Mixed-mode size-exclusion silica resin for polishing human antibodies in flow-through mode.

The polishing step in the downstream processing of therapeutic antibodies removes residual impurities from Protein A eluates. Among the various classes of impurities, antibody fragments are especially challenging to remove due to the broad biomolecular diversity generated by a multitude of fragmentation patterns. The current approach to fragment removal relies on ion exchange or mixed-mode adsorbents operated in bind-and-gradient-elution mode. However, fragments that bear strong similarity to the intact product or whose biophysical features deviate from the ensemble average can elude these adsorbents, and the lack of a chromatographic technology enabling robust antibody polishing is recognized as a major gap in downstream bioprocessing. Responding to this challenge, this study introduces size-exclusion mixed-mode (SEMM) silica resins as a novel chromatographic adsorbent for the capture of antibody fragments irrespective of their biomolecular features. The pore diameter of the silica beads features a narrow distribution and is selected to exclude monomeric antibodies, while allowing their fragments to access the pores where they are captured by the mixed-mode ligands. The static and dynamic binding capacity of the adsorbent ranged respectively between 30-45 and 25-33 gs of antibody fragments per liter of resin. Selected SEMM-silica resins also demonstrated the ability to capture antibody aggregates, which adsorb on the outer layer of the beads. Optimization of the SEMM-silica design and operation conditions - namely, pore size (10 nm) and ligand composition (quaternary amine and alkyl chain) as well as the linear velocity (100 cm/h), ionic strength (5.7 mS/cm), and pH (7) of the mobile phase - afforded a significant reduction of both fragments and aggregates, resulting into a final antibody yield up to 80% and monomeric purity above 97%.

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

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.

Novel peptoid-based adsorbents for purifying IgM and IgG from polyclonal and recombinant sources.

Polyclonal immunoglobulin therapeutics comprising dosed IgG and IgM combinations are powerful tools in fighting cancer and severe infections. The inability of protein ligands to produce polyclonal IgG- and IgM-enriched formulations and recover monoclonal IgM calls for novel ligands with superior biorecognition activity. In this study, a peptoid ligand discovered by our group, and integrated into affinity adsorbents LigaTrap Technologies' "Human IgG" and "Human IgM", were utilized to purify IgG and IgM from complex fluids. IgG purification from human serum using LigaTrap IgG afforded 94.6% purity and 62.9% yield, on par with Protein A/G resins. When challenged with CHO and HEK cell culture harvests with low IgG titer (<1 mg/mL), LigaTrap IgG returned values of yield and purity well above 60% and 90%. LigaTrap IgM was evaluated for purifying IgM in comparison with commercial adsorbents, and afforded a product purity of 93% from a CHO harvest (IgM titer of 1 mg/mL) and 75.1% yield from a HEK harvest (0.5 mg/mL). LigaTrap-M provided IgM enrichment up to 11-fold higher than HiTrap resin. The peptoid adsorbents separated IgG-depleted human serum into IgM- and IgA-enriched fractions. These results demonstrate the potential of the peptoid ligand for manufacturing polyclonal Ig formulations and monoclonal IgM therapeutics.

Late occurrence of transient advanced second degree atrioventricular block after successful transcatheter cryoablation of atrioventricular nodal reentry tachycardia.

Late occurrence of atrioventricular nodal block is an extremely rare occurrence after radiofrequency catheter modification of the slow pathway and has yet to be reported after cryoablation. We report a case of late transient advanced second degree atrioventriuclar block after cryomodification of the slow pathway.