Downstream processing is becoming a bottleneck as biopharmaceutical manufacturing shifts to more complex bispecific antibodies, FC-fusion proteins, and adeno-associated viral (AAV) vectors. A purification platform developed by a team of 24 researchers at North Carolina State University removes that bottleneck by providing both high flow-through and superior impurity capture.
For biomanufacturers, the combination of process intensification and more complex vectors has meant higher upstream titers that increased both product- and process-related impurities, thus increasing the downstream processing burden. The ramification is that some high-risk impurities, such as proteases, lipases, redox-active enzymes, and chromatin-associated species, have slipped through conventional capture and polishing steps and have been implicated in batch failures, clinical holds, and product recalls.
A recent paper outlines the strategies Wenning Chu, PhD, research scholar and first author, and colleagues devised.
Industry-relevant options
For proteins derived from Chinese hamster ovaries (CHO), the platform consisted of a resin-based pre-capture step that combines peptide ligand technology and a size-exclusion matrix, followed by a Protein A capture step, and a single-use size-exclusion mixed-mode resin for polishing. It enabled “product yields exceeding 70%,” final product pool concentration of 19–23 g/L/hour—notably more than the industry standard or approximately 15 mg/mL, “and monomeric purity of approximately 99%.” Host cell protein clearance ranged from 4 to 11 ppm.
“By shifting the impurity clearance from a bind-and-elute polishing step to a modular flow-through operation, this approach decouples impurity removal from product capture and reduces the process sensitivity typically associated with modality-specific polishing development,” Chu and colleagues wrote. “The productivity gains…were substantial.”
For AAV purification, the team used a mixed-bed absorbent followed by a capture step using either a platform AAV resin or a single-use, high-capacity chromatography membrane. By enforcing a 1:3 charcoal-to-resin ratio and optimizing the load volume, this method achieved an approximate 50% AAV recovery and a host cell protein level of 350 ng/mL—less than 100 ng per dose.
This fit-for-purpose platform purifies a wide range of products and impurities with modular flexibility and, the researchers note, “enables targeting impurity classes that often persist through conventional platforms and are removed only at the expense of yield, while still sustaining high productivity.”
These flow-through and pseudo-affinity technologies expand downstream processing options, “enabling more efficient and reliable manufacturing of complex biologics….” The team considers them “industrially relevant” particularly in light of feed variability.
Further research may focus on operations at low residence times and productivity enhancements, as well as scale-up and good manufacturing practice considerations.
