The smart biopolymer elastin-like polypeptide has been successfully employed previously for the purification of mAb and non-mAb biologics using affinity precipitation. However, for each target, this involves recombinantly cloning and expressing a fusion of ELP and the respective affinity ligand. In this work, we have examined the utility of streptavidin-ELP fusions as a platform for affinity precipitation. While streptavidin usually expresses as inclusion bodies that are relatively difficult to purify, fusions of streptavidin with other proteins can enable soluble cytoplasmic expression and therefore easier purification. A tetrameric streptavidin-ELP (SELP) construct was cloned, expressed and purified from the cytoplasm of BL21(DE3) <i>E. coli</i>, attaining yields of &gt; 10 mg/L of culture. Biotinylated CaptureSelect™ affinity ligands developed by ThermoFisher, were non-covalently conjugated with the tetrameric SELP, resulting in the formation of a larger macromolecular ligand-SELP complex (~190 kDa). Reversed phase UPLC, SE-UPLC and SDS PAGE analyses were employed to confirm the formation of the macro-ligand. The formed ligand complex was then evaluated for the affinity precipitation of mAbs in pure form and from a complex mixture. The results indicated 100% mAb capture in pure component as well as complete selective capture from a harvest supernatant, with greater than 80% recovery of highly pure mAb. A molar ratio of 16:1 of the ligand-SELP complex to mAb was required to achieve complete capture in from the complex feed. Importantly, no SELP was observed in the mAb eluate, indicating the preservation of the streptavidin-biotin interaction througout the process. Further, we showed that the purification efficiency of the ligand-SELP construct was maintained for multiple rounds of affinity precipitation. This proof-of-concept study demonstrates that SELP can be used in conjunction with biotinylated affinity ligands for mAb purification.

Streptavidin-ELP-based macromolecular ligand fusions for the purification of mAbs

ACS Fall 2020 Virtual Meeting

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