Exosomes are nanoscale bilayer membrane structure vesicles that cells actively secrete to extracellular secretion, carrying a large number of biologically active substances, which can play a role in material transmission and information exchange between cells as carriers. In recent years, exosomes have become the "rising star" of drug delivery carriers because of their advantages of good biocompatibility, low immunogenicity, and cross-blood-brain barrier. Both preclinical and clinical development of exosome technology as a delivery platform requires a large number of exosomes, and its isolation method needs to be easy to scale up and GMP compliant to support large-scale production (Figure 1). However, as mentioned in the previous article ( ), the establishment of an efficient exosome isolation and purification process is now one of the key bottlenecks faced in the industrialization of exosomes.
Figure 1 Schematic diagram of exosome production to clinical application[1]
Isolation and purification of exosomes
For therapeutic exosomes, the purity of the end product is critical for clinical application and regulation. The impurities that need to be removed in the production of exosome-based pharmaceutical components are mainly cell debris, proteins, host cell DNA, and non-exosome extracellular vesicles, such as apoptotic bodies and microvesicles. The size and physicochemical properties of exosomes overlap to varying degrees with lipoproteins, protein complexes, and chylomicrons (Figure 2), which also poses challenges for the isolation and enrichment of exosomes.
At present, there are some commonly used exosome purification methods on the laboratory scale, including ultracentrifugation, immunosegregation, polymerization precipitation, tangential flow filtration, molecular exclusion chromatography and microfluidic separation, but there is no consensus in the industry on the separation and purification process of large-scale production of exosomes. This is mainly because not all methods are suitable for the separation of large-scale samples, and different methods have their own advantages and disadvantages, and their separation purity and recovery rates are not the same (Figure 2) [2]. For example, the most widely used ultracentrifugation method can purify sample size and density, but is difficult to handle large samples; immunophilia capture acquires exosomes with high purity but can only specifically enrich exosomes expressing certain specific surface proteins; and microfluidic method is simple and fast, but is still only suitable for the extraction of small samples [3]. Recent studies have shown that tangential flow filtration and molecular exclusion chromatography (alone or in combination) can play a role in large-scale exosome purification [2], and we will combine the existing research data to introduce these two separation methods in detail.
Fig. 2 Yield and purity of different exosome isolation methods[2]
Tangential Flow Filtration (TFF)
Tangential flow filtration (TFF) is a form of filtration in which the direction of liquid flow is perpendicular to the direction of filtration, and is a pressure-driven membrane separation process based on molecular size [3]. At present, people use this technology to concentrate the cell culture supernatant, enrich exosomes while removing most of the protein impurities, and also achieve the purpose of liquid exchange in this process. In addition, hollow fiber ultrafiltration is a relatively gentle process that preserves the structural and functional integrity of exosomes while concentrating the fluid change [4].
Fig. 3 Large-scale exosome isolation by TFF method[5]
Gabriella et al. established a large-scale GMP-grade production method for preparing cardiac progenitor exosomes (Exo-CPC) as a pharmaceutical product, where they collected up to 8 L of exosome-containing medium after cell culture and isolated exo-CPC by tangential flow filtration [5]. For the specific operation, they first used a bladder filter (ULTA Pure HC 0.6/0.2 mm Capsule Filter, Cytiva) to clarify the supernatant, and then used a 300 kDa hollow fiber filter column (Cytiva) in the ÄKTA Flux 6 system to concentrate the sample to 300-500 ml, and added a 5-volume buffer for washing and changing the liquid, and recovered 200-300 ml of sample. Finally, it is filled after aseptic filtration to obtain GMP grade exosome products. As shown in Figure 3, after testing, it was found that this production method can achieve a recovery rate of 89%, and the removal rate of heteroproteins is as high as 97-98%. This suggests that the tangential flow filtration process can be applied to large-scale GMP-grade exosome production. Of course, there are also relevant studies that show that in order to obtain exosome samples of higher purity, tangential flow filtration technology is often used orthogonally with other technologies, such as ultracentrifugation, molecular exclusion chromatography and ion exchange chromatography.
chromatography
Molecular exclusion chromatography uses differences in molecular size between exosomes and other components in the sample to separate, a method that maintains sample integrity and biological activity, and can be scaled up well, with the ability to process a large number of samples. There are many studies that have successfully isolated and purified exosomes using this technique, however, this method also has its disadvantages, such as slow flow rate, volume limitation of loading, and sample purity that needs to be further improved.
Based on this, the multimodal chromatography filler Cytiva Capto core 700 has become a new favorite in the field of exosomes. Capto core 700 consists of a ligand-activated core and a 5 μm inert housing, the inert shell does not allow macromolecules exceeding 700 kDa to enter the nucleus, while the small molecules in exosome samples, such as host cell proteins, DNA fragments, etc., enter the nucleus and bind to octylamine groups with both anion exchange and hydrophobic effects to achieve the purpose of separation (Figure 4). This multi-mode filler separates exosome samples in flow-through mode for higher loading volumes, and the new generation of Capto scaffolding meets the requirements of high flow rates, efficiently capturing contaminants while saving operational time.
Fig. 4 Principle of Capto Core filler purification exosome technology[6]
Ryan et al. completed the isolation of cell culture supernatants and plasma samples using tangential flow filtration combined with PEG precipitation techniques and Capto core 700 chromatography [7]. After the completion of the pretreatment, the sample was used to perform approximately 10-fold ultrafiltration concentration using a 750kDa hollow fiber filter column (UFP-750-E-4X2MA, Cytiva) using the ÄKTA Flux S system, removing small molecule impurities and performing liquid exchange at the same time. Subsequent RNaseA treatment, PEG precipitation, and one-step Capto core 700 chromatography improved exosome purity as well as homogeneity. Their experimental data showed that this method obtained a higher concentration of exosome products compared with simple ultracentrifugation and PEG precipitation, and the size distribution was more uniform, with corresponding biological activity.
Of course, different exosome applications have different requirements for their separation and purification, in addition to the previously mentioned tangential flow filtration technology and molecular exclusion chromatography technology, other separation methods can also be combined. For example, two-step chromatography of Capto core 700 and Capto Heparin has been studied to isolate exosomes [8].
As mentioned earlier, the exosome production process includes upstream cell culture, harvest clarification, concentrate change, separation and purification, and sterilization filling. As shown in Figure 5, Cytiva can provide full-process equipment and consumables, such as bioreactors, HyClone media, 3D microcarriers for upstream cell culture, as well as downstream ultrafiltration equipment, chromatography equipment, hollow fiber membranes, and a variety of chromatography fillers. In process development, in order to meet different exosome purification needs, in addition to the preferred Capto core 700 multi-mode filler, chromatography fillers such as Sepharose 4FF/6FF, Capto Heparin and ion exchange can also be tried.
Figure 5 Schematic diagram of exosome process flow
References (swipe up and down to view):
- Jafari, Davod, et al. "Improvement, scaling-up, and downstream analysis of exosome production." Critical Reviews in Biotechnology 40.8 (2020): 1098-1112.
- Herrmann, Inge Katrin, Matthew John Andrew Wood, and Gregor Fuhrmann. "Extracellular vesicles as a next-generation drug delivery platform." Nature nanotechnology 16.7 (2021): 748-759.
- Wang Qian, Zheng Lei, editor-in-chief. Extracellular vesicles—basic research and clinical applications[M]. Beijing: Science Press, 2019
- Colao, Ivano Luigi, et al. "Manufacturing exosomes: a promising therapeutic platform." Trends in molecular medicine 24.3 (2018): 242-256.
- Andriolo, Gabriella, et al. "Exosomes from human cardiac progenitor cells for therapeutic applications: development of a GMP-grade manufacturing method." Frontiers in physiology (2018): 1169.
- Whitford, William, and Peter Guterstam. "Exosome manufacturing status." Future medicinal chemistry 11.10 (2019): 1225-1236.
- McNamara, Ryan P., et al. "Large-scale, cross-flow based isolation of highly pure and endocytosis-competent extracellular vesicles." Journal of extracellular vesicles 7.1 (2018): 1541396.
- K. Reiter, P.P. Aguilar, et al. “Separation of virus-like particles and extracellular vesicles by flowthrough and heparin affinity chromatography.” J. Chromatogr. A 1588 (2019) 77–84.
- Capto core 700 mutimodal chromatography datafile