Exosomes: An exciting new modality for drug delivery and regenerative medicine
Exosomes are membrane-bound nanovesicles that carry molecules from cell to cell as part of finely regulated communication mechanisms within the body. Work has been done on modifying exosomes to contain drugs that could then be delivered to cells in a targeted way. However, manufacturing these exosomes in large quantities is challenging. Here we see how the work of Lonza overcomes some of these challenges to enable the manufacturing of exosomes at scale.
Exosomes are small, membrane-bound vesicles which are usually between 30 and 150 nm in size, and are produced by most cells. The phospholipid bilayer that surrounds them protects molecules such as proteins, or DNA and RNA, from degradation without inducing an immune response. Exosomes are used in the body for cell-to-cell communication. A producer cell releases an exosome which can then travel to neighbouring or distant cells to deliver its cargo. This is an incredibly targeted interaction as the exosomes have specific surface markers that interact with receptors on the surface of a recipient cell. This interaction causes uptake of the exosome by the cell.
Once this interaction has occurred and the exosome has been taken up, its contents are released into the cell. The recipient cell has now been given information to be processed from the producer cell; an example of this would be delivery of messenger RNA which can be used to induce the recipient cell to produce specific proteins. These proteins can then be released to carry out their purpose, which can include augmenting an immune response to an infection.
Due to this targeted delivery system, exosomes could be used for therapeutic purposes. The naturally produced exosomes could be altered to contain and deliver drugs directly to specific cells. This would allow drugs to be delivered without any interference from surrounding cells or immune reactions within the body. This could drastically increase the safety and efficacy of payloaded drugs.
Lonza is a biotech company that is working on manufacturing exosomes to be used in precisely this way. The exosome therapy market is emerging but it has incredible potential with several companies in pre-clinical and early clinical stages working on determining the efficacy of their exosome-based therapies. While this emerging market is highly promising, the manufacturing process involves many rigorous steps, and there are many challenges associated with it. Analysis must be done at every step of the manufacturing process in order to assess the quality and quantity of the produced exosomes.
The manufacturing and purification processes
The key to enabling the large-scale manufacturing of exosomes is the characterisation of the particles and their subpopulations. At Lonza, researchers have combined their knowledge of cell, protein, and viral manufacturing in order to optimise the process for exosome manufacturing. By growing primary cells isolated directly from tissue or engineered cell lines in bioreactors, exosomes can be manufactured at large scale.
Lonza is currently optimising the process for the purification of exosomes from conditioned media from bioreactors. The purification of the extracellular vesicles is a multi-step process that starts with depth filtration to remove cell debris. This is followed by tangential flow filtration which uses transmembrane pressure to filter back larger particles, such as exosomes, whilst smaller particles pass through. These larger particles are then further purified using a process called anion exchange chromatography. In this process, the negatively charged exosomes bind to the positively charged solid phase of the chromatography column while impurities are washed away. Then, exosomes are released from the column using an appropriate buffer. The final purification step is 0.22 µm filtration which uses a pore that allows anything under 220 nm through, including the exosomes. Once all these steps have been carried out, the purified exosomes are placed into a formulation buffer ready to be used.
Process optimisation and analysis
These manufacturing and purification methods require careful characterisation and analysis at each step to allow for process development and to monitor product quality. At Lonza, this has been achieved by individually measuring each exosome using single particle analysis with nano-flow cytometry, a process whereby the exosomes are stained with fluorescent dyes so that they can be picked up when excited by a laser. This can be used to determine the percentage of recovered exosomes at each step of the purification process. Alternatively, the method can be used to characterise different exosome subpopulations using exosome-specific membrane proteins as bona fide markers (for example, tetraspanins). Double staining experiments have also been carried out to acquire further information on exosome subpopulations, and transmission electron microscopy analyses are then performed to assess the integrity of the purified exosomes.
All of this information can then be used to optimise these manufacturing and purification steps. For example, these methods supported the selection of the best filter for depth filtration, and helped decide which membrane to use in the tangential flow filtration step to improve the conditions throughout the purification process. These experiments have also led to the development of an improved method of purification to increase exosome recovery.
Exosome manufacturing challenges
There are many challenges associated with exosome manufacturing, and good manufacturing practices must be carried out. These need to be carefully tested to meet regulations and safety requirements, as the quality of the exosomes is of the utmost importance in order for the process to be effective and safe to use. A sufficient quantity of exosomes is also key for experiments. By extension, the costs of producing the exosomes is a factor which needs to be considered.
Innovation is the key to increasing quality and quantity as well as decreasing cost. At Lonza, the careful analysis and optimisation of each step of the manufacturing process has decreased these challenges. This allows for effective production, which in conjunction with increasing automation can help with decreasing costs. This means that exosomes can be produced successfully at high concentrations to be used in experiments. These experiments are the basis for future exosome treatment and provide a pathway to clinical use.
The future of drug delivery
The work that has been undertaken at Lonza has led to an optimised method of manufacturing and purifying exosomes. This is important in order to gain more clarity on how they work and to carry out experiments which test their safety and efficacy. All of this is done with the final aim of using exosomes as a novel and effective drug delivery system and regenerative therapy. Delivering drugs directly to cells in this targeted way can be more efficient as well as being cost effective. Modifying this naturally occurring process in a safe way could revolutionise the future of targeted therapy for many different diseases.
Lonza are working to optimise the exosome production process, with exciting implications for drug delivery.
Davide Zocco Head of Exosomes Development, Site Head
Lonza focus on developing and optimising treatments that prevent illness and support healthier lifestyles. They facilitate innovation and manufacturing technologies to enable pharmaceutical customers to serve their patients and consumers.
Marcos Langtry Director of Commerical Development, Allogenic Cell Therapy.
Creative Commons Licence(CC BY-NC-ND 4.0) This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Creative Commons License
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