So yes, thank you for the kind introduction, and thank you for all coming to the talk today. So I'm going to be talking about polysorbates and a little bit beyond that, and looking at different detergent strategies for biologic formulation.
The QR code you'll see on the front allows you to scan it and you can get a copy of the presentation. It will appear a few more times through the course of the presentation.
So the topics today, really around some of the proposed detergents for biopharmaceutical formulation stabilisation, go into a little bit on understanding polysorbates and a little bit on some of their alternatives.
We'll then talk a little bit to Super Refined Poloxamer 188 which is a launch, which is as recent as last October. And then we'll just summarise the talk.
Would you mind just pushing the slide forward?
Maybe that okay, okay, now it's working.
So, I mean, we've already had a little bit of discussion in this room earlier today on some of the issues which are reported with polysorbates, the fact it is quite a complex molecule, and that can lead some challenges in terms of the analysis of the product.
There are things around the stability of polysorbates in pack and around their oxidation then in use potential for some chemical instability, oxidation, enzymatic hydrolysis, which then can lead to fatty acid liberation, which then, consequently can lead to some sub visible or visible particulates, as well as, obviously the loss of the detergent.
Now personally, I think a little bit with this something along Jennifer Owens garden, many of you'll have never heard of this, but this is officially the most diverse environment in the entire globe.
So Jennifer Owen, she basically studied her garden for 30 years in absolute detail, she wrote two books, one at 15 years, one at 30 years, she discovered over 2,600 species in her garden, 741 square meters. She discovered 20 new species to the UK, and this was just a little garden in suburbia, and she discovered four new species entirely to science.
And I think this is a little bit where we are with some of this polysorbate investigation, where we've got a huge body of history of data that we can look into these things, and people have been looking into the minutiae and all these things in terms of degradation and effects that happen.
But actually, what we do know is just a hell of a lot, but we also know the good points and the bad points. And we need to kind of understand a little bit more where polysorbate really is and where it is to some of those alternatives. And you know, we're trying to do that with a little bit on Croda. So, you know, maybe at the minute, we're sort of maybe not quite at the 30 year point of knowing everything, but maybe we're at least at the 15 year point.
So really, what we're trying to do is look at understanding polysorbate understand its behaviour, how it works, consider what the impact of that is, and whether there are things we can do within the polysorbate chemistry.
But then also look at known chemical alternatives. And also, then sort of potentially look at what sort of things are there in novel chemistry which can stabilize pharmaceutical formulations. But obviously all of these come with, you know, increasing and changing levels of risk in terms of what we know about the products.
So in terms of what Croda offers, in terms of detergents in biopharma injectables, if we look at just a simple scan on biopharma circle for everything that was an injectable that was utilized in a biologic which also includes some of the RNA vaccines and so on,
You'll see that actually, obviously, it's no surprise that polysorbates are the most used and Poloxamer 188 but actually, a lot of the other surfactant type materials are things which are also on the Croda’s range, as you can see from having the Croda trade name in the in the final part of the table there.
So, we have an interest in looking at some of these different species, and we offer a lot of them.
This is what's kind of used actually commercially. And as I said, some of these are kind of more LNP related, or things that you can no longer use in Europe, like Triton X-100, or Octoxynol-9.
But then also, if you look in the literature, these are things where we actually sell a lot of these materials as well. So obviously, polysorbate and poloxamer are well known. But Croda also offer Poloxamer 407, we offer fatty alcohol othoxylates, and a number of different materials which have been sort of cited potentially for use within, you know, the biopharmaceutical formulation space.
So from a Croda perspective, it's kind of worth us looking at what all of these do, because we sell most of them, and it's good for us to obviously understand so we, as we said, looking to do this sort of combination of understanding polysorbate, but also exploring what else is out there.
So, in terms of understanding polysorbate, I've already mentioned that it's this complex molecule and sort of potential for some of these different stability issues, in terms of kind of that element around complex molecule analysis. That's something that's largely sort of now on top of in terms of the analytics which are available in terms of stability for polysorbates in pack. This is the kind of thing that generally we can solve by using the appropriate grade of polysorbate. So as Croda, we offer standard compendial compliance, monograph compliance polysorbate under the Tween brand 20 and 80. We sell high purity grade HPs, which essentially are kept under nitrogen and are made a little bit gentler, so they have slightly lower levels of impurities in them initially. And then we have super refined grades, where we
basically polish the product to remove some of those impurities. And those are our super refined grades of polysorbate 20 and 80. And then there's some around slightly different fatty acid distributions in our pure oleic acid range. And also, we have a low steric acid between 20 HP, which is more for a specific prevention and precipitation of fatty acids if they're liberated.
So we offer these different polysorbates, and essentially, by using particularly the super refine grades of these, we can negate those oxidation issues that we tend to see in storage because we've removed the impurities and we've removed the oxygen, which initiates the instability.
If we look at some of the in-use stability, we also see that some of our super refined grades can have a positive effect here. So, this is an example where we have standard between 20 compendial material and look at its stability at 0.04% in a histidine buffer, type one glass, so a very typical placebo formulation for a biopharmaceutical, and we see that there is, you know, an increase in non-ester product and corresponding decrease in ester materials and in free fatty acid liberation when we use the standard product, what we see no change when we use the super refined product.
And this is just an example of how that use of a higher quality polysorbate can prevent some of the hydrolytic stability that we see in, you know, more usage type conditions. So we see that with polysorbate 20, we also see that affecting the drug product, drug substance as well.
So this is an example where we've taken rituximab, we've stored it for six months at 25 degrees C, and when we use our super refined polysorbate 20, we see a much reduced change over six months under these kind of accelerator conditions, where we don't see a big spike in the DLs and aggregation of the product, which we do see if we use the standard grade of the poly sorbate 20, and we see some corresponding data that supports the second from SDS that they the protein is more stable.
If we then look at that same product in 12 month five degree storage, this just with the DLS data shown, we see again that corresponding increase in particle size due to aggregation with the standard grade of the polysorbate, whereas if we use the super refined we negate that.
In this particular case, actually, the control with no surfactant, was quite stable. But we do know if we agitated this particular formulation for about 24 hours, it also aggregated really badly.
So the surfactant was important to be there also, you know, we're also trying to understand more and more about the degradation enzymatically of some of these surfactants as well.
So this is a study where we've added 100 ppm of [unclear] triglyceride lipase and looked at the degradation of the polysorbate for 20 hours. Now this is a very high level, so it's really degrading the sample very quickly, which is what we intended to do, and looked at what happens in terms of the concentration effect.
And what we see is the effect in polysorbate 20 is that the triglyceride based lipase is degrading some of the higher order esters. But we also see, as we drop in concentration, actually the amount of detergent left starts to plateau out.
So it's not a case of, you know, you might see some very dramatic instability at 0.1% but actually, when you start going down in concentration, the amount that's actually remaining in the product is not necessarily tailing off quite so much.
But in terms of understanding, there are still things that you know we need to get our head around. So one of the confusing things if we do the same study with polysorbate 80, even though the enzyme is designed to degrade some of the triglycerides, what we actually see here is the monoesters are the least stable component of the ester matrix, and actually the diesters and the triesters are the ones that remain.
So there is a little bit more understanding, but we still see that same effect, that actually, as you drop in concentration, you still see a significant proportion of the polysorbate actually remaining. And in terms of which part is left and which, how much is left, actually something to stabilize the product is probably a very small amount.
We know polysorbates are incredibly efficient. And we also know in terms of kind of the components that are actually used, that actually all of them have a stabilizing effect. And this is just an example of a paper we did with BI where we looked at different fractions of polysorbate 20, and we saw that maybe there was some slight differences in terms of the efficiency of a few of those different components.
But ultimately, if it's a soluble ester component, so a mono-ester, or a di-ester component of the polysorbate, it successfully stabilizes the formulation at still relatively low concentrations.
So, in terms of alternatives, we're also looking at sort of screening different surfactants and seeing their physiochemical behaviour, and then looking at how those affect aspects like interfacial clearance of protein and surface interactions and stability of model IgGs, and also looking at degradation, potentially on some of those excipients.
So we looked at one example, sort of under static conditions where we look at the stability the IgG. We did this predominantly through looking at thermodynamics and shifting to a less stable form of the IgG, and also by DLS, and then these sorts of instances where you have quite static data, static conditions, we actually see that, you know, in this particular case, we didn't really need the surfactant in terms of stabilising the protein, but we also saw there were not necessarily any negative impacts from some of these other detergents that we could potentially use.
And we saw that, apart from a through erroneous results in the DLs data as well, but if we look at some of the more kinetic elements and look at some of the surface interactions, this is a study where we did interfacial rheology of BSA solution as a model protein and we looked at the elastic modulus of the surface and what we would do is set up the study look at how elastic the surface was, and then dose in the detergent, and actually look at what the drop off in the elastic modulus was.
So, demonstrating that the surfactant got between the solid surface and the protein interaction surface of the of the solution. So, kind of trying to model what might happen at the, you know, wall of a vessel.
If we do this study with polysorbate, we dose it in the elastic modulus. Well, we get the elastic modulus goes up. We dose it in after three minutes, and we see down to very, very low concentrations, this immediate clearance of the protein interaction with the interface. It's incredibly interfacially active.
And the study runs for two hours, and we see that that effect remains. If we look at different types of surfactants, so things like poloxamer 188, 407 and here at half their CMC concentration, based on CMC by DLS.
Then we see that pretty much the polysorbates are still the only ones that have that clearance effect that actually remains for the full two hours of the test, and what happens is there's a re-displacement of the protein with the interface for some of these other surfactants.
Now, if we actually go to higher concentration and go to twice the CMC, we actually see a more permanent clearance of the interface, so it's dropping down to a very low elastic modulus equivalent to water. So there is a capability of surfactants to do this kind of activity, and other surfactants to do it, but it probably isn't as efficient as what we tend to see from what polysorbate would do.
And if we kind of look at agitation studies with IgG over 24 hours. This is the stability of the main peak that we see for the protein by DLs. And we see that the polysorbates are sort of relatively unaffected by whether they're half or twice the CMC and the blue and the green bar, but we see some of the other species far more affected, some of the other detergents, so things like Poloxamer 188 and some of the fatty alcohol PEG based materials as well.
And if we look in lyophilisation again, we see that kind of same sort of story there. We do the reconstitution. Obviously, you want it to look the same.
At point 0.01% polysorbate 80, we can use that to give a successful reconstitution.
The light green is a little bit difficult to see, but it's basically the same before and after. Whereas we can do that again with sort of poloxamers, but we often need some slightly higher concentrations.
But just to take that forward a little bit, we've been looking at some of these different ranges. Last year, as I said, we launched our super refine poloxamer 188. Primarily, we've launched this for cell culture.
So we've basically been looking to optimize the product and the species to ensure high viable cell density and a high percentage of viable cells for CHO and HEK based bioprocessing and other mammalian cells.
But we also kind of looking at how this might perform in some formulation type studies. In terms of stability, we know that the core poloxamer chemistry there is stable at 25 degrees C for six months in a 10% solution.
Again, this kind of works come more from pre solutions that we might use for cell culture, but we're understanding what the stability is, and see that it's still good at formulation type conditions for long periods of time.
And also looking at some of it in terms of the stability for the drug substance as well. So here was a study we did with adalimumab, and we looked at all these different detergents that we kind of been looking at, and we saw actually the most stable product in this particular example was with our Super Refined Poloxamer 188 which is the line that you can see across the top.
So yeah. In summary, there are obviously some risks for hydrolytic polysorbate degradation, but this can be largely mitigated by using super refined polysorbate whether that's in the neat material in the container, or whether that's also carried through to what we see in use at some relevant concentrations in solution.
We've also shown how it can de risk drug substance degradation by utilizing the super refine polysorbate. We've shown that there's still some things to kind of understand around polysorbate degradation in terms of enzymes.
But maybe, you know some of the studies where it's often used at quite high concentration and with model enzymes, maybe doesn't tell us the sort of full story of what's going on, and a lot of the time these, there'll be a significant residue which still stabilizes the product.
We've looked at a range of different alkoxylate based protein surfactants, which can stabilise proteins.
And these need, potentially a little bit more optimization versus polysorbates, but they are also an option for looking at some stability within those criteria. And we also have the super refined poloxamer 188 which is another addition to what can potentially be used for protein stabilisation, as well as its applications and cell culture.
And with that, thank you, and I'll take any questions.
