresis), SEC-HPLC (for aggregate detection).

Use chemically defined media to avoid animal-derived components and ensure batch-to-batch consistency.

Transient expression: Fast (1-2 weeks), suitable for small-scale screening.

Stable expression: Requires 2-3 months for clone screening, suitable for large-scale production.

Chemical conjugation (e.g., NHS ester labeling), enzyme labeling (e.g., HRP/biotin conjugation), genetic engineering labeling (e.g., AviTag biotinylation).

Consider instrument excitation/emission wavelengths (e.g., FITC-488 nm, PE-561 nm), fluorescence intensity, photostability, and cost.

Lysine (random conjugation), cysteine (site-specific conjugation), engineered sites (e.g., non-natural amino acids).

By optimizing reaction stoichiometry and purification steps (e.g., hydrophobic interaction chromatography) to separate DAR variants.

MMAE, DM1, calicheamicin, etc., conjugated via cleavable or non-cleavable linkers.

Introduce Fc mutations (e.g., YTE to enhance FcRn binding), PEGylation, or fuse with albumin-binding peptides.

Yes, for example, switching IgG1 to IgG4 can reduce ADCC/CDC effects, making it suitable for blocking applications.

Via scFv tandem fusions (e.g., BiTE), Knob-into-Hole technology, common light chain designs, etc. Formats include DVD-Ig, Tandem scFv, and others.

Introducing point mutations (e.g., L234A/L235A) to eliminate Fc binding to immune cell receptors, thereby reducing side effects.