0:00
Yes, good morning.
0:00
So my name's Mark McDonald.
0:02
I'm a Senior Business Development Manager with EUROAPI working in our CDMO division.
0:07
I'm pleased to say we've got some of our leading chemists and scientific minds with us as well this morning to help talk about peptides and oligonucleotides and some of the recent developments in our R&D area.
0:24
Just as a brief introduction, I promise I won't take too long.
0:27
A bit of background on EUROAPI.
0:29
So we're, were sort of born in May 2022, so coming up to our 2nd birthday headquartered in France, but were a spin out from Sanofi.
0:41
So we've got a long history in the pharmaceutical areand a lot of expertise and knowledge still retained in our in our manufacturing sites.
0:51
We have an extensive API portfolio, so with over 200 APIs manufactured.
0:58
And also what we'll talk about a little bit more today is the CDMO platform. In terms of footprint…
1:06
So as the name might suggest, we're heavily based in Europe.
1:10
So we have sites in the UK, France, Italy, Hungary and most of our focus this morning will be in our sites based in Germany.
1:20
So we have Frankfurt, we have quite a large site there centred around oligonucleotides and peptides.
1:28
So scales from R&D up to commercial.
1:31
And also, we've recently acquired a site in Gera also in Germany, which used to be BianoGMP.
1:37
So focused on really small scale R&D activities, but also GMP, but again focused on I think bringing products through very quickly on, on short timelines in that facility.
1:52
So to support all the sites we have a number of different modalities and technologies that we cover.
1:57
So the R&D platforms are split essentially by product type or by activities.
2:04
So we cover small molecule activities, so traditional APIs and chemistry, peptides and oligonucleotides.
2:15
Also we have a strong biotechnology area in fermentation and enzymes, and we have a platform in particle engineering.
2:23
So we have very large spray drying capabilities in the UK and we have some micronisation capabilities as well.
2:33
I think just briefly, we have a very strong focus on ESG as well and some strong performance in this area.
2:41
The key points to pick out would be 100% of our sites will have renewable energy sources by 2025 and what we'll touch on in the presentation later.
2:54
Certainly on solvent recycling and solvent minimisation, it's a key topic in the peptide and oligo space and something we're focusing on very strongly.
3:07
Thank you very much.
3:07
So I'll hand you over Torsten to talk about our capabilities.
3:11
Good morning everybody.
3:12
My name is Torsten.
3:13
I'm heading the R&D activities for EUROAPI in Germany and be the platform lead for oligonucleotides and peptides and give you a little bit of a glance what we are capable to do in oligonucleotides and peptides.
3:25
And then we will follow up with some R&D activities we've done for innovations and for pushing the borders.
3:33
So one big thing we have a long tradition.
3:37
So in the 70s we started synthesis of peptides in solution phase in the industry park in Hoechst.
3:45
And we already had several decades on solution and solid phase synthesis in the industry and have far more experience and more than two decades already on oligonucleotides, peptides in solution and solid phase activities.
3:59
And last year we bought a company as Mark said BianoGMP in order to complement our activities from really the late research stage up to the commercialization and industrialization and have a nice story together with our colleagues and Gera.
4:14
So close connection in the Frankfurt side and Gera our equipment is mostly relying on Cytiva.
4:22
We starting with the early capabilities.
4:25
We do have development capabilities starting from the milligram scale app to the multi kg scale.
4:31
We have industrialised already oligonucleotides, ASOs up to commercialization stage and have a long experience on doing all clinical development stages including the analytical method development, validation phase appropriate through all the phases in clinical and being part of a lot of filing dossiers and also regulatory meetings.
4:55
As were embedded in the pharma companies.
4:57
So the teams are used to being part of a pharma team.
5:01
So they used to be more or less with our customers and speaking the same language.
5:06
That's one of our great pluses in the facilities.
5:08
We will shortly implement a new Asahi Kasei synthesiser in the facility which will complement our capabilities.
5:15
This will be online this year which is ranging that up to 600 gram scale and complementing the equipment from the OP400 in the oligo process in our facilities. Same activity we have for the peptide synthesis, we have the capabilities doing solid phase synthesis from the milligram scale up to the kg scale where we have also commercialised one large peptide.
5:42
For those who don’t know of peptides, if it’s above 40 amino acids it means it's already a BLA in the US.
5:48
So we are used already to dealing with these new kinds of regulations on BLA standards for peptides above 40 amino acids. And we can range again here from the analysis of the method development throughout the whole development of synthesis chromatography and then final release.
6:08
So we are capable of doing that also for the peptides here we have scaling up with a stainless steel up to 280 and we are about to extend our capacities to multi-hundred kgs at the moment.
6:20
That's the outspent for the next years.
6:24
And we are capable of running multiple kgs at the moment with that in our commercial environment or in the pilot environment where we do have two CS Bio 936s.
6:34
That's the capability - which the 936 could also be switched to commercial as they are embedded in a commercial plant.
6:43
As a chemical company as Mark said we are spread out with a large number of people and the tradition of medicinal chemistry and chemistry APIs.
6:53
So we do have a large capability in terms of vessels.
6:56
So we can do a liquid phase peptide synthesis in our EUROAPI network which is capable of doing that to a multi-hundred kg scale.
7:05
So we are having the sites in Frankfurt, Vertolaye, and Budapest, which has the capability to welcome large peptide projects, and we have the capability to do development also in our pilot scale facility in Budapest.
7:21
And then handing over to any of the larger scale vessels either in Budapest, Vertolaye, or Frankfurt. We will set up a new chromatography line which is state-of-the-art, which will be a two 80cm column set up chromatography for oligonucleotides fully embedded all the environment in Class C, it will be state-of-the-art.
7:50
At the moment you can see it's still some of the vessels are in plastic.
7:54
So that's the picture from last week.
7:58
So we are in the preparation phase for the running the PQ.
8:02
So it will be ready operational and welcome products in Q3.
8:07
So we will run the PPQ and the PQ readiness until the Q3.
8:12
And then you do have a state-of-the-art environment where we can handle in principle everything which is even going to low endotoxin measures for like CNS projects.
8:24
So that will be a really nice facility and will be set up in this year.
8:32
One short example of that, what we can deliver, I can't reveal the product, and can't reveal the customer, but it's a recent example of an oligonucleotide phase two project, which was transferred to us.
8:45
And this project was coming out of phase two readiness running already the clinical trials with an optimised process, we thought.
8:53
So were pretty capable of by the tech transfer and just an implementation.
8:58
We didn't do any big development scale.
9:01
We just used our experience and our standard settings where we treat ASOs.
9:06
It's not a small ASO, it's a longer chain ASO and treated that within two months in our lab feminization and welcome the product into our facility and could be able to already get it down from the synthesis time from 4 to 2 days.
9:21
We're able to reduce the solvents with our settings and our knowledge about how to reduce solvent reaction type down between 30 to 40% of the solvent consumption.
9:33
We're able to slightly improve the yield not too much, but slightly improved.
9:37
But it was also good is we could skip one cooling step which we didn't understand in the cleavage phase.
9:48
And also, that was the main goal of the company to reach 90% purity of their product because it's not easy to purify and we are the first time they’ve ever seen any kind of clinical batch purify up to 90%.
10:00
And that's just with implementation of the standard experience of the team.
10:05
So quite a good success.
10:08
Not every project is running like that, I must admit, but this is a nice example of what experience means in that field, even if it's a standard technology which is out there for 20 years.
10:20
Innovation is one critical point for us.
10:22
We dedicate 5 to 10% of our R&D people to innovation.
10:27
We really would like to push the borders for the field of oligonucleotides and peptides.
10:33
And so we do that in the areas of green chemistry, analytical chemistry, process optimization, and continuous process.
10:39
So we try to push in all areas the classical synthesis types, but also, we are trying to push the border in towards a new kind of technology.
10:49
So we are collaborating a lot of academia or companies.
10:53
I think here are two well-known companies mentioned where we look for continuous chronography and multi column chronography plus a new peptide synthesis flow capability with PeptiSystems.
11:05
But we have other collaborations which we can't reveal to push the border in the into the science in order to overcome some of the limitations of the technology means high solvent consumption, high energy consumption, low batch sizes.
11:19
So that's where we push the border and with our innovation teams.
11:25
And we will also see that we try to do that also for our continuous activities on the current technologies.
11:31
And part of that will be seen as R&D example by our scientist colleague coming as the next part.
11:39
And that's the part where we can't over then really to the to the things where the science is done in our journeys.
11:45
Giving you the presentation.
11:48
Thank you, Torsten.
12:00
Hello everyone.
12:01
I'm Arjun.
12:02
I'm a scientist in EUROAPI at one of the sites.
12:06
And today I'm just going to present you a case study that how can we make peptide synthesis more sustainable, especially the by improving the processes.
12:20
So solid phase peptide synthesis is the centre of peptide manufacturing.
12:24
But we can't deny the fact that the solid phase peptide synthesis still has existing challenges, especially in the spectre of green chemistry, particularly the DMF, because that's the solvent which is used for the solid phase peptide synthesis.
12:39
And it's a restricted chemical and will be banned in the future, for example.
12:45
And it's definitely hazardous to health and as well as involvement.
12:49
Second, because you are more focused towards delivering the peptide.
12:55
So in that and also the chemistry demands sometimes you use reagent in large excess.
13:02
And when needed many times you also have to repeat the coupling reaction so that you can achieve more efficiency and better peptide.
13:13
And given that you have to do multiple steps of these couplings, and each of the steps are involved in some reactions.
13:21
So you have to do multiple washing as such you involve lot of solvent consumption as well.
13:27
So all these are a big challenge for in terms of green chemistry. Also, because different peptides will have different kind of sequences.
13:38
So it's going to be taking a time if you want to really optimise the process.
13:43
So that's also a very time consuming process.
13:46
And because it involves multiple steps. To tackle the green chemistry problem, there is a huge interest in developing different kind of green solvents and making it more the peptide synthesis is more sustainable.
14:01
And in that regard, DMSO ethyl acetate mixture is kind of coming as a promising choice.
14:07
But still it's going to be challenging if you start manufacturing peptides using these green solvents.
14:15
The reason is you will have to take a risk of doing the optimization of any new coming peptide sequences using the solvent and because the chemistry is not well known for all kind of reagents and the sequences which will be behaving with the DMSO ethyl acetate solvent.
14:37
And one way to make it faster is to understand the chemistry, how it is happening and stuff.
14:42
And for that, we believe that process analytical tool is one way to go forward because it's very under explored and that can give you more information about what's happening.
14:54
And I'll this is what we have done in this case study, and I'll be talking more about this.
15:00
So here in this work we try to combine three things, green solvent, process analytical tool, and SpinChem.
15:06
For the green solvent we chose the DMSO and ethyl acetate because it already meets the requirements as a chemical as well as the logistic requirements for solid peptide synthesis.
15:17
For the process analytical tool I'd like to highlight that we are analysing the solution, not the reagents.
15:26
So we are analysing the solution in real time.
15:29
So every six seconds we have been analysing the solution in this case.
15:32
And for this we have select selected two tools, Raman spectroscopy and refractive index.
15:38
Raman spectroscopy because it can give you multiple information about the reaction at the same time.
15:44
Because if you are using two reagents, those reagents can have different footprints and fingerprints.
15:49
And you can get the information about these reagents with the with the Ramen. Refractive index and is easier to use.
15:57
It's very user friendly and can give you similar kind of information.
16:01
So we are also going into more mechanistic insights as well as having a way so that it can be applied easily to the GMP and stuff.
16:10
SpinChem is an alternative way to do solid phase peptide synthesis.
16:15
It's a kind of rotating bed reactor.
16:18
So you have you can see the cylindrical thing where inside it you put your reagents in your reactor and do same thing as a solid phase biosynthesis here.
16:31
An advantage of using SpinChem for our case is because it facilitates the assembly of the process analytical tool in our case.
16:45
So in case of peptide synthesis, you do assembly of amino acid one by one and each amino acid has to go through a synthetic cycle, and each in synthetic cycle involves two reactions.
16:56
And of course after that you have to do the washing.
16:58
So the reaction is the coupling reaction, then you will do the coupling washing, you do deprotection and then you have to do the deprotection washing.
17:05
So our first step was to test that our PAT can give a response to each of these steps.
17:15
In case of coupling reaction, the initial coupling solution includes your amino acids and the DIC which is a coupling reagent and Oxyma as your additives.
17:30
So why do you need to use a coupling reagent? Because you have to activate the carboxylic acid group so that it can react.
17:37
So if you see in here that this reagent will always be present because you try to take in a bit of excess.
17:44
So you can track that along with that Oxyma which gets consumed first and regenerated.
17:50
So you have two reasons for which you have the possibility to look into your spectra. Here this was the first thing.
17:58
So we tried, and we can see the couple of distinctive peaks which in the case of amino acids, it corresponds to the Fmoc group and all the amino acids we are using for our solid phase synthesis generally have the Fmoc group.
18:13
So that has been a kind of fingerprint for it.
18:15
And in case of Oxyma, were also getting a peak at around 2200cm.
18:22
So this was the other peak.
18:24
So we had basically two pieces of information about a coupling reaction if you want to get a better insight about it.
18:33
So we tried checking the coupling reaction with various reagents.
18:37
And this is what the pattern we have been observing.
18:40
For example, the red one here is for the amino acids.
18:45
So you can see over the period of time, you can see the reaction is completing and the depletion, the consumption of the amino acids and you can see over period of time it's plateauing.
18:56
So which means the reaction is the kind of complete.
18:58
It's always good to have another reagent for further validation and that in our case is Oxyma because you can see that Oxyma is quickly getting consumed.
19:07
But over the period of time it starts forming back.
19:11
And here you can see that that's also start plateauing.
19:13
So to information, we had two markers just in case of coupling reaction which is confirming that our reaction is complete.
19:22
In case of deprotection basically you try to remove the Fmoc group and for that you need a basic condition.
19:40
And when we looked at the spectra, we can see that both the peaks have a distinctive feature in the spectra.
19:47
So for PP reading, we see in this reason and for this we see in around this reason.
19:51
So this again become a new marker to get inside to our deprotection reaction.
20:00
And this is how the graph looks particularly in the deprotection case because it is a general trend of doing deprotection in two cycles.
20:17
So in the you can see consumption happens very fast like within two minutes is done.
20:24
And then the product, which is DVF adduct also starts forming very quickly and then it's saturated.
20:32
So basically from here you can get the information and then maybe in many cases you don't need to have the second cycle because it's already complete.
20:40
But still you we are doing it.
20:42
It's not just that you get a lot of information here and you can tweak your parameters in real time if you need.
20:51
Along with these reactions, piperidine washing is particularly of more interest because if you don't wash your piperidine very nicely, then you are basically making your purity worse.
21:05
So it's better to have a better piperidine washing.
21:09
So for this we have done multi variant analysis and made a regression model which can predict the piperidine residue of up to 0.02%.
21:22
In our case we consider that OK, the maximum will be trying to have is 0.2%.
21:28
So that was kind of marker in our case that we decided to have.
21:32
So with all for all these steps, when we had the marker, now our next step was to go for the peptide synthesis.
21:40
And in this case, this is what the SpinChem can help us.
21:42
Because with using the SpinChem, were able to have our own setup.
21:47
No need to have any synthesiser.
21:51
We made our own synthesiser, which is equipped with the online PAT.
21:55
So in PAT you can see that we had the raman and the refractive index when you are doing the reaction, the solution is being pumped through this tube with the tube says connected with the refractive index and raman and you are getting the information every six seconds in our case.
22:13
So as a model substrate, we chose Aib-enkephalin because it is a short model peptide and one of the reason people choose is because it is challenging synthesis.
22:23
The reason because of the stearic hindrance presents here.
22:26
And then second, we chose a longer chain peptide Gonadorelin because it is quite active API. In both the synthesis we saw that there was a tailoring needed for many of the steps and particularly the coupling reaction was the one where we needed a lot of changes.
22:51
So here you can see especially in the reaction time that the first three couplings were totally fine but as soon as you have to do another Aib coupling then it requires far longer time.
23:06
And this is the information which if you try to do in normal lab then you have to do an offline analysis.
23:12
Every time you will be taking some solution and checking if the reaction is complete or not.
23:17
But here we are getting this information real time whether the reaction is complete or not.
23:21
And along with that you also get impact on the last synthesis because Aib still has the steric hindrance, and this is what you can directly see it.
23:34
Same thing in the case of Gonadorelin, we saw that every coupling has different reaction time, which is not something unusual.
23:43
Of course, every reaction is going to be different.
23:46
But you can see that we can tailor the reaction time because some is taking more than two hours, some is being complete under 90 minutes for example.
23:54
And in doing general optimization, the pattern would be like OK, you will be either doing the whole coupling for two hours or three hours in case of for example, our greener solvent.
24:05
So if you are taking two hours then basically still you are saving 11% of the time.
24:10
But if you are doing three hours then you are saving 67% of the time.
24:14
Even though if you are putting two hours you are compromising with your quality because there are still couple of reactions which is taking more than two hours.
24:22
So this way in real time you can make the decision that how long you want to have a reaction to have.
24:28
But this is not the only case.
24:30
I would also like to highlight that you can instantaneously flag any deviation.
24:35
So here what we observed there was sudden its temperature changes and we saw that there was temperature change for one of our coupling reactions and particularly it impacted quite a lot for the deprotection.
24:48
So we saw that in this case, even two deprotection cycles were not enough to do the full Fmoc removal and we needed to do the third one.
24:56
And this kind of deviation, you will never be able to find it out if you are just doing a normal synthesis because here it’s giving us real time and we can quickly make the decision that oh yes, 2 cycles are not enough, let's do the third one.
25:09
But on the other side, as I said, piperdine washing is also very important.
25:14
We saw that the five washes are not enough to reach our maximum residual limit, which is 0.2.
25:20
So we had to do one more wash.
25:22
And this kind of information is very important if you are doing a optimization or like in general synthesis.
25:30
And once we have done the synthesis with SpinChem, then we use that PAT process condition and applied it on classical peptide synthesiser.
25:43
And what we observed that the purity was kind of quite similar, definitely bit better than in case of classical peptide synthesiser.
25:52
But I would also like to highlight here is that this was just in one attempt.
25:56
Usually if you get a new peptide, so with a different sequence and you are doing even just with DMF, you may have to do the reaction multiple times.
26:06
I mean the whole synthesis multiple times to know that OK, which coupling has which is causing issue and then you're going to repeat it.
26:13
But in this case, you just know the information in one go that which coupling has problem.
26:18
And I would also like to highlight that Aib-enkephalin has as far I know has the highest purity we have got just in one attempt. With this, I think I might have been able to convince you that PAT monitoring is a quick solution and good solution to push the boundary of sustainability in solid phase peptide synthesis because it can enhance the efficiency, improve your quality, it can give you instant flagging of deviation and it will let you make a quick decision.
26:51
Along with that, of course, SpinChem, you have seen that it the yields was not as great as the classical peptide synthesiser, but this is the first work.
27:01
Every, every research takes time.
27:02
Every technology takes time.
27:04
So of course if we try to do more development, we may find that SpinChem gives us a better yield and better way of doing solid peptide synthesis over a period of time.
27:16
So it was good working even with SpinChem.
27:19
But last but not the least, I like to highlight that this PAT technology is not just restricted to greener peptide synthesis.
27:27
You can apply it to even your existing classical peptides synthesiser if you want and it can be also transferred to oligonucleotides.
27:35
Thank you for listening.
