0:00
Thanks a lot for this nice introduction.
0:01
And yeah, welcome to my talk.
0:04
With the title: Trehalose, Sucrose, Amino Acids: Essential components of platform biopharma formulations.
0:12
I will definitely focus my talk on applications related functionalities, performance in biology, drug products, but also, you know, recently also with a pandemic being behind us basically on mRNA technologies and potential applications there as well.
0:30
That's the outline.
0:31
It looks a bit crowded, so don't worry about this.
0:34
I will basically talk about, you know, physical chemical properties of sucrose and trehalose, how they, you know, differentiate with regards to characteristics and how they behave and under certain conditions and also which type of implications that might have with your biopharma, you know, platform formulations.
0:55
I will also give you some examples about sucrose and trehalose in COVID-19 related formulations applications.
1:02
We talk about liposome mRNA vaccines and the utilisation of carbohydrates there.
1:07
And we also recently entered the market.
1:10
I would say recently, I mean basically five years back the amino acid market products which are really truly designed to develop for the utilisation Aas as excipients biopharma applications.
1:20
We'll talk about that and our quality approach for those and we'll sum up basically with some kind of conclusions and definitely also with our product range which we are offering to formulators for the biopharma market.
1:34
Let me just briefly introduce Pfanstiehl to you.
1:36
We are actually quite an old company.
1:38
We are one of we are 104th year of existence.
1:43
The company was founded in 1890 by Carl Pfanstiehl.
1:45
That's where the company name comes from and you see here an advertisement back from 1920.
1:52
Basically we're doing the same stuff as we did almost one hundred years ago and we are really focusing on high purity, low endotoxin components, specific excipients for biologics, vaccines, cell culture media and also other injectables.
2:09
Even in the small molecule area you know liquid and the lyophilised form.
2:16
We are a true US based manufacturer.
2:19
We are based in Illinois, FDA approved and FDA inspected and you see here main, our main products like trehalose, sucrose, galactose, maltose, mannose, mannitol, adenine base monohydride, histidine base monohydrochloride, methionine, glutamine and sodium succinate.
2:37
We don't have thousands of products.
2:39
We are very focused on excipients which are really focusing on stabilisation technologies as well as utilisation even also in upstream like galactose and mannose really focused and designed for biopharma pharma use.
2:54
All these products are manufactured in robust validated GMP processes at our FDA approved site in Waukegan, IL.
3:03
These are tried and true parenteral excipients for a long time.
3:06
So they are considered to be very safe.
3:09
Those products are utilised in the majority of top 10 biotech blockbusters and we do see us as really market leaders in terms of manufacturing capabilities and capacity, but also in terms of quality for you know, trehalose and sucrose specifically designed for the utilisation in biologics as you might know.
3:31
And then I think just go briefly about that is that trehalose as well as sucrose are very commonly used carbohydrates in biopharma formulations and very much you know, utilised to stabilise different modalities starting from human IgGS, ADCs, but also even bispecific and other more complex biopharma modalities.
4:02
The same is also valid for sucrose on the lyophilised drug products as well as liquid drug products.
4:11
You see also different routes of administrations intravenously, subcutaneously and so forth, even intravitreal administration.
4:19
There are examples for where products like sucrose and again low endotoxin high quality are utilised as a stabiliser.
4:30
I think it's not a secret, it's not a miracle for you that if I tell you that a typical bio platform antibody formulation consists of three major components.
4:41
The buffer.
4:42
I mean, it can be either phosphate citrates, succinate acetate, histidine surfactant, polysorbates to prevent aggregation as well as sugar components, carbide components, you know, utilised to stabilise the molecular antibodies due to the fact that of course a protein is a big buffer system.
5:06
Most of these antibody formulations are formulated in a slightly acidic environment, liquid environment.
5:16
And that's important to understand and once again to make this fact aware because that has also implications on about stability.
5:23
For example, of the excipients, which I will tell you right after. In this presentation and in this specific slide, if you look at the chemistry of trehalose and sucrose, I mean, this is very simple chemistry, so nothing very fancy, but it's always good to understand is that trehalose really consist of two glucose scaffolds, whereas basically sucrose consists of glucose and the fructose scaffold.
5:54
That's not the only difference.
5:55
The difference is also the thermodynamic instability of the bonds between those scaffolds is alpha-alpha 1-1 glycosidic bond in trehalose is extremely stable.
6:07
It's really not subject to hydrolysis even under very extreme chemical conditions in terms of heat or pH value, which is different to sucrose that won't is subject to hydrolysis and can hydrolyze into glucose and also free fructose.
6:28
That means that there is a risk that due to this hydrolysation, glucose can be formed and that is something which is not really beneficial because glucose is a reducing sugar which you don't want to have in your formulation.
6:41
Anyhow, that's something I will come back to later on.
6:45
If you compare the physical chemical properties of trehalose and sucrose, I think there's some differences.
6:52
Of course, we talked about the chemical structure, but we should also talk about the solubility.
6:58
Sucrose has a five times higher solubility at room temperature than trehalose.
7:07
I think the most compelling difference is here the difference in the glass transition temperature of the pure substance, which is basically for trehalose 60 centigrade higher than sucrose.
7:20
Then as I said, the difference in the rate of hydrolysis is also very significant.
7:28
Basically, trehalose cannot really, under normal conditions can really hydrolyse and can form free glucose.
7:36
It's important to understand because that can also have implications on your formulation, specifically if your protein is subject to glycation.
7:44
So whenever you're formulating a protein which in like a slightly acidic environment, there's a potential issue that free glucose can be formed.
7:59
That can also increase the risk of glycation that regards basically you should also consider trehalose as an alternative.
8:09
Some literature is given here.
8:13
I won't go through that in more detail.
8:15
So glucose is that also a topic for trehalose.
8:21
And as a matter of fact the compendium monographs allow basically glucose is a concomitant component and impurity up to levels of not more than 0.5% which is quite high.
8:34
Our trehalose is very significantly lower than that.
8:39
So the glucose contents of our trehalose is significantly lower than the compendial specification limits.
8:44
And even at the 0.5% there, we had examples in the past and also with collaboration with customers where even this compendial allowed glucose levels really to cause some issues with regards to protein glycation.
9:03
We have with our standard product, this is really the product which is really having a glucose level which is the lowest on the market.
9:10
We even have specific customised solutions where we are even coming to non-detectable limits here when talking about trehalose in terms of physical chemical characteristics, I think you are well aware that also in the literature there are reports that there's a risk of crystallisation of trehalose specifically in frozen conditions.
9:37
And one of these two of these literature speak to address this.
9:44
And as a matter of fact, the phase distribution of amorphous and crystallised trehalose in frozen solution, it really depends on the ratio of trehalose to the molecular antibody.
9:58
And that's very important to understand.
10:00
There's only in a certain area of the phase diagram, there's a risk of crystallisation.
10:07
And the crystallised trehalose definitely is something you want to avoid because this allows aggregation.
10:13
So I think the preferred state is amorphous less state.
10:17
And the results of these literature really identify an optimal range of the trehalose molecular antibody weight: weight ratio of 0.2 to 2.4, which is capable of physically stabilising molecular antibody formulations during long frozen storage.
10:39
Basically these are the experiments basically which have been performed in that literature where you can see here that the amount of the amount of crystalline trehalose, 2 amorphous trehalose here in blue and crystalline red really depends on the trehalose concentration prior to freezing.
11:01
But not only that, but also in terms of the concentration of the molecular antibody.
11:08
That means basically with the same concentration of trehalose prior to freezing, with the increasing amount of molecular antibody, you offend a higher amount of, you know, amorphous trehalose.
11:19
So you can, I definitely identify with this type of experiments also an optimal range that definitely also depends on the protein concentration in general, also other formulation components compositions, which I cannot really generalise here.
11:35
The phase diagram, as you know, is also a function of temperature.
11:42
So they're very, you know, very famous examples in literature where basically protein aggregation can occur at -20 centigrade but not at 10 or -40.
11:53
The crystallisation at -20 centigrade really deprives the protein of the cryoprotectant, leading to a slow increase in aggregation.
12:02
However, the aggregation of the protein raises the glass transition temperature and reduces the drive for crystallisation.
12:13
The extent and rate of crystallisation depends on the protein content and its distribution, the trehalose metrics.
12:19
So what we are saying, yes, we are aware about the risk that they are there might be, you know, crystallisation of trehalose.
12:27
On the other hand, you know, in terms of the right manufacturing controls in place and also being in the right part of the phase diagram, you can avoid that.
12:35
And we are definitely trehalose is a very good stabiliser from molecular antibodies.
12:42
We also do see some as I said, I mean in comparison without taking into account all these, you know, chemical physical properties.
12:51
We also see some advantages of trehalose over sucrose means that we talked about, I mean I talked about that in terms of stability of trehalose at low pH, no risk of hydrolysis in forming of free glucose.
13:09
It once again, I think there's no risk of being there.
13:12
So there's no risk of glycation of the protein.
13:17
I think you might have heard also in the literature that due to the fact that sucrose is a plant derived natural occurring product there might be also the presence of nanoparticulates in place in sucrose from a natural source bleeding in cane.
13:35
We definitely have to have process measurements in place to reduce that to the absolute minimum.
13:42
But there always will be some other particulars which should be avoided due to the fact that those can occur and basically can induce aggregation.
13:53
There's all, you know, discussion about beta glucans in sucrose which are not present in trehalose.
13:59
Beta glucans are concomitant components of cellulose containing plants, which can first, you know, cause false positive response to endotoxin testing.
14:10
There's something you don't want to do, we don't want you to have in your formulation.
14:14
On the other hand, they can also induce an immune response, which is also to be avoided.
14:20
So it's very important either not to use an excipient having better glucans or if you're using like sucrose, it would be always better glucans in place.
14:30
However to have a product, an excipient being utilised which is really characterised and with regards to better glucans and also at very low levels.
14:40
We also see that, you know, trehalose does have an advantage in terms of life cycle management.
14:47
A lot of lyophilized products, drug products can be easily also transferred into without really changing a lot of excipients from the lyophilized from in the liquid form.
15:00
Whereas basically if you start from sucrose, transferring that from the lyophilised version into a liquid versions, you can come into issues regarding once again, free glucose and so forth.
15:13
We also talk I mean, I heard yesterday a lot of about spray drying antibodies and definitely I mean trehalose with a high glass transition temperature is ideal with regards to the physical chemical properties also for spray drying applications.
15:28
And last but not least, once again, crystallisation of trehalose is a known biophysical, physical chemical behaviour which can be overcome if you put the corresponding, you know measurements in place with regards to protein trehalose concentration or ratio as well as controlling rate of freezing and flowing and so forth.
15:52
In the next slides, I will show you a bit basically about our quality approach and key quality attributes for our excipients and basically which are really based on key data or let's say key data, we achieved.
16:06
So what you see here is basically the tests we have been performing in terms of your endotoxin beta glucans, TAMC, TYMC.
16:14
And then we have basically a particle size distribution in another particular range measured by NTA and you have you know, the limit of quantification, the specification limit set by us.
16:31
And then we have this is cane sucrose, beet sucrose, trehalose, agenine base, agenine monohydrate, histidine monohydrate and this is nitrosamine.
16:43
And actually the data below averages over different batches.
16:49
What is missing here is a standard deviation.
16:51
I think I might get this comment anyhow, but it's something we have in place and we are very confident that we are very consistent in our measurements.
17:00
And what you can see is that you know for the majority of our achieved averages about for example for [unclear] were was an average about 355 batches.
17:12
So a very tremendous database we have in place.
17:16
Those are really even lower than the limited quantification.
17:20
So it's very high quality products, very consistent in terms of quality.
17:28
Another example is basically we are testing for each of our batches impurity levels and we have set up very tight specifications in the PPD range and we are testing all 29 limit impurities which you can test basically here.
17:51
Once again, this is limit of quantification.
17:53
I do apologise the unit is not really here in this tables, but it's all in PPP.
18:00
This is limit of quantification of the test method.
18:03
This is a set specification.
18:04
So what the means spec 20 means that lower than 20 and the results below once again averages over let's say a certain amount of batches we produce.
18:19
And what you can see is actually that we are yeah, either very low in once again, very low in the elementary impurities over as an average even below the limit of quantification.
18:32
And the only one where we are buffer basically is for iron, even though very low.
18:37
Yeah, and chromium because we are still working in this TMST environment the same we are also doing, I won't go through that all, but also for the amino acids once again, more and more biopharmaceuticals are really just based formulated based on amino acids no longer with carbohydrates.
18:58
So it's also important to have the products utilised which are really characterised and low in limitable impurities.
19:06
This is a typical COA of our beet sucrose.
19:11
Of course, you see actually 61 testing items if you look at some generic APIs, they are not characterised like this.
19:21
I mean the message what we, our quality approach to the exipient market is really to go beyond the pharmacopeia requirements because most of them are when the monographs have been developed for oral drugs or for our applications and for parental ones, not specifically for biopharma.
19:39
And there are two aspects to consider, of course, the safety of the patient, the other inside the safety, I mean, basically we have to protect your drug substance.
19:46
So if there's any potential impurity being present in the excipient which can, you know, can harm your drug substance with regards to any induced degradation.
19:58
We just want to avoid that by really providing a very highly characterised materials, specifically if it comes to an impurities, specifically if we talk about, you know, methods like manganese, copper, iron and so forth.
20:15
But also, you know, the amount of sugar going into a lyophilized drug product is quite high.
20:25
So you know the weight is quite high, the weight share is quite high.
20:31
So it's important also to have a product you know utilised which is the extremely low in the endotoxins here with the specification of limit of 0.2 U per gramme, not milligramme even programme which is important to say it out.
20:44
And we have a very highly characterised material.
20:50
The same is valid for once again, I think also for trehalose as well as you know, our newly launched Methionine , which is characterised in the same way as our [unclear] the same quality approach we are applying for our carbohydrates.
21:07
We are also doing that for the amino acid excipients.
21:11
And then with that I think we really consider us as a global quality standard setup for excipients going beyond pharmacopeial requirements.
21:22
Some of the compendial monographs are even based on our products in some way.
21:28
We can sponsor monographs, for the sucrose we even if implemented an HPLC test, which is so far up to no, not required by them pharmacopeial requirement compendial.
21:44
But there will be a change coming up shortly where basically you know an HPLC assay an impurity assay is being required once again.
21:53
For us, it's important lowest endotoxin, there is lowest bio burden lowest and complete coverage of limit impurities.
22:01
We have a process in place which is designed to minimise any nanoparticular implication and we are also developing internal capabilities to attract trend control and quantitate neutralism mean as well as any choice of structural impurities.
22:20
So that's something which we are doing even now and we are doing also beyond what we are putting on the COA.
22:29
Now, I'm switching gears a bit.
22:31
Just wanted to mention that.
22:32
Yeah, as you might, you know, I think we already heard about that, I think also yesterday about stabilisation of [unclear].
22:41
So mRNA or lipid nanoparticles.
22:45
I mean sucrose and trehalose have been widely used also for the stabilisation of COVID-19 applications with different functionalities.
22:51
And I won't go through that in detail, but stabiliser for protein milk antibody survival vectors cryoprotectant once again. Yeah, even let's say in the final stage and excipients in lyophilised vaccines and malformations.
23:07
So once again, I think these products I mean have been applied to billions of people.
23:15
So quality of the utilised sucrose and trehalose really highly matches in terms of yeah, full GMP endotoxins due to risk mitigation in terms of keeping the number of patients, side effects, information of particulates under consideration.
23:33
And but also in terms of the quality of utilised sucrose and trehalose which highly matters with regards to the instability of the mRNA.
23:45
So basically products need to need to be utilised, which are RNAse free.
23:52
And yeah, due to the instability of lipid nanoparticles as well related lipids, which can also impact basically, you know, this potential in impurities being present in the exit.
24:06
I think that's one of the overview basically with different, you know, COVID-19 vaccines and therapies which have been stabilised by sucrose and trehalose.
24:16
I mean, on mRNA viral vector vaccines, but also welcome antibody formulations and so forth.
24:26
Just a quite, you know, very small comment on the history.
24:32
I mean, why have been these mRNA vaccines?
24:35
So, yeah, so proof that quickly in the pandemic place because there was of course knowledge around basically on mRNA as well as on diapers homes from basically starting from the early 60s.
24:49
And basically this prior understanding of mRNA, its stabilisation and lipid nanoparticle stabilisation and delivery technology really helped to accelerate the development of emergency approval of both COVID-19 vaccines specifically from Pfizer, BioNTech.
25:05
And there were also already some liposomes and lipid nanoparticles, you know, products on the market which also have been stabilised with carbohydrates such as sucrose, trehalose from in the middle of 90s where this Doxil, the, this lipolyposomal drug.
25:24
And then coming back to 2020, also the application fields.
25:29
And there of course there have been also some key players in the market.
25:32
We have also the manufacturing capabilities as well as the knowledge how to produce lipid nanoparticles only in a consistent way and so forth.
25:40
Once again, coming back to the field of mRNA and how sugars play a very important part with regards to the functionality of stabilisation.
25:52
I think you are pretty much aware that such lipid nanoparticles basically have a component active ingredients, the mRNA cholesterol to modify the, you know, the lipid bilayer basically in terms of its transition temperature, pegylated lipids to basically to protect the LNP, helper lipids, cationic in other lipids to stabilise the MRA buffer salts.
26:23
And once again, most of the time sugars are being utilised to as a cryoprotectant or stabiliser basically to protect the structural integrity of the lipid nanoparticle and to ensure the certain stability.
26:41
However that still for both Moderna as well as Pfizer products that requires cold storage.
26:50
I think what you see here is sucrose in both cases has been utilised.
26:55
The difference irrespectively of the lipids being utilised is also here the buffer component.
27:00
I think that's also very important to understand that I think Pfizer BioNtech start actually with a phospho buffer, Modena with a Tris buffer, which also has some kind of implications with regards to the stability.
27:11
And they are different aspects to consider in terms of the shelf life and storage conditions of mRNA vaccines and therapeutics considering mRNA stability, LNP stability, once again, sourcing and quality control of excipients including carbohydrates.
27:28
And if it's critical, once again, I already touched based on the factor here, the RNAses, once again the buffer components going into that.
27:38
And specifically if you talk about pH fluctuations during freeze and thaw cycles and once again the cryoprotectants and of course, I mean on long term stability there might be also the possibility of a lyophilised version.
27:55
I think that was also covered in some of the talks yesterday.
28:00
Yeah, in terms of enhancing mRNA RNA stability, all excipients have to be RNAse free.
28:08
That's what we can tell for our products, below 0 centigrade storage conditions.
28:16
You know the utilisation of the buffer selection is crucial once again, where phosphate buffer tends to have a drift in bulk pH by 3.5 units when cooled down, it can cause mRNA stability.
28:32
Whereas the Tris buffer seems to be more stabilised, more stabilising effect as and known as the hydroxyl freeze radical scavenger.
28:43
Once again, sucrose trehalose utilised as lyoprotectants, cryoprotectants and also pH optimisation of the final formulation is also crucial for mRNA vaccine stability.
29:01
I think let's say it's also commonly known that we that sucrose and trehalose in formulations have some beneficial effects in terms of long-term stability, but also in delivery efficacy.
29:16
And yeah, there's a lot of literature around we had some talks about that yesterday as well.
29:26
Once again, I think the message here we would like to say is that, you know, quality definitely matters because mRNA is inherently sensitive.
29:36
Lipids of high purity are essential for the LNPs.
29:40
Lipid nanoparticles can also undergo chemical and physical instability and chemical instability comprises the degradation of the lipids and the LNPs that are can also undergo hydrolysis and oxidation.
29:56
So any minor impurity from process or the materials including CPN can lead to instability in an undeserved product profile.
30:04
So once again, the more you know confidence you have with regards to the characterization levels of the excipient you are utilising, the more you know, the higher, the lower the risk is basically that there's any interaction which can cause instability.
30:23
Just yes, I'm just going through that.
30:26
Sorry for that.
30:31
There are different applications once again for amino acids and basically you know it's viscosity modifier specific for high concentration water antibodies, we have the corresponding solutions in place.
30:52
I mean, this is basically our product range, which I would like to offer to you and to present to you once again the classical excipients here as well as amino acids arginine, histidine, methionine and glutamine all compendial USP, EP, JP Chinese pharmacopoeia compliance.
31:16
Once again, some new product launches around methionine glutamine once again.
31:24
Last inspection of France has been in January 2023, ended up with No 483 oppositions.
31:29
We have also the corresponding regulatory support in place globally.
31:32
We have fire Chinese drug master files for all of our products.
31:36
And of course, I mean, we are really convinced that we are offering products which are really truly fit for purpose, carbohydrates and I mean for the development in manufacture of biologics based on three pillars, product quality, regulatory support and also global world class product supply.
31:54
Also in terms of I mean capacity, but also dedication to inventory, risk mitigation.
31:59
And yeah, we have definitely proven that during the pandemic.
32:03
With that, I'm coming to the end of my talk and thank you so much for your attention.
32:08
Thank you.