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.