Probably the most costly steps in manufacturing protein medication equivalent to antibodies or insulin is the purification step: isolating the protein from the bioreactor used to supply it. This step can account for as much as half of the full price of producing a protein.
In an effort to assist scale back these prices, MIT engineers have devised a brand new technique to carry out this sort of purification. Their strategy, which makes use of specialised nanoparticles to quickly crystallize proteins, might assist to make protein medication extra reasonably priced and accessible, particularly in growing international locations.
“This work makes use of bioconjugate-functionalized nanoparticles to behave as templates for enhancing protein crystal formation at low concentrations,” says Kripa Varanasi, a professor of mechanical engineering at MIT and the senior creator of the brand new examine. “The objective is to scale back the price in order that this sort of drug manufacturing turns into reasonably priced within the growing world.”
The researchers demonstrated that their strategy can be utilized to crystallize lysozyme (an antimicrobial enzyme) and insulin. They consider it is also utilized to many different helpful proteins, together with antibody medication and vaccines.
MIT graduate scholar Caroline McCue is the lead creator of the examine, which seems at this time within the journal ACS Utilized Supplies and Interfaces. Henri-Louis Girard PhD ’20 can also be an creator of the paper.
Antibodies and different protein medication are a part of a rising class of medicine referred to as biologics, which additionally embrace molecules equivalent to DNA and RNA, in addition to cell-based therapies. Most protein medication are produced by residing cells equivalent to yeast in giant bioreactors.
As soon as these proteins are generated, they should be remoted from the reactor, which is normally carried out by a course of referred to as chromatography. Chromatography, which separates proteins primarily based on their measurement, requires specialised supplies that make the method very costly.
Varanasi and his colleagues determined to attempt a distinct strategy, primarily based on protein crystallization. Researchers typically crystallize proteins to check their constructions, however the course of is taken into account too sluggish for industrial use and doesn’t work nicely at low concentrations of protein. To beat these obstacles, Varanasi’s lab got down to use nanoscale constructions to hurry up the crystallization.
In earlier work, the lab has used nanoscale options to create supplies that repel water or to change interfaces for injecting highly viscous biologic drugs. On this case, the researchers needed to adapt nanoparticles in order that they may domestically improve the focus of protein on the floor and in addition present a template that may permit the proteins to align appropriately and type crystals.
To create the floor they wanted, the researchers coated gold nanoparticles with molecules referred to as bioconjugates — supplies that may assist type hyperlinks between different molecules. For this examine, the researchers used bioconjugates referred to as maleimide and NHS, that are generally used for tagging proteins for examine or attaching protein medication to drug-delivering nanoparticles.
On this time-lapse video, protein crystals type extra quickly on nanoparticles functionalized with molecules referred to as bioconjugates (high). The nanoparticles on the backside would not have the bioconjugate molecules.
Credit score: Courtesy of the researchers
When options of proteins are uncovered to those coated nanoparticles, the proteins accumulate on the floor and bind to the bioconjugates. Moreover, the bioconjugates compel the proteins to align themselves with a particular orientation, making a scaffold for added proteins to return alongside and be part of the crystal.
The researchers demonstrated their strategy with lysozyme, an enzyme whose crystallization properties have been nicely studied, and insulin. They are saying it is also utilized to many different proteins.
“It is a normal strategy that might be scaled to different techniques as nicely. If you already know the protein construction that you simply’re attempting to crystallize, you possibly can then add the appropriate bioconjugates that may pressure this course of to occur,” Varanasi says.
Of their research with lysozyme and insulin, the researchers discovered that crystallization occurred a lot quicker when the proteins had been uncovered to the bioconjugate-coated nanoparticles, in comparison with naked nanoparticles or no nanoparticles. With the coated particles, the researchers noticed a sevenfold discount within the induction time — how lengthy it takes for crystals to start forming — and a threefold improve within the nucleation charge, which is how shortly the crystals develop as soon as began.
“Even at low protein concentrations, we see much more crystals forming with these bioconjugate-functionalized nanoparticles,” McCue says. “The functionalized nanoparticles scale back the induction time a lot as a result of these bioconjugates are offering a particular web site for the proteins to bind. And since the proteins are aligned, they will type a crystal quicker.”
As well as, the workforce used machine studying to research 1000’s of pictures of crystals. “Protein crystallization is a stochastic course of, so we wanted to have an enormous dataset to have the ability to actually measure whether or not our strategy was enhancing the induction time and nucleation charge of crystallization. With so many pictures to course of, machine studying is one of the best ways to have the ability to decide when crystals are forming in every picture with out having to undergo and manually rely each,” McCue says.
This undertaking is a part of a Invoice and Melinda Gates Basis effort to make biologic medication, equivalent to prophylactic antibodies which were proven to forestall malaria in medical trials, extra broadly accessible in growing nations.
The MIT workforce is now engaged on scaling up the method in order that it might be utilized in an industrial bioreactor, and demonstrating that it could possibly work with monoclonal antibodies, vaccines, and different helpful proteins.
“If we are able to make it simpler to fabricate these proteins wherever, then everybody on the earth can profit,” Varanasi says. “We’re not saying that that is going to be solved tomorrow due to us, however this can be a small step that may contribute to that mission.”
Along with the Gates Basis, the analysis was partly funded by a Nationwide Science Basis Graduate Analysis Fellowship.