Today, crystallization is used primarily to analyze the structures of proteins. It also is gaining favor as a purification method for elastase, proteases, albumin, and lipase, in addition to industrial-scale insulin purification. Now it can be used for more cost-effective downstream mAb purification.
Replacing protein A chromatography with a single crystallization step greatly reduces the financial, human, and environmental costs of downstream purification, according to a team of European researchers led by Gianluca Di Profio, PhD, director of research, Consiglio Nazionale delle Ricerche, Istituto per la Tecnologia delle Membrane in Rende, Italy.
His team replaced protein A with a continuous template-assisted membrane crystallization (TMC) technique in which templates were embedded in a porous, hydrophobic, polyvinylidene fluoride membrane containing a layer of polymerized polyvinyl alcohol. This water vapor-permeable composite membrane allows osmotic flow of water vapor from the protein solution, which facilitates crystallization.
“The templates…serve as nucleants for crystallization,” Di Profio and colleagues explain in a detailed paper in Nature Protocols, so crystallization occurs at low protein concentrations.
Easy scaling & automation
Although optimizing crystallization conditions for mAbs is challenging, especially at large scale, the scientists produced gram-scale quantities of mAbs in an economical, sustainable, continuous flow production environment. The method is easy to scale up and down and to automate, says the Italian team.
“Further optimization of this method would, potentially, allow the replacement of all chromatographic steps, including ion exchange,” Di Profio tells GEN.
Cost savings could be significant, he says, as protein A resin costs $10,000 to $15,000 per liter and the polymeric membrane replacement costs less than $100 per square meter.
“Furthermore,” Di Profio says, “membrane-assisted crystallization does not require the extensive use of chemicals and buffers that are necessary for column regeneration (up to 200 times) in the chromatography process.” This also significantly reduces water usage and other environmental concerns, and generally yields proteins that are purer and more stable than other downstream purification methods by multicycle crystallization.
The current method has been validated for the anti-CD20 mAb. Adapting the protocol to other biomolecules is expected to involve relatively minor adjustments.
To apply this method, Di Profio advises developers to prevent the membranes from wetting. He defined “wetting” as flooding the insides of the membrane with solutions that migrate through it. Those solutions sometimes contain impurities that change the properties of the membranes from hydrophobic to hydrophilic. “Wetting can be prevented by designing specific membrane properties before the process, or by pre-treating the mAb solution to remove the interacting materials,” he says.
