0:01
I always like to start my talks with a little teaching of history.
0:05
So if you go back to the ancient Stone Age and we all know how important the Stone Age was over here in Europe, we basically start moving from hunter gatherers to the agrarian type civilization that we have today.
0:20
And when you consider what that leads to, which is a prevalence of grain based foods, we can see that the seeds of type 2 diabetes were established about 10,000 years ago, right at the end of the last Ice Age.
0:33
So this was a disease 10,000 years in the making.
0:37
And now the pharmaceutical companies are making the big money to try to sort this out and give people back the quality of life that they need.
0:47
And being one of those patients that takes Mounjaro, I can tell you that it really does work and it's pretty amazing.
0:55
And the fact that it suppresses your appetite.
0:57
So I always like to say I'm a living example that these things really do what they're supposed to do.
1:03
And I'm happy to be an advocate for that.
1:07
So what is type 2 diabetes and how is it linked to obesity?
1:13
It's really interesting to consider that the two are very closely related.
1:18
In fact, most people that are considerably overweight, I being one of those can look at that and say, well, I had some obesity indication.
1:27
So it's likely that I may develop type 2 diabetes.
1:30
And in fact, there's a three X Factor there that you're likely to develop type 2 diabetes if you're obese.
1:38
And generally speaking, we have overall obesity about 70%.
1:43
And then the truncal type obesity, which is where people store their weight around their midsection, which is more like what I do.
1:52
This is the type of obesity that leads to a heavy indication of type 2 diabetes.
2:00
So what's interesting about type 2 diabetes related to type 1 diabetes is 1 is completely preventable.
2:08
If we modify our lifestyle and obtain a higher level of exercise, we can actually reverse type 2 diabetes.
2:16
So insulin resistance is something that can be undone.
2:20
Whereas type 1 diabetes are typically children that experience the destruction of their islet cells and their pancreas.
2:28
It's an autoimmune disease.
2:29
So once those cells are destroyed, typically those patients need insulin.
2:34
And it was more or less a death sentence and all the way up until around the 1920s when Lily was the first company to put out insulin.
2:43
That helps sort out and give people back some quality of life as long as they were tolerant to insulin injections.
2:50
And that really kind of changed diabetes care.
2:54
But as more grain based eating habits became prolific in in the world, we saw the development of a new type, new form of diabetes, which was the adult onset, which later became referred to as type 2 diabetes.
3:11
Overall, you have major considerations with the effects of having high blood sugar and what these can lead to is blindness or diabetic retinopathy.
3:24
You can have a chronic kidney failure nephropathy where you're basically promoting in state renal disease, have a higher level of cardiac ischemia and strokes due to the higher levels of sugar in the blood.
3:40
Cardiovascular disease is the leading cause of death in most type 2 diabetics.
3:47
And peripheral neuropathy, you see that's a lot the people that get the tingling in their limbs due to poor circulation.
3:53
Circulation issues can lead to amputations which really affect people's lives.
3:58
So it's a significant disease with major consequences in lifestyle and it's something that the pharmaceutical industry has put a lot of effort into trying to get on top of this.
4:10
You can see this with reflection towards the development of sales of Type 2 diabetic products since 2022 to 2032.
4:22
You're going to see about a 2.5 fold increase in sales from 32 billion up to $70 billion for these compounds.
4:31
It's expected to even be higher than that due to some of these newer Type 2 diabetes GLP agonist that are really taking off with respect to treatment of obesity as well as type 2 diabetes.
4:45
So it's growing rapidly.
4:47
And if you consider that compared to the type 1 diabetes market, which are the insulin analogues that people are taking such as detemir, aspart, and lispro, these have stable annual sales in the range of around 20 billion and it's not changing.
5:06
So clearly the Type 2 diabetes market is one that's driving the intense interest that we see in peptides around the world and is constraining the capacity uniquely and all of the peptide CDMOs.
5:22
In fact, there was a shortage last year and the year prior to these Ozempic and Mounjaro compounds because the supply just could not keep up with the demand, especially as they became popularized for treatment of obesity.
5:40
Peptide therapeutics are really critical towards treating type 2 diabetes.
5:46
There's four major areas with regard to the peptides that are potentially therapeutic and those are the GIP or gastric insulinotropic, or used to be called gastric inhibitory, polypeptide.
6:02
Then you have the GLP-1s themselves.
6:04
You have glucagon, amylin and PYY.
6:08
All of these are modalities that are interesting for the development of potential treatment of type 2 diabetes or appetite suppression.
6:17
Some have been more successful than others.
6:19
For example, amylin pramlintide was developed initially back in the early 1990s and it never really took off in terms of sales because it wasn't something that was capable of being delivered in a sustainable and regular manner that would have an achievable therapeutic benefit.
6:41
It needed to be dosed three times a day.
6:44
That wasn't convenient for patients, but with adaptations to better delivery systems, as well as the ability to modify the peptide with some of these unique lipids that increase the serum half-life, suddenly amylin has a new life of its own.
7:00
But right now what's leading most of the effort with regard to treatment of obesity and type 2 diabetes are these GLP-1 agonists.
7:09
So if you consider the timeline of what's going on in the development, the first extracts from the duodenal area of the stomach led to the discovery of these pancreatic secretions that had a benefit with regard to delivering things like insulin and greater understanding.
7:30
Of course, insulin then was discovered and sequenced etcetera in the early 1940s.
7:36
It was discovered in the 20s and ultimately this is what paved the way for treating diabetes.
7:43
But what really took off in the 1980s was a further understanding of these incretin peptides.
7:49
And the incretins are the peptides that stimulate when you take them orally.
7:55
When you take sugar orally, it stimulates the release of greater levels of these sugar sequestering peptides like GLP-1.
8:05
And what really got me interested in this particular area was I've been a guy that's worked with peptide toxins my whole life.
8:13
They've always fascinated me.
8:15
And the fact that the first one of these came from the Gila Monster was what really interested me, that the saliva of the Gila Monster had this exenatide 4, which was the first of these GLP-1 agonists that was commercially developed by the company Amylin along with Eli Lilly.
8:33
This was the first of these to be approved back in 2005.
8:37
They coupled that with a better drug delivery system so it wouldn't have to be dosed twice daily to create Bydureon, which was basically a PLGA combination with the exenatide.
8:51
The first real blockbuster to take off and really succeed in this area was Nova Nordisk's Victoza.
9:00
It really kind of took over the marketplace.
9:04
Eli Lilly had developed Trulicity at that point as well, which was a much longer version of a GLP coupled to an FC fragment of an antibody.
9:13
But Liraglutide was a simpler molecule to produce.
9:17
It's basically a GLP analogue with a lipidation and it extended the single day dosing which really made it possible to improve the compliance of the patients and ultimately succeeded in being one of the first truly commercially successful of the GLPs.
9:37
Byetta was not nearly as successful in terms of the overall treatment options for patients, but Victoza are really kind of took off as well as Trulicity.
9:48
But the real blockbuster came when Novo introduced there follow up compound which was semaglutide and this particular product now had a longer fatty diacid isolation, a few changes within the molecule to stabilize the molecule to dipeptidyl peptidase-4, which is the key way that these molecules are deactivated.
10:11
And with that, they now developed a product that also had a weekly benefit of dosing, which improved patient compliance.
10:20
And they also developed formulations of this for better treatment of weight loss as well as oral delivery of a AGLP agonist.
10:30
These are the Holy Grail for peptides.
10:33
Can you make something easier to take?
10:35
A patient would much rather take a pill than take a shot.
10:39
And I can tell you taking the shot is really inconsequential.
10:42
You actually don't even feel it.
10:44
So it's, it's pretty amazing.
10:46
But the, the key thing is Ozempic began the trend of seeing real patient loss of weight.
10:54
It got celebrity endorsements, etcetera.
10:57
And once you hit that, especially in the American marketplace and the American marketplace has the commercial TV advertising effect of patients seeing this on TV and asking for it from their doctor where it's different around the world.
11:11
But that really stimulated this tremendous demand that we're seeing.
11:16
And now the last entry to this was Lily's tirzepatide, which was approved in mid-2022 and then a follow up approval for obesity control in November of last year.
11:31
This is Mounjaro and Zepbound, which are tirzepatide formulations.
11:36
This is the first of the dual agonist.
11:39
So it's not only just the GLP-1, but it actually has a GIP-1 component as well, which has more appetite suppressing effects.
11:48
So let's look a little bit at the structures of these molecules, and you can see that they're all basically around 31 to 40 residue peptides, with exenatide being the first.
12:00
The key substitution that established it to be more effective than just GLP-1 by itself was a substitution in the position #2 where you had a glycine instead of the normal serine that was there in the regular molecule, the naturally occurring molecule which made it more stable to DPP4.
12:21
Liraglutide was the fatty acylated version with alanine in the C4 position where a couple of other amino acids were modified to allow the lipidation to be done on a recombinantly derived material so that they only had a single lysine that was reactive and capable of being isolated.
12:43
We'll skip the albiglutide because that really didn't last long on the market.
12:49
Smith Kline kind of pulled that back.
12:51
Dulaglutide is the IgG fragment has GLP conjugated to the n-terminus.
12:58
It's a recombinantly produced product has that same glycine in the position #2 to improve stability.
13:08
Lixisenatide is a exenatide variant with a bunch of lysines at the C terminus to improve solubility and help to reduce fibrillation.
13:18
Semaglutide is the one that you see with the C18 fatty diacid.
13:24
It also has a couple of mini pegs in there to improve solubility as well.
13:28
Because one of the interesting components molecularly that occurs in many of these GLP analogues is the tendency to want to aggregate and fibrillate just like the beta amyloid type peptides.
13:40
So when you're producing these commercially, that's something that you have to deal with, which is when you're doing large scale purifications, how are you going to handle these large volumes of material that are coming off that you can't lyophilize until you've got your main pool that's ready for lyophilization?
13:57
How stable is that product going to be for a long period of time?
14:01
So it's really critical to have a molecule that's as stable as possible and to minimize those aggregating effects which affect the long term stability.
14:12
And Mounjaro is our tirzepatide is the one shown on the far left on the bottom.
14:19
That's the Lily compound that also has the C18 diacid mini PEG.
14:27
But interestingly, this product has two AIBs positioned in the molecule.
14:32
And because of that, the entire chemical process for developing this was the only viable way to proceed relative to Novo's recombinant process for making semaglutide.
14:45
So this molecule is the one that really is driving the chemical synthesis part of the industry to adapt and even look at different options for making this molecule truly large scale and adapting total solid phase synthesis to more of the hybrid technologies where we take fragments and put them together to try to improve the overall crude quality.
15:10
So let's just talk briefly about how each of these molecules were made. In the early 2000s with Fmoc/tBu solid phase synthesis.
15:21
Exenatide was the primary molecule that was put out and the demand for this molecule was only in the mid-10s of kilos per year and because of that total solid phase synthesis was more than amenable and worked well.
15:36
Dulaglutide, Liraglutide, Semaglutide are all injectable products that are made recombinantly.
15:43
And because of that the scale up of those is a little bit more simple because you simply have an increase in your fermentation tanks without the issues that we have when we now are dealing with massive amounts of resin and sampling and having to deal with all of the solvent waste it's generated.
16:03
Semaglutide is the oral form Rybelsus that came out.
16:08
Once you move into oral dosing of peptides, what you find is that the dosing requires about 8 to 10 fold increases in the dosing to overcome the poor absorption rates that you see in the stomach.
16:21
And because of that, now we're looking at needing quantities up into the metric tons.
16:27
Tirzepatide because it's a total solid phase synthesis or a hybrid synthesis, chemical synthesis needs to be adapted.
16:35
And that's the one right now that's kind of really revolutionizing how we're all going to work in this space.
16:42
All of the CDMOs that are working for large scale production, how are we going to handle making metric ton quantities?
16:50
And if we learn the lesson of Fuzeon, which was Roche's compound that they developed back in the mid-1990s for treating AIDS, it was projected that enfuvirtide or Fuzeon was going to need metric tons.
17:04
But patients didn't really like taking that because it required dosing twice a day.
17:10
It was a gluteus injection, so they weren't very compliant, and it never really materialized more than maybe 50 kilos a year.
17:18
So metric tons weren't necessary.
17:21
But what that did for our industry was the supply chain to make the enfuvirtide is what brought the Fmoc amino acids and solid phase resins to make this product in that potential quantity down to a reasonable level, making all of these molecules now accessible.
17:40
And all of the people working in the development of peptide therapeutics benefited from Roche's investment into that technology.
17:49
So now we move into what we're really wanting to focus on is how do you make products like tirzepatide, chemical synthesis and what can we do to improve that overall yield so that we can get the cost of goods down while getting the purity up.
18:08
And the way that most companies are looking at this, and AmbioPharm is a specialist in this particular area, which is developing a hybrid based strategy.
18:19
So we all know how solid phase synthesis works.
18:23
You have a simple cycle of making the peptide where you have the C terminal residue linked to a resin.
18:30
You go through a cycle of deprotection, you add in an activated amino acid.
18:35
You couple that, and you repeat the cycle until you've completed the peptide.
18:40
When you're making a longer peptide you can repeat this, but you start to see decreasing crude quality due to low efficiency couplings.
18:51
We can get around that by using certain building blocks like pseudoprolines and even other dipeptides.
18:58
Try to minimize the problems that we see as we're making a longer peptide.
19:03
But some peptides are just intrinsically very difficult to make.
19:07
GLPs are one of those.
19:09
There very similar to beta amyloid.
19:10
That's just a tough synthesis.
19:13
You can use heat, but heat can do other things and stimulate other side reactions.
19:18
So the real optimal way to scale something up and improve your overall crude quality is to take peptide and break it into pieces because it's a lot easier to make three 10 residue peptides than to make one 30 residue peptide.
19:34
And this is what we call the hybrid peptide synthesis approach.
19:38
So we look for the disconnection positions in the peptide sequence, which means where we can break that peptide efficiently and then use that as a potential site for coupling.
19:49
So we look for things that are non-racemic.
19:52
We can look at glycines and prolines to control that process so that we don't generate racemic products in the coupling.
20:00
But when we don't have that, we have to look for alternatives.
20:03
So that's the key thing is where are those disconnection points?
20:07
Once you've made the three fragments, you typically will take fragment 1 and couple it to fragment 2 and then conclude that by coupling fragment 3.
20:16
And this is usually done all in the liquid phase.
20:19
So you have more control of your stoichiometry, you're using lower equivalence of each of the components, and because of that you can keep your cost of goods controlled.
20:30
So for tirzepatide, for example, from Eli Lilly, they presented this in a paper two years ago.
20:37
They broke the peptide into four fragments that you see here.
20:41
You see the C terminal 30 to 39 up top, it's terminated with a C terminal amid which is perfect.
20:48
It allows you to easily do this as a solution based coupling for liquid phase based strategies.
20:56
Then we have the next fragment 22 to 29 that's coupled on to that.
21:01
Then we have the middle fragment 15 to 21 and then lastly 1 to 14.
21:06
If you look at those, you can see that two of the peptides are terminated in glycine.
21:11
That's the optimal residue that you want for coupling.
21:15
It allows you to have low racemisation when you're coupling into that and the other ones have aliphatic, but the minor aliphatics that don't contribute much to racemic problems.
21:26
You want to avoid residues like isoleucine, phenylalanine, those which have a high propensity to racemise.
21:34
So they chose the alanine and the leucine at those two positions.
21:38
And with that they were able to have a very nice process where at each of the intermediate liquid phase condensations, they would use nanofiltration as a means of removing all the byproducts and all of the coupling reagents that had been spent and move that sample to the next step.
21:59
And they did this basically in a sequential fashion and we're able to establish a method that they could produce kilo quantities of tirzepatide using this strategy.
22:13
At AmbioPharm, we have the same type of philosophy that our goal was always looking at very large quantity peptide synthesis.
22:22
And we excel when we're working in these quantities greater than 100kg.
22:28
And that's because we've always believed that the hybrid technology is that which gives us that competitive advantage.
22:35
So for peptides with the annual quantities greater than 100 kilos, this is typically the strategy that we have pursued.
22:43
Several of these GLP agonists that are currently in development in our generic part of our company called Ambio have been things that we've been working on internally.
22:55
One of these I've taken as a case study.
22:57
I'm sorry, I can't tell you exactly which one it is, but you can guess, and you'll probably be right.
23:04
I just, I have to refer to it only as GLP agonist.
23:09
So our goal was initially to try to develop a solid phase synthesis because that's the fastest way to get to the final product.
23:19
So we developed an Fmoc standard solid phase and then we had a subsequent acylation step that was completed off the resin using an orthogonally positioned protecting group.
23:31
Once we did that, we had a 2 step purification process before the acylation and after the acylation.
23:40
What that results in is a long sample time of having to hold the fractions, which makes it pretty untenable to do that for the long term viability of a commercial scale production.
23:52
You can see that the crude quality that was achieved for this particular product was only around 38%, which is still quite decent for the product overall, but solid phase derived material.
24:07
You can see a lot of very close impurities eluting close to the main peak there.
24:12
So it was selected was a general hybrid approach as an alternative and this was basically to break that peptide into four fragments much like tirzepatide and then assemble two of those peptides using liquid phase and two of them using a Chlorotrityl based strategy and then put those together.
24:33
This is the overall process.
24:35
You can see that the two fragments one to four are made using LPPS and the C terminal which basically is 27 to 31, that's the C terminal fragment 4 are done using solution phase, which we are well equipped in AmbioPharm to work with LPPS type approaches.
24:54
And the middle 2 fragments were made using 2-CTC base strategies.
25:00
Much the same type of connection point, one was with a leucine and the other with a glycine.
25:05
And then three to four was assembled and then two to three to four.
25:09
And then lastly, the full length molecule was then put together 1, 2, 3, 4, followed by a cleavage step and final HPLC processing.
25:20
And we achieved a far superior crude quality at 63% with a lot fewer mainline of baseline impurities that were much simpler to remove.
25:33
So overall the conclusion was that we were able to improve our crude quality by nearly twofold over the solid phase process.
25:42
The purification of the hybrid product was done in less than half the time that it took to do the solid phase product.
25:49
And ultimately it was a much more scalable result and has allowed us to have much shorter fraction holding times, which greatly improve the overall stability of the product.
26:03
And with that, I'd like to thank my collaborators from our Shanghai site, Guoqing Zhang over in AmbioPharm, Shanghai and our North Augusta research and development team, as well as Sherry Li, who heads our QC group over in Shanghai.
