Science Pool

Children diagnosed with TUBB4A leukodystrophy (Hypomyelination with Atrophy of Basal Ganglia and Cerebellum) experience a progressive and severe deterioration of recently acquired motor skills. Currently, there is no available cure for this condition. Evotec, in collaboration with SynaptixBio, is developing antisense oligonucleotides (ASOs) targeting TUBB4A mRNA to suppress gene expression, irrespective of specific mutations.

The research presented here represents a crucial segment of Evotec’s oligonucleotide-based drug discovery initiative. This study employs a combination of in vitro, in vivo, and bioinformatic methodologies to minimize the synthesis and testing of acutely toxic molecules in murine models.

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Eloi Haudebourg¹, Yvan Eb-Levadoux¹, Catherine Pech¹, François Autelitano¹, Berenice Rotty¹, François Lantreibecq¹, Yannick Cogne¹, Navratan Bagwan¹, Alessia Cavaliere², Rossella Cardin², Alberto Vezzelli², Alessandro Greco²,
Philipp Ellinger³, Wiebke Afhueppe³, Karoline Droebner³, Winfried Wunderlich⁴

_{1 Evotec (France) SAS Toulouse, France
2 Aptuit (Verona) Srl, an Evotec Company, Verona, Italy)
3 Bayer AG Pharmaceuticals Research, Berlin, Germany
4 Evotec SE, Goettingen, Germany}

Introduction: While proteomics tools are increasingly used in the early steps of the drug development journey, its use remains limited to exploratory endpoints in clinical phase. Successful MS methods used in the exploratory and preclinical phases are adapted to antibody-based assays. For the present study, no antibody of the target engagement biomarker could be validated for animals in preclinical phase. An MS-based protein quantification method was therefore developed in Research Use Only (RUO) and ultimately transferred to a Good Clinical Practice (GCP) environment for validation and application to support preclinical development and clinical trial Phase 1 as a primary endpoint.

Methods: First, a high purity Stable Isotope Labelled (SIL) protein was produced in HEK293 cells cultured in SILAC medium and purified (anti-poly-histidine followed by IMAC and SEC). Second, a targeted MS assay was developed for the absolute quantification of biomarker of interest. Briefly 2 µL of plasma was processed on an iST kit (Preomics), peptides were injected using a microflow LC (M Class, Waters) on a C18-CSH column (Acquity, Waters) and quantified in MRM mode on a triple quadrupole (6500+, Sciex).

Preliminary data: Acceptable protein purity (> 80 % based on MS intensity) and excellent heavy label incorporation (>98 %) was archived. A targeted proteomics assay was first developed in RUO and transferred to a GCP facility where the method was validated according to FDA validation of bioanalytical methods for Industry and ICH-M10 guidelines. Specifically, the method validation included the assessment of precision and accuracy of the assay, surrogate matrix equivalence, lower limit of detection (100 ng/ml), concentration range, short- and long-term stability of the samples.
The validated, targeted MS method is routinely used to quantify a target engagement biomarker in a currently ongoing Phase 1 clinical trial.

Novel aspects: First time for the use of MS-based targeted proteomics to quantify proteins in clinical samples as primary endpoint.

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Oliver Kardell¹, Till Kindel¹, Frank Rolfs¹, Samira Vautrin¹, Barbara Kracher¹, Thomas Gronauer², Christine von Toerne² , Andreas Tebbe¹, Stefanie M. Hauck², Carleen Kluger¹

<sub>1. Evotec München GmbH, Anna-Sigmund-Str.5, D-82061 Neuried (Germany)
2. Helmholtz Zentrum München, Ingolstädter Landstraße 1,  D-85764 Neuherberg (Germany)</sub>

Liquid biopsies like plasma, cerebrospinal fluid (CSF), or urine are a less invasive and inexpensive alternative to tissue biopsies. Additionally, these body fluids are known to contain a multitude of known and yet to be revealed biomarkers that can be detected using mass spectrometry (MS) based proteomics. There are several distinct advantages of this technology: (I) it gives unbiased access to the biofluid proteome, (II) it is species independent, (III) it can reveal information about isoforms and post-translational modifications and (IV) it does not require availability of antibodies. Furthermore, MS based proteomics has been rapidly evolving over the last years due to the development of new generations of MS instruments such as timsTOF or Thermo Astral, advancements in sample preparation like nanoparticle-based protein enrichment, as well as novel AI based tools for data processing. The continuous progress results in increased depth and throughput of clinical proteomics.

However, this highly dynamic nature of the field can also be a drawback, as applications of mass spectrometry-based proteomics for larger clinical trials is often hindered by a lack of standardization across laboratories and studies. To address this problem and to enhance the integration of MS-based proteomics into medical research the CLINPSECT-M consortium and its MS laboratories conducted a round robin study on clinical specimens such as human plasma and CSF.

Here, the data from the round robin study is compared to internal benchmark studies on human biofluids that were performed at Evotec. In addition, while the focus of the round-robin study was to harmonize workflows for undepleted plasma and CSF and track interlaboratory reproducibility, the Evotec study also measured longitudinal platform stability and shows how nanoparticle-based proteomics using the Proteograph^TM can provide deeper coverage of the plasma proteome and at the same time overcome some of the challenges associated with undepleted workflows. 

Multicenter Collaborative Study to Optimize Mass Spectrometry Workflows of Clinical Specimens Oliver Kardell, Christine von Toerne, Juliane Merl-Pham, Ann-Christine König, Marcel Blindert, Teresa K. Barth, Julia Mergner, Christina Ludwig, Johanna Tüshaus, Stephan Eckert, Stephan A. Müller, Stephan Breimann, Pieter Giesbertz, Alexander M. Bernhardt, Lisa Schweizer, Vincent Albrecht, Daniel Teupser, Axel Imhof, Bernhard Kuster, Stefan F. Lichtenthaler, Matthias Mann, Jürgen Cox, and Stefanie M. Hauck, Journal of Proteome Research Article ASAP, DOI: 10.1021/acs.jproteome.3c00473

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Carleen Kluger, Evotec (München) GmbH

The advance of nanoparticle-based technologies (e.g. ProteographTM)  in combination with high-end mass spectrometry (MS) has increased the depth of proteome coverage for biofluids like plasma, serum or cerebrospinal fluid (CSF), making it possible to routinely analyze 2.000-3,000 proteins per individual biofluid sample. Over the last two years we have extensively used this technology at Evotec to screen multiple different patient cohorts, each containing up to a few hundred serum or plasma samples from patients with very different disease backgrounds, including patients with multiple co-morbidities and a dedicated cohort of healthy subjects that were hospitalized over-night to ensure maximum control of biofluid and data collection.

While in the past, most activities in the field of Clinical Proteomics were limited to a specific patient cohort in a clearly defined disease context, the next challenge is to integrate proteomics signatures from multiple cohorts into a unified framework. Here, we show how this can be achieved by minimizing technical variability during sample collection, sample preparation and MS measurement using highly automated nanoparticle-based proteomics approaches. 

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Data Integration: With Bioinformatics to Biological Knowledge

Tim Blokker, Christian Schiffmann, Micael Fernandes dos Reis, Christopher Bruhn, Christiane Honisch, Carleen Kluger, Barbara Kracher, Erik Schliep, Evotec International GmbH

PanHunter is a user-oriented and interactive web application allowing for in-depth data discovery across multi-omics datasets without the need for coding skills. The platform was originally built by Evotec’s bioinformatics team to handle large transcriptomics datasets generated by our industrialized high-throughput transcriptomics platform. In recent years, with the advancement of high-throughput mass spectrometry-based proteomics, major advances have been made to tailor PanHunter towards handling massive datasets from different proteomics data formats.

The strength and novelty of proteomics analysis in PanHunter is founded in the holistic approach to integrate multiple levels of data (e.g. gene, protein, and post-translational modification (PTM)) through the whole workflow starting from quality control (QC) over exploratory analysis, differential expression analysis down to functional analysis, and incorporation of biological knowledge from various sources. PanHunter can map between PTM IDs, UniProt IDs, and on transcriptomics level Ensemble IDs. This mapping allows the user to analyze features simultaneously in linked plots, with data stemming from transcriptomics, proteomics or PTM data.

Here, we provide examples to highlight how the expansion of PanHunter’s proteomics capabilities facilitates data integration and interpretation in the context of precision medicine, thus driving easier access to novel medicines across various therapeutic areas.

E.MPD, Evotec‘s molecular patient database, combining patient and omics data across multiple disease areas has been extended to cover autoimmune diseases in recent years. Integration of proteomics data from Evotecs’s high-throughput mass spectrometry-based proteomics platform with patient data in PanHunter enables novel insights into the regulation of autoimmune diseases on the level of the proteome.

Evotec’s ScreenPep platform allows for high-throughput screening of compounds of interest. To enable seamless data analysis after the screening, PanHunter’s signature database has been broadened with proteomics signatures from Mitchell et al. 2023. These signatures can be used to investigate toxicological effects and help decipher the mechanism of action of novel compounds.

Mitchell, D.C., Kuljanin, M., Li, J. et al. A proteome-wide atlas of drug mechanism of action. Nat Biotechnol 41, 845–857 (2023). https://doi.org/10.1038/s41587-022-01539-0

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Evotec is investing in the strategy, to transfer progressively Evotec’s collection compounds into acoustic tubes (AT) to support our partner’s screening needs. Compounds will still be distributed from Master stock plates for Assay Validation (AV) and Primary Screen (PS) steps. However, for the Hit Confirmation (HC) and Hit Profiling (HP) phases, compounds will be prepared directly from AT. 

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Evotec’s Sample Management aims to deliver the highest quality service while maintaining efficiency. By striving for continuous quality improvement and minimizing equipment errors, we build trust with our partners through high-speed turnaround and a ‘right first time’ approach.

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After 25 years of innovative compound management support, Evotec’s sample management has expanded its operational presence by adding capacity and capabilities in Toulouse, and now offers the storage of biological material in a dedicated facility to ensure sample security and integrity.

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