Development of peptide affinity ligands for the purification of polyclonal and monoclonal Fabs from recombinant fluids

•FRWNFHRNTFFP targets both the heavy and the light chain of human Fab.•Fab binding capacity of FRWNFHRNTFFP-Toyopearl resin is ∼20 g per L of resin.•FRWNFHRNTFFP purified Fab from CHO fluids with yield >90%, HCP LRV>1.9, and DNA LRV>2.•FRWNFHRNTFFP-Toyopearl resin maintains its purification activity over 20 re-uses.•The cost of FRWNFHRNTFFP-Toyopearl resin is $6K/liter against $15K/liter of Capto™ L. Engineered multi-specific monoclonal antibodies (msAbs) and antibody fragments offer valuable therapeutic options against metabolic disorders, aggressive cancers, and viral infections. The advancement in molecular design and recombinant expression of these next-generation drugs, however, is not equaled by the progress in downstream bioprocess technology. The purification of msAbs and fragments requires affinity adsorbents with orthogonal biorecognition of different portions of the antibody structure, namely its Fc (fragment crystallizable) and Fab (fragment antigen-binding) regions or the CH1-3 and CL chains. Current adsorbents rely on protein ligands that, while featuring high binding capacity and selectivity, need harsh elution conditions and suffer from high cost, limited biochemical stability, and potential release of immunogenic fragments. Responding to these challenges, we undertook the de novo discovery of peptide ligands that target different regions of human Fab and enable product release under mild conditions. The ligands were discovered by screening a focused library of 12-mer peptides against a feedstock comprising human Fab and Chinese hamster ovary host cell proteins (CHO HCPs). The identified ligands were evaluated via binding studies as well as molecular docking simulations, returning excellent values of binding capacity (Qmax ∼ 20 mg of Fab per mL of resin) and dissociation constant (KD = 2.16·10−6 M). Selected ligand FRWNFHRNTFFP and commercial Protein L ligands were further characterized by measuring the dynamic binding capacity (DBC10%) at different residence times (RT) and performing the purification of polyclonal and monoclonal Fabs from CHO-K1 cell culture fluids. The peptide ligand featured DBC10% ∼ 6-16 mg/mL (RT of 2 min) and afforded values of yield (93–96%) and purity (89–96%) comparable to those provided by Protein L resins.