Downstream process innovation is alive and kicking into high gear — especially when it comes to chromatographic purification as new therapeutic modalities and economic pressures are challenging old ways of thinking. This month’s insert includes discussions of mixed-mode chromatography, a potential platform for adenoassociated viruses, and antibody-fragment purification. First, BPI’s senior technical editor reviews recent developments in data science, chromatographic chemistry, and process engineering. Then, managing editor Brian Gazaille discusses the ins and outs of mixed-mode chromatography with the University of Virginia’s Nick Vecchiarello. A contract manufacturer and technology supplier report on their collaborative work toward a purification platform for adenoassociated viruses. And Tosoh Bioscience highlights the benefit of converting the capture step for antibody fragments to a continuous process.

Introduction: Data Science, Chemistry, and Process Engineering Are Driving Bioprocess Innovation Forward
by Cheryl Scott
Creativity and problem-solving are very much alive and well in downstream processing of biopharmaceuticals. With upstream production groups outputting an increasingly diverse set of product streams, downstream process engineers have been under mounting pressure to devise and implement new purification solutions to the downstream bottleneck problem. Many groups are turning to technology suppliers and even academic researchers for help, and the results have been impressive so far — with more advancements to come. Here, BPI’s senior technical editor focuses on three areas of innovation in her review of recent developments:  data science, chromatographic chemistry, and process engineering. The intersection of information technology and life science brings many benefits to the biopharmaceutical industry, including for process modeling, monitoring, and control. Meanwhile, great strides and important refinements also are emerging in the chemistry and materials of chromatographic purification. Many drug developers partner with technology suppliers and academic researchers to help them solve problems and find new approaches to separation and purification. Certainly, new therapeutic modalities and economic pressures are challenging old ways of thinking in purification, and thanks to technological advances such as those highlighted herein, some of the most creative problem-solving is happening in the realm of process engineering.

Demystifying Mixed-Mode Chromatography Resins: Emerging Applications for Purification of Non-MAb Protein Therapeutics
by Brian Gazaille, with Nick Vecchiarello
Drug-development pipelines are bringing forth a breadth of nonantibody proteins and fragments as well as variations on the conventional-antibody theme. Such formats leverage the efficacy advantages of antibody binding while surmounting key technical and economic obstacles associated with monoclonal-antibody manufacturing. Some of these alternatives might enable use of microbial and other expression systems and could exhibit improved stability. But questions remain for their downstream processing. Wherever protein A affinity resins are unsuitable, developers will require novel separation techniques. Here, BPI’s managing editor talks with Nick Vecchiarello, an assistant professor of chemical engineering at the University of Virginia, about the distinctive purification requirements of non-MAb therapeutics and the potential advantages of mixed-mode media. Vecchiarello has presented at recent conferences about opportunities for purification using mixed-mode chromatography media and described his laboratory’s efforts to perform high-throughput screening of candidate resins toward developing comprehensive ligand libraries to facilitate selection of the best mixed-mode options.

Platform Optimization for Efficient AAV Purification: Insights from a CDMO
by Vincent Ravault, Hélène Lebas, Cedrick Rousseaux, Adrien Auffret-Cariou, Brian Mullan, Nicolas Laroudie, Alejandro Becerra
Application of different AAV serotypes has enabled gene-therapy developers to target a wide range of tissues and organs and address a number of diseases — but each serotype necessitates its own specific purification process. Such development work requires considerable resources and takes time, which can delay a product’s clinical progress. Contract development and manufacturing organizations (CDMOs) such as Yposkesi seek to establish platform purification processes that will require minimal adjustments between batches and products, as is the norm for monoclonal antibody development. Such a platform approach will help to reduce delivery times by leveraging high productivity, rapid turnover, and standardization of production methods. Thermo Fisher Scientific’s POROS CaptureSelect AAVX chromatography medium lends itself to platform use for AAV capture, whatever the serotype. Here, authors from the two companies report on their investigation into the influence of critical operational parameters such as residence time and loading density on binding capacity, vector recovery, and product purity using different AAV serotypes. They also compare the capture performance of different affinity adsorbents and demonstrate resin scalability at laboratory scale.

Intensification of Fab-Fragment Purification: Multicolumn Chromatography Using Prepacked Protein L Columns
by Sebastian Thürmann, Patrick Endres, Jonas Wege, Egbert Müller
Antibody fragments — such as fragment antigen-binding (Fab) domains, single-chain variable fragments (ScFvs), and heavy-chain variable domains (nanobodies) — have emerged as increasingly important therapeutic and diagnostic alternatives to full-length monoclonal antibodies (MAbs) for a multitude of diseases. Whereas MAb downstream processing is well established and easy to scale based on protein A capture, the purification of antibody fragments is just on the verge of standardized processing. As authors from Tosoh Bioscience explain herein, the most promising candidate for effective capture of those fragments containing a kappa light chain is protein L affinity chromatography. To establish an effective capture process for ScFv purification, they compared the capabilities of two different protein L chromatography resins in batch mode and then investigated their performance using multicolumn chromatography to enhance process performance further. The higher capacity use, increased productivity, and reduced buffer consumption all improve process economics and increase the feasibility of purifying large quantities of Fab-containing fragments relatively quickly.