QCM-D is a real-time surface sensitive technology that is widely used to study interfacial phenomena at the nanoscale. In a recent webinar, Prof. Jackman, Associate Professor in the School of Chemical Engineering and Translational Nanobioscience Research Center at Sungkyunkwan University in South Korea, shared his experience using QCM-D for various applications in the field of membrane biophysics. As case example, he shared the story around a recent study where he and his team took on a membrane biophysics approach to identify better and functionally equivalent detergents to replace Triton X-100, an industrially important detergent which is currently being phased out due to safety concerns. This is what he said.
Using detergents to inactivate membrane enveloped viruses
Based on the early studies of anti-microbial lipids, lauric acid (LA) and glycerol monolaurate (GML) [1], I want to turn our attention to the analysis of surfactant-membrane interactions in a more applied context, Prof. Jackman says. I want to focus on what I call a detergent application, and this example focuses on how we can inactivate membrane enveloped viruses.
Many different types of viruses have lipid membrane coatings which are necessary for their structural integrity and for their functional behavior such as infectivity. The most widely known example these days is perhaps the COVID-19 virus, SARS-CoV-2, which is membrane enveloped. Other important examples include influenza and dengue, and on the agricultural side, African swine fever virus. One of the most important ones from an industry perspective is HIV, which is also an enveloped virus.
It is a big issue how we can use detergents to ensure safe blood supplies. For example, when we have blood donations, we can use detergents to ensure that there is no contamination or pathogenic viruses in them. Similar thinking has also been widely used in cell-based manufacturing, where often a million cell lines are used to produce biopharmaceuticals of choice like, for example, antibodies. When using cell-based manufacturing, there is always some concern about viruses being present in the system. This has led to a huge industry interest in viral inactivation in cell-based manufacturing, Prof. Jackman says.
Detergent-based virus inactivation
There are many ways to do this virus inactivation, including solvent-detergent mixtures using an acidic pH environment, irradiation of various sources, or virus removal strategies, Prof. Jackman continues. One of the most common approaches, which is widely used in ensuring safe plasma supplies as well as in cell-based manufacturing, is what we call “detergent-based virus inactivation”. This is particularly effective for inhibiting membrane enveloped viruses, i.e., viruses that have a lipid bilayer coating. How this works is that the detergent is membrane active, i.e., it disrupts membranes or solubilizes them. So, if an enveloped virus comes into contact with this detergent, it will be inactivated, Prof. Jackman explains.
The most widely used detergent Triton X-100 is being phased out
In the industry, for several decades, Triton X-100, which is a nonionic detergent, has been the most widely used one for these kinds of applications, Prof. Jackman says. However, there has been a problem recently and that problem is that the Triton X-100 is being phased out, especially in the European Union, where certain regulatory concerns have popped up in terms of safety.
Worldwide efforts to find compounds to replace Triton X-100
There are several pros of Triton X-100, Prof. Jackman says. It enables fast inactivation of enveloped viruses, and it is easy to use in manufacturing processes. However, the problem is that a phenol containing metabolic product of Triton X-100 is a known endocrine disruptor, and because of this, it is being phased out in the European Union. This has led to wider efforts worldwide to find good, environmentally friendly replacements that work similarly, but do not have these hazardous issues that Triton X-100 poses. So, there has been a huge need to find replacements. In the literature, many companies such as Eli Lilly, Biogen, and Takeda have published reports recently searching for compounds to replace Triton X-100.
The challenge of finding a similar working replacement for Triton X-100
One of the challenges of finding a replacement for Triton X-100 is that we want it to work similarly, i.e., it should have similar efficacy and ideally a similar mechanism, Prof. Jackman says. But it is challenging based on traditional bio-based discovery to search for detergent candidates that meet those criteria. Current experimental approaches are quite slow and expensive. They usually involve virus- or sometimes bacterial inactivation assays and require strict safety requirements. Also, they typically focus on endpoint rather than mechanism. So, they look at how much the virus infectivity decreases by, for example, rather than how virus effectivity decreases. And they often demand time intensive expert labor, he says.
Using QCM-D and a biophysics-based approach to complement bio-based discovery
So, we were interested in seeing whether we could complement bio-based discovery efforts by focusing on a more biophysics-based approach, Prof. Jackman continues. To do this, we were particularly interested in a paper by Eli Lilly [2] that was published a few years ago talking about the identification and characterization of a Triton X-100 replacement for virus activation. In this paper, it was discussed how a particular glycosides surfactant called Simulsol SL 11W had similar virus inactivating properties to Triton X-100 and therefore could potentially be a suitable replacement.
Comparing how Triton X-100 and Simulsol SL 11W interact with membranes
These conclusions were based on biological data, specific virus inactivation data, and we were interested in considering this further in the case of how both compounds might interact with membranes, Prof. Jackman says. TX-100 is known to interact with membranes, and Simulsol SL 11W also disrupts membranes. Since TX-100 and Simulsol SL 11W could be considered to have similar biological properties, we were interested in seeing it from a biophysical perspective - do they also disrupt membranes similarly? We wanted to look at factors like concentration dependance and mechanism of action to see how similarly they really work at a biophysical level to complement what is known from their biological activities. So, when we took this membrane biophysics approach [3], what we were interested in doing was studying the interactions of Triton X-100 and Simulsol SL 11W with supported lipid bilayers, SLB platforms, on silica surfaces using QCM-D technology, he says.
Different CMC values
Before we did the QCM-D measurements, we measured the CMC, the critical micelle concentration, of these two compounds, Prof. Jackman continues. The CMC is important because it is oftentimes related to the onset concentration of a detergent’s membrane-disruptive activity. The results showed that Triton X-100 has a CMC in PBS of around 300 uM, whereas Simulsol has a CMC around 2300 uM. So, even before looking at the membrane interactions, we saw that the CMC values were very different.
QCM-D tracking shows that Triton X-100 causes complete SLB removal with rapid disruption
We then proceeded to look at QCM-D tracking of the detergent-SLB interactions and here we saw very different behavior in terms of how TX-100 and SL-11W work in terms of disrupting membranes, Prof Jackman says. What we saw in the TX-100 case was that when we added detergent to the supported lipid bilayer, we saw a rapid increase in the frequency change corresponding to lipid mass loss from the sensor surface. And after we did a washing step, all the mass was removed from the surface. So, this led us to conclude that Triton X-100 causes complete SLB removal with rapid disruption, he says.
QCM-D tracking shows that SL-11W causes negligible SLB removal with slow interaction kinetics
On the other hand, in the SL-11W case, when we added the detergent to the bilayer platform, we saw a slow and gradual decrease in the frequency, which is suggestive of changes in the membrane budding but not solubilization, Prof Jackman explains. After we washed, the signals went back near the baseline values but did not indicate solubilization. So, we saw a completely different type of interaction with SL-11W. Membrane solubilization was not observed, instead we saw negligible SLB removal with slow interaction kinetics.
QCM-D reveals that Triton X-100 and Simulsol SL 11W interact with membranes in different ways
So, these two compounds disrupt membranes in very different ways, Prof. Jackman says. This was further confirmed through looking at concentration-dependent behaviors and a pie-to-pie comparison. We saw in terms of the maximum interaction responses, that SL-11W caused extensive membrane remodeling, whereas TX-100 did not directly cause solubilization. But in terms of final measurement responses, the QCM-D results supported that TX-100 causes membrane solubilization and SL-11W does not. So, we saw very, very different changes in terms of how TX-100 and SL-11W interact.
Using a membrane biophysics approach to identify better and functionally equivalent detergents to replace Triton X-100
Based on the QCM-D results, we were able to see that TX-100 causes membrane solubilization of the SLB platform as a model system, whereas SL-11W did not, Prof. Jackman says. We think that this membrane biophysics approach can help us identify better and functionally equivalent detergents to replace Triton X-100. We have now expanded this type of research to look at over 15 different types of detergent replacements and are building out a mechanistic model of what works not only biologically, but also biophysically to retain the efficacy of TX-100 as well as identify detergents that work similarly mechanistically, he concludes.
To learn more about how QCM-D is used the field of membrane biophysics, watch the webinar by Prof. Jackman.
