0:02
Thank you very much for giving me the opportunity to speak and giving the bio analytics area some space here at the meeting.
0:13
I think it's an interesting topic.
0:16
My topic will be on the clinical and the bio analytics of oligonucleotides. Specifically how to quantify with high sensitivity and how to assess immunogenicity in complex biological matrixes.
0:51
Yeah, just a few words on the company.
0:54
FyoniBio is located in Germany, Berlin.
1:00
We are a service company ISO 9001: 2015 accredited. The next slide gives you an overview on what we are doing.
1:15
So besides clinical bioanalytics, we have services around the development of biopharmaceuticals such as cell line development, and upstream and downstream process development and also a lot of MS based analytics during process development.
1:52
We have a booth outside and we probably met you earlier.
2:01
So my topic is clinical bioanalysis.
2:07
We’ve been doing this for a long time and our focus is client customised assays.
2:15
So, developing assays in pharmacokinetics, PD and immunogenicity for sponsors of clinical trials to support clinical trials.
2:28
But of course it starts earlier, in the preclinical phase.
2:33
We develop these assays, validate assays and analyse samples under ISO 9001: 2015 and GCLP.
2:43
GCLP means good clinical and laboratory practise.
2:46
Not that familiar but it's the guideline for the clinical lab.
2:56
We have experience now in phase I, II, and III clinical trials mostly in Europe.
3:04
Several years ago we received samples from Russia and we also analysed samples from Japan.
3:20
Our focus in the beginning was on antibodies and glycoproteins, but in the last years we’ve dealt a lot with oligonucleotides.
3:29
OK, this is gives just a brief overview of the assays we are running under GCLP.
3:40
I mentioned PK immunogenicity.
3:43
When you start immunogenicity, at one point you need to analyse the neutralising activity of the others.
3:50
The nAb assay, and we also have the immune cell function.
3:57
So we have immune assays that can be customised and established for such as CDC or ADC or other antibodies.
4:07
But we have other possibilities.
4:09
We can analyse facts by using phenotype cytokine releases. Biomarker assays is a niche product.
4:22
Its something a little bit different used to analyse single nuclear type polymorphisms by melting curve analysis.
4:29
A project starts typically from the consultancy, so to open with a protocol and to give writing the protocol some support regarding when the sample should be taken.
4:50
Then as I mentioned, we establish assays, we validate these, we analyse clinical samples and of course we manage the samples.
5:02
OK, this is a short list of instruments we have.
5:07
Mostly we use the electrochemiluminescence technique from MSD.
5:13
So that would be then the electrochemiluminescence ligand immunoassay, ECLIA.
5:18
Also we use ELISA assay.
5:20
So these are the workhorses.
5:22
Let's say we do a lot on that.
5:24
For cellular essays, we use the time-resolved fluorescent measurements. We also use mass spectrometry for PK analysis in certain areas.
5:33
I will come to that. We also use flow cytometry high resolution melting curve analysed by the Lightcycler.
5:41
I mentioned that for the SNPs.
5:45
So now about oligonucleotides. Our field is the quantification, the immunogenicity assessment to analyse metabolites by LC-MS.
6:02
What do we consider, what is our understanding of oligonucleotides? Therapeutic oligonucleotides are also called ONDs, we have lot of experience with siRNA with lengths of 16 to 21 nucleotides or base pairs with various internal modifications or external modifications such as GalNAC fatty acids.
6:34
When it comes to the quantification of therapeutic oligonucleotides, this is a short list of applications.
6:43
We just heard about several applications in Vincent’s talk, so it's ADME.
6:52
And to analyse the concentration in other liquid biological samples to estimate or to calculate the elimination half-life, to analyse, the metabolites or truncated oligonucleotides and bio distribution of target binding is something also in this area in for animal studies.
7:23
The challenges we see in PK assays is clearly matrix effect.
7:29
Of course, sensitivity is always a topic drug metabolite specificity.
7:34
This is something do we can detect truncated oligos.
7:39
The techniques should be very robust and capable of high throughput, and we would always prefer assays that have low sample or no sample processing.
7:54
So with this in mind, we compared, or I listed here 3 techniques to compare them and we have chosen this hECLIA, which is a combination of hybridization and the hECLIA technique.
8:15
I’ll explain it more in a moment. It's compared with the LC-MS technique which is often used for quantification.
8:23
We heard about it and the Stem-Loop qPCR, I think we didn't hear about that.
8:32
It's very sensitive compared to comparable with the Stem-Loop qPCR.
8:38
But in, in contrast to the Stem-Loop qPCR, it's much easier to perform.
8:45
It's a rapid technique and capable of high throughput and has big advantages.
8:53
Of course, also its transferable to other analytes and other tissues, other sample types. And no sample processing is also important.
9:04
The disadvantage is clearly that we can't analyse truncated oligonucleotide.
9:11
For that we use and the LC-MS technique.
9:18
This explains a little bit about the technique.
9:21
I think a different technique to what Vincent described for quantification because we use two probes to immobilise the analyte, the oligonucleotide.
9:38
First, this is a biotinylated probe and a detection probe is then used for detection with an antibody that is routinely labelled.
9:50
And then you can have a read out in the MSD instrument.
9:58
And with this technique, you have here.
10:01
It's an example of nice dilutions curves, but this is shown also in the next slide.
10:08
This is already where we get with this with this assay.
10:12
So we have an assay range.
10:14
Typically this is here for an ASO and for siRNA round 1 picomolar.
10:22
So sometimes less, sometimes a little more.
10:26
And this translates then depending how much you dilute the sample to a sensitivity of 40 or 6.5 in this area picomolar of the oligo in the sample, the liquid sample. You get very acceptable accuracy and precision.
10:51
And you don't we didn't see in our assays any cross activity with other unrelated sequences.
11:00
And of course, it's important that the dilution is very linear and that shows also that the assays can be easily transferred to other sample types on both sides.
11:20
First one point also that the terminal congregation of an oligonucleotide doesn't hurt.
11:30
So for example, fatty acids we had GalNAC conjugation. Also, the sense strand of the siRNA doesn't block or disturb or affect the assay.
11:48
But I didn't show you, what I focused on here is the different tissue types and sample types for you see very nice overlying curves for different human liquid sample types and also tissue types.
12:11
And here we have two different animal types from mice to monkeys.
12:20
And the siRNA range always starts below 1 picomolar and increases to about 10.
12:34
This is the summary about the quantification of assays of oligonucleotides.
12:42
I've briefly shown that we can quantify oligonucleotides in the low picomolar range in with this very good accuracy and precision.
12:54
It's very robust and fast assay.
12:57
We don't have any sample processing.
13:01
It's capable of high throughput as an ELISA similar technique and it can be transferred to other sample types as also shown here.
13:13
Yeah, we're working on CSF.
13:20
It's giving us a headache but we hope to solve that soon.
13:27
About immunogenicity, it's about the detection mainly of anti-drug antibodies and the titration.
13:40
The titration assay is about analysing the neutralising activity, you also want to see eventually whether the ADA you observe is binding at a conjugated structure or at the oligonucleotide itself.
14:07
Cytokine release is always a topic and the activation of the immune system by TLR receptors, but my focus is clearly the other detection here in this talk.
14:21
Why should we assess immunogenicity? Oligonucleotides or the therapeutic oligonucleotides are not that well known for immunogenicity, but RNA is indeed very immunogenic and looking at the spectrum of approved oligonucleotides, we see a range of oligonucleotides that don't have any other response or it's unknown. For Mipomersen 72% of patients responded with ADA, to this drug.
15:04
Another example.
15:05
So there is there are clearly therapeutic oligonucleotides that are able to induce anti-drug antibodies.
15:16
Patisiran, for example, 4% of patients are reported to develop ADA in that case against the PEG as part of the LMP.
15:29
And what more often observed is that there might be a trend that ADA occurrence does not start in the beginning of the treatment, but at a late stage.
15:46
So here in that case, 202 days after the first administration of the drug in 30% of the patients.
15:59
And FDA and all authorities need to see the other analysers and in case of rare diseases they request post marketing approval.
16:15
But not only to see anti-drug antibodies, but also to look at antibodies against the target protein.
16:35
The last point is that all these ADAs for all these products did not affect any pharmacokinetics or clinical efficiency.
16:46
So this is also one discussion point for why it should be analysed but the authorities need to see it on one side.
16:56
And I think also yesterday someone pointed out that we need to know how the patient responds to the therapeutic oligonucleotide.
17:08
The first thing is to start with risk assessment.
17:13
So the question is which administration route should be considered as well as drug modifications.
17:21
Phosphorothioat in all therapeutic oligonucleotides was developed for CPGs to induce the immune system.
17:34
It would be also interested to see whether other modifications we saw may have an impact on immunogenicity, we don't know, but congregation of macro molecules like PEG, GalNAC proteins, lipids may change the situation.
17:57
Of course patient status is also important.
17:59
The possibility of pre-existing ADAs is very interesting to consider.
18:06
And with this in in mind, one should always bank samples to analyse later.
18:15
So if you don't start to analyse ADA in Phase I, you should at least take the sample to analyse it.
18:22
When should ADA be analysed in clinical samples?
18:27
Generally before drug administration.
18:29
So when you have a multi dose administration, then it should be always before because otherwise you have the very high drug level and it's difficult to analyse anti-drug antibodies.
18:45
And then you should start two and four weeks after first administration to get the onset.
18:54
But as I mentioned before, probably it's more important to look at the long run of the clinical trial, at least to the end of the study.
19:08
Yeah, yeah.
19:11
This is how anti-drug antibodies are analysed.
19:18
You establish a screening assay, a confirmation essay and at the end you will analyse the titre of the titration essay.
19:28
Confirmation assay means basically that you use the screening essay and compete the signal away with the drug.
19:41
So when you get a reduction in the signal then you confirm that the antibody you see is indeed against the drug.
19:51
All the assays are cut point based.
19:55
So you have a cut point and if the signal is above that cut point, you have a potentially positive sample that goes in into the confirmation assay.
20:07
If it's then negative or then positive according to the cut point again.
20:14
Then try it again.
20:20
So the challenges for the ADA assay are the generation of positive controls.
20:28
The assay design is different to other proteins
20:39
We did a lot of ADA assays also for antibodies and glycoproteins.
20:44
I mentioned that, and it makes a big difference for oligonucleotides.
20:50
Cut point determination and drug tolerance is also always a challenge.
20:56
For other assays.
20:57
This is how we approach it.
21:00
So we mostly use the bridging format.
21:05
So you have the ADA here and it's immobilised by the biotinylated drug and detected by routine new labelled drug.
21:15
But for oligonucleotides this format doesn't work very well in most cases.
21:21
So we had to develop a sandwich format, and this gives you the assay range which goes down to 10 nanogram per ML to up to 10 microgram of ML with this sandwich format.
21:38
So for the assay designs.
21:39
The format is an important point that needs always to be checked for, for oligonucleotides and the positive control mostly arised in rabbits.
21:53
You want to have a polyclonal positive control because that resembles more the human situation.
22:03
And with that case we also controlled the positive control with control oligonucleotides so the specificity of the positive control.
22:14
And we could see that the positive control didn't recognise oligonucleotides with the same sequence, but unmodified that one or modified or different sequences.
22:33
Only the drug itself is able to compete here.
22:36
It reduces the signals.
22:42
Another challenge is the cut point determination especially for oligonucleotides because we see in several cases, we see a big variation of the signal in pre dose samples.
22:57
So patients that were not treated or, in volunteers that were not that didn't get the drug, so far they have already very high variance in the signal the screening assay and when it comes in to confirmations assay.
23:15
So after inhibition with a drug, the inhibition signal is still very high.
23:24
So that makes it difficult to calculate a cut point by the positive samples or negative ones.
23:32
And our solution for that is the specific normalisation for patient specific cut points. Now with that we get pretty sensitive assays with 19 nanogram per ML in normal serum and diseased patients with low background or high background.
23:58
also the confirmation assay shows similar sensitivities in case of diseased patients with high background.
24:11
We need to have a closer look high BG and low BG.
24:15
I should also mention that these variations are not due to pre dose of anti-drug antibodies that are present.
24:26
These are unspecific signal.
24:28
So inhibition you see with the drug, you see that the signal remains still high on the variation. The precision is very acceptable and drug tolerance in our hands works also it's also very nice.
24:53
So you look at increasing amount of the drug if you LQC samples are still positive and you do that to drug concentration that are maximally to be observed in the clinic and that works very well with these assays.
25:15
So in summary, we for oligonucleotides. We can have cut points.
25:24
We have a very sensitive assay also in place.
25:30
We can perform also the intra assay procedure which is very good and has high drug tolerance.
25:39
And with that just a summary.
25:42
I didn't get to talk about all these assays here, such as LC-MS to analyse the oligonucleotides, so I would like to stop and thank you.
