Predicting DILI (drug-induced liver injury) is challenging compared to other organ-specific toxicities. Translation from animals to humans is poor and, mechanistically, DILI can be complex. As a consequence, DILI continues to be one of the leading causes of attrition during drug development. Better human relevant models are required to improve early stage DILI prediction. Cyprotex is committed to researching and developing approaches to improve the prediction of DILI using human cell-based models in combination with novel techniques such as toxicogenomics and artificial intelligence (AI).

At the Society of Toxicology (SOT) conference on March 10-14, 2024, Cyprotex presented a poster titled, ‘An AI Approach to Drug-Induced Liver Injury Risk: Prediction of Safe Maximum Doses from Toxicogenomics Profiles’. The research evaluated 128 compounds from the FDA Liver Toxicity Knowledge Base – 68 of these compounds were associated with DILI and 60 of these compounds were not associated with DILI. Transcriptomics profiles were generated after dosing primary human hepatocytes in triplicate at 8 concentrations over 24 hr.

Machine learning is a subset of artificial intelligence which is used to find patterns, make decisions and optimise outcomes. In this study, the high throughput transcriptomics profiles of a set of known DILI-positive and DILI-negative compounds were used to train a supervised machine learning model to predict a safe maximum C_max for novel compounds. When interpreting the results, a compound was predicted as DILI-positive if the true C_max was above the predicted safe C_max, and a compound was predicted as DILI-negative if the true C_max was below the predicted safe Cmax. The model achieved the following metrics on the test set (assuming 40x C_max level and 90% DILI score threshold):

The poster provides a detailed insight into two DILI-positive (TAK-875 and bosentan) and two DILI-negative compounds (dopamine and caffeine) to demonstrate the power of the transcriptomics and AI in predicting DILI as well as identifying specific mechanisms of toxicity. The model was able to capture the importance of cholestasis-associated genes in DILI.

To learn more about the use of transcriptomics and AI in DILI prediction:

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Just - Evotec Biologics is constructing a new biomanufacturing facility in Toulouse, France. The facility applies the company’s successful J.POD design featuring a single-use continuous cell culture manufacturing platform set inside production-on-demand modules within a ballroom manufacturing space. The investment of approximately €150 million was announced in April 2021 and the company broke ground on the project in September 2022. In October last year the building shell was completed and the autonomous cleanroom POD installation occurred at the beginning of this year. Now the equipment will be installed into the cleanroom PODs ready for the facility to be operational in the second half of 2024.

At the core of this endeavour lies the innovative J.POD biomanufacturing facility developed by Just – Evotec Biologics. J.POD facilities contain the company’s continuous manufacturing platform for antibodies and other therapeutic proteins with an Fc-region. The continuous process is so highly intensified that it can be contained within production-on-demand modules that sit within a ballroom cleanroom space. The design is central to the company’s mission: to design and apply groundbreaking technologies that dramatically expand global access to biotherapeutics.

The J.POD design is commercial biologic manufacturing-ready but can also easily deliver batches for clinical trials. The ability to modulate capacity easily depending on the lifecycle stage of the molecules is one of the advantages of continuous manufacturing.

Transitioning to Continuous Manufacturing

Traditional fed-batch manufacturing methods have long been the standard for producing biologics. However, switching to continuous manufacturing with a high degree of intensification reduces the cost of goods manufactured (COGM) of biologics to less than $50 per gram by reducing the cost of building and running biomanufacturing facilities.

The use of pod modules in the design of J.POD Toulouse allow for greater agility, readily expandable facilities, and lower risk. Unlike traditional methods that require scaling up to larger production trains, the J.POD approach ensures flexibility in meeting demand fluctuations. This is extremely valuable to companies that launch new products and find it difficult to predict the ramp up in market demand.

By expanding into Europe, J.POD Toulouse enhances the company’s ability to support customers based in Europe, effectively. With facilities on both sides of the Atlantic, the company is providing supply chain security by having duplicated capacity in two geopolitically stable regions.

Just - Evotec Biologics' announcement last year of a multi-year, long-term tech partnership with Sandoz to develop and manufacture multiple biosimilars in J.POD facilities demonstrate the industry’s readiness to embrace continuous production methods.

Process Development Capabilities

To support manufacturing operations J.POD Toulouse facility houses robust process development capabilities, including:

1. Cell Line Development: Streamlining the creation of high-yield cell lines for antibody production.
2. Upstream & Downstream Process Development: Optimizing the entire continuous production process, from cell culture to purification.
3. Formulation Development: Crafting stable and effective formulations for therapeutic molecules.

Conclusion

J.POD Toulouse prepares to open its doors with its commitment to cost-effectiveness, scalability, and supply chain security. This facility stands poised to transform the way we produce lifesaving biotherapeutics. Watch this space—J.POD Toulouse is about to make waves in Europe and beyond.

This project benefits from French government funding as part of the Investments for the future Programme (programme d’investissements d’avenir in French) and is also supported economically by the Occitanie Region.

Patients around the world need access to affordable biopharmaceuticals to treat life-threatening conditions. High manufacturing costs can make these medicines unaffordable and limit their use amongst global patient populations. Historically, the biopharma industry has manufactured these medicines in large-scale stainless steel production facilities. Such facilities take years to construct and require over $500M of upfront capital investment. The cost of manufacturing biologics in these production plants is high and especially inefficient when asset utilization is low.

To reduce production costs, the industry increasingly recognizes that the next generation of production facilities must break with existing manufacturing paradigms. New facilities must be small and agile with intensified manufacturing platforms that allow extremely high productivity to meet late phase clinical and commercial demand.

Agile J.POD Facilities

Just-Evotec’s J.POD facilities apply modular technology to reduce the footprint of cleanrooms. Our facility design minimizes the expensive utilities needed to run a stainless-steel plant and instead leverages fully single-use and continuous biomanufacturing platforms. We culture mammalian cell hosts in perfusion bioreactors that we connected to a continuous purification train. In this way we can sustain volumetric productivities of over 2 g/L/day and continuously purify antibody during the production run making efficient use of production equipment.

The CAPEX associated with a J.POD facility is less than $200M. Our J.POD Redmond facility is operational in Washington, USA while we will bring a new European facility online in Toulouse, France in 2024. These facilities in two geopolitically stable locations will provide our customers with additional supply chain security.

Comparing J.POD to Traditional Facility Designs

Just-Evotec Biologics production engineers use models and associated visualization tools to optimize production costs. These tools show the relationships between facility design, production demand and drug substance manufacturing costs. Our engineers created mathematical models of a large-scale stainless steel and our J.POD facility. They used Net Present Cost (NPC) to compare scenarios. NPC estimates cash flows by computing operational costs and discounting over time using a capital parameter. It does not include revenues in the accounting of cash flows and assumes capital costs incurred at the beginning of the project are sunk costs.

Engineers took the following approach to compare the stainless steel and J.POD facility designs:

1. They generated 512 different patient population curves. (example is shown in the lower graph of Figure 1)
2. They estimated bioreactor capacity and utilization needed to deliver the mass of product required by these patient population curves for both facility types. (example is shown in the middle graph of Figure 1)
3. The engineers ran the model to estimate the manufacturing costs associated with each facility production mass output. (example shown in in upper graph of Figure 1).
4. The team used NPC calculation to produce a concise estimate of cash expenditures over time and normalized these values by their corresponding mass outputs. They assembled histograms to visualize the underlying statistical distributions behind a particular facility configuration (Figure 2).

Figure 1. The determination of manufacturing costs associated with two different biopharmaceutical manufacturing facilities producing sufficient drug product to meet the needs of a specific patient population curve.

Figure 1 shows that a stainless-steel facility has a higher initial cost at Year 0 because of the high capital expense allocation and has higher fixed costs than the J.POD facility over the operating period.

Figure 2 shows the benefits derived from the agility of the J.POD facility design. The NPC over the operating period was lower in the J.POD facility than the stainless-steel facility in every scenario modeled. The width of the NPC distribution for a POD-based facility is narrower than that of a stainless-steel facility. The production costs associated with a J.POD facility are largely independent of capacity utilization because of the lower upfront capital costs. Furthermore, managers have the option to expand capacity if needed by reacting to market demand estimates on a yearly basis. This is a significant advantage of the J.POD facility because managers can tailor plant capacity within the network to the latest market projections, making it more capable of reacting to disruptions or demand fluctuations.

Figure 2: Histogram depicting normalized Net Present Cost (NPC) estimates for both facility types.

Agile Efficient Biomanufacturing

J.POD facilities are inexpensive to construct and commission due to their small size and use of single-use technologies that limit the amount of plant utilities needed. We can quickly deploy these assets in response to fluctuations in demand forecasts. Production within J.POD facilities is very efficient due to the continuous manufacturing platform and the ease with which we can modulate capacity to maximize asset utilization. J.POD facilities are leading the transition away from expensive large-scale stainless steel production assets towards more agile and lower cost biopharmaceutical manufacturing. Access to these remarkable facilities is available to Just-Evotec Biologics customers through our innovative partnering arrangements. Together with our partners we are reducing the costs of biologics and making them more accessible to patients around the world.

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How to optimize your hit identification strategy 

What is hit identification and why is it important?

Hit identification (Hit ID) is an important step in the development of new medicines. It is the process of identifying molecules with desirable biological activity, such as the ability to bind to the target and modify its function. As hit ID is one of the first steps in drug discovery, optimizing this process is essential to provide the best possible chemical starting points for your drug discovery and development process. A successful hit ID campaign will maximize the output in terms of the number of high-quality hits that enter the hit-to-lead and lead optimization stages, saving you precious time and resources further down the line. 

However, identifying high-quality, validated hits is no easy feat. There are several components of hit ID campaigns that need to be considered, including the selection of the compound library, and the development of pharmacologically sensitive and robust assays for screening, triage, and validation. Hit ID also requires a broad interplay of disciplines, like reagent production, in vitro biology, medicinal chemistry, and statistical data analysis. These interact across several approaches, such as target-directed, structure-based, in silico, and phenotypic hit ID. With this in mind, extensive multi-disciplinary expertise is applied to design and implement the right hit ID campaign for your target.

To help simplify this pivotal stage in the drug discovery process and ensure that you get the most high-quality hits for your target, here are our top considerations for hit ID campaigns. Across those, we explore how an integrated end-to-end platform is key to supporting all stages of the hit ID process, and beyond.

Considerations to help you optimize your hit identification campaign

Screening strategies

There are several well-established screening approaches for hit ID, including target-directed, structure-based, in silico, or phenotypic high-throughput screening routes. Different approaches can be run in parallel or individually, depending on your target.  

Choosing the right screening strategy is one of the most important considerations for the hit ID process, and will largely determine the success of your campaign. To learn more about the different screening approaches, visit our hit identification webpage.  

Assay development

Primary assays are used for the detection of on-target activity or binding in high-throughput screens. There are different types of primary assays, such as cell-based, or biochemical assay systems. While cell-based assays provide a more physiologically relevant context, biochemical assays can give you deeper insight into target binding or modulation of the target’s function in a cell-free environment. Therefore, the type of assay you choose depends on your target, and the specific goals and requirements of your campaign. Visit our in vitro biology webpage to learn more about the different phenotypic and cellular-based assay technologies. 

When optimizing primary assays, it is important to verify the relevance of the assay to your specific disease state and target. Additional factors such as robustness, pharmacological sensitivity, reproducibility, scalability, and cost efficiency are taken into account. Development of such primary assays can be a complex and lengthy process that involves many steps, from initial setup, optimization, and pharmacological characterization, through to the adaptation to the screening system and pre-screening.

Further to the primary assay, the development of an appropriate readout counter assay is strongly recommended. Such assay applied at later stages of the screening process enables the identification of potential readout-interfering compounds that could result in false positive hits.

Compound libraries

Compound libraries are collections of small molecules used to identify hits in high-throughput screening assays. The success of your hit ID campaign relies heavily on your chosen compound library. To maximize your chances of success, such compound libraries should consist of highly attractive, chemically diverse compounds with proven lead-like properties, but also good solubility and stability. Thus, quality, size, and diversity of the compound collection will impact the success of your hit ID campaign.  

In high-throughput screens, large libraries of several hundreds of thousands of compounds are screened to cover as much of the available chemical space as possible. However, in specific cases, a more tailored screening approach might be more appropriate, using a smaller, either diverse, or focused compound library.  

Screening and hit triaging

The screening process involves several steps, starting with a pilot screen using a representative subset of the screening collection. Upon definition of the final screening conditions including the compound concentration, the primary screen is then performed on the selected screening deck. The primary hits identified are confirmed by testing them again in replicates before the final step of concentration-response profiling is started. 

The concentration-response relationship of confirmed hits is tested against the primary assay and the readout counter, as well as against relevant selectivity targets, if applicable. A diligent, data-driven analysis of the results, involving a medicinal chemistry review and assessment, enables the prioritization of compound series with both a desired biological profile and attractive chemistry.  

Figure 1. A typical hit ID workflow, illustrating the various steps involved in high throughput screening and hit triaging. 

Hit validation 

Following hit triage, prioritized hit series are validated to confirm their biological activity through the application of secondary assays. These are carried out using orthogonal readouts like biophysical methods to confirm on-target activity, or are conducted in more physiologically relevant systems like cell-based assays. Secondary assays are used to assess several crucial properties of the hits, including functional response.  

Medicinal chemistry efforts during hit validation focus on the analysis of the hit’s structure-activity relationship (SAR). Such analysis aims to identify the structural elements that are associated with its biological activity. To further the assessment of on-target activity and selectivity, additional in vitro assays are commonly conducted at this stage. These evaluate the absorption, distribution, metabolism, and excretion (ADME) properties of the hits. You can visit our DMPK and ADME-Tox webpage to read more about secondary ADME-Tox assays for hit validation. 

Using all this information together, the hit series that will progress to the hit-to-lead phase are identified. The number of series that are taken forward will depend on resource availability, although around two to three hit series are usually recommended. 

Mastering hit identification  

When optimizing your hit ID campaign, there are many factors to consider, including the quality of your compound library, and the strategies for primary screening, triage, and validation. The most successful hit ID campaigns implement a data-driven approach that utilizes techniques across a broad range of disciplines, including biochemistry, computational chemistry, in vitro biology, and medicinal chemistry.  

Figure 2. A checklist for successful hit ID campaigns, from screening strategy through to hit validation, to help you increase the number of high-quality hits obtained. 

View our webinar to learn more about the essential considerations for successful hit identification by high-throughput screening, including planning, compound collections, infrastructure, assay formats, and the benefits of an interdisciplinary approach. 

Externalizing your hit identification 

At Evotec, we provide industry-leading hit ID services through our decades of experience, state-of-the-art screening technologies, and a diverse, high-quality compound collection. This utilization of a broad range of expertise, facilities, and technologies takes away the stress of hit ID, minimizing the risks of attrition, and even allowing you to hit previously undruggable targets.  

When partnering with Evotec, our expert screening team will help you design and conduct a bespoke hit ID campaign that is optimized for your target, giving you the best possible chemical starting points. And once the best hit series for your target have been identified, our integrated, end-to-end R&D platform can support you from drug discovery, right through to drug development and manufacture, helping you get your drug to market in the simplest, most time, and cost-effective manner. 

For more information on our hit ID services, and to learn how we can support your projects from concept to clinic, visit our website.

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Also, join our ongoing webinar series on accelerated hit identification through innovative high throughput screening approaches.

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“The intracellular exchange assay design enables studying the cumulative effect of modulators from the intracellular face of ion channels.” 

Background:

Intracellular binding sites of ion channels are targets for many drugs (example: anesthetics targeting voltage gated sodium channels). Pharmacological manipulation of the intracellular (IC) environment is as critical as the extracellular (EC) environment.  

In manual patch clamp, once the gigaseal is established and the patch is in whole cell mode, manipulating the IC environment by pipette solution exchange is a technical feat few can achieve (reference).  

This is where the intracellular exchange (ICE) assay developed by Evotec on Sophion Qube changes the game. 

The assay design: 

As is illustrated below (fig. 1), an EC salt solution is added to each well in a 384-well QChip via an inlet well and the same can be removed via a waste well, both from the top (fig. 1). On the other hand, the IC salt solution has only one inlet/outlet channel i.e. for both perfusing in and out. 

In the ICE assay, cells are patched on this QChip in the whole-cell mode using a negative pressure via the patch hole. The cells are thus surrounded by the EC solution while the cytosol is accessed via the IC solution.  

Figure 1: Architecture of each well in a 384-well Qchip (left). A single hole Qchip with the indicated intracellular (IC) solution inlet.

Hereafter, the magic begins: after one or more stabilization stages to obtain baseline activity, the IC solution is completely replaced with a new IC solution in sequential liquid periods. Our ICE assay has been developed to execute such IC exchanges up to four times (fig. 2), replacing the previous solution completely with a new solution each time.  

Figure 2: Liquid periods in the ICE assay with the stabilization periods to obtain baseline activity followed by four rounds of IC solution exchange. 

Thus, modulators can be delivered to the cytosolic face of the ion channel cumulatively.  

All the while, the cells are maintained in whole cell configuration in the QChip and the seal resistance does not drop below the QC threshold (40 MΩ, fig. 3). The ion channel target is observed to respond to each increasing concentration of the modulator post-exchange (fig. 3). 

Figure 3: (a) Seal resistance during sequential liquid periods interspersed with removal of the Qchip from the bio-chip interface, (b) Current vs time (IT) plot. Signal from the target ion channel responds to cumulatively increasing concentration of the modulator in cytosolic side. 

Achieve more with less reagents and resources: 

• With four rounds of exchange, the volume of the IC solution required is only 4 ml for 3-4 experiments.  
• The assay has a throughput of up to 26 compounds per Qchip for a 3-point dose response profiling.  
• The assay is very resource-friendly also in terms of chemistry, since only minute amounts of compound are required. 
• An experiment with four exchanges lasts only about 90 minutes.  

In a proprietary assay developed by Evotec, it was possible to profile about 700 compounds in 70 runs performed over 64 days.  

Key learnings: 

• The IC exchange assay can cumulatively test three increasing concentrations of the same compound, delivered to the cytosolic face, providing pre- and post-compound data.  
• A complete solution exchange is possible between each concentration, thus preventing compound cross-contamination.  
• The assay can also be adapted to different intracellular ion concentrations, pH values, or signaling molecules.  
• Druggability of ion channels as a family of proteins is greatly enhanced. 

No matter whether your drug discovery program is in the hit ID, hit-to-lead, lead optimization or early safety assessment, the ICE assay is useful for high-throughput hit profiling at any point.  

Companies interested in using Evotec’s technology are welcome to contact Evotec at info@evotec.com. 

In many diseases, genes are not altered, but their regulation is disturbed, and only recently have approaches emerged to target gene regulation as a therapeutic tool.

How are genes and their expression regulated? While the central dogma of the 1960s DNA->RNA->protein still holds true, recent advances in molecular biology have provided much insight into how cells regulate which genes are transcribed into mRNA and how translation of RNA into proteins is regulated. It is now known that mRNA can undergo many chemical modifications, induced by a variety of enzymes. These modifications affect mRNA maturation, stability, and lifespan, as well as the rate and duration of translation and mRNA degradation. Likewise, some small modifications can either prematurely terminate protein synthesis, reduce peptide yield, or alter the amino acid sequence of the translated protein.

While the existence of post-transcriptional RNA modifications has been known for more than 30 years, their mechanisms, functional consequences and connection with human diseases including cancers have only recently been elucidated, making RNA epitranscriptomics an unexplored and exciting field for drug discovery. Meanwhile, more than 150 RNA modifications have been reported and approximately 300 RNA-modifying enzymes have a potential as novel therapeutic targets.

Evotec's partner Storm Therapeutics is amongst the first companies to pursue RNA-modifying enzymes as drug targets. The company identified RNA methyltransferases, a class of approximately 75 enzymes, as the most promising target class. Some have been identified as important regulators of cancer development and progression and thus represent promising novel antitumoral targets. Storm focused on METTL3, an enzyme involved in the co-transcriptional methylation of internal adenosine residues in eukaryotic mRNAs. This enzyme regulates fundamental aspects of mRNA life cycle, such as splicing, transport to the cytoplasm, stability, and translation into protein. It was known that in AML cell lines, knocking out METTL3 leads to a pronounced antiproliferative effect associated with a reduction of the BCL2 (anti-apoptotic factor). In other models, a marked upregulation of genes associated with innate immunity, such as those in the interferon (IFN) signaling pathway was demonstrated following METTL3 depletion.

However, tackling new classes of enzymes such as RNA methyltransferases requires breaking new ground in assay development, screening, and downstream hits to guide progression. To address these challenges, Storm and Evotec began collaborating in 2016, which later evolved into an integrated drug discovery and development alliance focused on novel small molecule RNA epigenetic drugs for oncology and other diseases. Using Evotec's fully integrated small molecule drug discovery and development platform, including biomarker support (development of a m6A-mRNA level evaluation technic), STC-15 was identified. STC-15 is a potent, selective small molecule inhibitor of the mRNA modifying enzyme, METTL3. STC-15 was developed from high throughput screening to candidate nomination in less than three years.

In preclinical cancer models, treatment with STC-15 significantly inhibits tumor growth. In addition, a profound cell-intrinsic interferon response was observed, following an accumulation of double-stranded RNA. In mouse models, the induction of innate immunity mechanisms, such as the interferon pathway, enhanced T-cell mediated cancer cells killing. This work has been recently published in Cancer Discovery, a leading cancer journal (Guirguis, Ofir-Rosenfeld et al., 2023). Notably, it activated innate immune pathways and inhibited tumor growth as effectively as anti-PD1 therapy in some models. In addition, the data showed that the combination of the two agents resulted in significantly greater activity, leading to tumor regression and durable anti-cancer immunity. Detailed investigation of the mechanism of action of the two treatments revealed that they act independently, providing a strong rationale for their combination. This added significantly to previous studies where STC-15 demonstrated efficacy in leukemia models through mechanisms such as inhibition of leukemia stem cell function (Yankova et al., Nature, 2021). Furthermore, additional combination studies revealed a high degree of synergy between STC-15 and Venetoclax, a BLC2 inhibitor and standard of care therapy for acute myeloid leukemia (AML) patients.

This data provided the rationale for the development of STC-15 both as a monotherapy and as a combination partner for immune checkpoint inhibitors or with BCL2 inhibitors for the treatment of solid tumors and leukemias, respectively. Following the selection of STC-15 as a first-in-class development candidate in 2020, the seamless integration from project initiation to IND using Evotec's INDiGO platform led to the entry of STC-15 into Phase I clinical trials in 2022. The orally bioavailable, highly selective METTL3 inhibitor is being developed for the treatment of solid tumors and may also have potential in AML.

A phase 1, multi-center, open-label, first-in-human study is evaluating multiple ascending daily oral doses of STC-15 in a 3+3 cohort design. The study is designed to systematically evaluate the safety and tolerability, pharmacokinetics, pharmacodynamics, and clinical activity of STC-15 in adult patients with advanced malignancies. Dose levels for further evaluation in expansion cohorts will be selected based on all available PK, PD, target engagement (including m6A-mRNA level evaluation), efficacy, safety, and tolerability data, including long-term safety data beyond dose-limiting toxicities (DLTs).

Patient enrolment started in November 2022, and the company anticipates top-line results in 2024.

The development demonstrates the benefits of Evotec's integrated, accelerated IND-enabling platform to support the exploration of novel and exciting biology to maximize innovation, to execute efficiently with rapid and seamless integration from target to IND.

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A recent publication in Nature Medicine further validates Just – Evotec Biologics’ J.MD^TM Molecular Design suite. 

Children under the age of 5 years account for more than 75% of deaths attributable to malaria. The World Health Organization (WHO) has recommended the vaccine Morsquirix for paediatric use, but it has modest efficacy. Monoclonal antibodies and other complementary strategies are critically important in efforts to eradicate this disease. 
 
A multi-disciplinary team of scientists from multiple organizations collaborated on a project to select and engineer a potent and long-lasting antibody drug with anti-malaria activity. Key to this mission was to develop an antibody with developability properties amenable for cost-effective manufacturing and dosing in paediatric populations. 
 
Scientists from Just – Evotec Biologics played a crucial role in the project. They helped with the lead candidate selection by ranking a panel of human-derived potential antibody candidates for developability using Just - Evotec Biologics’ in silico Abacus tool. Abacus is a component of our J.MD^TM Molecular Design suite of tools for antibody research and development. Once the team had selected a lead and backup candidates, Just - Evotec Biologics scientists designed optimized variants of the candidate antibodies with greatly improved developability properties informed by stability violations found with the Abacus tool. 
 
Scientists used stable pools to express the optimized designs and generate material for biophysical characterization and activity assays. This enabled the final lead selection of AB000224 and AB007088 for advancement as a clinical lead and backup. The team engineered the variable domains of both antibodies to enable low-cost manufacturing at scale for distribution to paediatric populations. 
 
Just-Evotec Biologics scientists identified the best-producing clonal cell line, expressing the candidate molecule in continuous-perfusion bioreactors at twice the original titre. They advanced the candidate into production following good manufacturing practices. The material generated from this production run is being used to support studies for clinical development of an antimalaria drug suitable for use in paediatric populations living in Low to Middle Income Countries. 

Learn more in the following article published in the prestigious journal, Nature Medicine. 

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The biopharmaceutical industry must break with existing manufacturing paradigms if it is to reduce the cost of biological medicines and ensure they are accessible to patients around the world. The industry’s next generation of production facilities will use innovative approaches to minimize clean room space utilization, reduce the footprint of facilities and lower production costs.

In order to meet the global demand for biological medicines in these small and agile productions facilities, intensified manufacturing platforms are being developed that allow very high productivity to meet late phase clinical and commercial demand. These platforms utilize mammalian cell hosts in perfusion bioreactors linked to continuous downstream trains. Cell culture productivities have reached a point where a bioreactor can sustain a volumetric productivity of more than 2 g/L/day. This, coupled with the development of low-cost media and advancement in process automation technologies around the bioreactor, has translated to an ability for companies to operate a single-use bioreactor uninterrupted for over 15 days and execute harvest and Protein A capture on a continuous basis.

Optimizing Continuous Biomanufacturing Operating Conditions

Engineers at Just-Evotec Biologics were challenged to find the optimum operating conditions to enable the lowest production costs irrespective of facility mass output. To achieve this aim, they developed process models for our continuous process contained within a J.POD® facility. 

J.POD facilities apply modular cleanroom pod technology arranged in a controlled, non-classified ballroom to minimize the cleanroom footprint of operations that would have previously taken place in a large ballroom. Media and buffer preparations, cell expansion, upstream, downstream and post viral are all housed in separate pods. The design minimizes fixed utility infrastructure and instead relies on single-use continuous upstream and downstream operations. 

Figure 1 shows the relationship between Cost of Goods Manufactured (COGM) of a therapeutic antibody, volumetric productivity and culture duration. The results indicated that COGM is strongly inversely correlated to volumetric productivity between titers of 0.5 and 3.0 g/L/day, as shown by the sharp drop in COGM with increased volumetric productivity. The inverse correlation is not as strong when comparing COGM and culture duration, which is likely because most of the cost reduction benefits are attained when the first few kilograms of product are manufactured (e.g., because of the high cost of shared downstream disposables). Marginal increases in culture duration will generate higher amounts of product and allow the production of metric tonne-quantities of antibody but are accompanied by a proportional increase in media and downstream buffer costs.

Figure 1. Sensitivity plot illustrating Cost of Goods Manufactured (COGM) versus bioreactor design variables. Color coding was used to layer in throughput as a measure of the three variables studied.

Expanding Access to Life-Saving Medicines

Just-Evotec Biologics has two facilities in North America that are operational and manufacturing biological medicines for clients. Our European facility will be brought online in Toulouse, France in late-2024. 

These facilities in two geopolitically stable locations will provide our customers with additional supply chain security. They feature our intensified manufacturing platform allowing the agile and low-cost production of biopharmaceuticals.

The cost-modelling described in this article shows the potential for the J.POD facilities to be capable of delivering metric-tonne quantities of biological medicines for late phase clinical and commercial manufacturing. Furthermore, it illustrates how the COGM decrease dramatically as titers increase so that extremely low production costs can be achieved. We believe that this provides an opportunity to reduce the overall costs of biological medicines and will allow patients around the world greater access to these life-saving medicines.

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Cardiotoxicity is one of the most reported adverse effects that leads to pre-clinical and clinical drug failure. To tackle this, the International Conference of Harmonization (ICH) S7B guideline in 2005, proposed a non-clinical assessment of new drug entities using in vitro electrophysiology studies (typically hERG ion channel) and in vivo telemetry in animal models. Although these are very sensitive approaches, they may have also led to unwarranted drug attrition of many potentially valuable therapeutics due to the low specificity nature of the assays.

More recently, the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative was proposed by experts in the field and was established to move safety pharmacology towards in silico and in vitro approaches utilising new and emerging technologies such as stem-cell-derived cardiomyocytes. Building on this new safety paradigm, colleagues from Evotec and Cyprotex have worked collaboratively to develop a non-clinical model using cutting-edge techniques with improved predictive power to de-risk cardiotoxicity in early drug discovery. We have presented the output of this work in a research article titled “In-depth mechanistic analysis including high-throughput RNA sequencing in the prediction of functional and structural cardiotoxicants using hiPSC cardiomyocytes” published in a recent edition of Expert Opinion on Drug Metabolism and Toxicology.

In this article, we describe the use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as an in vitro model system together with three high-throughput technologies incorporating structural assays (high-content imaging, HCI), functional assays (Ca²⁺ transience, CaT) and high-throughput RNA-sequencing (ScreenSeq) for the pre-clinical risk assessment of novel compounds. The transcriptional responses of hiPSC-CMs to 24 h treatment with 33 cardiotoxicants (12 structural cardiotoxicants, 14 functional cardiotoxicants, 7 structural/functional cardiotoxicants) and 9 non-cardiotoxicants of mixed therapeutic indications were investigated and compound-induced differential gene expression (DEG) was calculated in comparison with vehicle treated controls. Likewise, the hiPSC-CMs responses to six structural readouts (cell count, cellular ATP, mitochondrial mass, mitochondrial membrane potential, calcium content, DNA structure and nuclear size) and four functional readouts (amplitude, frequency, peak width and decay time) were analysed. In summary, hiPSC-CMs recapitulated expected structural and functional toxicity mechanisms, validating their use as in vitro model system to detect and characterize modes of toxicity. ScreenSeq identified several molecular mechanisms of toxicity such as alterations in cardiac pathways, genotoxicity, ER stress and mitochondrial toxicity. Together, HCI, CaT and ScreenSeq provided the best cardiotoxicity prediction metrics (10x C_max: 100% specificity, 82% sensitivity, 86% accuracy; 25x C_max: 89% specificity, 91% sensi-tivity, 90% accuracy).

This study not only provides invaluable cardiotoxic mechanistic information of the drugs tested, but it also demonstrates the potential of this mechanism-driven risk assessment approach in predicting drug-induced cardiotoxicity in hiPSC-CMs.

Read the paper.

Read more about our Cardiotox Screen assay consisting of both a functional assay (examining the mechanical function of the cardiomyocytes) and a structural assay (assessing morphological changes and loss of viability).

In addition, discover more about our transcriptomics offerings here.

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Patients around the world have limited or restricted access to biopharmaceutical medicines. Reducing production costs while still maintaining high quality standards will help increase the affordability of biologics and ensure more patients benefit from these life-saving medicines.

Traditional biomanufacturing facilities have failed to deliver biopharmaceutical products with sufficiently low Cost of Goods Manufactured (COGM) to allow greater patient access. These facilities have been built with fixed capacity and a focus on large-scale fed-batch manufacturing. Scaling-up processes to large-scale fed-batch manufacturing facilities involves considerable risk, resource, and upfront costs. Such facilities often lack flexibility which limits the products that can be produced within them and can leave valuable production assets idle for periods of time.

Manufacturing costs link directly to capacity utilization and product demand. There is a historical precedent within the biopharmaceutical industry of operating with excess capacity but we must recognise this comes with a financial penalty. We must address this challenge if we are to respond to global healthcare emergencies, changes in the way healthcare systems are managed and greater demand for global access to biotherapeutics.


Continuous Bioprocessing Platforms and Modular Facility Designs

The industry needs new flexible biomanufacturing concepts to quickly react to market fluctuations and achieve a higher predictability of costs. Modern biopharmaceutical productions facilities use building and manufacturing technologies, such as modular construction, to minimize clean room space utilization and reduce footprints. They allow faster speed to market with a lower upfront capital investment and are readily expandable when product demand is better understood. Continuous manufacturing platforms can be integrated into these facilities for low-cost bioprocessing using mammalian cell hosts in perfusion bioreactors linked to continuous downstream trains. Production costs remain low irrespective of facility mass output, the product quality attributes are consistent and manufacturing footprints are minimized.

Just-Evotec Biologics has developed a low-cost manufacturing facility design utilizing modular cleanroom pod technology that we call J.POD®. The J.POD facility design features individual pre-fabricated cleanroom pods arranged in a controlled, non-classified ballroom to minimize the cleanroom footprint of operations that would have previously taken place in a large ballroom. Media and buffer preparations, cell expansion, upstream, downstream and post viral are all housed in separate pods. The design minimizes fixed utility infrastructure and instead relies on single-use continuous upstream and downstream operations.

Biomanufacturing Facility Cost Comparisons

We developed process models for a fed-batch process in a traditional stainless-steel facility, a fed-batch process in a single-use facility and three continuous processes in a J.POD facility. The models were created using the Biosolve software (Biopharm Software Ltd) to show the benefit of the J.POD facility design on the COGM of an antibody biologic. We used Net Present Cost (NPC) to compare scenarios. NPC estimates cash flows by computing operational costs and discounting over time using a capital parameter. It does not include revenues in the accounting of cash flows and assumes capital costs are sunk costs incurred at the beginning of the project.



Figure 1 shows the expected costs from operating the different facility types and assumes their throughput increases at a rate of 250 kg/year up until a peak value. The range selected was representative of market demands for typical biopharmaceutical manufacturing facilities. Jumps in the NPC correspond to points when the capacity of a facility is reaches and new builds are needed.

We can draw the following conclusions from the results.

1. J.POD facilities achieve remarkable production outputs despite their small footprint because of the high productivity of the continuous perfusion process.
2. Fixed CapEx comprises a large proportion of the total costs in a stainless-steel facility. J.POD facilities apply single-use technologies resulting in a shift from fixed to variable costs.
3. The fully continuous J.POD facility gives the lowest costs with the stainless-steel facility returning the highest expenditure over the range.
4. The stainless-steel facility is not being operated at its optimum utilization rate over the production output range modelled that leads to inefficiencies.

Driving Up Access to Biotherapeutic Medicines

We believe that modern biomanufacturing facilities must have smaller processing spaces, higher production throughputs and lower production costs. Modelling shows how our J.POD facilities have the lowest initial build and operating costs as well as the ability to control operating costs. These facilities are outperforming older manufacturing platforms in terms of cost and utilization. They are becoming an essential component of strategies for reducing costs and driving up access to biotherapeutic medicines. 

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