CUSTOM DEVELOPMENT CAPABILITIES

A Streamlined Approach for Customized Cell Line Generation

Eurofins DiscoverX utilizes a vast array of cell line engineering technologies, e.g. vectors and viral transduction methodologies, to deliver program-specific assay design requirements. Cell lines can be engineered with unique cell backgrounds (different from the existing catalog products) and native receptors, natural variants or mutant receptors, or various non-human species (orthologs). Assays can be designed to enable a functional readout as proximal or distal to the target receptor using Enzyme Fragment Complementation (EFC) or non-EFC technologies.

Assay Platforms Supported by Core Proprietary Technologies

Physiologically relevant assays can be designed using the EFC platform (summarized below) or other non-EFC technologies. These robust, reproducible assays are easily implemented in screening, characterizations, investigational new drug (IND)-enabling studies, profiling, and other applications.

PathHunter^® Cell-Based Assay Platform has been successfully employed to develop validated assays for over 700 targets spanning most druggable target classes, including GPCRs, kinases, checkpoint modulators, cytokine receptors, nuclear proteins, and more. Innovative applications of this core technology have enabled the development of robust, reliable, and high-throughput-friendly chemiluminescent assays to measure protein-protein interaction, protein translocation, secretion, degradation, receptor dimerization, cytotoxicity, and more.

HitHunter^® Biochemical Assay Platform is a validated platform for quantifying second messenger signaling for cAMP and cGMP. This platform is ideal for identifying unique ligand pharmacologies (agonist, antagonist, allosteric, partial agonists, and inverse agonists), developing novel assays (e.g. cleavage assays to test protease and substrate activity), monitoring G_i and G_s GPCRs, discovering PDE inhibitors, and more. HitHunter assays are formatted as competitive immunoassays with gain-of-signal for precise, sensitive analyte detection without separation or wash steps, providing improved sensitivity and precision compared to conventional immunoassays.

KILR^® Cytotoxicity Assay Platform is a simple, non-radioactive, and dye-free method to specifically measure target cell death in a co-culture with effector cells. Designed to quantitatively measure direct cell death mediated by target-specific antibodies, complement, immune effector cells, and more, this cell-based assay platform has broad applications for immuno-oncology drug development from screening to QC lot release testing. The assays can detect cancer cell death through various mechanisms of action such as ADCC, CDC, ADCP, ADC, CTL mediated death, bi-specific antibody mediated T-cell redirection, CAR-T, and adoptive T-cell therapies.

InCELL Compound-Target Engagement (TE) Platform includes InCELL Pulse^™ and InCELL Hunter^™ assays to determine compound cell entry and quantitatively measure compound binding to an intracellular protein of interest in the native cellular environment. This universal and powerful development platform is broadly applicable to several target classes (e.g., kinases, epigenetic proteins, and more) and bridges between in vitro binding assays and cellular functional or phenotypic assays. Both platforms allow for evaluating compound cell membrane penetration and target engagement for intractable targets, even where other technologies have been unsuccessful.

SPRINTer^™ Targeted Protein Degradation (TPD) Platform utilizes biosensors to quantify endogenous protein turnover in disease-relevant cell models induced by targeted degrader molecules such as PROTACs and molecular glues. SPRINTer assays can detect and quantify changes in endogenous protein levels with higher sensitivity and more rapid kinetics than phenotypic endpoint assays (e.g. cell proliferation). In drug discovery and development, these assays can identify and rapidly screen therapeutics targeting endogenous proteins and perform potency ranking of small molecule degraders.

Bioassay Development and Implementation

An ideal potency bioassay should mimic a given therapeutic molecule’s MOA while producing highly precise, accurate, and reproducible data as per International Council for Harmonisation (ICH) and United States Pharmacopeia (USP) guidelines under a quality-controlled environment compliant with Good Manufacturing Practices (GMP) and Good Laboratory Practices (GLP). Most assays developed during early development or characterization phases require significant re-optimization or extensive development into phase-appropriate formats before these can be implemented for routine QC batch release/validation testing, resulting in significant delays for the drug program.

Eurofins DiscoverX offers the largest menu of ready-to-use qualified bioassays in a complete kit format. However, for several programs, an optimized bioassay may not be available in our catalog or be qualified with your specific clinical molecule. Our custom assay development team will work closely with you to understand and address the requirements to deliver the best fit-for-purpose solution for your program, from implementation for potency testing in QC lot release to long-term support for the life of your biologic therapeutic.

Bioassay Development and Implementation Workflow: A Holistic Approach Supporting Biologics Programs

Assay Development

Based on an agreed work plan, the Eurofins DiscoverX development team members, with their extensive and deep expertise, designs the assay based on the desired MOA and target biology. Stable cell pools are established, and the functional performance of the selected clone is confirmed with the reference standard or the clinical molecule of choice. The cell line is confirmed over 50 passages to ensure long-term assay reproducibility and converted to the ready-to-use cryopreserved bioassay format. The bioassay is tested to ensure tolerance to complex matrix, delivering the flexibility to develop the cell-based assay for detecting neutralizing antibodies.

Bioassay Qualification

The ready-to-use bioassays are developed and optimized following systematic qualification studies to meet regulatory expectations for potency testing. These qualification studies are designed to evaluate the critical quality attributes (CQA) for a bioassay, as defined by the International Council for Harmonisation (ICH) and United States Pharmacopeia (USP) guidelines that include accuracy, precision, linearity, range, specificity, and robustness, for implementation for potency testing in QC lot release. Bioassay requirements are defined during project discussions to establish specifications for these attributes, including relative potency range, number of analysts, and more. The bioassay is also evaluated for stability-indicating properties. The study report is provided for review and approval to generate a pilot lot for in-house bioassay testing. Optionally, the study data can be delivered in an ICH study report format. The bioassay is delivered as a complete kit with a simple, detailed protocol, optimized reagents, and assay plates, enabling fast and easy implementation in a QC environment.

Method Transfer

The qualified bioassay is transferred for implementation to your partner testing sites, including Contract Development and Manufacturing Organizations or Contract Research Organizations (CDMO/CROs) through an established streamlined method transfer process. The transfer is initiated via discussion between Eurofins DiscoverX and the receiving site(s) to present the development data, followed by classroom and lab training. Once the assay team at the testing site has successfully run the method with your clinical molecule per the provided protocol, your team will review the data for approval. Once the method has been successfully transferred, Eurofins DiscoverX remains available for any technical support during the validation study as well as post-commercialization.

As your program expands and enters additional markets requiring you to release the biologic drug at new sites, our team ensures the seamless method transfer to each of your new testing sites. To further support the global implementation for biologics programs, we are continually certifying CRO/CDMOs through our Bioassay Certification Program on the use of our platforms, delivering you the confidence in selecting and working with your outsourcing partners.

Bioassay Production

As part of bioassay validation at the testing site for QC lot release, the study may require multiple lots of the bioassay cells, which are considered as critical reagents. Once implemented as the potency release assay, sample size for testing as well as stability testing is used to determine the volume of the bioassay cell vials required. This usage determines the production lot size that can be delivered on a pre-determined shipment date or triggered on-demand. Our flexible and agile manufacturing operations can meet the requirements of up to 1,000 vials per lot and multiple lots of the bioassay.

Long-Term Support

Biologic therapeutics, originator or biosimilar drugs, deliver innovative care to patients and remain in use for decades. This necessitates consistent batches released using robust bioassays and implementing a critical reagents management program that ensures long-term assay reproducibility and supply assurance of critical reagents such as the cryopreserved bioassay cells. Eurofins DiscoverX has implemented programs to support your critical reagents management programs. The Cell Banks for Bioassays Program focuses on the generation and storage of analytical master cell banks. These dedicated, well-characterized 2-tiered cell banks ensure long-term assay reproducibility and supply of the bioassay cells as well as removing the need for lot-to-lot bridging for each production lot. Additionally, Eurofins DiscoverX offers long-term supply and technology transfer agreements. Contact a custom assay development specialist for further information.

Accelerate Your Drug Discovery Project with Custom Recombinant Proteins Tailored to Your Program’s Needs

Whether your program necessitates the creation of a protein from the ground up (when an off-the-shelf protein is not available) or the customization of an existing protein (e.g. sequence modification), Eurofins DiscoverX & Eurofins CALIXAR Custom Protein Development teams are here to collaborate closely with you. We value your input and will work diligently to deliver your custom protein of choice in a cost-effective manner.

Let us help you generate active enzymes and complex proteins, including kinases, phosphatase, epigenetic proteins, ubiquitins, and many others. With over 25 years of protein and enzyme development experiences, our dedicated team of experts from the original Millipore/ Upstate kinase enzyme development team will deliver high-quality enzymes per the specification required to meet your research goals.

For your membrane protein needs, Eurofins CALIXAR proudly offers 10+ years of expertise and patented technologies (innovative solubilizing and stabilizing reagents). Rest assured, our team provides you with fully native, functional, and stable membrane targets, including GPCRs, ion channels, transporters, and more.

Quality Assurance

Quality is our focus when developing recombinant proteins, ensuring the highest purity, stability, homogeneity, functionality, and lot-to-lot consistency. We will provide a detailed certification of analysis (CofA) indicating batch-specific product performance. During product development, our QC validation includes rigorous assessments of purity and homogeneity, thermal shift and cryostability assay performance, and functionality evaluations (ligand binding and specific activity assessment, including Km substrate, Km ATP, Z’, and inhibitor response studies).

Custom Protein Development and Production Process

Protein Construction

With a focus on preserving protein integrity and functionality, our team of scientists offers comprehensive protein construction starting from a full-length, wild-type sequence of any protein target. This ensures you are provided with natively folded proteins while maintaining unmodified binding sites and epitopes. Based on the specific requirements, precise protein tagging (His-tag, FLAG-tag, HA-tag, GFP-tag, GST-tag, Avi-tag, or other) is applied for various purposes, such as protein purification, localization, or immobilization. Lead sequences are then selected for optimal expression. Cleavage sequences (HRV3C, TEV, Thrombin) are incorporated into the protein construct to facilitate tag removal after purification. Ultimately, we will design the most suitable protein construct for your project.

Protein Expression

Various cell expression systems, including bacteria (E. coli), yeast (Saccharomyces cerevisiae, Pichia pastoris), insect cells (sf9, sf21), and mammalian cells (HEK293, CHO), are employed to synthesize recombinant proteins, each offering unique advantages. With expertise in diverse systems, we will ensure high expression of native and functional recombinant proteins by employing cutting-edge solutions to advance discovery programs to move to the next phase. Applicable for pharmaceutical, biotechnology, and academic research, our adaptable approach spans from rapid and cost-effective microbial systems to precise mammalian expression systems, including transient expression (PEI/ lipofectamine transfection, BacMAM) systems to generating inducible stable cell lines.

Protein Solubilization and Stabilization

Our proprietary protein solubilization process employs cutting-edge methods to extract proteins from the cell membrane. These methods involve the screening of 20-40 conditions combining proprietary molecules, commercial detergents, and polymers of diverse chemistry. Optimal solubilizing conditions are selected based on the extraction rates and thermostability of the protein target.

For stabilization, we utilize a range of solutions, including stabilizing reagents (proprietary or commercial reagents, amphipols) and lipid bilayer reconstitution, such as MSP-based nanodiscs and proteoliposomes. Our expertise in selecting a mix of lipids adapted to the protein target ensures optimal stability, integrity, and functionality, helping you accelerate your drug discovery programs.

Protein Purification

Depending on the initial protein construct design, various purification techniques and principles are utilized to purify your tailored proteins. Multiple affinity chromatography methods are applied: immobilized metal affinity chromatography (IMAC) for His-tagged proteins isolation, immuno-affinity chromatography for FLAG/HA/or other-tagged proteins, and Strep-Tactin® resin chromatography for strep-tagged targets. The native and functional recombinant proteins can also be purified by the proteins overall size (by SEC – Size Exclusion Chromatography), charge (by IEX – Ion Exchange Chromatography), and hydrophobicity (by HIC- Hydrophobic Interaction Chromatography). Purified protein quality is assessed by determining purity (SDS-PAGE and Coomassie blue staining), quantity (protein assay), and homogeneity (native-PAGE and western-blot). Cryostability and thermostability are also evaluated by monitoring intrinsic fluorescence changes of pure proteins during a change of temperature.

Protein Characterization

Our approach to protein characterization encompasses a range of techniques and technologies, including ligand binding and thermal shift assays using cutting-edge systems like Monolith X and Andromeda. These methods allow for the determination of ligand binding parameters, comparison of ligands, and evaluation of thermal stability, among other critical analyses. We also apply a comprehensive suite of enzymatic assays and stability tests, meticulously designed to provide unparalleled insights into the functionality and endurance of your proteins. Our advanced methodologies ensure that properties are evaluated thoroughly, setting the gold standard for precision in drug development and vaccine discovery.

Large-scale Production

For milligram-scale production of recombinant proteins, we have developed a process combining large-scale cell biomass production (25L-bioreactors) with an automated purification system. This combination ensures high-quality, consistent output, particularly when handling substantial volumes. Accurate, standardized operating procedures guarantee homogeneity and consistency in each batch supported by certificates of analyses.