Science Pool

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 To obtain plasma, blood is generally withdrawn by a conventional venous collection method.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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Autotaxin (ATX) is a secreted glycoprotein that hydrolyzes lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA). LPA signalling directly modulates tumour cell function and contributes to the development of the fibrotic tumour microenvironment, resulting in reduced host immunity and impaired response to therapy.

iOnctura, a clinical-stage oncology company, based in Switzerland (Genève), targeting core resistance and relapse mechanisms at the tumor-stroma-immune interface, has developed a potent and orally available autotaxin inhibitor, IOA-289, as a novel treatment for pancreatic cancer and other highly fibrotic tumours.

The first Phase I clinical study in healthy volunteers has been successfully completed. Evotec is very proud to have actively contributed in conducting the trial and analyzing the results that led to the achievement of this exciting goal.

Read the poster presented by iOnctura at the ESMO Immuno-Oncology Congress held in December 2021 to learn more about IOA-289 and its activity as autotaxin inhibitor. IOA-289 was also presented by iOnctura at the recently held AACR Annual Meeting in New Orleans.

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Evotec sample management team is responsible for the long-term storage and distribution of Evotec and partners compound collections.

In 2019, Evotec decided to invest in acoustic tubes technology in order to support future customer need and plan for potential transfer of our libraries into acoustic tubes. In parallel to this decision, Evotec sample management decided to initiate an internal project to evaluate the impact of acoustic tubes usage on compound integrity.

This poster highlights and describes the experiments performed, which substantiate and validate the use of acoustic tubes.

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In this poster we focus on:

• Development of robust feeder-free 3D differentiation protocol to reduce iNK cells
• Transnational validation of iNK functionality with freshly isolated CLL patient tumor cells
• Enhances CLL patient tumor cell killing with CAR19 iNK
• EVOcells Oncology programs

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Chikungunya virus, which is transmitted by mosquitoes, can cause disabling chronic arthritis. There are currently no medicines for prophylaxis of Chikungunya, or other viruses transmitted by Aedes mosquitoes, such as Dengue and Zika. Potential therapeutics have been identified, but to perform a successful chemoprophylaxis trial during a short Chikungunya outbreak requires an identified at-risk population. We examined the potential for application of a household transmission model, as used in testing prophylactic drugs against respiratory viruses, included influenza and COVID-19.

In this poster we:

• Set out the evidence for multiple Chikungunya infections occurring per household
• Describe how we are validating the secondary household infection rate
• Show how this could be used in future clinical trials to demonstrate prophylactic efficacy against chikungunya virus infection and other Aedes-mosquito-borne viruses

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Reactive metabolites play a role in drug-induced toxicity. Early stage in vitro screening for electrophilic reactive metabolite formation involves the use of trapping agents such as glutathione (GSH) in the presence of a drug metabolising system.

In this poster, we focus on:

• the use of an alternative GSH trapping method using combined stable labelled GSH (GSH-¹³C₂,¹⁵N) with unlabelled GSH
• the advantages of the stable labelled GSH method over common approaches for the detection of GSH conjugation such as GSH neutral loss post acquisition
• analytical conditions including the HPLC and mass spectrometric methods 
• validation data for a set of literature compounds

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Lipophilicity is an essential physicochemical property used to predict compound behaviour with respect to pharmacokinetics, pharmacodynamics and safety. 

In this poster, we focus on:

• the use of the chromatographic hydropobicity index (CHI) as an alternative to LogD for lipophilicity determination
• the use of LC Time-of-Flight mass spectrometry for CHI determination and its advantages with respect to reduced inference from impurities, decreased compound requirements and improved throughput
• applicability of CHI for large scale screening projects
• the presentation of the reproducibility and robustness of the method along with comparison with literature values

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Cardiotoxic drugs can display acute alteration in the mechanical function of the myocardium (functional changes) or morphological damage to cardiomyocytes and/or loss of viability (structural changes).   

In this poster, we focus on:

• the detection of functional cardiomyocyte changes through monitoring of calcium transients using human iPSC-derived cardiomyocytes
• the analysis of structural morphology using high content imaging (calcium homeostasis and mitochondrial function) as well as cellular ATP in human iPSC-derived cardiomyocytes
• the presentation of a strategy for understanding cardiotoxicity risk for novel compounds enabling in vitro to in vivo translation at an early stage in preclinical screening.

Read our poster to learn more about our research!

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