Science Pool

Cardiotoxicity in response to pharmaceutical drugs, chemicals and environmental toxicants can develop as a result of changes in structural integrity of cardiac tissue or functional changes in cardiac electrophysiology. The development of new approach methodologies (NAMs) is important in predicting these cardiotoxic liabilities.

In this poster, we focus on:

• the use of human iPSC-derived cardiomyocytes in combination with a panel of in vitro toxicology assays including:
  • calcium transient monitoring
  • high content imaging of morphological changes
  • cellular ATP assessment
  • high throughput transcriptomics using RNA-seq
• the results from profiling 42 compounds in the assessment of structural and functional endpoints along with predictive outcome (sensitivity, specificity and accuracy)
• mechanistic information including the detection of biological pathways associated with the cardiotoxic effects

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The cytochrome P450 enzyme, CYP46A1, is predominantly expressed in the brain and plays a major role in cholesterol metabolism by converting cholesterol to 24-hydroxy cholesterol.

In this poster, we focus on:

• the development and validation of an in vitro high throughput screening platform for characterisation of inhibitors and activators of CYP46A1
• a comparison of testosterone with cholesterol as a potential replacement probe substrate for studying CYP46A1 metabolism
• data generated from a screening cascade of 2321 FDA approved drugs

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The tuberculosis lesion environment is highly complex. Mycobacterium tuberculosis (M.tb) reside in various niches and their metabolism and other characteristics, including drug tolerance depend on the specific environmental characteristics of those niches. So-called persister bacteria refer to those that are difficult to eradicate with drug treatment and that may contribute to the long durations of TB treatment required for cure. Many in vitro models have been developed in attempts to mimic these niches with the objective of determining potential anti-TB compound activity in models that reflect M.tb’s characteristics in vivo.
Foamy macrophages Foam-M are characterized by lipid body accumulation induced by Mtb infection and they may be an important niche for Mtb during infection.

In this poster we:

• We describe the development of a robust 96 well plate Foamy macrophages assay designed to mimic this niche, and suitable for medium-high throughput evaluation of compound activity during drug discovery.
• We describe TB compounds activity in the foamy macrophage assay compared to other TB current assays
• We show that infection of foamy macrophage does not trigger an anti-inflammatory response in host cells

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Hepatitis B is a serious liver infection caused by the hepatitis B virus (HBV). It is still a major public health problem with 250 million chronically HBV-infected patients worldwide with none of the current treatments leading to a cure.
More effective treatments are needed to achieve HBV cure in a large proportion of patients.
We aim to develop a treatment, combining simultaneous stimulation of CD40 and IFN-I pathway, which leads to a strong anti-HBV effect with minimal inflammation.

In this poster we:

• Present the key results that demonstrated the potential of such combination to induce anti-HBV effects in vitro and in vivo
• Introduce the design of our new antibody-based therapeutic that combine the two modalities in one single molecule
• Demonstrate the efficacy of the molecule in HBV infection system in Primary Human Hepatocytes
• Show key results from our preclinical safety assessment in vitro and in vivo in Non-Human Primate

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Full poster title: Development of recombinase-based targeted integration systems for production of exogenous proteins using transposon-mediated landing pads

Summary of the poster:

• We demonstrated proof of principle of targeted integration systems in our CHO host cell line with consistent genome integration into expected landing pad sites
• Test cases using three antibody or antibody-fusion therapeutic molecules showed similar levels of productivity
• We also show preliminary data from ongoing work to build upon these targeted integration systems, which includes isolating a single-copy landing pad cell line and developing a CHO display platform

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In this poster, presented at SfN 2018, Sessolo et al. present 3Brain high-density multi electrode array (HD-MEA) as a system to monitor and characterize seizure-like activity in hippo-cortical slices induced by different compounds.

The high system resolution allows to monitor in detail the entire slice and through the software showing the activity map (in real-time) the sign of compounds' action is easily found.

The technology allows to acquire Local Field Potential (LFP), Multi Unit Activity (MUA) and Single-Unit Activity.

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This poster includes information about:

• Functional brain slice electrophysiology by HD-MEA platform
• Combined neuronal circuitry studies through functional brain tissue imaging

Initially presented at FENS 2018 by Ugolini et al., 3Brain high-density multi electrode array (HD-MEA) as a system for long lasting monitor and characterize spiking activity of hundreds Purkinje cells simultaneously by using different positive and negative Ca++-activated K+ channels. Responses can be evaluated though different analysis. It is a useful tool for compounds validation on cerebellar slices.

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Toxicokinetic evaluation is a regulatory and scientific requirement in the drug development process. To obtain plasma, blood is generally withdrawn by a conventional venous collection method. Microsampling is a less invasive sampling technique, which allows to reduce the stress correlated to the conventional blood sampling and to decrease the number of rodents for a preclinical study. The implementation of microsampling in particular, in non-human primate can reduce the stress and promote a positive interaction with technical staff which improves the overall well-being of the animal (refinement).

In this poster we summarise the work done to evaluate the possible influence of the blood sampling method on drug plasma concentrations, using LC-MS/MS methods in non-human primate for four drugs selected based on acid-base and volume of distribution properties.

The poster was presented by our expert Rossella Cardin at the 24th International Reid Bioanalytical Forum held in Cambridge, UK, on June 13-16, 2022.

 

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Key Takeaways:

• We have developed an AI-generated antibody library platform, which we call J.HAL^®,  utilizing a Generative Adversarial Network (GAN) that generates novel sequences which mimic natural human response, as well biasing toward diversity and developability features.
• The resulting Humanoid Antibody Library was successfully screened to obtain a panel of novel, diverse and pharmacologically active human antibodies against SARS-CoV-2.

 

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Key Takeaways:

• Non-infectious, purified RVLPs can be used in place of model viruses to predict process performance for viral clearance.
• Initial screenings show that clearance also trends similarly between bench screenings and higher throughput plate screenings.
• Plate-based screening of RVLPs in-process can examine up to 24 different run conditions simultaneously and uses less viral surrogate compared to bench scale runs, allowing for greater evaluation and confidence going into formal viral clearance studies.

Improving virus clearing studies in recombinant protein production

Chinese hamster ovary (CHO) cells are the most frequently used mammalian host cells for the industrial manufacturing of recombinant protein therapeutics. They can produce recombinant proteins on the scale of up to 10 gram per liter of culture. However, they are also known to contain type‐C endogenous retrovirus (ERV) sequences in their genome and to release retroviral‐like particles. Although evidence for their infectivity is missing, this has raised safety concerns, and regulatory agencies require demonstration that the purification process removes or inactivates viruses.

Viral clearance validation is assessed through “spiking studies”, whereby model mammalian viruses are introduced into process material which then undergoes the purification technique to be tested. Viral quantity before and after processing is determined through infectivity or qPCR assay. As these studies use live viruses, they require specialized Biological Safety Level laboratories (BSL) and experienced personnel and can create a substantial bottleneck because typically only 3rd party facilities are qualified to perform these studies.

As an alternative, Just - Evotec Biologics is in the early stages of establishing a high-throughput process using commercially available purified retrovirus-like particles from Cygnus Technologies LLC. These particles are non-infectious and mimic the physicochemical properties of live infectious viruses. By using these particles as spiking agents, the retroviral clearance capability of downstream unit operations can be studied, assessed, and quantified by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Usually, this is performed at bench scale using chromatography columns.

In a poster presented at this year’s ACS spring conference entitled High throughput optimization of chromatography steps for viral clearance using retrovirus-like particles (RVLPs), researchers from Just-Evotec Biologics detailed the high-throughput workflow for the analysis of RVLP content for rapid analysis of in-process samples.

The research team compared common bench scale chromatography runs with a plate-based screen using resin-loaded filter plates and a liquid handling robot. While at bench scale, only a single set of run conditions can be tested at a time, the plate-based screening can examine up to 24 different run conditions simultaneously. It also uses less RVLP stock solution. The researchers expect that plate-based screening of RVLPs will not only save time and costs, but also allows for better evaluation and confidence before formal viral clearance studies.

 

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