The Global Impact of Pharmaceuticals

There is no doubting the importance and role that pharmaceuticals play in treating and preventing disease. This is an especially evident reality faced by the pharmaceutical industry in the recent years of the COVID-19 pandemic. As of September 2023, approximately 13.5 million vaccines have been administered globally as reported by the WHO1.

What if this scale of biomanufactured vaccines was applied to other pharmaceuticals and biologics?

Our team was inspired by seeing the scale that a bio production process can achieve, and have set our sights on integrating a highly scalable synthetic biology platform for producing biologics. Biologics are defined as products derived from living cells or components of a living cell. They range from vaccines, genetic therapies, proteins, nucleic acids, small molecules, or a combination of biomolecules, among others for various biotechnology applications to diagnose, treat, or prevent disease or illness2.

Figure 1: Chart demonstrating sale of biologics in Canada over 5 years. Adapted and modified from NPDUIS Canada.

Each day, researchers across the globe are racing across the globe to develop more efficacious biologics for a vast number of incurable diseases or illnesses. From tackling the heterogeneity of cancer, microbial resistance to antibiotics, viral diversity in infection, and genetic predisposition has led to increased patient demand for more diverse and affordable drug treatment options. The high cost and demand for resources in developing biologics has patients and health care professionals calling for the ease of developability of these life saving therapeutics.

Adapting biomanufacturing to synthetic biology systems

While there is no shortage of innovation for biologics, our team this year is focusing on tackling the biologics production process as the main bottleneck hindering the development of cheaper and more efficient therapeutics for patients and simultaneously, developing a platformed bioproduction toolkit for use by other researchers and industry professionals in their own labs By using foundational advance principles to underpin this synthetic biology approach, we also hope to create a more sustainable future for the drug discovery pipeline by reducing its impact on the environment.

The "current" manufacturing standard

Figure 2: Illustration demonstrating current challenges faced in the biomanufacturing industry

Difficult-to-express proteins include cancer therapeutics, antimicrobial peptides/proteins, toxins, membrane proteins, antibodies, and others. Current biomanufacturing platforms are limited by their slow turnaround rates and their reliability on host cell cohesiveness. High throughput production of proteins is often limited by cytotoxicity and deleterious effects that arise when high concentrations of these proteins are produced in a host cell. Many protein families are difficult to express due to inclusion activity formation, protein inactivation and other reasons3. Innate mechanisms inhibit the production of toxic proteins and inactivate protein toxicity to avoid cellular death. However in doing so, the active portion of the protein is significantly decreased resulting in not only lower purity, but also lower protein yield4. Thus conventional methods for producing therapeutic proteins are energy-intensive and entail slow turnaround times for optimized protein production. With the depletion of natural resources on a global scale, there is an urgent need to develop sustainable and low-waste methods for expressing and purifying proteins efficiently and rapidly.

The problem with current platforms

Synthetic biology technologies have emerged to eliminate such barriers to whole cell protein production in vitro by harnessing cell free protein synthesis (CFPS). Cell free protein synthesis, or in vitro protein synthesis refers, to the production and folding of proteins using biological machinery in a cell free environment, i.e., without the use of living cells. Due to the open nature of this system, a cell membrane is not required resulting in the elimination of the need to maintain cellular viability.

The traditional way of manufacturing would need 40,000 m2 of greenhouse growth to grow what we need. Our CFPS platform combines pre-existing advantages making it a simpler, faster and more versatile form of protein production.

Janis Schleusner - LenioBio, specializing in plant based CFPS systems

However, while CFPS is the natural route to take for high throughput protein production and purification, every platform comes with its limitations. Current cell-free systems suffer from low efficiency energy regeneration and reaction duration. Conventional systems rely solely on substrate-level phosphorylation; which involves the direct phosphorylation of ADP to ATP via a coupled reaction and phosphorylated intermediate5. This provides a short burst of energy but is expensive to use resulting in longer production times and lower yields. Our team has engineered new foundational advance approaches to underpin the CFPS approach and combine protein biosynthesis, purification, and preliminary validation into one harmonious process.

How can we merge intrinsic genetic tools with CFPS?

While CFPS is not a newcomer to foundational advanced biomanufacturing platforms, our team hopes to highlight the magnitudes of difference in adapting production systems to be scalable, modular, and multi-faceted using refined, engineered genetic elements. The key player here is the use of natural and precise protein splicers: Inteins.

Our Solution

We are excited to present PILOT - Platformed Inteins: A Linked Orthogonal Toolkit a synthetic biology based tool aimed at developing an orthogonal, intein based platform for protein expression. By utilizing intein protein technology, a natural self-cleaving post translational modification endogenous to a subset of all domains of life6, we created a modular CFPS production platform allowing simultaneous biosynthesis, extraction, and purification of proteins. Through the creation of inverted inner membrane vesicles (IMVs), complemented by the use of cheap and energy dense carbon sources such as methanol, we aim to increase the efficiency of CFPS energy generation. Our usage of intein mediated modularity coupled with the creation of an optimized CFPS reaction has the potential to accelerate the research and development pipeline of biologics.

Figure 3: Graphic summarizing UBC iGEM Vancouver 2023 project

Footnotes

  1. World Health Organization (2023). WHO Coronavirus (COVID-19) Dashboard Overview. https://covid19.who.int/ ↩

  2. U.S. Food & Drug Administration (2023). Biological product definitions. https://www.fda.gov/files/drugs/published/Biological-Product-Definitions.pdf ↩ ↩2

  3. Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in microbiology, 5, 172. https://doi.org/10.3389/fmicb.2014.00172 ↩

  4. Jin, X. & Hong, S.H. (2018). Cell-free protein synthesis for producing ‘difficult-to-express’ proteins, Biochemical Engineering, 138 (156-164). https://doi.org/10.1016/j.bej.2018.07.013. ↩

  5. Feher, J. (2012). ATP Production i. In Elsevier eBooks (pp. 171–179). https://doi.org/10.1016/b978-0-12-382163-8.00020-7 ↩

  6. Shah, N. H., & Muir, T. W. (2014). Inteins: Nature's Gift to Protein Chemists. Chemical science, 5(1), 446–461. https://doi.org/10.1039/C3SC52951G ↩

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