Primary Separation Methods of Exosomes from Cell Culture

We first discussed the untapped and exciting potential of exosomes in our blog, The Potential of Exosomes, asking the question, could they be the next level of targeted cell therapies? We then considered cell culture conditions and scale-up of exosomes utilizing upstream bioreactor technologies. Here we explore primary separation methods with one eye focused on the end game – commercial production of life-changing biotherapeutics.

Over the years, several methods of cell culture clarification from the exosome of interest have been developed. For exosomes to be manufactured for clinical use, an ideal clarification method would have to balance the key parameters: high yield, process time duration, and scalability.

Traditional Methods for Exosome Separation: Differential Ultracentrifugation

Differential ultracentrifugation, or pelleting, is considered the most prevalent method for separation. With differential centrifugation, a cell culture sample is separated based on density, size, and shape. Differential ultracentrifugation is performed in a string of cycles at various speeds to remove cells, cellular debris, and apoptotic bodies. The final material obtained is the pellet which contains the exosomes and contaminants such as protein aggregates, viruses, and other microvesicles. 

Although differential ultracentrifugation is the fundamental method of separation it is considered time-consuming and labor-intensive. There is an equipment requirement with the possibility of mechanical damage and low portability¹. Most importantly, exosome isolation through differential ultracentrifugation may end up with low purity. At certain centrifugal forces, there is a high probability of extracting/pelleting other particles with the same buoyant density, size, and mass as the target exosomes. 

Traditional Methods for Exosome Separation: Density-Gradient Centrifugation

Researchers have attempted to overcome the low-purity challenges of the differential ultracentrifugation method through density-gradient centrifugation. The density differences between extracellular components can be exploited to separate exosomes from contaminants.  There are two types of density-gradient ultracentrifugation typically employed for exosome isolation: isopycnic density-gradient ultracentrifugation and moving-zone gradient ultracentrifugation.

Isopycnic density-gradient ultracentrifugation is a density-based separation technique whereby a medium, such as sucrose, is added in layers of increasing density from top to bottom to cover the range of densities found in the sample. The sample is then added to the top of the gradient medium, and typically centrifuged at a very high speed over a long period of time. The exosomes and contaminants, including protein aggregates, apoptotic bodies, microvesicles, and viruses, will settle in a static position in the correlating layer of the same density¹.

Isopycnic has its disadvantages because separation is based on density differences, but there is a risk of co-isolating exosomes and microvesicles since they may share similar buoyant densities¹.  This problem can be overcome with the use of moving-zone density-gradient centrifugation, a method that separates particles by size and density. 

The moving-zone gradient ultracentrifugation method has two gradient medium layers – a low density top layer, lower than all the solutes in the sample, and a very high-density bottom layer which acts as a cushion to protect against potential exosome pelleting. After centrifugation, the particles will be divided into different heights in the medium gradient¹. In this way, particles with the same density can be separated based on size. 

Although more intricate ultracentrifugation methods can produce sufficiently pure exosomes, it has many limitations. It generally processes low volumes, has expensive equipment requirements, can be extremely time-consuming and labor-intensive, and requires highly trained technicians¹. Additionally, the constant centrifugal forces can potentially impact the structure and function of the exosomes. Most significantly, these methods are not scalable for manufacturing. A scalable clarification process is necessary in order to successfully transition from research/pre-clinical development stages to a workable clinical commercial process.

Direct Flow Filtration: An Alternative to Ultracentrifugation

Direct flow filtration or direct interception is a separation process in which filters retain particles that are larger than the nominal retention. In this process, fluid flow is applied parallel to the filter or membrane to capture contaminating particles in the filter layer. The pressure from the driving force of the peristaltic pump drives molecules smaller than the filter pore size including the target exosomes through the filter resulting in clarified material that will be processed downstream to obtain purified exosomes. There are several types of filters that can be used for direct flow filtration such as prefilters, depth filters, bioburden reduction filters, and sterilizing grade filters. Depth and sterilizing grade filtration are two prominent types of filters that are used in the separation of exosomes from cell culture.

Exosome Separation Scalability with Depth Filtration

Depth filters are comprised of cellulose fibers, additives, and resins. These components create a porous matrix of randomly distributed fibers that result in a tortuous path for particles to be removed through adsorption and size exclusion. Filter additives are naturally occurring materials that aid in filter adsorption. Resins are macromolecules that act as a binder that allows the formation of filter sheets, increases the wet strength of the filter, and can impart unique charge properties.

Depth filters are also available as single-use options that are easy to operate and readily scalable from lab to commercial stage. They have been shown to provide high yields and high filtrate quality when processing exosome feed streams. One of the challenges of direct flow filtration is pre-mature fouling. Prolonged use of the filter during clarification will decrease the filter capacity and potentially result in clogging of the filter and membrane pores. Development scientists should consider retention ratings and characteristics such as charge when deciding which filters would be most suitable for their process. In order to avoid filter exhaustion, the clarification process must also be optimized to have a defined operational flux and an appropriately sized filter for a specific working volume.

Direct-flow filtration using depth filters can be used in line with a bioreactor as an efficient and effective option for the scalable clarification of exosomes. This technique of separation is widely used in exosome bioprocessing at the pre-clinical and clinical stages.

In comparison to centrifugation, direct flow filtration is cost-effective, scalable, has high yields, and can process larger sample volumes with a relatively shorter process time. To transition from early, to late-stage development and the ultimate goal – commercialization of life changing drugs and invaluable diagnostic tools – exosomes developers clearly benefit from high-performing clarification methods that scale up efficiently.

View this on-demand webinar to gain further insight into exosome production challenges and thoughts on how to overcome them.