![]() ![]() Mixed‐mode (MM) chromatography has shown to be a highly selective technique for the separation of proteins and commonly used MM adsorbents include cation hydrophobic Capto MMC™ 10, anion hydrophobic Capto Adhere™ 11, 12, 13, ceramic hydroxyapatite 11, 14, and hydrophobic charge induction MEP HyperCel™ 11, 13, 15, 16, 17, 18. However, to avoid additional protein engineering, expensive Protein L‐based adsorbents as well as laborious purification processes, an alternative purification approach is needed. If the scFv contains kappa light chain II, it is possible to use Protein L‐based affinity adsorbents only by grafting Protein L binding activity 9. In the former case, the purification is limited to fragments containing the V kappa light chain (I, III, or IV) domain. Current conventional methods for purification of scFv preparations from feedstocks include the use of Protein L‐based affinity adsorbents or alternatively affinity tags 6, 7, 8. Production of scFv is currently particularly challenging from a downstream point of view in terms of achieving high product recovery and purity. Its application ranges from therapeutic drugs in tumor therapy and diagnostic probes in imaging to affinity ligands in chromatographic processes 1, 2, 3, 4, 5. Single chain variable fragment (scFv) is a widely used format in both basic and applied clinical research. 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. ![]() The optimized conditions enabled binding of the scFv to Capto Adhere™ below its theoretical p I, while the majority of HCPs were in the flow‐through. Totally, 258 different HCPs were removed, corresponding to 84% of identified HCPs. Chromatography with Capto Adhere™ in binding‐mode 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. The DoE designs of the polishing step included both binding and flow‐through modes, the latter being the standard mode for HCP removal. Capture of scFv from human embryonic kidney 293 (HEK293) cell feedstocks was performed by hydrophobic charge induction chromatography (MEP HyperCel™), whereafter polishing was performed by anion hydrophobic MM chromatography (Capto Adhere™). In this work, a purification process for a scFv using mixed‐mode (MM) chromatography was developed by design of experiments (DoE) and proteomics for host cell protein (HCP) quantification. However, they can be challenging to purify unless using expensive Protein L‐based affinity adsorbents or affinity tags. Single‐chain variable fragments (scFv) are widely used in several fields. ![]()
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