Developing a Sustainable Source of Squalene

The molecule squalene is widely used in the personal care and cosmetics industries and is increasingly being included as an immunological adjuvant in vaccines. Currently, the majority of the world’s supply of this in-demand product is sourced from the livers of deep-sea sharks. However, squalene is in short supply, and alternative, more sustainable sources are desperately needed to meet demand and protect shark populations. 

Two companies passionate about improving this situation – Synshark and Phylloceuticals – recently entered a strategic partnership to produce pharmaceutical squalene from plants. Through the partnership, the two companies will develop manufacturing processes for squalene production in Lemna sp. (duckweeds) and Nicotiana benthamiana (N. benthamiana), a small, Australian ornamental plant.

Technology Networks spoke to Jason Ornstein, executive director of SynShark, and Barry Holtz, PhD, chief scientific officer of Phylloceuticals, to learn more about the collaboration and how it could help to address current challenges associated with squalene supply and access.

Anna MacDonald (AM): Can you give us an overview of what squalene is and the role that it plays in vaccines? 

Jason Ornstein (JO): Squalene is a molecule found in most living organisms but in very trace amounts. It is a long-chain hydrocarbon that is created in the human liver and transferred through our blood system to our skin where it plays a role in our natural protection from ultraviolet rays from the Sun. As an antioxidant, it also helps humans protect themselves from pollutants on the skin. Industrially, it is used as an emollient in personal care products because it is colorless and odorless. Unfortunately, the most plentiful source of industrial squalene is the liver of deep-water sharks.

A squalene-based vaccine adjuvant is delivered as nanodroplets emulsified by a phospholipid or non-ionic surfactant through injection, which increases the cellular uptake of the antigen in the vaccine. Once present, squalene recruits immune cells to the site, carrying the antigen into the lymphatic system and elicits an aggressive response. It’s the “why” of the response of the immune system to squalene that remains unknown. While there are other adjuvants, squalene is the only adjuvant ingredient that is perfectly biocompatible.

AM: What current challenges are associated with squalene supply and access? Are there any initiatives in place to address these issues? Can you tell us more about the use of plants to produce squalene?

JO: Most of the volume of industrial squalene is sourced from shark livers. While many personal care consumers are unaware of this, environmentalists have raised awareness and many cosmetic companies have sought alternative sources. One natural source is the by-product of olive oil production. This is limited to the total harvest each year. To consider U.S. Food and Drug Administration (FDA)-regulated medical use as an adjuvant, this source would require indoor growth of olives, which is limited by the years it takes to develop an olive patch and the total harvest time.

Another initiative is the novel Amyris product of synthetic squalane (an oxidized version of squalene). Unfortunately, this compound lacks the bioactivity of squalene. Amyris has proposed its compounds for adjuvant use but has been rejected by pharmaceutical companies. There is a desperate search for a new plant source of squalene that can be harvested indoors and fast.

AM: Why were tobacco and duckweed species chosen for this initiative? Why are both transgenic and transient approaches being developed?

Barry Holtz (BH): Both species have inherent advantages in the expression of some kinds of heterologous proteins. Since both have almost identical downstream purification steps, it is prudent to have both species to evaluate.

In general, it is customary to use the transgenic approach in Lemna sp. since the inherent gene stability allows constant recycling of some of the biomass as inoculum in a flow-through system. Typically, N. benthamiana utilizes the rapid and stable transient system. Only five to seven days are required to get to maximum expression. However, for complex polygenic systems, some of the genetic information can be a permanent transformant and additional genetic input can be inserted as a transient construct.

AM: What are the advantages of manufacturing products such as squalene in this way? Are there any challenges to overcome?

BH: The production of multiple products, especially a lipid intermediary metabolite and protein in the same plant by multi organelle directed (MODä ) biosynthesis, represents huge savings in upstream cost and high return from a low cost of goods. 

We have developed a proprietary separation technique early in the process that segregates discrete sub-cellular fractions so that multiple products can be concentrated and sent on their individual process paths.

AM: What about regulation? How soon could reliance on unsustainable sources of squalene become a thing of the past?

JO: The product would be regulated by the FDA and with the experience of the Phylloceuticals team, plans are being put in place to develop the regulatory path. During the current pandemic, the FDA has been actively prioritizing vaccine tools such as this which could increase the strength of SARS-CoV-2 vaccines. It is commonly known that the European Union is considering regulation against shark-sourced squalene.

Jason Ornstein and Dr. Barry Holtz were speaking to Anna MacDonald, Science Writer for Technology Networks.