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. [Courtesy of the researchers, colorized by MIT News]
Engineers at MIT developed a new way to accomplish purification while manufacturing protein drugs, potentially reducing costs of production.
The researchers say the manufacturing process represents one of the most expensive steps in manufacturing protein drugs. These kinds of drugs include antibodies or insulin. Purification involves isolating the protein from the bioreactor used to produce it.
According to an MIT website post, the step can account for up to half the total cost of manufacturing a protein. In order to reduce these costs, the MIT team developed an approach using specialized nanoparticles to rapidly crystallize proteins. Such a method could make protein drugs more affordable and accessible, especially in developing countries.
The researchers demonstrated that their approach can crystallize lysozyme and insulin. However, they believe it could extend to other useful proteins like antibody drugs and vaccines.
MIT graduate student Caroline McCue served as lead author for the study. It appeared today in the journal ACS Applied Materials and Interfaces. Henri-Louis Girard is also an author, along with senior author and MIT mechanical engineering professor Kripa Varanasi.
“This work uses bioconjugate-functionalized nanoparticles to act as templates for enhancing protein crystal formation at low concentrations,” said Varanasi. “The goal is to reduce the cost so that this kind of drug manufacturing becomes affordable in the developing world.”
The protein purification process
According to the researchers, most protein drugs are produced by living cells, such as yeast, in large bioreactors. Once generated, the proteins must be isolated from the reactor through a process called chromatography. This step, which separates proteins based on size, makes the overall process “very expensive,” according to MIT.
Varanasi and colleagues say that researchers often crystallize proteins to study their structures. However, many consider the process too slow for industrial use with issues at low concentrations of proteins.
To overcome this, the laboratory aimed to use nanoscale structures. They wanted to adapt nanoparticles to locally increase the concentration of protein at the surface. This also provides a template for the proteins to align correctly and form crystals. The researchers coated gold nanoparticles with bioconjugates to create a surface. These bioconjugates — called maleimide and NHS — most commonly tag proteins for study or attach protein drugs to drug-delivering nanoparticles, MIT said.
Coated nanoparticles allow proteins to accumulate at the surface and bind to the bioconjugates. The bioconjugates also compel the proteins to align themselves with a specific orientation. This creates a scaffold for additional proteins, the MIT team explained.
“This is a general approach that could be scaled to other systems as well,” Varanasi said. “If you know the protein structure that you’re trying to crystallize, you can then add the right bioconjugates that will force this process to happen.”
How this influences rapid crystallization
The researchers said that, in studying lysozyme and insulin, crystallization occurred “much faster” with protein exposure to the coated nanoparticles compared to bare nanoparticles or no nanoparticles. They observed a seven-fold reduction in the induction time for crystals to begin forming. MIT said the team also observed a three-fold increase in nucleation rate (the rate at which crystals grow once started).
McCue said the crystals formed even at low protein concentrations, eliminating one of the previous stumbling blocks for this method.
“The functionalized nanoparticles reduce the induction time so much because these bioconjugates are providing a specific site for the proteins to bind. And because the proteins are aligned, they can form a crystal faster,” she explained.
MIT’s engineers also used machine learning to analyze thousands of images of crystals. McCue said the large dataset allowed for the team to “really measure” the improvement of crystallization.
The Bill and Melinda Gates Foundation funds this project as part of an effort to make biologic drugs, such as prophylactic antibodies that could prevent malaria. A National Science Foundation Graduate Research Fellowship also partly funded the research. The MIT team now plans to scale its process for use in an industrial bioreactor. Researchers also aim to demonstrate its capabilities with monoclonal antibodies, vaccines and other useful proteins.
“If we can make it easier to manufacture these proteins anywhere, then everyone in the world can benefit,” Varanasi said. “We are not saying that this is going to be solved tomorrow because of us, but this is a small step that can contribute to that mission.”