Depending on the process and who provides the statistics, downstream processing accounts for between 50–80% of the cost of producing biotech drugs. Whatever the exact number, protein purification looms large in the economic viability of a product. Hence the interest in systematizing and streamlining recovery and purification, from discovery scale to multiton production.
Marcel Ottens, Ph.D., assistant professor in the department of biotechnology at Delft University of Technology, sees growing interest in QbD and design of experiment (DOE) approaches that incorporate process analytic technology.
Dr. Ottens, who specializes in high-throughput experimental and mathematical approaches to optimizing chromatographic separations, predicts ongoing improvement in higher-flow and -capacity chromatographic resins, membranes, and monoliths, innovation in ligand chemistry (e.g., mixed-mode resins), affinity antibodies, antibody fragment-based chemistries, and greater reliance on operational modes like continuous and simulated moving-bed chromatography. Augmenting these advances will be greater adoption of disposables and an emphasis on facility fit to accommodate higher expression levels.
Shifts in expression systems with direct impact on purification will occur as well. “Although mAbs produced via mammalian cell culture currently dominate the protein therapeutic market, simpler expression systems like yeast and E. coli will become more significant,” he says.
Despite these innovations, Dr. Ottens predicts that the industry’s innate conservatism, forged by regulatory issues, will continue to hamper process improvements. Other hurdles include the high cost of capture resins, higher titers, and the complexity of therapeutic biomolecules. “We still lack sufficient knowledge of the physical-chemical properties and phase behavior of proteins, particularly with respect to adequate purification process design.”
Recognizing these changes are coming, vendors are viewing separation scale as a continuum. As QbD concepts infiltrate protein development and manufacturing, small-scale and early-stage protein isolation will be viewed as a warm-up for pilot and large-scale purification. DOE is already helping processors identify binding and elution characteristics with an eye toward scaleup.
GE Healthcare’s recent introduction of PreDictor™ plates and the ÄKTA™ avant line of chromatography systems exemplify this.
PreDictor Plates, which are 96-well microtiter plates charged with resins for rapid screening of conditions critical to binding, elution, and washing, show excellent correlation with chromatography column results.
From these miniaturized experiments process developers can move on to PreDictor RoboColumn™, consisting of miniature columns packed with resin. Another option is the column-based ÄKTA avant instruments, which help fine-tune conditions derived from PreDictor plates in small-column formats and provide robustness testing.
“Together, these products help process scientists characterize their design space efficiently, and thereby, develop a safe and robust process,” comments Eric Grund, Ph.D., senior director, biopharma applications. “DOE software used with these products allows you to do fewer experiments and get better understanding of the process before scaling up.”
Dr. Grund recognizes that it’s possible to overthink and overcharacterize processes before scaling up. “You have to be smart to know when to move on to the next study. We’re still all learning how to use these tools.”
Novel antibody formats like fragments and IgMs that lack Fc regions are changing the notion of platform separations. At the same time, therapeutic molecules are becoming more complex, for example biobetters with extended plasma half life, cell culture-manufactured vaccines, virus-like particles, and other molecules for which platform separations do not exist.
“If you don’t need an Fc region, why produce a 120 KDa molecule when you can achieve the same result with an antibody fragment?” asks Laurens Sierkstra, CEO of BAC, a specialist in high-affinity protein purification products.
BAC in essence creates purification platforms for complex molecules by designing protein A-like affinity resins for initial capture from fermentation broth, cell culture, or plasma. Other steps, such as ion exchange, polishing, ultrafiltration, and virus inactivation are performed as needed.
With GE Healthcare, BAC has introduced products for purification during discovery and preclinical stages through large-scale production: KappaSelect for antibody fragments, VIIISelect for therapeutic proteins, and AAV for viruses. “We are building a product portfolio that can act as the ‘protein A’ for the purification of all major classes of new biotherapeutics,” Sierkstra says.
The technology is based on single-domain antibodies, which are 110-residue peptides consisting of one variable domain of a heavy-chain antibody. Single-domain antibodies exhibit the same affinity as intact antibodies but are more resistant to heat and cleaning regimens. The antibodies are generated in llamas then cloned and expressed in large quantity in baker’s yeast. Protein A is produced similarly in E. coli. The ligands are attached to a suitable bead or surface to form the capture resin. According to Sierkstra, resulting resins are stable to base and strong acid.