Purification is one of the most expensive methods of manufacturing protein drugs. To make the method cheaper, MIT engineers have devised a new way to perform this type of purification.

In a new study, they reported their new purification method, which uses specialized bioconjugate-functionalized nanoparticles to rapidly crystallize proteins. These proteins act as templates for enhancing protein crystal formation at low concentrations.

During the research, scientists applied them to crystallize lysozyme (an antimicrobial enzyme) and insulin. They believe their approach could be used for many other beneficial proteins, including antibody drugs and vaccines.

In previous work, the group has adapted interfaces for injecting highly viscous biological drugs or created polymers that reject water using nanoscale characteristics. The goal in this case was to alter nanoparticles to increase the protein concentration locally at the surface and act as a template for proper alignment and crystallization of the proteins.

The scientists coated gold nanoparticles with bioconjugates, chemicals that can help form bonds between other molecules to create the required surface area. Maleimide and NHS bioconjugates were used, which are often used to label proteins for research or attach protein drugs to nanoparticles for drug delivery.

The proteins collect on the surface and bind to the bioconjugates when protein solutions are exposed to these coated nanoparticles. In addition, the bioconjugates force the proteins to arrange themselves in a certain direction, creating a framework for other proteins to attach to the crystal.

The scientists used the enzyme lysozyme, whose crystallization properties have been extensively studied, and insulin to demonstrate their methodology. They found that crystallization occurred much faster when the proteins were exposed to the bioconjugate-coated nanoparticles than bare or no nanoparticles.

Countless other proteins could potentially benefit from it.

The induction time, which measures how long it takes for crystals to start forming, was increased sevenfold with the coated particles. The nucleation rate, which measures how fast crystals develop once they start, was increased three times.

MIT graduate student Caroline McCue said: “Even at low protein concentrations, we see many more crystals forming with these bioconjugate-functionalized nanoparticles. The functionalized nanoparticles shorten the induction time because these bioconjugates provide a specific site for protein binding. And because the proteins are tuned to each other, they can form a crystal faster.”

“In addition, the team used machine learning to analyze thousands of images of crystals. Protein crystallization is a stochastic process, so we needed a huge data set to measure whether our approach improved the induction time and nucleation rate of crystallization. With so many images to process, machine learning is the best way to determine when crystals form in each image without having to manually count them all.”

The study appears today in the journal ACS Applied Materials and Interfaces.