New purification method could make protein drugs cheaper

MIT engineers have developed a new method for protein purification that could help to reduce the cost of producing protein drugs such as antibodies and insulin. This new approach uses specialized nanoparticles to rapidly crystallize proteins, which is a more affordable way of isolating the protein from the bioreactor used to produce it. The purification step is one of the most expensive steps in manufacturing protein drugs and can account for up to half of the total cost of manufacturing a protein.

Protein purification and biologics

Antibodies and other protein drugs are part of a growing class of drugs known as biologics, which also include molecules such as DNA and RNA, as well as cell-based therapies. Most protein drugs are produced by living cells such as yeast in large bioreactors. Once these proteins are generated, they have to be isolated from the reactor, which is usually done through a process called chromatography. Chromatography requires specialized materials that make the process very expensive.

New approach based on protein crystallization

Researchers at MIT have developed a new approach to protein purification based on protein crystallization. Protein crystallization is a process that researchers often use to study protein structures, but it is too slow for industrial use and does not work well at low concentrations of protein. To overcome these challenges, MIT researchers used nanoscale structures to speed up the crystallization process.

In this new approach, the researchers used bioconjugate-functionalized nanoparticles to act as templates for enhancing protein crystal formation at low concentrations. The nanoparticles are coated with molecules called bioconjugates, which can help form links between other molecules. The researchers used bioconjugates called maleimide and NHS, which are commonly used for tagging proteins for study or attaching protein drugs to drug-delivering nanoparticles.

A microfluidic device was designed to combine protein solution with nanoparticles and then form thousands of tiny, identical droplets. Inside each of these droplets, the proteins interact with the nanoparticles, which help them to form protein crystals. Credit: Massachusetts Institute of Technology

Rapid crystallization with functionalized nanoparticles

In their studies with lysozyme and insulin, the researchers found that crystallization occurred much faster when the proteins were exposed to the bioconjugate-coated nanoparticles, compared to bare nanoparticles or no nanoparticles. With the coated particles, the researchers saw a sevenfold reduction in the induction time and a threefold increase in the nucleation rate. The induction time is how long it takes for crystals to begin forming, and the nucleation rate is how quickly the crystals grow once started. The bioconjugates provide a specific site for the proteins to bind, and because the proteins are aligned, they can form a crystal faster.

Machine learning to analyze crystals

The team also used machine learning to analyze thousands of images of crystals. Protein crystallization is a stochastic process, so a large dataset was necessary to determine whether the new approach was improving the induction time and nucleation rate of crystallization. Machine learning was used to determine when crystals were forming in each image without having to go through and manually count each one.

Impact on developing countries

This project is part of a Bill and Melinda Gates Foundation effort to make biologic drugs more widely available in developing nations. Protein drugs such as prophylactic antibodies have been shown to prevent malaria in clinical trials, and the new approach to protein purification could help to make these drugs more affordable and accessible.

Scaling up the process

The MIT team is now working on scaling up the process so that it could be used in an industrial bioreactor. They are also demonstrating that it can work with monoclonal antibodies, vaccines, and other useful proteins. If successful, this could help to make protein drugs more affordable and accessible to people around the world.

Journal Information: Caroline McCue et al, Enhancing Protein Crystal Nucleation Using In Situ Templating on Bioconjugate-Functionalized Nanoparticles and Machine Learning, ACS Applied Materials and Interfaces (2023). DOI: 10.1021/acsami.2c17208