0:21
Good morning, everyone.
0:22
I'm happy to be here.
0:23
I'm happy to be able to present you some of the work we've been doing with our clients at Gyros Protein Technologies.
0:29
And I'll be talking about our advanced technology platform to manufacture GLP-1 analogs but also other complex molecules.
0:39
And I would actually like to start with the most striking finding that was, for me, the proof that peptides have finally become mainstream, which I found that South Park giving a full episode on that topic, which is really funny.
0:53
I encourage you to see it.
0:55
But yeah, more serious stuff.
0:59
So what's the role of peptide synthesis within the field of obesity treatment?
1:03
And we did some data mining on clinicaltrial.gov and enriched it a little bit with AI to get some more data.
1:12
If you're interested in the raw data, please just connect with me, I can share it with you.
1:15
And so what we see is that we have a split of small molecules versus peptides in development, which is all GLP-1 like all anti-obesity treatment distributed across the different stages with the slides or plus peptides in the later stages.
1:31
And if you look at the way that these peptides are manufactured, we see that solid phase peptide synthesis is the majority of those underscoring the need to develop novel and better solutions to meet the demand.
1:44
Now GLP one analogues are not formidable challenge themselves, but also represent common synthesis challenges for peptide synthesis.
1:52
So they're long accumulating impurities, which makes downstream processes more complicated.
1:57
They're often prone to aggregation and gelation, which is requires certain knowledge and expertise and handling up and downstream.
2:05
And also they're often modified.
2:06
It's not only lipids, not only pegylations, it's also sometimes, you know, conjugations to proteins and all that stuff that I've been hearing a lot in this conference to make a hit to a decisive lead through modifications.
2:23
But what are the more process related problems that scientists are facing when you follow the molecule from early stages to late?
2:31
And you know, let me introduce you to Belinda, my virtual friend, a peptides full stack scientist who wants to follow the molecule through this pipeline.
2:41
And so initially she would encounter problems with throughput, right?
2:46
You need a lot of synthesis throughput to get all your hits tested.
2:50
Then they become more complex with all those modifications and with complexity, the request for higher purity goes up.
2:57
So it's actually two difficult things in one go.
3:00
And when you start, you know, moving towards the clinic, you have to also anticipate scale up.
3:04
And that does not necessarily mean always like the kilogrammes, but also a few grammes that you would need in your tox studies, in your studies to advance fast through this process.
3:14
And you also have to anticipate ecological impact.
3:16
The higher the scale, the more impactful this will become.
3:19
We've heard a lot about green chemistry and the importance.
3:23
And I would assume that a candidate which is now in the earliest stages, we'll have much more requirements to be manufactured with green chemistry in the future.
3:33
So at this point here, Linda really has to anticipate what's coming next.
3:41
Now very simplified, the workflow is simple.
3:44
Upstream you synthesise your peptide, you cleave it, and then downstream you purify it.
3:48
I think Walter, he's not here this morning, but he showed it very nicely.
3:53
You have it like silos, right?
3:55
You have the upstream, you have the cleavage, it's like a bridge, and then you have purification.
4:00
Sounds simple in the first place.
4:05
And we asked our database what's the problem?
4:08
What's your biggest bottleneck within this workflow?
4:11
And every second scientist will tell you it's purification.
4:14
Now let's try to understand what is actually the problem.
4:18
I think it's fair to say that, you know, HPLC represents like 90-95% of the purifications being done for peptide purification.
4:26
It's an established gold standard.
4:27
Everyone uses it, everyone knows how to use it.
4:30
You can buy HPLCs with automation, sample orders, even auto collectors.
4:36
You can scale your column from grammes, micrograms to kilogrammes.
4:40
It's all possible.
4:41
So it's not really the capability of the HPLC that poses the challenge.
4:46
But that's my hypothesis and I'm more than happy to discuss what you think about it.
4:50
I think the major problem is that it's distinct technology, right?
4:55
What does it mean?
4:56
So if we look at Belinda here, she does the synthesis.
5:00
And I think the picture from Walter was so nice because it's actually separated from purification.
5:07
That means you need both infrastructure, you need synthesis infrastructure and you need purification infrastructure.
5:13
You actually need to bring your peptide from a very favourable, highly acidic environment into acetonitrile water.
5:21
You have to transfer it from the one to the other.
5:23
That also poses challenges, draws like takes time to actually get to that point.
5:28
And it's a process handover.
5:30
If there are two separate teams, you know, it's not like the HPLC is waiting for you, right?
5:34
So it will probably be queued up in a pipeline.
5:37
And so again, you lose time and that's what you really don't want during your development.
5:42
So it's either Belinda, if she does both, she does a lot of work on purification and that takes away a lot of time that she can actually spend on better stuff thinking about the synthesis and developing a better drug.
5:54
Or the purification team is overloaded with all those crude mixtures that they have to purify.
5:59
And maybe that's not necessary because there is kind of a solution and it's where we're catch and release purification comes into play because it's basically empowering the synthesis scientist to be able to do part of the purification, like do part of it.
6:14
Purification is the first line purification because it integrates seamlessly into the work of the upstream scientist.
6:21
And actually the synthesis know how, if you understand how to do peptide synthesis, you'd be able to do to get really good results with this technology as well.
6:30
And the master himself, the inventor of solid phase peptide synthesis, already knew that this is actually a good idea.
6:38
So what he was doing is Bruce Merrifield.
6:40
He tagged the target peptide and then purified on a separate media and then cleft of the purified peptide that was bound to that media and got a purified peptide out.
6:54
It's wild chemistry.
6:55
So it's not really applicable in sense, but it's the same concept that we're doing now at Gyros Protein Technologies as well.
7:03
The chemistry has evolved a lot and what we use here is a reductively cleavable linker molecule, but it follows the same concept.
7:09
You take your target peptide at the end of your solid phase peptide synthesis, you cleave it off and you continue working in fritted cottages, but you just swap the resin and get your purifications done.
7:23
Now, how does the workflow look now for Belinda?
7:25
Actually she owns part of the downstream work in her lab, right, because she basically extends the synthesis with this PEC technology because it integrates nicely into the synthesis workflow.
7:39
You don't need any additional instrument like infrastructure.
7:41
You can basically do it in a few more in micrograms to grammes.
7:45
You can do it in parallel.
7:47
So you can do it a few peptides.
7:49
You can do it in high throughput and one of the nice features as well as you can stick to your better solvents like DMSO, HFIP, TFA or even sometimes you can use your TFA cleavage cocktail to go right into the purification to get rid of precipitation entirely.
8:07
So it's either blend up, being able to focus more on the synthesis work or if you act work in two separate teams, it's less burden for the purification team and less burden for the HPLC column as well.
8:17
Because you go in with what we call a PEC grade peptide, which is just a purity enriched crude, if you will.
8:23
And that goes from 80 to 95%.
8:26
And if necessary, of course, sometimes you need those higher purities, you can go to your purification team and they will be more happy to get a 95% pure peptide for polishing than to start all from the start with a very messy crude.
8:40
I want to recall some of the data that has been already shared by Lorenzo 2 days back.
8:46
They use catch and release to manufacture liraglutide.
8:49
So here you see the concept, you start the synthesis, you tag it with a linker, you load it on the agarose beads or PMA beads, which is an oxyma ligation between the linker and the aldehyde modified material, and then you reductively cleave off the peptide.
9:04
In their case, they got 86% purity in a well, decent yield.
9:09
They also explored another route where they actually wait for lipidation until the peptide is bound to the agarose material.
9:16
So it's kind of a late-stage modification that you can chew on the of the unprotected peptide on the agarose resin.
9:22
And in this case they got a way higher purity and a similar yield.
9:28
Now what I would like to show you some data that we did in our own labs.
9:33
So we wanted to go to semaglutide with the intention to go into larger scales.
9:38
And I will focus on that like let's say before going to the CMC team.
9:43
But when you're actually your design of the peptides already done, when you need a synthesis that gives you enough material for your studies.
9:52
We explored both approaches like direct synthesis with a building block, a late-stage modification on PEC, but also compared it to late stage modification on the solid phase peptide synthesis resin.
10:04
So we started with an investigational run.
10:06
We checked all three approaches.
10:07
We very simple, 5 equivalents, room temperature, single couplings.
10:12
We just wanted to understand the synthesis, not looking so much into a good purity here, just to understand where do we need to get better in terms of coupling.
10:21
We use our chorus system which is parallel synthesiser and can give us UV monitoring data.
10:27
So to really understand what the system looks like.
10:29
And then we applied heat to the difficult coupling sites and added the PEC to the process very simply.
10:37
And this is how we get for the approach to, it's the direct approach where we had the lipidated building block to 93% purity and with a pretty good recovery.
10:48
Then we went for one millimole, which is still doable on the small-scale Chorus system where we chose only the best approach and went down to two equivalents having like a scale up in mind.
10:58
So we wanted to do something that is very like low in resources.
11:02
So it's better to transfer to larger scales.
11:07
The UV purity went down a little bit, mostly of course, because we went down in equivalents.
11:11
But this is something we're working on right now.
11:14
We didn't polish.
11:16
You can see if this is not enough, it's way easier task to go into a polish with HPLC to get to the real high purities you might need in your work.
11:27
Next is to transfer this to the Sonata system, which is our pilot scale synthesiser to get really into the multi gramme scale.
11:34
It's currently ongoing, but what I would like to show you that it's possible and we're pretty sure that we will get good results here is to show you another example of a collaboration where we worked with the Sherry T Berlin to get this disulfide and modified peptide of the contrast agent here in a high amount and good purity.
11:56
So the request was over 5 grammes with over 90% purity, but we wanted to have this with PEC like no HPLC needed.
12:05
And so we started with the synthesis and that did full PEC purification in the system.
12:10
So it's fully automated on the Sonata to get to the purity we wanted.
12:15
And we were able to deliver in time and very, you know, in a lean process, if you will, to get this material out.
12:23
So the beauty of this is once this works, you're really set up for scale up because one thing for with the PEC purification, the resin has a very high loading capacity and you work in your SPPS reactors basically.
12:34
So consider a 400 litre SPPS reactor.
12:37
You can purify a considerable amount with this technology while saving a lot of solvents and applying a truly orthogonal approach, right?
12:47
It's not just another chromatography, it does a chemo selective isolation versus a physical chemical separation, which is good because also the guidelines ask for orthogonal purity methods analytical wise.
13:01
But if you design in an orthogonal purification method from the start, this will give you huge advantage to actually build in like a truly orthogonal way to get your peptide done.
13:13
Note it's always needed some optimization; it's not something that really works out-of-the-box for all of the peptides.
13:20
That's why we offer services to design PEC into your process.
13:27
We do this in four steps, which is a process audit.
13:29
We look at the process, how does PEC fit into there?
13:32
Do a feasibility study just to see, OK, does it work?
13:34
Does the quality meets your requirement?
13:37
Then go into development, actually scale up to the requested scale and application.
13:41
And then we can either transfer the technology to the client, we can do it.
13:46
We can also transfer to CMOs if you need it in larger scale this way.
13:51
I want to highlight one of our long-standing collaboration.
13:54
I'm really happy Mitra that you made it here.
13:57
CEO of Pplus Medical, a company which we've been working for a quite some time to develop pretty sophisticated collagen related peptide which we actually were able only to get with this PEC technology.
14:12
And the modification patterns that we use here and the CRP-A as we call it or as Pplus Medical is calling, it's really great compound to work up.
14:23
I'm not sure if Cosmos Utical would be a good name for it, but it's a really a good molecule.
14:31
And if you want to learn more about it, you can check some information here or talk to Mitra, of course, directly.
14:41
So this is the team in Berlin that works on these PEC projects.
14:52
They're basically all representing one Belinda because each of like all of these projects are always done by one person because we're able to actually build in both synthesis and purification in a very lean fashion and with low, let's say requirements of infrastructure and capacity.
15:11
So, and this allows us using this technology to explore multiple modification pathways to find the best possible solution for the candidate they are looking at.
15:21
Depending on the purity requirements, might be enough to use PEC only, which is great because you didn't really get rid of the entire HPLC downstream work.
15:31
Or we can include it as a complementary autonomous system to get higher purities.
15:36
And it's designed for manufacturing as we call it, because once you are able to work with PEC in your smaller scales, you'll be able to upscale easily because it's doable and it's actually green and it gives you benefits across the board when going into larger scales.
15:53
So get in touch.
15:54
I'm happy to take any questions.
15:58
Thanks to the team, of course, thanks to the collaborators, thanks to our partners and clients and you for the attention.
16:03
And yeah, happy to take any questions.
