Recombinant Human Collagen: Plant-Based Production Addresses Growing Demand for a Vegan OptionRecombinant Human Collagen: Plant-Based Production Addresses Growing Demand for a Vegan Option

From cosmetics to dietary supplements to regenerative medicine, the global market for collagen — and increasingly, vegan and/or plant-based collagen products — has expanded dramatically in recent years (1). Some biotechnology entrepreneurs and researchers are responding by leveraging improvements in heterologous protein expression technologies to manufacture recombinant human collagen at commercial scale.

In Canada, PlantForm Corporation is working to meet the growing demand for collagen products by using synthetic biology to “grow” bioidentical human collagen at commercial scales. This company aims to share in the global market for vegan collagen, which is valued at over US$6.4 billion and expected to reach $11.4 billion by 2030 (2).

“By using plants as biofactories to produce the target proteins,” says Don Stewart, PlantForm’s president and chief executive officer (CEO), “we can provide a low-cost and highly scalable source of collagen that matches the mechanical properties and functionality of natural human collagen while eliminating concerns about immunogenicity and disease transmission that come from animal sources of collagen.”

Plant-based protein-production systems offer several advantages over methods that use microbes or mammalian cells and require large bioreactor/fermentation vessels and specialized growth media (3). Plant-based systems are far simpler and more easily scalable without requiring the complex and expensive equipment and multiple steps of cell-culture operations.

Stewart cautions that human collagen is a complex and challenging protein for recombinant production, involving a large number of enzymes that regulate its expression and maturation. His company addresses those issues with its vivoXPRESS platform using proprietary Nicotiana benthamiana plants to grow proteins for antibody drugs, vaccines, and now collagen.

“It’s a complicated molecule to make,” he says. Human collagen is heavily hydroxylated, which is important to the stability of its triple-helix structure, collagen fibril assembly, and how the fibrils interact with other proteins. “With the vivoXPRESS system, we’re able to produce collagen that’s properly hydroxylated; other expression systems that use insect cells, Escherichia coli, or yeast can’t do that. We’re able to grow, extract, and process the proteins quickly — within a few weeks — and we can scale up [production] easily by simply growing more plants” (Figure 1).

^{Figure 1: Comparing recombinant-protein production scale-up using a classical cell-culture process* and a plant-based system.}

^{* Wright B, et al. A Novel Seed-Train Process: Using High-Density Cell Banking, a Disposable Bioreactor, and Perfusion Technologies. BioProcess Int. 13(3) 2015: S16–S25.}

Biological Building Blocks

Collagen is the most abundant protein in a human body. As the primary building block for skin, muscles, bones, tendons, and ligaments, collagen accounts for about 30% of our total protein content. Although humans make their own collagen naturally, our innate production slows down over time, which leads to injuries, aches and pains, and wrinkled skin as we age. The desire to mitigate the effects of aging, treat injuries, and repair joints damaged by arthritis has spawned a multibillion-dollar collagen industry.

Most collagen used in consumer goods is extracted from fish scales or the skin and bones of pigs and cattle. In recent years, consumer demand has been growing for plant-based or vegan alternatives to animal-derived collagen — for various reasons, including animal welfare and sustainability concerns, as well as the growing popularity of vegetarian/vegan diets. However, even though most “vegan collagen” supplements on the market that are made from natural plant sources and are rich in amino acids, vitamins, and minerals purported to stimulate collagen production — they contain no collagen themselves.

In addition to lifestyle products such as cosmetics and dietary supplements, collagen and collagen-based materials have been used in medicine for over 50 years in many applications, including treating wounds and burns, cartilage repair, bone and vascular grafts, tissue scaffolds, plastic surgery, and more (4). Historically, collagen used for medical and pharmaceutical purposes has been extracted from animals or human cadavers. However, just as skin creams and other products containing animal-sourced collagens can cause allergic reactions, nonhuman collagens used in medicine present risks of allergenicity, prion/pathogen transmission, and other forms of contamination. Batch-to-batch variability issues also may arise due to source diversity and harsh purification processes that modify the protein and impair its functionality.

Plant-based recombinant expression systems could provide a highly reproducible platform to overcome the shortcomings of both animal- and cadaver-derived collagens (5). Recombinant production could represent an ideal source of collagen for tissue-engineering and regenerative medicine applications, in particular.

Advantages of Recombinant Production

Scientists have identified 28 known types of collagen (6). Each of those has some unique components, depending on its function and anatomical location, but all share a similar quaternary protein structure that features three repeating protein chains assembled and folded into a triple helix.

Collagen types 1–3 make up about 80–90% of collagen in our bodies:

• Type 1 is most abundant and thus is commonly used in medical applications such as wound care and orthopedics

• Type 2 provides flexibility and support to cartilage and ligaments in our joints

• Type 3 is found in muscles, blood vessels, and organs, where it plays a key role in wound healing.

“The vivoXPRESS system has been adapted to make types 1, 2, and 3 so far,” Stewart says. “We don’t see any reason why we couldn’t make any of the other types.” That’s because plant-based systems provide a large amount of genetic control and flexibility of expression, both of which facilitate protein processing, posttranslational modifications (PTMs), proline hydroxylation, and assemblage of peptide chains into functional three-dimensional structures.

Stewart explains that the transient expression platform allows for the coordinate provision of a variety of supplementary activities (e.g., proline hydroxylase or lysine hydroxylase/glucosyl transferase) that may not be present or functionally active in the native N. benthamiana plant. The plant also naturally expresses an array of endoplasmic-reticulum–resident chaperone proteins that are homologous to mammalian counterparts and provide appropriate functionality to enable assembly of hetero- or homomeric proteins comprising several polypeptide chains.

“Highly specific chaperones can be introduced as required along with the target protein during transient expression,” Stewart says. “For example, the vivoXPRESS system has been demonstrated to express a variety of complex multimeric proteins such as monoclonal antibodies, plant viruses, laminin, and virus-like particle vaccines.”

How It Works: In the vivoXPRESS system (Figure 2), the leaves of four-week-old N. benthamiana plants are infiltrated with a recombinant agrobacterium solution containing the genetic sequence of collagen. The plants express target proteins in their leaves as they continue to grow. The plants are harvested in a week or two, then those leaves undergo a simple purification process for recovering the protein at high purity levels. A key advantage of this approach is that depending on which PTMs are needed, the agrobacteria can be designed to direct expressed proteins into different plant-cell compartments, where they can be protected from the extracellular environment.

^{Figure 2: Timeline of plant-based recombinant-protein manufacturing.}

“The end result,” Stewart says, “is a pure product that’s inexpensive to produce and easy to scale, has no animal components and therefore [raises] no concerns about possible disease vectors, is properly processed and assembled, and is virtually identical to human collagen.”

To adapt the vivoXPRESS system for collagen production, the protein sequence for human collagen was back-translated to a DNA sequence using codon bias for N. benthamiana, with the addition of a plant-specific signal sequence to direct the recombinant protein into the extracellular compartment. The gene was synthesized and cloned into PlantForm’s proprietary expression vector, and sequence fidelity was confirmed. The resulting vector was transformed into Agrobacterium tumefaciens EHA105.

To produce the collagen product, N. benthamiana plants are coinfiltrated with the collagen vector and auxiliary vectors for suppression of gene silencing and for PTMs (including the enzymes for proline hydroxylation and lysine hydroxylation/glycosylation) according to PlantForm proprietary protocols. Following that procedure and a period of incubation, the plants are harvested and collagen is extracted for purification using a number of steps based on the protein’s physicochemical characteristics.

A Partnership To Advance Collagen-Fiber Manufacturing

The flexibility of the vivoXPRESS platform has attracted interest from companies that are interested in manufacturing collagen for global markets. For example, 3DBioFibR Inc. (Halifax, Nova Scotia, Canada) and PlantForm recently were awarded $1.3 million from Next Generation Manufacturing Canada (NGen) to support their codevelopment of the world’s first scalable dry spinning of recombinant human-collagen fibers. This collaborative project will combine PlantForm’s recombinant expression system with 3DBioFibR’s patented, fully automated collagen-fiber manufacturing technology to produce such fibers at commercial scale.

When collagen is extracted from animal sources using harsh chemical methods, it is broken down from its higher-order fibrous form into individual collagen proteins. Those are used in various biomedical applications such as tissue engineering.

3DBioFibR’s collagen-fiber spinning platform is the first technology to demonstrate an ability to spin those collagen molecules back into fibers that closely match both the biochemical and biomechanical properties of natural collagen structures.

“This exciting project demonstrates the versatility of our vivoXPRESS platform,” Stewart says, “and it has the potential to make new life-saving and life-changing treatments available for millions of people.”

References

1 Vegan Collagen Market Size, Share and Trends Analysis Report By Source (Fruit, Vegetables), By End-Use (Food & Beverages, Healthcare, Personal Care & Cosmetics), By Region, and Segment Forecasts, 2024–2030. Grand View Research Inc.: San Francisco, CA, 2024; https://www.grandviewresearch.com/industry-analysis/vegan-collagen-market-report#.

2 Plant Collagen Market Analysis. Coherent Market Insights: Pune, India, November 2023; https://www.coherentmarketinsights.com/market-insight/plant-collagen-market-6016.

3 Gazaille B, Stewart D. Leveraging Plant-Based Protein Expression: Implications for Biomanufacturing and Biodefense. BioProcess Int. 21(11–12)i 2023; https://www.bioprocessintl.com/expression-platforms/leveraging-plant-based-protein-expression-implications-for-biomanufacturing-and-biodefense.

4 Shekhter AB, et al. Medical Applications of Collagen and Collagen-Based Materials. Curr. Med. Chem. 26(3) 2019: 506–516; https://pubmed.ncbi.nlm.nih.gov/29210638.

5 Shoseyov O, Posen Y, Grynspan F. Human Collagen Produced in Plants. Bioengineered 5(1) 2013: 49–52; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4008466.

6 Ricard-Blum S. The Collagen Family. Cold Spring Harb. Perspect. Biol. 3(1) 2011: a004978; https://doi.org/10.1101/cshperspect.a004978.

Barry Gunn is a freelance writer contributing on behalf of PlantForm Corporation, 1920 Yonge Street #200, Toronto, ON, M4S 3E6, Canada; 1-416-572-7795; https://www.plantformcorp.com. vivoXPRESS is a registered trademark of PlantForm Corporation.