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Well, good afternoon everyone and thank you for joining.
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Really appreciate you giving us some of your time here.
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So I'm I basically represent the TA instruments division of Waters and what I'm going to do today is introduce you to a new high throughput screening platform for looking at the thermal stability of protein therapeutics and oligonucleotides.
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Before I do so, however, I just want to highlight that Waters have a very nice analytical portfolio for biopharmaceutical research.
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I'm sure a lot of you know Waters for their LCMS systems.
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You probably are aware of Wyatt technology for light scattering techniques, which are excellent for characterising biologics.
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But maybe some of you don't know that Wyatt were bought by Waters two years ago.
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TA instruments on the other hand have been part Waters since 1996 and our focus is materials characterization techniques as a function of temperature.
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Specifically in the pharmaceutical and biopharma arena.
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We do a range of techniques for the biophysical properties characterization.
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And then obviously Waters have precision chemistry products as well as informatics platforms.
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So here we see an overview of the TA instrument solutions for biotherapeutics.
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One thing I want to highlight in particular is calorimetry.
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So techniques such as ITC, isothermal titration calorimetry and differential scanning calorimetry are considered gold standard techniques because they allow the analysis of biomolecules in their native state.
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They also contain a lot of other additional useful information because they track thermodynamics and this can provide very useful insights into the solution environments for a biomolecule.
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The rapid screening DSC, which is the platform I'm going to talk to you about shortly, is specifically targeted at higher concentration therapeutics and I'll come on to talk more about that in a second.
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We have rheometers to accurately measure the viscosity of biotherapeutics, which help in designing delivery systems as well as optimising manufacturing processes.
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And then finally, our traditional thermal analysis techniques such as DSC and TGA, while they generally applicable for small molecule characterization in biopharma research, they're very good for optimising freeze drying cycles, for example, and understanding the stability of a lyophilised product.
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And then if you're working in a GMP environment, we have software solutions to support you in that.
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I think it's clear that the use of antibodies, engineered proteins and oligonucleotides is on the rise specifically because it reduces the pressure on healthcare professionals by making a lot of these therapeutics administrable by patients themselves either subcutaneously or ophthalmically.
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However, developing high concentration therapeutics, I'm sure you will know better than I do, is not easy and there's a lot of challenges including stability issues where you can get protein aggregation, denaturation, which will affect and shelf life issues, as well as viscosity.
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Generally, as you go to higher concentrations, you're dealing with higher viscosities, which can be difficult to manufacture as well as administer.
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So as scientists, you need to have robust analytical solutions and techniques to really help you guarantee the quality of the therapeutics you're making.
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I'm going to focus here on stability.
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And when I mentioned stability, I'm talking about short term thermal stability.
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So how do we currently measure this?
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Well, there's two techniques really.
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There's differential scanning calorimetry, DSC and nano DSF, which is a fluorescence based technique.
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Both of these are good.
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They have their advantages.
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They also have their downsides.
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DSC generally provides high precision quality data.
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However it's a relatively slow technique because you can only run 1 sample at a time and you need relative relatively large amounts of sample and you often need to dilute your samples which further complicates the workflow.
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Nano DSF on the other hand is very high throughput technique and it is kind of accepted that the data quality is not as information rich as DSC, but it depends where you are and whether this is suitable or not.
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So what we set about trying to do was see if we could bridge the gap between these two and this is where the RS-DSC fits in.
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So the RS-DSC is essentially a high throughput platform, but we've retained the quality of data that you would come to get from a DSC.
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And the other advantage is it really gives you the ability to measure formulation strength concentrations, which can provide some interesting new insights.
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So just a little bit about the platform, it can analyse up to 24 samples at the same time.
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So you can imagine compared to traditional DSC, this could really accelerate, you know, the development process.
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It's a dilution free technique and it allows you to test, as I said, formulation strength concentrations, which can provide new insights.
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You don't need to clean anything.
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The chips are disposable, so they're single use and more importantly, they this eliminates run to run contamination.
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The technique only the chips only require about 11 micro litres of protein or sample.
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And then finally, we've got some nice, automated analysis features in our software, which really speed up the processing of the data and access to the critical stability information.
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So in summary, the RS-DSC really simplifies as well as accelerates thermal stability screening.
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For those of you maybe not familiar with DSC, just a quick recap.
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DSC basically measures the difference in heat flow rate between a sample and an inert reference as a function of temperature.
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So in this example data set we can see a monoclonal antibody being scanned up in temperature and we see peaks as different domains unfold essentially.
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So what's happening is the samples absorbing energy and to go from the native to the unfolded state.
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So the critical parameters we use are the maximum peak temperature, which usually signifies the thermal stability.
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We also capture T onset, which is the start of the unfolding process.
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DSC is a gold standard technique as I mentioned earlier.
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It really is an information rich technique which can provide a lot of useful information as well as just these thermal stability criteria.
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And as you can see from the bottom, what I'm talking about is thermodynamics.
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Because we're measuring the delta H, the enthalpy, we can determine thermodynamics, which can be useful indicator of the solvent or the, you know, solution environment of the biomolecule.
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TA instruments have a family of DSCs which are all suited to particular points in, you know, in the workflow.
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So our most sensitive DSC is our nano DSC which works with liquid samples.
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It's got fixed in place capillary cells which you can see hopefully in the picture top.
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So you prepare your sample into these cells.
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It uses relatively large amounts of sample, but it is very sensitive and it can work with anywhere from sort of .1 mg/ml up to about 20 mg/ml.
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But you do have to be careful with these type of design of DSC because if you go too high in concentration and your protein, the nature's while it's in the cell and it forms a gel like structure, it can cause a problem because it's often very difficult to clean.
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So scientists and users often have to worry.
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Watch out for that.
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If you were to screen samples on the Nano DSC, you can automate it so you can use 96 well plates and it would typically take probably about a week to screen 96 samples.
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On the other hand, the RS-DSC could probably screen that in a working day.
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The RS-DSC like the Nano uses liquid only samples only 11 micro litres of sample and it's really targeted from sort of for a typical antibody 20 mg/ml up upwards 200 mg/ml.
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With oligonucleotides, we can generally get down to about 55 mg/ml in concentration.
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And then finally, our discovery DSC platform is ideal for optimising lyophilization cycles and it's also used for small molecule characterization.
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And obviously it can deal with solid as well as liquid samples.
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So thermal stability is a critical parameter in biologic drug development.
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And as we've, as I've already kind of mentioned, DSC is an excellent gold standard technique for assessing the thermal stability.
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And DSC is a modality agnostic technique.
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So it works with a range of biologics and modalities.
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You know, there's no restriction really.
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And obviously, it's a very powerful technique to understand the structure of your biomolecules, which dictates their function.
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And it's applicable in a wide range of areas, including candidate selection, formulation, optimization and so on.
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Just wanted to kind of share a couple of example data sets measured on the RS-DSC.
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So protein mutation is a commonly used technique for optimising structure and function proteins, and a single amino acid mutation can have a measurable effect on the thermal stability.
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In the first example, we see a small panel of antibodies which were tested on the RS-DSC.
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Firstly, we see the parent molecule which basically contains a single transition which encompasses the unfolding of the CH2, Fab and CH3 domains.
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When we add a single amino acid mutation 1, the blue data, it doesn't have a marked effect on the thermal stability and the general profile.
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However, in additional samples, mutations 2 and three, we where we've again just a single amino acid modification, we can see a marked effect in the stability.
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So we're seeing an evidence of shoulder in mutation 2, an actual peak which probably represent the CH2 domain, and then the RS-DSC, because of its ability to do parallel screening, is excellent for formulation screening studies.
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So in this example we have an antibody trastuzumab in a range of different buffers and you can see quite clearly that for the CH2 domain, there's no real difference in terms of stabilisation between the histidine, borate, or PBS buffers, whereas the succinate buffer has a dramatic effect.
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And it's no surprise that the most stabilising buffer histidine shown here is one that's used in the final formulation for this approved drug.
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Because of the concentration ranges it can cope with, the RS-DSC is also excellent for looking at the thermal stability trends as a function of concentration.
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In the first example on lysozyme, we ran a series of different concentrations, and we ran triplicates at each concentration.
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So firstly you can see the excellent reproducibility of the technique, and we can see here trend.
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As we increase the concentration, we see an increase in thermal stability to a point when we get to around 280, we start seeing the peak shift to a lower temperature and also evidence of an additional peak which indicates some sort of degree of higher order structure.
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Chymotrypsin on the other hand, shows a kind of reversed effect whereby as we increase the concentration we shift to we get a thermal stability decrease.
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In this example, we ran a pair of oligonucleotides and first thing we noticed was that as you increase the oligonucleotide length, you get an increase in concentration.
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I think that's probably would be expected as well as sorry increase in stability.
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And then also as we increase the concentration, we see an increased thermal stability as well.
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And as I mentioned earlier, we can reliably typically get down to five mg/ml concentration generally because oligonucleotides tend to involve higher energy transitions.
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In the final example, we're looking at ADCs and basically in a couple of different buffers.
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So firstly we have the parents parent antibody and then we've got two different systems with different drug loadings, both anti-cancer drugs and we can use the RS-DSC very effectively to screen for the most stable formulation.
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And we can see here that the ADC 2 which has positive impacts in of thermal stability on both the domain unfoldings is the most stable formulation.
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Just wanted to kind of show you a little video of the hopefully this works.
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So you can see the loading of the chips here.
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And then that box on the left hand side is self-adhesive glass cover slips, which are placed on top of the chips using the tweezers supplied.
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And then these are punched to seal them and then they're loaded into the calorimeter.
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You can see on the right hand side the reference chips, which are reusable.
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And then a cover lid is placed on top to ensure the chips are in good contact with the calorimeters.
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Lid is closed and then you're ready to go.
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Here we see an overview of the calorimeter blocks.
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So essentially there is 4 calorimeter blocks and there's six calorimeters in each block.
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As a reference to give you an idea of relative size, we put a penny in the picture and you can see that basically on the positions 3 and 4, you can see the cover lids, which are there to make sure that the chips are in good contact with the calorimeters.
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And you see a side view of the calorimeters, one of the calorimeters there and the calorimeters have integrated and arrays of thermal piles to ensure accurate temperature measurement.
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Here we can see the just a screenshot of the analysis software where you've got.
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Obviously we're dealing with much larger data sets, so you need a more streamlined way to look at this.
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So we've introduced automated peak detection, up to five transitions, baseline detection and so on.
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If you wanted to go in and do a bit more detailed tweaking of the parameters, you can use our wizard to have a step by step approach to looking at the baselines and the peaks and T onsets.
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And then we've got some quite nice visualisation tools to enable looking at, you know, the large data sets that are generated.
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So in summary, the RS-DSC is an excellent approach to look at challenges for stability testing in high concentration biotherapeutics.
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It's tailored to high concentration formulations.
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It can handle high viscosities, typically 80 centipoise or less.
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There isn't really a limit, but obviously it's about the pipettability.
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Then we can do truth formulation strength testing.
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So there's no dilution required.
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This really simplifies the workflow.
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We use single use microfluidic chips, so there's no cleaning necessary, low volumes and it integrates well into complementary workflows.
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And that's pretty much all I have to say.
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Thank you for listening.
