California-based startup Triplebar Bio has teamed up with FrieslandCampina Ingredients to develop a cost-effective approach to scaling up the production of the bioactive milk protein lactoferrin via precision fermentation.
An iron-binding anti-microbial protein found in mammalian milk, lactoferrin has benefits around immunity, iron regulation, digestive health, endurance, and muscle strength, but has historically only been available in small quantities owing to the costs and challenges of isolating it from milk (thousands of liters are required to make a single kilo).
By producing lactoferrin in fermentation tanks using genetically engineered microbes optimized by Triplebar Bio, FrieslandCampina will be able to unlock new markets beyond infant formula from adult nutrition to performance nutrition.
‘Hyper-throughput’ screening platform
Founded in 2019 by biochemist Dr Jeremy Agresti, Triplebar has developed a ‘hyper-throughput’ screening platform deploying microfluidics and rapid testing. This is coupled with AI and machine learning to develop more efficient biomanufacturing platforms utilizing microbial or animal cells.
The cells can be optimized to produce everything from lactoferrin to superior cell lines for cultivated meat products and novel therapeutic biologic drug candidates, says the firm, which has raised $25 million to date from backers including Synthesis Capital and The Production Board.
Lactoferrin: Demand exceeds supply
While the unit economics of manufacturing whey and casein proteins via precision fermentation can be challenging, bovine lactoferrin currently sells for $700-2,000/kilo, making the economics of producing it with microbes attractive, Triplebar CEO Maria Cho told AgFunderNews.
“There are other players working on lactoferrin via precision fermentation [such as TurtleTree and De Novo Foodlabs], but FrieslandCampina Ingredients [a subsidiary of Dutch dairy giant FrieslandCampina] is already a leading player in lactoferrin (which it currently isolates from milk) and other dairy bioactives. Its ability to scale and distribute gives it a clear advantage in the market.”
Triplebar’s rapid testing platform “allows us to find those kind of needle in the haystack strains that can produce bio-equivalent proteins at high titers that are properly folded with the right efficacy,” explained Cho.
“Lactoferrin is a difficult protein to express. That’s in part because it has anti-microbial qualities [making it inherently more challenging to produce in a microbial system] but also because there needs to be certain glycosylation patterns or sugars on the outside of the protein.
“And historically, it’s been challenging to make this protein in an organism because you’re not seeing that particular pattern.”
That said, as lactoferrin is secreted into the fermentation broth, downstream processing is somewhat easier as you don’t have to rupture the microbial cells into order to get at the protein, she said.
“The key is evolving these cell lines to have the properties to scale, so it’s not just about achieving high titers [the amount of an expressed target molecule relative to the volume of liquid].”
‘A microprocessor for biology’
While scores of companies now use synthetic biology to program microbes to produce everything from dairy proteins to sweeteners via precision fermentation, the unit economics are challenging if strains are not optimized for productivity, titer, and yield, said Cho.
According to Cho, Triplebar’s system enables it to screen millions of mutations and optimize a microbial strain, for example, over many generations at record speed, effectively “running evolution at hyper-speed.”
She added: “Our expertise enables the product and process discovery to scale at orders of magnitude, faster and cheaper than any other company that’s out there. We take a hypothesis-free approach and say what’s the phenotype, the output of the thing that we’re looking for? And then Triplebar technology enables us to see how biology did it.”
Triplebar’s screening platform acts as a “microprocessor for biology,” integrating hardware, software, biology and biochemistry to miniaturize and accelerate evolution, explained Cho.
“Essentially we take a test tube and miniaturize it into a microreactor. We create tens of millions of microbial strains [for example], encapsulate them in our microreactors and add test reagents. And then we take tens of millions of microreactors and put them on a chip that fits in the palm of your hand.
“We then take that chip and run it on our benchtop system looking at thousands of tests per second. This is three to four orders of magnitude faster than what is currently found in terms of throughput and it allows us to determine what is the genetic profile of that organism that is creating that end product phenotype that we’re interested in?”
Collaboration model to get products to market more rapidly
She added: “We’re getting a ton of inbound inquiries. I think in the food space in particular there’s definitely been a shift towards collaboration and partnerships to try and get products to market faster. I think there’s less of that ‘not invented here’ syndrome.”
In some cases, a company will approach Triplebar with a target bioactive in mind, and Triplebar can help develop a host microorganism or animal cell optimized to produce it. In other cases, companies may already have a production strain in place, but it needs further optimizing, she said.
While standard liquid handling robotic technology can enable high-throughput screening of samples, Triplebar can assess “orders of magnitude” more, claimed Cho, noting that if you want to assess the impact of a genomic edit “at every possible position” in a given organism, the number of potential tests is enormous.
In the case of animal cells, “which like to grow in animals, not in tanks,” she noted, “We have to evolve those cells to have the properties needed to grow in tanks at cost points that could allow them to be broadly marketed, so that might mean increasing cell density, or getting them to grow in suspension [rather than having to adhere to something].”
Commercial partnerships
Other partners Triplebar works with include UMAMI Bioworks, which is developing optimized cell lines suitable for large-scale cultivated seafood production.
With Triplebar, UMAMI is testing “millions of potential phenotypic solutions in the time it normally takes to search mere hundreds” to accelerate cell line development and optimization without the need for genetic modification, said Cho.
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