Producing Human Milk Sugars for Use in Formula

• Certain types of milk sugars, called oligosaccharides, form the third largest component of human milk.
• These human milk oligosaccharides (HMOs) have been shown to positively influence the gut microbiome and immunity.
• These sugars are structurally complex and diverse and, as a result, extracting or synthesizing them for use in formula has been challenging.
• Researchers are studying different ways to obtain these sugars, including extracting them from cow milk, chemically or enzymatically synthesizing them, or using microbes to produce them.
• At the moment, only a handful of simple HMOs have been produced at a large scale, but many others have been synthesized in smaller amounts.
• Extraction and synthesis techniques are continuing to improve, but we are still a long way from replicating the full diversity and complexity of sugars in human milk.

It’s well known that human milk is good for you (1-5). Sugars, called oligosaccharides, form the third largest component of human milk and have been associated with many beneficial effects. These human milk oligosaccharides (HMOs) have been shown to influence the composition of the gut microbiome, modulate the immune system, and help protect against pathogens (6-11, 22). HMOs act as prebiotics, promoting the growth of certain beneficial bacteria while suppressing the growth of other disease-causing bacteria (12-18). In addition, some HMOs have been found to mimic the attachment sites of harmful bacteria and thus block their ability to attach to and invade cells in the infant intestine (19, 20). HMOs may also be involved in the development of the infant gut, immune system, and brain (8-11).

Given the various benefits of HMOs, there has been a lot of interest in figuring out how to introduce HMOs into formula. However, more than 200 human milk oligosaccharides have been discovered so far, and their variety and complexity makes them challenging to synthesize (21-23). “Right now there are no formula where human milk oligosaccharides are being added,” says Geert-Jan Boons, Professor of Chemistry at the University of Georgia.

In an effort to deliver some of the benefits of HMOs, current dietary products sometimes include simpler oligosaccharides, often derived from plants (19, 24). Some of these simpler oligosaccharides have been reported to have prebiotic effects, but they do not have the structural complexity and diversity of HMOs. The effects of HMOs are considered to be highly structure-dependent, so in order to better replicate their function researchers are trying to produce oligosaccharides more similar to those in human milk.

“The bottom-line is that the carbohydrates that are being added right now to formula are not the carbohydrates found in human breast milk,” says Boons.

Extracting HMOs from milk

One way to obtain the same oligosaccharides found in human milk would be to purify them directly from breast milk. About a year ago, a press release by startup Medolac Laboratories announced its ability to commercially purify large amounts of native HMOs from donor human milk (25). But the difficulty of obtaining large amounts of human milk for commercial HMO production means that the majority of efforts have focused on other approaches to obtaining these molecules.

One such approach involves concentrating and extracting HMOs from cow milk. The oligosaccharides in cow milk are structurally similar to those in human milk, but their concentration is much lower (24, 26). Cow milk is already the most common milk used for infant formula in the U.S., so oligosaccharides extracted from it would be expected to be safe for human consumption. Researchers are trying to use filtration techniques to remove most of the lactose and salts from cow milk and increase the concentration of oligosaccharides.

The University of California, Davis milk processing lab is developing methods to extract large quantities of both human and bovine milk oligosaccharides from cow milk, according to Daniela Barile, an Associate Professor of food science and technology at UC Davis. These sugars could be tested in animal studies to determine whether they provide the beneficial effects associated with HMOs. Cow milk, and in particular whey—the liquid part of milk that separates from the curd during cheese production—could thus potentially serve as a way to produce commercial oligosaccharides with similar benefits to those in human milk.

“The technology’s in place, so we should be able nowadays to isolate oligosaccharides from whey,” says Barile. “Whey is a great source, but there are still great challenges if you want to really reach good purity and have a reproducible process batch to batch,” she says.

Individual oligosaccharides from cow’s milk are not exactly identical to those in humans, but an advantage of this technique is its ability to replicate some of the oligosaccharide diversity found in human milk, says Barile. Other methods have so far only been able to produce a handful of these sugars, she says. “If you really want to say that you want to mimic human milk, instead of having just one or two oligosaccharides you want to have the full complement,” says Barile. “The synthesis approach has been making a lot of progress, and they can now make bigger quantities, but it’s not representing the very complex constellation of different structures that is found in human milk,” she says.

“Right now, the isolation process can yield a better diversity than synthesis gets. So you can have more structures, you can have more molecules, so it’s more similar to human milk,” says Barile. “But there are still many challenges,” she says. “There is not a single product in the market right now made of oligosaccharide isolated from whey, so it’s all in the future,” says Barile. “We are at the beginning of the process, there’s still a long way to go.”

Using chemistry

Oligosaccharides can be synthesized through a series of chemical reactions, and that’s another approach that researchers have been pursuing. “The challenge is, we do not have robust technology to make complex carbohydrates at this time,” says Geert-Jan Boons. Unlike the process by which DNA is used to produce RNA and RNA is used to produce proteins, carbohydrates are not biosynthesized through a template-mediated process, Boons says. “If DNA goes to RNA goes to protein, that gives exact copies. When carbohydrates are being biosynthesized, because it’s not a template, you create heterogeneity,” he says.

“There are laboratories that are trying to automate chemical oligosaccharide synthesis in the way a peptide can be synthesized on machines off a standardized protocol,” Boons says. “The protocols are still not very robust, but progress is being made,” he says.

Glycom is one company that is using chemical processes for HMO synthesis, although the company also uses other production methods. However, using chemical synthesis to create commercial quantities of HMOs without making them prohibitively expensive could be a challenge. “The beauty of chemical synthesis is, they can make any HMO,” says Yong-Su Jin, Associate Professor of Food Science and Human Nutrition at the University of Illinois. “However, the cost would be much, much higher,” he says.

Stefan Jennewein, Managing Director and cofounder of one of Glycom’s competitors, Jennewein Biotechnologie, believes there are multiple issues that make chemical synthesis impractical for commercial production of HMOs. “At the time we founded the company, several chemical processes were established relying on chemical synthesis, which however are based on the use of toxic reagents like pyridine and chloroform and other noxious chemicals,” says Jennewein. While these processes might work fine at a small scale with extensive purification, they have high costs and lack scalability, he says. “Many in the industry are convinced that these processes are not compatible with food production,” Jennewein says.

Harnessing microbes

Instead of chemical synthesis, Jennewein Biotechnologie and many other companies and researchers use genetically engineered microbes to produce HMOs. “Microbial production is a very stable and safe method,” says Yong-Su Jin. It’s similar to techniques already used for large-scale production of amino acids and vitamins, he says. “It is a very robust and safe way to mass-produce food quality material,” says Jin.

Jin genetically engineers microbes to introduce the enzymes necessary to produce HMOs. At the moment he is using either the bacteria Escherichia coli, which is often used to produce proteins and metabolites, or the yeast Saccharomyces cerevisiae, used in baking, winemaking and brewing.

“So far we are using these two microorganisms to produce human milk oligosaccharides,” says Jin. “In particular we are making 2’-Fucosyllactose (2-FL), which is one of the most abundant HMOs in human milk,” he says (27). “We did very subtle chemical analyses, and we are very confident that our 2-FL is the same as 2-FL in human milk,” says Jin.

“That’s one advantage of biological production,” Jin says. “With chemical synthesis, you may have some minor modification somewhere, but enzymatic or microbial production have high fidelity in the reaction,” he says.

Jin says he is able to use genetically engineered E. coli to make up to 2-3 gm/L of 2-FL in the medium, similar to its levels in milk. Even though the E. coli strain he is using is very different from the ones that cause food disease, Jin says that, due to negative public perceptions about E. coli, he is now switching to using yeast. “Because they drink wine and beer or eat bread everyday, people believe that this strain is safer, so we are trying to make 2-FL in yeast right now,” he says. He says he is still optimizing 2-FL production in yeast to produce similar levels to that in E. coli.

Companies including Glycom, Jennewein Biotechnologie, and Glycosyn LLC are working on producing simple HMOs at a much larger scale for commercial use. “Several companies are currently developing formula containing 2’-fucosyllactose, but also other HMOs will soon enter the stage,” says Stefan Jennewein. Jennewein Biotechnologie produces 2-FL at a commercial scale using genetically engineered E. coli, and has been seeking market approval for their products. “We were the very first who filed for a Novel Food application in the EU for a food ingredient originating from a recombinant bacterial process,” says Stefan Jennewein. “In 2014 we obtained GRAS (Generally Recognized as Safe) status for Infant and Toddler Nutrition and General Nutrition in the US,” he says. The company is filing for registration in other major markets, and has been building production capacity for large-scale production of HMOs. “We completed the world’s first commercial multi-ton facility for HMOs in 2014, which is fully certified for food production,” says Jennewein.

Although microbial production can produce HMOs at a large scale, it has so far only been used to make a few of the simpler HMOs. Researchers are still trying to figure out how to expand the repertoire of HMOs that can be produced using microbes. “The good news is, although we have these 180 or so HMOs, they are not random chemical structures,” says Jin. “If we look at the basal structures, only 2-3 different sugars are connected with some rule,” he says. “So it doesn’t mean that we need to construct 180 strains with different biochemical pathways. Maybe if we make only 6 or 7 pathways, by mixing and matching combinations we will be able to create 180 different HMOs.”

“It’s like Lego blocks,” says Jin. “If you have 3 Lego blocks, then you can create 20 or 50 different shapes,” he says. “So in the future, we should be able to make any desired HMO by microbial production,” says Jin. “But I think it’s still very far off,” he says.

An enzymatic approach

To create some of the more complex oligosaccharides, Geert-Jan Boons and others have been focusing on enzymatic methods. “We have also very complex oligosaccharides in milk, and it is our belief that they are actually the compounds that perform very specific biological functions,” says Boons. “Those are not easily accessible right now,” he says.

Boons says his research group has been able to express almost every mammalian enzyme involved in modifying complex sugars, and he is working on using these enzymes to produce almost every human milk oligosaccharide. “The caveat is, we can produce only small amounts,” he says. Although the technology may not be able to produce commercial levels of HMOs, Boons says it will be very helpful for research purposes, to find out which HMOs are beneficial and what their functions are.

“I think that discovery, what these molecules actually do and which ones are the interesting ones, that will be done through chemical and enzymatic synthesis,” says Boons. “So, we will go through a discovery phase, find out how these molecules actually perform their beneficial properties, and which are really the active components, and create a mixture that can make a big difference for humans,” he says.

“Large scale production will be done through biotechnology, with cells that are engineered to produce these oligosaccharides,” says Boons. “I think what will be done in the next couple of years is, the relatively simple oligosaccharides, which can now be produced at a relatively large scale, they will move into the clinic,” he says. “Basic scientists like me, we will develop protocols to make the more complex ones, and they will be examined in cell culture and animal models, and when we begin to understand how they work, they will move into the clinic,” says Boons. “So, a lot of exciting things are happening,” he says.

However, it’s going to take a while before scientists or companies are able to produce formula that contains all the oligosaccharides found in human milk. “We are still a long way from making an artificial human milk oligosaccharide composition,” Boons says. “What we can do is begin to supplement cow milk with the main simple oligosaccharides found in human milk,” he says. “To make the whole structural diversity found in human milk, that is still quite far away,” says Boons.

Yong-Su Jin is confident that a combination of academia and industry will figure out ways to produce HMOs in the same manner they were able to achieve the production of many other oligosaccharides over the last five or 10 years. “The last 2-3 years have been very exciting,” he says. “Before that, although we had publications about the beneficial effects of HMOs, I didn’t see that much commercial activity, but now I see more and more infant formula companies interested in adding HMO into their product,” Jin says. “So, it’s a very exciting time,” he says. “I’m very optimistic, but we are still at a very early stage.”