Singleâchain variable fragments (scFv) are widely used in several fields. However, they can be challenging to purify unless using expensive Protein Lâbased affinity adsorbents or affinity tags. In this work, a purification process for a scFv using mixedâmode (MM) chromatography was developed by design of experiments (DoE) and proteomics for host cell protein (HCP) quantification. Capture of scFv from human embryonic kidney 293 (HEK293) cell feedstocks was performed by hydrophobic charge induction chromatography (MEP HyperCelâ˘), whereafter polishing was performed by anion hydrophobic MM chromatography (Capto Adhereâ˘). The DoE designs of the polishing step included both binding and flowâthrough modes, the latter being the standard mode for HCP removal. Chromatography with Capto Adhere⢠in bindingâmode with elution by linear salt gradient at pH 7.5 resulted in optimal yield, purity and HCP reduction factor of 98.9 > 98.5%, and 14, respectively. Totally, 258 different HCPs were removed, corresponding to 84% of identified HCPs. The optimized conditions enabled binding of the scFv to Capto Adhere⢠below its theoretical pI, while the majority of HCPs were in the flowâthrough. Surface property maps indicated the presence of hydrophobic patches in close proximity to negatively charged patches that could potentially play a role in this unique selectivity.