INDUSTRY EXPERTS ON MAM IN DEVELOPMENT AND QC

At the CASSS 2017 WCBP conference, a session was held on “streamlining biopharmaceutical development and quality control by multi-attribute methods,” which included presentations by three industry experts on MAM: Just Biotherapeutics Scientist Richard Rogers, Amgen Attribute Design Scientific Director Jette Wypych, and Roche Diagnostics Principal Scientist Patrick Bulau. The following are the remarks by Rogers and Bulau. Rogers covered: ● what the MAM is – the workflow, search algorithm, attribute analytics and new peak detection ● Just’s use of MAM in molecular optimization and real-time monitoring, and ● the MAM consortium. Bulau honed in how MS is currently being used in QC at Roche Pharma’s site in Penzberg, Germany, where he plays a leadership role in its analytics program.

JUST’S RICHARD ROGERS First of all, I would like to thank the organizers for inviting me to speak today. It is a great opportunity to talk about the progress we have made over the past couple of years developing the multi-attribute method. And today I am going to focus on how we are actually using it throughout process development. Just Biotherapeutics was started in 2014. It was spun out of Amgen when the Seattle site was shut down. Jim Thomas, Dean Pettit and Victor Fung were instrumental in putting this group together. The goal of Just is to leverage the technology development we are doing at each stage of process development to actually lower the cost of biotherapeutics. And the goal is to then pass that on to the patient to make these amazing drugs available to the developing world. In order to implement the multi-attribute method and actually use it to characterize our biotherapeutics, we need some core components, the first of which is high-resolving/high-mass-accuracy mass spectrometry. And we now have an industry where every vendor has this type of capability. We have choices on which mass spec platform to use. The second is accurate and fast data-dependent search software, which every vendor also has, and we have many academic options as well for searching these data. So we have these two key components that are readily available. The last thing is actually the most important component of the multi-attribute method – leveraging mass spectrometry to understand these molecules. Everybody has a different definition of what a multi-attribute method is. I am going to talk to you about what I think the multi-attribute method is today. And I think that you absolutely have to have this key component of that method to make it successful. So it is being able to detect things that you did not know were present in your molecule by leveraging software.

Today I am going to tell you what the multi-attribute method is to me and to Just. I will go through what our work flow is and how are preparing samples, how we are analyzing them. And then, I will talk about the search algorithm that I am using for the data that I am presenting today. And then the two components of the multi-attribute method – attribute analytics and new peak detection. Then I will go into some case studies as to how we are applying this to stressed antibodies. I will then go a little bit deeper into how we are leveraging the MAM for molecular optimization of our molecules and then moving the MAM into real-time monitoring in the manufacturing space. And lastly, over the last year, we formed a consortium for the multi-attribute method. It consists of biopharma leaders as well instrument vendors and software vendors.

The Multi-Attribute Method Here is a slide outlining a handful of assays that we use to characterize our molecules or release our molecules. Most of these things are single attribute methods. Some may give you more than one attribute, but most just do one. But by leveraging mass spectrometry, we can actually get rid of six of these [those outlined in red below] and get all of those data from a well-characterized mass spectrometry-based MAM.

Here is a slide going a little bit deeper into that. We know there are very important attributes on our molecules or associated with our molecules, including clips, charge variants, glycosylation, actually being able to show the identity of our molecule, and then process impurities. And we have assays specific for each of these things that we currently use in process development. But through the development of the multi-attribute method, we can leverage the high-resolving, high-mass- accuracy mass spectrometry to monitor all of these things in one method.

Workflow The workflow is very simple. I benefitted from a very strong platform that was developed before I even got to Amgen. I was able to leverage research done by Da Ren’s group at Amgen to implement a 30-minute digest – the overall method is about two hours – that gives us a very robust digest, reproducible, which is absolutely essential for leveraging the multi-attribute method. And then we use mass spectrometry to characterize that digested sample. Biopharma Finder Search Like I said before, you can leverage many different types of search algorithms to look at these data. Here I am showing results from BioPharma Finder. Once we characterize our molecule and understand where those modifications are, we actually don’t need MS 2 to do that monitoring, which makes it very easy to make this a QC-friendly method. Once we have our attribute list, we can transfer that to an MS 1-only method and use software – for instance here I am showing Pinpoint – to monitor the known attributes. But that is not sufficient, because something could happen to the molecule through cell culture or through process development that we did not know we should monitor. And so to actually have that purity component, to give us confidence that we are able to detect things that we did not know, we apply – here I am showing the software Sieve – to do differential analysis. And that gives us our new peak detection component. Then, once we have that method developed in process development, we want to take that and eventually use it to release molecules in QC. And so to do that in collaboration with Thermo, we put each of these components into Chromeleon to give us a fully compliant software package. I also want to point out that we have published the attribute analytic component of the method two years ago in mAbs. To do this search of the digested sample, we typically digest with trypsin, and then look for normal modifications including glycosylation, cyclization, C-terminal lysine, oxidation, deamidation, glycation, and isomerization. But a key component to this search is this variable mass search. Most search algorithms allow you to do this now. This comes in handy when you are doing forced degraded samples. That does a lot of weird things to our molecules, and we do see new peaks show up under those circumstances.

But applying this sort of criteria to our search, we are able to identify what those peaks are and then go through and interrogate what the mass shift could be. We typically get greater than 98% sequence coverage using a tryptic digest, but every single molecule is going to be unique. So you have to optimize your digest or enzyme depending on the amino acid sequence of your molecule. Attribute Analytics So now moving into the attribute analytic portion of the presentation: Leveraging Pinpoint, it is a very simple workflow. What we do in the analytical group is we identify those critical quality attributes using our search algorithm, and we leverage Orbitrap technology, either using a hybrid Orbi or the Exactive platform. We enter those peptides into Pinpoint, either the unmodified peptide or the oxidized or modified peptide. And now, here is a very key component of the multi-attribute method: You want to include all the tactical charge states and all the tactical isotopes. So anything you are able to detect reproducibly for these species correlate with the abundance of that species. If you are only monitoring one charge state, you are not getting the full picture of the abundance of that species in your sample. So we include all of those isotopes and charge states. Then the output from Pinpoint actually gives you the parent peptide, the oxidized peptide, the retention time. It shows you the isotopic distribution vs. the theoretical. So it gives you confidence that the peptide that you have identified is in fact the correct one. We also show the extracted ion chromatogram, as well as giving you the area underneath that peak. And the area is what we use to calculate the relative abundance of that modification – so dividing the area of the modified peptide by the sum of the modified peptide vs. the parent peptide. Now I am going to go through a bunch of examples of how we have compared the multi-attribute method to those classic assays. The first example here is the release glycan assay, the HILIC assay. We look at the HILIC data and then compare that to the glycopeptide that we detect using the multi-attribute method. As you can see here, we are getting excellent correlation with the HILIC data, even down to the low-level glycans. And there a number of glycans that you can see here that we are able to monitor reproducibly with this method. This becomes even more important when we start to look at fusion molecules. That previous example was a monoclonal antibody. It had a single site of glycosylation. But what do you do if you have a molecule that has one or more sites of glycosylation? You can’t get that information from a release glycan assay. You get a global picture of what the glycans look like. But if you leverage the multi-attribute method, you can actually look at site-specific glycosylation and start to get at structure function. How are these specific glycans affecting the ability of my molecule to perform its function? Next we did a comparison of our charge variant assay to a specific modification. Here we are looking at just the single light chain deamidation. Over the days of culture for this molecule, you can see that the acidic variants change. They actually go up over time. And we are able to track that change in the acidic variants with this single light chain deamidation. But I will point out that there is an offset here. Just monitoring that one deamidation is not actually giving us the whole picture. It is when we go in and leverage the multi-attribute method that we start to understand that there are glycations, there are isomerizations, there are other deamidations that are actually contributing to this change in the acidic variants, which we can detect very easily and reproducibly with this method.

The next thing we showed was the ability to measure and quantify clips. This is an in vitro assay where we actually took an antibody and clipped it completely with the IdeS enzyme, and then spiked it back into unclipped molecule. We looked at that spiked series with the reduced CE-SDS assay, and then also looked by MAM. As you can see here, as we go from 100% unclipped to fully clipped, we are able to track that very well. Even at a 1% clip level we can see that, and lower than that I might add. The next example I want to show is detecting host cell proteins and actually monitoring host cell proteins. Typically we use process-specific ELISAs or kit ELISAs to measure host cell protein through our process. What we are showing here is that at different steps in the process development pipeline, we see by ELISA, the host cell protein total ppm level dropped considerably once we get to the AEX step. But, if we go through and actually characterize what whole cell proteins are in our sample at each of these steps, we then realize that it is specific whole cell proteins that may be lingering longer and binding to our molecule directly, and that the host cell protein ELISA may not be able to detect those proteins because they are bound to our molecule. So understanding how these proteins are interacting with our molecule is important for understanding potential safety risks associated with these host cell proteins. Just to summarize, to date we have monitored multiple things using this method including – like I showed – deamidation, all the way through isomerization, non-glycosylated heavy chain. And you can also do sequence variants. You identify a sequence variant that you have in your molecule. You can also track that very easily with this method. Mass Spec-Based New Peak Detection Now I want to focus in on the most important component of this method – new peak detection. I can’t stress enough that this is absolutely essential for using this method to release biologics from QC. We have to have that purity component. We cannot ever get rid of our current purity methods without this method having that purity component. The nice thing about this method is that it is automated. You are leveraging the ability of this software to interrogate your mass spec data. There is no bias. You are not relying on a person to look at a UV tracer or an electropherogram. You are leveraging the ability of the software to do this for you. The thing that we have realized, once we started using this method, is that we are actually detecting new critical quality attributes by doing this. The way that we do this is by applying filters to eliminate false positives. It is a very simple workflow…. The way the software works is that it goes through and does a base peak alignment, and then we apply filters or criteria for doing peak detection, including how wide a peak can be, what the accuracy of that peak should be, as well as the retention time and the M/Z range. This was the first group of concept slides that I threw up back at Amgen, and then we have reproduced this many times just to show proof of concept. I presented this at another meeting, and there was a regulator there and they jumped all over it. I will explain why in just a second. What we are showing here is…the spiked peptide and the control trace. We spiked in 15 peptides. Of those 15 peptides, we detected 13. The beauty of this method is that we do not have to resolve them. We could see four of those peptides in the base peak chromatogram. But six of them co-elute with the lgG1 peptides. This method can detect them. Three of

those peptides are actually below the base peak threshold, so they would be below that limit of detection if you are using a visual method. And then two of those peptides were not detected, and that is where the regulator is like, ‘you have false negatives.’ But the beauty of this method and the role of the process development scientist is to figure out where to set those thresholds. What you do is that you actually change that threshold. Your safety criteria says that you need to be down to this level to be able to detect when this molecule falls out of spec – when you might have a safety risk. The cool thing about doing that is when you apply a different threshold, you know that then you are able to monitor everything that you need to monitor on this molecule. Instead of detecting 13 out of 15, we are actually seeing 24 new species now. The very cool thing about that is when we go in to look at what those other nine things are – they are actually contaminants from the peptide mix that we spiked in. And we can show that through a dilution series. So it is a very powerful method, and we are able to leverage it by applying these filters and thresholds to get at the purity of our molecule. Here is an example as to how we apply new peak detection to different formulations looking at clips here. We have formulation 1 vs. formulation 2. We looked at a stability series, where we are looking at -80C storage, 25C or 40C. You can see in either formulation that our clips are increasing over the temperature series. If we look at the total clips detected, we see those increase. Now, we did not know where these new peaks were occurring. We apply new peak detection and we see new peaks come up. And then we go back and characterize what those new peaks are, and we start to realize that there are clips that are occurring in places that we did not know would clip in the first place. So we can then identify exactly where those clips are, as well as quantify how much of our molecule is clipping at each different temperature. And this is just a snap shot. I am only showing four clips here. There are other clips that we can monitor that I am just not showing just to keep the figure small.

Using the MAM at Just In the second to last part of the talk, I want to show how we are applying the multi-attribute method to different core components of what we are doing at Just. Molecular Optimization The first one is molecular optimization. Part of what we are doing through molecular optimization is looking for hot spots in the amino acid sequence of our molecule and how do we take that amino acid sequence and make the molecule better for manufacturing. So you can see this is actually an example of a very hotspot-heavy molecule. We can look at this and try to figure out what amino acids or what components of this molecule might cause issues for manufacturing. I just want to focus really quick here: We have an aspartic acid at position 58, which is actually solvent exposed. And so you might run the risk of isomerization with this site. So then we do stability experiments and actually see that, yes. over different temperatures and different formulations, this is a hot spot for isomerization. So this would be a site that we would want to target for molecular optimization and potentially change it, because the isomerization of this could affect the potency of the molecule.

Real Time Monitoring The next part of applying the MAM is actually in the manufacturing facility. It could be a 500 liter…or a two liter bioreactor, but we use aseptic sampling. This is being done currently in collaboration with Merck and Bend Research to pull cell free samples from bioreactors and apply the MAM to them, and then take those data to actually make cell culture changes so that we can get the molecule we want. The process no longer defines the molecule. We define what the molecule is. So this is a snapshot of what the Merck PRO Lab looks like. And it is a very cool lab to be in. Over here on the left is the bioreactor. It actually has the ability of pushing a sample all the way through the different unit ops, all the way to single pass UF. So you get drug substance out at the end. In the middle here of this room, is where the cell removal system sits. And then right on the other side of this equipment is where the Exactive Plus sits for doing the MAM.

What we have done in this lab is actually apply MAM to each unit op and looked at how the attributes are behaving at each step. I would point you to Doug Richardson’s poster today. It is number 221.

MAM Consortium I want to finish up by explaining what we are doing with the MAM consortium. The purpose of this consortium is to bring the biopharma community together so that we can understand how each of us are using the multi-attribute method, and come to a consensus on how we want to apply this in process development and for QC release, and come up with a common data type. So that when we go to the regulators, they are seeing the same data from all these different companies, so that they are comfortable with how we are presenting it and how we are using it for each molecule. Then, we have shown that we are able to characterize our molecules better with this. And then the goal is to reduce the number of assays out of QC. We meet roughly every other month. We have one group present and it is usually followed by discussion. This year we are actually embarking on a very fun experiment. We are going to do a new peak detection round robin. And this is in collaboration with NIST. We are going to create a stressed NIST molecule as well as a peptide spike NIST digest, and then send that out to all the consortium members and ask them to apply new peak detection, and get a gauge on we are as an industry for applying new peak detection to our molecules.

Summary In summary, I hope I have shown you how we are leveraging the multi-attribute method for characterizing our biotherapeutics. We really benefit from the technology that we have access to now as well as the great search algorithms that are available. But I want to focus in again on new peak detection. When you hear multi-attribute method, I also want you to hear new peak detection. That has to be a key component of the method to be successful in QC. We have shown that this method is extremely precise and can track attributes very similarly to our conventional assays. This is a direct measure. We are directly measuring the attributes that are important to our molecule. It is not an acidic variant or a basic variant. It is a specific modification, or a specific clip to our molecule, or a specific impurity. Automated new peak detection is actually more sensitive than the current purity assays. We are not limited by what we see visually. We can actually go deeper into the data and actually learn what is going on in our molecule when it actually falls out of speck. We are identifying new modifications with this method. And I showed how we are leveraging it for molecular optimization, PAC, and real-time release, hopefully. In the consortium, we are bringing together leaders from the biopharma community to make MAM successful for QC release. So, this project is huge. There are so many people that have been involved over many different companies. Here is a snapshot of all of those people. Currently at Just, I work with Nancy Nightlinger and Randal Bass. But this product started back at Amgen, and all these people currently at Amgen or alumni of Amgen were instrumental in making this method a reality. Then I also want to focus attention to Merck. We also have a great collaboration with Merck right now – Doug Richardson, Yi Wang, Bhumit Patel, and Dave Pollard.

ROCHE’S PATRICK BULAU

Today I would like to summarize how we apply mass spectrometry for quality control purposes at Roche Pharma in Europe.

Initially I would like to introduce what we do at the Roche Pharma site at Penzberg, Germany. In the main part of my presentation, I would like to show you one example where we use mass spectrometry for the quality control testing of trastuzumab.

In the second part of my presentation, I would like to discuss with you some points to consider when applying quantitative LC-MS peptide mapping for QC purposes.

Roche’s Penzberg Operations

At the Roche site in Penzberg, Germany we do have research, development, and manufacturing of diagnostics and pharma products. In our global network for biologics, we do work closely together with our US colleagues from Genentech and our Japanese colleagues from Chugai Pharmaceuticals.

At the Penzberg production site we do manufacture drug substance for multiple marketed products such as Herceptin (trastuzumab) for the treatment of HER2 positive breast cancer, EPO, in its pegylated form Micera, for the treatment of anemia, interferon alpha 2A, also in its pegylated from Pegasys for the treatment of hepatitis C. But also the recently licensed glycol-engineered antibody Gazyva for the treatment of leukemia.

In principle, we do apply both techniques for ionization with mass spectrometry, [MALDI-MS] and also electrospray-based methods. We do this not only to assess the primary structure of proteins by intact analysis of peptide mapping, but also to assess the degree of glycosylation pattern by peptide mapping, direct analysis of reduced or intact protein samples. But we do also directly assess glycan heterogeneity by MALDI-TOF mass spectrometry.

In addition, we also assess chemical and post-translational amino acid modifications, such as deamidation of asparagine procedures, and isomerization of aspartate to isoaspartic acid. We also analyze oxidation processes resulting in the formation of oxidized methionines and tryptophan procedures. We also assess N- and C-terminus modifications of our antibody polypeptide chain. And we do also identify glycation site by LC-MS peptide mapping.

We do also apply sensitive peptide mapping to identify product-related lower band variants like sequence variants or process-related variants such as host cell proteins. We do assess higher order structure by applying HDX mass spectrometry. And for the assessment of non-covalent interaction of our products, we do also apply native MS conditions.

We do also assess unexpected modifications – for example, derived from the application of E coli to bioprocessing.

Application of MS Methods in the Laboratory

In general, we can split our efforts into two parts: The extended characterization and release testing of drug substance.

In the area of extended characterization, we do support comparability studies by analyzing chemical and post-translational modifications, and also glycosylation pattern. We do support isoform characterization, charge and size variants, process development activities by assessing host cell proteins or sequence variants content. We support our marketing organization in the case of troubleshooting activities, and also in the case of in-process analytics. Other applications like CQA assessment, structure/function relationships are also supported by extended characterization studies.

In the field of the release testing [of drug substance], we have been using Maldi-TOF MS for development products since 2009 – mainly for the identity testing using a peptide mass fingerprint approach. That is an approach developed by Genentech and already presented at several conferences. But we use also Maldi-TOF MS for discriminating protein products with identical amino acid sequence, but different glycan distribution, for example, which isn’t the case for glycol-engineered antibodies.

As we will show in a minute, we also use electro spray-based mass spectrometry of reduced antibody samples for QC testing of trastuzumab since 2004. And we also use LC-MS peptide mapping for identify testing of interferon alfa-2a drug substance.

Trastuzumab Example of ES-MS in Release Testing

As mentioned before, we do use electrospray-based mass spectrometry for QC testing of trastuzumab – and here, not only to confirm the molecular mass of the light and main heavy chain variants, but also to do a quantitative determination of the main galactosylation variants (G0F, G1F, the 13016 connection, or G2F). We do so because we were requested by the Japanese health authorities to assess galactosylation of the heavy chain not as a safety parameter, but as a process consistency parameter.

Originally we established a QC test system on an old Q-TOF 1 instrument from Micromass Waters. Your operators can see here in a direct infusion mode.

The test principle is relatively easy. We first apply a denaturation and reduction step for trastuzumab, followed by rebuffering into a suitable electrospray medium using gel filtration columns. And then we perform an offline electrospray measurement of the reduced trastuzumab sample by Q-TOF or LCT mass spectrometry.

Then finally we perform an automated calculation of the molecular masses for the light and heavy chain, and also we do a relative qualification of the main heavy chain gylactosylation variants.

You can see a sum mass spectrum of reduced trastuzumab. And for better inspection of the charge state I will zoom in on this graph. And you can see different charge states for the light chain, and also the different charge state for the main galactosylation variants Q0F, Q1F, and Q2F.

In order to get a more accurate mass determination and quantification, we created derivative spectra out of the data, using a validated in-house software.

Acceptance Criteria and Validation

Since it is a QC assay, that also defines system suitability and acceptance criteria for the batches. The accuracy of the molecular mass is [determined for the light chain and main heavy chain variants for the reference material]. They must be in a certain range. And also we had to define a threshold for the intensity [of the light chain signals].

Acceptance critieria for release of batches: The batches for release must have mass values that are highly comparable to the mass values for the reference material. And the relative amounts determined for the main heavy chain variants must be within the defined limits of the production history.

We had also to validate our method according to the guidelines of the International Conference on Harmonization. Just briefly, we had to: ● assess the accuracy of the mass determination for the light and heavy chain variants for the quantification ● define/elucidate precision (repeatability and intermediate precision) ● analyze the robustness of the test system ● describe the linearity of the quantification for the main heavy chain variants, and finally ● identify the limit of detection and limit of quantification. If you are more interested in the details, the validation data is attached in the backup slides for your inspection.

Just briefly with regard to accuracy, we compared our quantitative data with data sets obtained by two alternative methods: one is the high-performance anion-exchange chromatography with pulsed amperometric detection [HPAEC-PAD], and the second method was LC-MS peptide mapping of the respective glycosylated Fc peptide. By plotting here the data obtained for the QC method against the…data for the HPLC method, you can see for all three main glycosylation variants, a high correlation. The same was also true by comparing the data with LC-MS peptide mapping.

With regard to robustness, you can see a plot where we displayed the qualification data for these three galactosylation variants, G0F, G1F, and G2F for the reference material over a period of about five years. We are convinced that truly is robust, and we have never seen significant variations in the qualification results for our reference material.

In conclusion, we do believe that direct assessment of not only the molecular masses for identity purposes but also for the quantification of the main protein variants can be assessed by a direct application of electrospray-based mass spectrometry.

For trastuzumab, we have analyzed more than 500 drug substance batches since 2004, and we have just observed three system suitability deviations and five out of trend results.

Recent improvements for lifecycle management for mass spectrometers: We had to bring in a new type of mass spectrometer within our quality control system.

Points to Consider When Applying LC-MS for QC

In the second part of my presentation, I would like to discuss with you some points to consider when applying quantitative LC-MS peptide mapping for QC purposes.

Here you can see a high-level summary of the current control strategy of trastuzumab for product-related variants.

We do not assess free thiols because we have never seen significant content, and more than 99% of the results are oxidized…We do assess deamidation and isomerization events by cation exchange chromatography because in the CDRs of trastuzumab we have three susceptible degradation sites, namely: light chain asparagine-30 in the CDR-1, heavy chain asparagine-55 in the CDR-2 – they are both prone to deamidation – and heavy chain aspartic acid in the position 102 which is prone to isomerization.

Fortunately we don’t have accessible [Met or Trp] residues within the CDRs [complementarity-determining regions] of trastuzumab. And therefor it is also not monitored at the release drug substance level.

Also for the well characterized Fc oxidation sites we have never seen significant alterations in the content. We also do not assess n-terminal bioburden information, glycation and acetylation events because we have never seen significant levels. We do assess, as mentioned before, glycosylation. Historically at Genentech, they used the [CE-LIF] for glycan assessment. At Penzberg, for a long time we have used an electrospray-based method as described before, and since two years ago we do at both sites in the US and Europe apply a more detailed characterization of the Fc glycans by [2 AB HPLC].

Of course, aggregation and fragmentation events are monitored by size exclusion chromatography.

[A technical comparison of the MS methodology to the traditional methods made by Bulau at this point in his presentation is not included.]

Summary

Finally I would like to summarize my presentation.

We do believe that both MALDI and electrospray methods can be used for robust and specific identification purposes, especially for the ID testing of biologics. In our experience, those systems are even more robust than classical…assays.

I hope I could convince you that robust determination of main product variants can be conducted by electrospray- based mass spectrometry. Here we have demonstrated glycosylation variation. But our new high-resolution mass spectrometers can for sure also assess oxidation events, but maybe not deamidation or isomerization events.

Multi-attribute monitoring of multiple CQAs by quantitative LC-MS peptide mapping should be carefully established with respect to robustness, method transfer to other mass spectrometers, but also to other production sites for drug substance or even filling sites for drug product, and acceptance criteria.

For sure if you would apply a multi-attribute method in the QC, it would have a strong impact on the specification. Typically we have specified something like total HCP content, or the charge variants acidic region. If you would now specify individual peptides, there is a certain risk that you end up with more deviations compared to the old control strategy.

Neverthelesss, we do believe that mass spectrometry can be realized and conducted in a QC environment and should really add value to the control strategy.

Finally I would like to thank a lot of people for their contribution [● Katrin Bomans ● Lea Bonnington ● Manfred Wozny ● Markus Haberger ● Marco Thomann ● Maria Maier ● Susanne Eltner ● Niklas Engler ● Markus Dembowski ● Dietmar Reusch.]

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