Continuous precipitation-filtration process for initial capture of a monoclonal antibody product using a four-stage countercurrent hollow fiber membrane washing step.

The significant increase in product titers, coupled with the growing focus on continuous bioprocessing, has renewed interest in using precipitation as a low-cost alternative to Protein A chromatography for the primary capture of monoclonal antibody (mAb) products. In this work, a commercially relevant mAb was purified from clarified cell culture fluid using a tubular flow precipitation reactor with dewatering and washing provided by tangential flow microfiltration. The particle morphology was evaluated using an inline high-resolution optical probe, providing quantitative data on the particle size distribution throughout the precipitation process. Data were obtained in both a lab-built 2-stage countercurrent washing system and a commercial countercurrent contacting skid that provided 4 stages of continuous washing. The processes were operated continuously for 2 h with overall mAb yield of 92 ± 3% and DNA removal of nearly 3 logs in the 4-stage system. The high DNA clearance was achieved by selective redissolution of the mAb using a low pH acetate buffer. Host cell protein clearance was 0.59 ± 0.08 logs, comparable to that based on model predictions. The process mass intensity was slightly better than typical Protein A processes and could be significantly improved by preconcentration of the antibody feed material.

Self-assembly of globular proteins with intrinsically disordered protein polyelectrolytes and block copolymers.

Intrinsically disordered polypeptides are a versatile class of materials, combining the biocompatibility of peptides with the disordered structure and diverse phase behaviors of synthetic polymers. Synthetic polyelectrolytes are capable of complex phase behavior when mixed with oppositely charged polyelectrolytes, facilitating nanoparticle formation and bulk phase separation. However, there has been limited exploration of intrinsically disordered protein polyelectrolytes as potential bio-based replacements for synthetic polyelectrolytes. Here, we produce negatively charged, intrinsically disordered polypeptides, capable of high-yield expression in E. coli and use this intrinsically disordered peptide to produce entirely protein-based polyelectrolyte complexes. The complexes display rich phase behavior, showing sensitivity to charge density, salt concentration, temperature, and charge fraction. We characterize this behavior through a combination of turbidity assays, dynamic light scattering, and transmission electron microscopy. The robust expression profile and stimuli-responsive phase behavior of the intrinsically disordered peptides demonstrates their potential as easily producible, biocompatible substitutes for synthetic polyelectrolytes.