Trastuzumab biosimilar (Anhui Anke)

We developed a method to produce a soluble form of a single‐chain fragment variable (scFv) targeting human epithelial growth factor receptor 2 (HER2) in Escherichia coli. By optimizing the orientations of the variable heavy (VH) and variable light (VL) domains and the His‐tag, we identified the HL‐His type antibody with the highest HER2‐binding activity. Purification of HL‐His yielded 40.7 mg from a 1 L culture, achieving >99% purity. The limit of detection was determined to be 2.9 ng, demonstrating high production yield, purity, and sensitivity. Moreover, we successfully labeled HER2+ cell lines with fluorescent dye‐conjugated scFv, resulting in a significantly higher observed signal‐to‐background ratio, compared to that of HER2− cell lines. This highlights the potential of these fluorescent scFvs as valuable probes for HER2+ breast cancer diagnostics. Notably, the process for the complete scFv production was streamlined and required only 4–5 days. Additionally, the product maintained its activity after freeze storage, allowing for large‐scale production and a wide range of practical applications.