Therapeutics and cosmeceutics industries are increasingly recognising the potential of extracellular vesicles (EVs) as biologics. As cell-derived, nanoscale phospholipid bilayer vesicles, EVs carry a diverse assortment of proteins, nucleic acids, and small molecules that can influence recipient cells in a huge variety of ways. The specific characteristics of EVs, which are dictated by their origin environment (i.e., cell of origin and that cell’s environment), define their impacts on recipient cells, making EVs highly malleable.
Therefore, there is great potential for identifying and even engineering EVs which will have a positive effect on specific cosmetic or medical conditions. However, the journey of creating EV-based biologics poses complex challenges, particularly in terms of production, isolation, and quality control.
The batch problem: the inconsistencies of scaling up
Scaling up EV production requires culturing donor cells (i.e., cells of origin) in either a bioreactor or other large cell culture vessel. Regardless of whether your cells need passaging or exist in a continuous or near-continuous culture system, conditioned culture media will be extracted for EV isolation in batches of some form. Inevitably, there will be some between-batch variation in EV size, concentration, zeta potential or composition. Other parameters will vary to different extents such as soluble protein levels, pH, endotoxin levels, or even the presence of dead cells. Although these differences are somewhat unavoidable, monitoring these fluctuations is essential; deviations beyond acceptable parameters may necessitate the rejection of a batch or even the retirement of the bioreactor or culture system. This testing, and the resulting decisions, are critical for producing a consistent, safe, and effective product.
Monitor, monitor, monitor: the importance of quality control
The size, concentration, and zeta potential of particles can be monitored both within the raw conditioned cell culture media and once the EVs have been isolated. These two timepoints in the process give two different pieces of information.
The first, taken on raw samples, indicates if the EV characteristics of the sample fall within acceptable parameters. Whether EVs need to be concentrated for this measurement depends on their concentration in the initial sample. This preliminary measurement serves as a checkpoint, giving you the yes/no signal of whether to isolate EVs from that batch. As isolating from an unsuitable batch is a waste of staff time and consumables, significant resources can be saved. Additionally, if EV parameters are subpar, this assessment could indicate that it is time to retire the bioreactor from which that particular batch. Such real-time monitoring of bioreactor quality is essential for efficient EV production at scale.
Next, the EVs should be monitored for size, concentration, and, if desired or required for your product, zeta potential after isolation and any post-isolation concentration. The acceptable parameters here will be different from pre-isolation parameters and likely more stringent as this is closer to the final product. For example, you may have a narrow EV concentration range which is acceptable, as well as a minimum particle-to-protein ratio which will indicate isolate purity.
A range of particle measurement technologies can be employed for these important monitoring steps, but not all are created equal. Only Tunable Resistive Pulse Sensing (TRPS) instruments can measure all three factors – and they do so with only 35 µL of sample for each measurement.
Tuning into TRPS for large-scale EV isolation
TRPS uses the Coulter principal to measure the size, concentration, and zeta potential of nanoparticles such as EVs. As particles pass through an aperture – which we call the nanopore – they disrupt the electrical current running through the nanopore. The magnitude of the resulting disruption – referred to as a blockade – is directly proportional to the size of the particle. Concentration is determined by the rate of particles passing through the pore, and the zeta potential is determined by measuring the electrophoretic mobility of particles in a zero-pressure state. TRPS is capable of measuring size and concentration or size and zeta potential in a single measurement. If all three are important, all three parameters can be collected in two successive runs using the same 35 µL sample.
The principles and mechanisms of TRPS makes it a data-rich technique as you can identify both size and zeta potential information on each individual particle in the measured population, giving you a wealth of important information. Such capabilities provide a key advantage over other methods, all of which can only ever give you either size or zeta potential information on your particles. Many methods, especially those based on light scattering cannot do this on an individual particle level. Instead, they give ensemble measurements on a whole population level.
Ensemble measurements therefore are often unable to identify all or even any subpopulations in polydisperse samples such as EVs. Failing to identify the presence of an unwanted subpopulation (e.g., a new, uncharacterised population) could present a major issue for your product’s performance. This makes TRPS not only your best bet, but also your only bet if you value the accuracy of the measurements you take on your product.
TRPS guiding EV isolation in the field
Our latest TRPS instrument, the Exoid, is currently in the field in several therapeutics and cosmeceutics companies doing just the kind of quality control jobs discussed above.
Figure 2 shows purity data for one of our commercial customers who were aiming to isolate functional EVs from very large quantities of conditioned media. As you can see, the Exoid was able to accurately measure the concentration of EVs (which was then used to determine purity) at the stage of the raw conditioned media, following concentration by tangential flow filtration, and post size exclusion chromatography qEV isolation. This shows the suitability of the Exoid for quality control measurements at every step of the EV collection and isolation process from large volumes of conditioned culture media.
The development data (anonymous for confidentiality reasons) shown in Figure 2 is from what is now known as the qEV PurePath for Therapeutics service (Figure 3).
The future of EV therapeutics: when resolution counts, count on the Exoid
The rate at which the EV therapeutics field is growing seems almost exponential. The demand for our qEV PurePath for Therapeutics service attests to that, as do the number of clinical trials and publications on the topic increasing year on year. As the industry races towards the clinic or even cosmetic shelves, TRPS quality control measures are essential in making sure EV therapeutics reach – and remain – on the market.