0:30
Thank you very much for that introduction.
0:36
Yeah, I would like to introduce you to what we are doing in clinical bioanalytics for oligonucleotides in complex biological matrices.
0:44
But before that, I would, I'd like to I would like to introduce give shortly introduce you to Fyoni Bio.
0:54
We are located in Berlin.
0:56
35 people are working there and we develop biotherapeutics as a service company and do clinical bioanalytics for customers.
1:11
Just a brief overview.
1:14
We do a lot on biotherapeutics, proteins, cell line development.
1:41
So I was just mentioning the cell line development for biotherapeutics.
1:46
We have there just a few words and number of CHO cells with very high productivity.
1:52
So we are very proud of that something like 5 grammes per litre in the pool just for you to know if somebody is interested.
2:03
We developed the process and on the GMP and this is a component of course with a lot of MS based characteristics and ambiguities to characterise the protein.
2:14
And we need bio assays to look at the activity of the proteins.
2:19
But the topic of today is clinical bio analytics where we develop mostly PK, PD and other assays to assess the bio therapeutic from protein to oligo.
2:42
So not only proteins, but also oligonucleotides, which is a topic today in complex market cases.
2:53
We did that for our clients for many different clinical phases from phase one to phase four in trials spread over whole Europe and sometimes also an overseas and are doing that for antibodies, glycoproteins and oligonucleotides. Often or regularly we are audited.
3:17
This is an overview of techniques we are running under GSLP.
3:23
So the company is ISO certified and the biologics are done under GSLP.
3:30
And I mentioned already the PK and immunogenicity assays.
3:36
We do of course also not assays and analyse single nucleotide polymorphisms but also develop biomarker assays.
3:46
Cytokine release we analyse, sometimes we do some immune phenotyping by fax and analyse the activity of cells like CDC assays or ADC assays.
4:01
The project starts typically from consultancy and ends up then in analysis of clinical samples with assay establishment and assay validation according to guidelines.
4:13
And also, we need of course to have a good central management in house.
4:18
So this is some kind of overview of what we are doing in general.
4:23
Now I focus a little bit on oligonucleotides, therapeutic oligonucleotides and what I mean with that are ASOs or double stranded siRNA in the length of 16 to 21 nucleotides or base pairs with various chemical modifications and with conjugates, terminal conjugates from proteins, peptides, fatty acids and GalNAc.
4:52
This is the assay we are using for quantification of oligonucleotides.
4:59
It's a combination of the hybridization and electrochemiluminescence immunoassay.
5:04
So we use two probes to hybridise them with the analyte.
5:15
One probe is biotin related and is used for capturing them and the other one has an immune tag that is used for detection with an antibody and everything is read out in the equipment.
5:30
So we don't need any sample pre-treatment and we can run this assay in a kind of high throughput assay it was 96 well plates.
5:44
And this shows a little bit the data about these assays 2 case studies one with an ASO and one with an siRNA ASO was conjugated both are conjugated one with GalNAc and one with a lipid.
6:01
And you see a nice assay range from 1 picomolar to 10 nanomolar about with a good sensitivity and all the accuracy and precision results match very well with just which you need for clinical application.
6:26
And also the specificity to analyse that.
6:31
We don't analyse other DNA, but we just discussed already looking at metabolites with more selectivity.
6:39
That is of course different in these assays.
6:44
This is a ligand binding assay.
6:45
So you usually detect -3 -, 2 -, 1 metabolite also.
6:51
But on the next.
6:52
Yeah, I come back to that in the next slide.
6:56
On that slide, I wanted to show that this assay is very easy to handle.
7:01
It's a very robust assay and we can use it for any kind of matrix we tested so far.
7:08
So liquid matrix is and tissue matrices from animal from human.
7:14
CSF was always described to be not that easy.
7:18
It works fine after some optimization to say in these assay runs fine in this matrix and many tissue samples also and many types.
7:37
Yeah.
7:38
And now I'm coming back.
7:39
The advantages I mentioned already what how we would like to improve and what we are doing about this assay is one we would like to have a surrogate matrix so we see differences in the matrices.
7:52
We would like to have one matrix and so we don't need to develop for each matrix and you the assay from scratch again, we want to improve the sensitivity.
8:03
We have the technique in house now to improve the sensitivity but this is still ongoing in research.
8:11
And the improvement of N - 1 to N - 3 metabolites, they still they interfere.
8:17
This is a problem with the ligand binding assay.
8:22
But with some playing around with the probes, you can improve that a lot at least that you don't detect anymore N – 3, N -2 probably a little bit N - 1 still.
8:34
So the goal is to quantify only the intact molecule.
8:42
OK, this was about quantification.
8:44
Now some words about the anti-drug antibody assays we go the typical approach which we heard yesterday already.
8:56
So screening confirmation and titration assay on top, of course, and the neutralising assays and we characterise the order for domain specificity and isotype.
9:15
So in case there are different conjugates on the oligomer.
9:20
It's interesting to see, it's important to see whether the antibody, the ADA antibody recognises either the sequence of the oligo or the attached conjugate.
9:35
And we also analyse cytokine release and TLR binding.
9:41
Next slide.
9:42
This gives us a little bit an overview about anti-drug antibodies observed against therapeutic oligonucleotides.
9:54
In general, the FDA says you should do a risk assessment because it's considered not as this risky but when you elongate, when you attach any conjugate to it becomes a different story.
10:16
And also what is considered of lowest of course that so far no influence of observed ADA on the pharmacokinetics or clinical efficiency was observed.
10:30
Nevertheless it needs to be analysed.
10:34
And but if you have a low risk oligonucleotide then FDA says just do biobanking and analyse at a later stage short run if you have other appearance.
10:50
But there are also oligonucleotides.
10:52
I have here a few examples maybe you know about them that show ADA response for example pneumonia, the first generation oligonucleotide.
11:05
Also 72% of patients responded with ADA.
11:09
Another example is Patisiran where they observed ADA against the PEG 2000 of the LNP and also interesting they observed with Inotersen at a very late onset two days after first administration in 30% ADA.
11:32
So there are anti-drug antibodies against oligonucleotides for sure.
11:39
This is approach I think I don't need to explain it in detail.
11:43
The screening confirmation titration approach but for oligonucleotides in detail there are differences and these are shown here on this slide.
11:54
Usually you use the bridging format where you use the anti-drug antibody to bridge the biotin related drug and the label drug for detection.
12:11
And then you immobilise everything and can detect and quantify the anti-drug antibody on the right side.
12:21
Here in this graph, it shows that it's just ADA concentration against the signal that this approach, the bridging approach doesn't work very well.
12:30
And this we saw for many other assays against oligonucleotides, which works very fine for proteins.
12:38
Instead, you need to go or we have the experience that we use sandwich format.
12:55
With the sandwich approach we get pretty decent assay with a good sensitivity.
12:59
But what we see still as a problem is that we have a pretty high variance in background, which is unspecific backgrounds or no pre dose antibodies.
13:12
And so it's given here that the sensitivity is also lower for some patient samples with high background.
13:22
And this is my last slide is about this problem that we are still on research and to better understand this background is variability especially in oligonucleotides.
13:37
We don't see that for proteins to improve that and to help of course in the cut point determination.
13:47
I think with that I would close already and I think I have plenty of time for some questions.
14:03
Thanks.
