Exosomes: Considerations for Cell Culture Conditions and Scaling-Up
January 17, 2022
The relatively untapped potential of exosomes, as considered in our first blog, brings about the need for continued research and development for new biotherapeutics and their manufacturing processes. In this blog we share some considerations for upstream processing and look at some of the challenges we currently face.
There are several parameters to consider when determining cell culture conditions and scale-up efforts when manufacturing exosomes and each should be carefully considered to retain cellular characteristics. Choices such as whether to use serum or serum-free media in the bioreactor, or understanding the effect of hydrodynamic forces from the bioreactor conditions on the product, make a difference.
It is important that the production process does not alter the characteristics of the parent cell. If these characteristics are changed, this could affect the production, size, composition, or marker distribution, and has the potential to alter the overall function of the exosome. This could lead to the production of a subpopulation that will have a significant impact on the safety or efficacy of the final drug product.
Traditionally, fetal bovine serum (FBS) is added to the growth medium in the bioreactor for the growth of anchorage-dependent cells. The challenge, however, is that FBS also contains its own exosomes. So, when scaling into production, the presence of exosomes from the FBS could contaminate the culture, with the possibility of being present in the final drug product. If utilizing FBS, this can be managed, and the adherent cells could be washed several times with a fresh medium and analyzed to quantify the presence of the FBS-derived exosome in culture. There are also some commercially available alternatives to using FBS, such as serum-free media, and exosome depleted serum. Previous studies have shown that the use of serum-free media can lead to an increase in the production of exosomes. However, in these studies a shift in the biology of the exosome was observed due to the stress induced from the absence of serum. If taking a different path, the use of exosome depleted serum does not mitigate all the challenges that are associated with the use of serum, such as source and lot-to-lot variability and virus contamination risk. So, there is no universal solution that works for all.
Suspension cell culture also carries limitations. If not controlled, the bioreactor parameters such as oxygen sparging and agitation may induce cellular stress that can lead to phenotypic alteration of the cells. This cellular stress could alter the exosomes that are produced. Under high stress conditions which affect cellular integrity, apoptotic bodies could be produced, introducing another class of contaminants with sizes partially overlapping with that of the exosomes.
Technologies Available for Cell Culture Production
For exosome production, cells can be cultured either in adherent mode, in which cells are attached to surfaces, or suspension mode, in which cells are suspended in the medium by agitation.
Exosomes that are designated for therapeutic or diagnostics are produced from stem cells, primary cells, or less established cell lines. The majority of these are anchorage-dependent and will need to be grown in a tissue culture flask (T-flask or multi-trays) but a high density of cells is needed for efficient production scale-up. For adherent cell culture, scaling-up requires a larger surface area. This can be achieved with large multi-layer T-flasks or fixed-bed bioreactors. A hybrid system utilizing adherent cell cultures on microcarriers in a stirred tank reactor can also be used for production. In comparison to suspension culture production, the scale-up of adherent cell culture in this way can be more time consuming, more expensive, and less efficient.
Advances in cellular engineering have made it possible for the use of cell lines such as human embryonic kidney cells (HEK293) to be cultured in suspension mode. These cells can be grown in serum-free media and are known for their rapid growth rate, ease of cultivation and high transfection efficiency. They also exhibit high productivity of exosomes. Several biologics have been approved by the Food and Drug Administration (FDA) and European Medical Agency (EMA), produced through this cell line.
Using modern upstream bioreactor technologies, scalable volumes of exosomes can be produced in suspension cell culture and experience from similar processes provides a solid foundation to support the journey to large scale manufacturing.
There are clearly specific challenges associated with the upstream production of exosomes. However, these challenges can be overcome as we continue to understand the underlying mechanism of how to produce exosomes with the use of native or engineered cell lines. The current research and market landscape reveals that these challenges are worth solving because the potential therapeutic or diagnostic capabilities of exosomes could be a game changing opportunity for the biopharmaceutical industry.
To learn more about exosomes, and how to overcome any potential challenges in producing exosomes you can watch our on-demand webinar.
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Juliet Kallon, Bioprocess Specialist, Scientific and Laboratory Services
Morenje Mlawa, Bioprocess Specialist, Scientific and Laboratory Services
Nebiyat Tsegaye, Bioprocess Specialist, Scientific and Laboratory Services
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