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Exosome Downstream Process Solutions


In 2013, the Nobel Prize in Physiology or Medicine was awarded to American scientists James Rothman and Randy Schekman and German scientist Thomas Zudhof for their work in the field of regulating mechanisms of intercellular vesicle transport. For making outstanding contributions, this award has made the interest in exosome research rise again.


With the deepening of research, exosomes have gradually entered the industrialization stage from the field of scientific research. The number and scale of start-ups in the field of exosomes continues to grow. Currently, there are nearly 50 companies around the world "competing on the same stage", focusing on the four major fields of tumors, brain diseases, vaccine development, and gene therapy/rare diseases. For example, Exosome Diagnostics and Codiak Biosciences, a benchmark company in the treatment field, are leading the field of exosome diagnosis and have launched two products developed for tumors, which have entered clinical phase I; the huge potential of exosomes in drug delivery has led many pharmaceutical giants including Roche, Takeda, and Eli Lilly to enter the market, and four large-scale cooperations worth US$1 billion have occurred one after another.


Exosomes are small cellular vesicles with a diameter between 30 and 150 nm and a phospholipid bilayer, composed of natural human proteins and lipids. Its structure can be seen in the figure below. Most cells can secrete exosomes. By carrying proteins, nucleic acids, lipids and metabolites, exosomes can be used as a drug delivery tool, a treatment agent for diseases, or for damage repair and medical aesthetics related to regenerative medicine.


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VesiCURE official website-Schematic diagram of exosome structure


At this stage, the biggest challenges facing the industrialization of exosomes are the technical difficulties in clinical translation and the standardization of the review system. At present, only a small number of companies can realize high-throughput, automated, high-purity, and low-cost exosome isolation and purification technologies; exosome research and development started late, and clinical application is very early. Exosome organisms with high disease specificity are independently developed. Markers, establishment of exosome drug delivery platform, and screening of effective exosome drugs are the key points for exosome biotechnology companies to achieve breakthroughs. Finally, domestic standards for the production, clinical and quality control of exosome drugs have not yet been established. Therefore, exosome companies need to assist national regulatory authorities in building a corresponding standard system for review and supervision.


Next, let’s analyze the production of exosomes and the construction of a process platform. Because exosomes come from diverse sources, there are many cell types, such as MSCs that can be cultured adherently, HEK 293 cells that can be cultured in suspension, etc., and their downstream There are also many options for process flow. Ultracentrifugation is usually used at the scientific research level, and industrial routes suitable for amplification and industrialization are also gradually maturing.


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Exosome production process


For the culture of exosomes derived from HEK293 or other suspension types, you can choose Bio-Link CytoLinX® WB and CytoLinX® BR, which have also achieved excellent cell culture performance on the 50L scale:


Schematic diagram of Bio-Link CytoLinX® WB and CytoLinX® BR equipment

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Performance of HEK293 cells cultured with CytoLinX® WB


In the downstream process, the combination of ultrafiltration and chromatography technology is a downstream process technology suitable for process amplification and capable of large-scale production. Since the diameter of exosomes is 30~150nm, the vesicle structure is sensitive to shear force. Therefore, when selecting hollow fiber ultrafiltration, attention should be paid to the pore size, material, and ultrafiltration conditions (flux, transmembrane pressure, loading capacity, shear force) optimization. The ultrafiltered samples are subjected to chromatography purification to obtain high-purity exosomes.


MaXtar COLL 700 or MaXtar COLL 400 is a multimodal chromatography medium with a porous passivation layer in the shell and hydrophobic and ion exchange in the core. Macromolecules above 700KD/400KD directly pass through the external water; impurities such as host proteins and nucleic acids enter the shell and combine to achieve the purification effect.


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Currently, COLL 700 has been used in a variety of macromolecule applications including exosomes. Usually, the yield and purity are improved by optimizing the loading conditions, loading volume and flow rate.


For example, the application cases of COLL700 in macromolecular virus vectors are as follows, ultimately achieving a purity of close to 80%.


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