Downstream Purification Solution of Exosomes

What is an Exosome?

Exosomes are cell-derived vesicles with a diameter ranging from 30 to 150 nm. They contain endogenous proteins and nucleic acids and can alter the biochemical characteristics of recipient cells through the transfer of biomolecules, thus playing a key role in cell communication [1].

Exosomes can serve as biocompatible drug carriers to combat various diseases, such as organ damage. However, the development of exosome-based therapies still faces numerous challenges, including their heterogeneity and low production yield etc.

Figure 1. Pathways of Apoptotic Bodies, Microvesicles, and Exosome Formation

Exosome Downstream Purification Solutions

Figure 2. Exosome Production Flowchart

The market demand for exosomes is continuously increasing. To achieve large-scale production of exosomes, it is necessary to develop and optimize production processes. Based on extensive data feedback from the application side, the technical team at Cobetter has made the following preliminary membrane filtration selection recommendations.

Figure 3. Exosome Downstream Purification

Clarification Procedure

1. MSC Cell Clarification Case 1:

Initial turbidity of cell supernatant: 23 NTU;
Model selection: CHT50x1;
Parameters: Final pressure differential 14.5 psi, loading capacity 925.4 L/m²;
Filtrate mixed turbidity: 10.1 NTU, yield >80%.

2.HEK293 Cell Clarification Case 2:

Initial turbidity of cell fermentation broth: 427 NTU;
Model selection: CL40x1;
Parameters: Final pressure differential 10.2 psi, loading capacity 69.17 L/m²;
Filtrate mixed turbidity: 19.2 NTU, yield 112.4%.

TFF Ultrafiltration Steps

Model Selection: Cobetter-Minilab-30 Hollow Fiber (300 kD, 0.5 cm², Diameter 0.5 mm)

Parameters: Shear rate = 4000 s⁻¹

Concentration Step:

• Transmembrane pressure (TMP): 2.20 psi
• Volume concentration factor: 18.51x
• Average flux: 42.22 LMH (liters per square meter per hour)

Dialysis Step:

• Transmembrane pressure (TMP): 2.15 psi
• Volume dialysis factor: 10.09x
• Average flux: 30.91 LMH

Nanoparticle Tracking Analysis (NTA) was used to measure the particle size distribution of the ultrafiltration concentrate, and the distribution was similar to that before the concentration. When comparing the vesicle count before and after concentration, the vesicle recovery rate in the concentrate was 103.37%.

Figure 4. Exosome Hollow Fiber Ultrafiltration

Compared to traditional centrifugation methods, using hollow fiber ultrafiltration for exosome concentration and collection offers several advantages, including simpler and more flexible operation, lower shear force, minimal vesicle damage, and higher product yield. Additionally, hollow fibers have better scalability, and our company's various product formats can also meet customers' needs for exosome production at different scales and volumes.

Sterile Filtration Steps

MSC Cell Ultrafiltration - Case 1:

Using the U25 SMDS sterilizing filter, under an average flux of 294.51 LMH, with a final pressure difference of 1.15 bar and a filtration load of 134.41 L/m², the vesicle yield was 45.547%.

Exosomes are present in all living organisms and are virtually ubiquitous, including in archaea, algae, mammalian cells, bacteria, higher plants, and fungi [3]. Cobetter's technical team has conducted extensive testing on exosomes secreted by bacteria, mammalian cells, and higher plants, continuously optimizing solutions to accelerate the research and development process for their clients.

Of course, based on the source of exosomes and their specific applications, targeted purification solutions can also be proposed. The above information is limited; for more detailed requirements, please feel free to contact Cobetter's technical team. 

Reference：

1.Possibility of Exosome-Based Therapeutics and Challenges in Production of Exosomes Eligible for Therapeutic Application ；Takuma Yamashita, Yuki Takahashi,* and Yoshinobu Takakura Graduate School of Pharmaceutical Sciences, Kyoto University; 46–29 Yoshida-Shimo-Adachi, Sakyo-ku, Kyoto 606–8501, Japan. Received February 16, 2018）

2.Exosome Theranostics: Biology and Translational Medicine；He C, Zheng S, Luo Y, Wang B. Exosome Theranostics: Biology and Translational Medicine. Theranostics 2018; 8(1):237-255. doi:10.7150/thno.21945.

3.DE S N.Enterotoxicity of bacteria-free culture-filtrate of Vibrio cholerae[J].Nature,1959,183（4674）:1533-4.