Bio reagents

Evotec provides access to a comprehensive portfolio of services and capabilities around cell and protein production. These can be accessed as a stand-alone function or as an integral part of a wider collaborative research programme accessing our drug discovery expertise.

Combining expertise in cell sciences and protein production methods with biophysics, functional assays, and HTS Evotec offers clients flexible and efficient platforms for timely delivery of high quality discovery reagents.

Cell line generation

Evotec offers stable cell line generation services through a customised offering tailored to our partner`s needs. Cell lines are produced from recombinant cells which have been either generated by stable transfection or viral transduction. Working from highly stable clones, a broad range of target expression levels and co-expression of multiple sub-units is available.
All cell lines are screened using immunofluorescence labelling, Western blot, or RT-PCR, and further validated in cell-based assays to confirm the functional expression of the target protein.

Cell line generation capabilities include:

• Custom expression strategies and vector design
• Constitutive or inducible target expression
• Target or reporter gene expression
• Polycistronic or independent co-expression of auxiliary factors
• Multiple subunits and resistance markers

Membrane preparation

Evotec offers cell membrane preparations in bulk in different purity grades e.g. for ligand binding assays or in vitro transporter activity assays. Cell membrane production can be accessed as standalone service or as integral part of wider collaborative programmes.

Membranes are produced in high quality, being obtained by gentle cell disruption methods. Customised aliquots are prepared to fit individual client’s projects’ needs. The membrane functionality is systematically validated by ligand binding assay:

• Cell membrane preparation from transfected cells, stable cell lines, primary cells, or tissues
• Large scale preparation of up to 10 billion cells in a batch
• Gentle and highly efficient cell disruption by nitrogen decompression using a different state-of-the-art cell disruptors (e.g. PARR-bomb^® or micro-fluidizer)
• Enrichment of membranes by differential ultracentrifugation
• Quality control by Western blot and ligand binding assay

Validation of membranes by radioligand binding assays:

• Filter binding or Scintillation Proximity Assay (SPA)
• Saturation binding (Bmax, Kd), Kinetic binding (Koff), competitive binding (EC50)
• Screening of agonists, antagonists and allosteric modulators

Assay Ready Frozen cells

Evotec’s cell culture unit has many years expertise producing assay ready frozen cells for our partners from leading pharma and biotech companies. Frozen cells are provided as a standalone service or as integral part of wider collaborative research programmes.
Frozen cell are bulk produced from up to 10 billion cells in a batch, validated by help of functional cell-based assays and provided in customised aliquots (e.g. in plates, vials, tubes, or bags).

Frozen cells service offering:

• Assay ready frozen cells from transfected cells, permanent cell lines, or primary cells
• Bulk production of suspension or adherent cells in screening batches
  Carefully monitored cell expansion in CellStacks^® or bioreactors
• Gentle harvest of cells preventing impact on target pharmacology
• Automated dispensing of cells into cryovials using Fill-It^® technology
• Controlled rate freezing of cells using a CryoMed^®
• Functional control of assay ready frozen cells in cell-based assays

Transient bulk transfections

Evotec offers transient bulk transfection services to overcome limitations with targets which are not stably expressed in mammalian cells.
Expertise is available with a variety of transfection technologies including lipid based and viral mediated delivery to achieve transient expression of toxic or complex targets. The transient transfection approach allows for instant access to cell-based reagent in bulk quantities with high target expression levels.

Scalable transient transfection by electroporation using MAXCYTE^® STX technology:

• Highly efficient transfection of up to 10 billion cells in a single batch
• Simultaneous co-transfection of multiple subunits, auxiliary factors or reporter constructs
• Adjustable expression levels for functional cell-based assays or high yield protein expression
• Downstream processing of transfected cells to assay ready frozen cells, membrane preparations or purified proteins

Global cell banking service

At its European and US sites Evotec provides cell banking or back-up storage service for our partner’s cell lines.
Besides storage of the cell lines the service package includes cells expansion and dry ice shipment to our partner’s global sites.

Evotec’s cell banking service includes:

• Cell expansion under controlled culture conditions
• Cell harvest applying gentle methods to maintain target activity
• Preparation of customised cell density compatible with assay requirement
• Automated dispensing of cells into vials
• Controlled rate freezing and storage of the cells in liquid nitrogen vapor phase

This optimised process ensures the shortest time from cell harvest to freezing which is critical to maintain the quality of frozen instant cells. Frozen instant cells are routinely tested for their viability and ability to adhere shortly after thawing.

Gene-to-protein

Evotec offers a broad expertise in providing recombinant proteins.  Working in partnership with our clients we offer customised solutions, bespoke and modular workflows, flexible production scale and access to a unique suite of integrated in-house technologies, to deliver projects in a time-efficient and cost-effective manner. Strong communication driven by scientist-to-scientist interaction. A core team of Bioinformaticians, Structural Biologists and Protein Scientists design protein constructs and production routes tailored to their final use, whether it be assay, biophysics, activity assays, structural biology or animal studies.

Steps in optimisation of construct design include: 

• Bioinformatics analysis including homology modelling and secondary structure prediction using state-of-the-art software
• Identify optimal tagging strategies, domain boundaries and mutagenesis
• Choice of expression host and desired cell localisation  e.g. secreted, periplasmic
• Custom vector design

Multiple expression systems:

• E. coli
• Yeast
• Baculovirus infected insect cells
• Mammalian cells
• Cell-free

Comprehensive purification platform:

• Multiple cell lysis options
• Protein refolding screen and scale-up by dilution/dialysis
• Chromatography including, affinity, ion exchange, hydrophobic interaction, size exclusion and native
• Endotoxin removal and confirmation by LAL assay
• Typical QC includes SDS-PAGE, western blot, Mass Spec, Dynamic Light Scattering, High-Throughput analytical/fluorescence size exclusion, biophysics and activity assays, SEC-MALLS

Membrane Proteins

Working with an optimised process for high-throughput screening of expression and solubilisation conditions  and large scale protein production, we deliver quality isolated target protein, amenable for assays, biophysics and structural biology, in fast turnaround times.

Scouting and sample optimisation:

• Fluorescence based SEC screening
• Automated microscale affinity purification
• Thermofluor based stability assay on membrane bound and solubilised sample
• Radioligand binding assay on membrane bound and solubilised sample
• LCP-FRAP and in situ screening through Diamond light source, UK

Large scale membrane protein production: 

• Heterologous expression in mammalian, insect, yeast and bacterial systems
• Production of Virus Like Particles (VLPs) and lipoproteins
• Enriched membrane preparation and purification
• Pharmacological and biophysical analysis for characterisation of proteins
• Crystallisation in lipid cubic phase (LCP), bicelles and vapour diffusion

Labelled Protein for Assays, NMR, SPR and MST

Labelling strategies include isotope labels for NMR and mass spectroscopy standards, SeMet enriched proteins for macromolecular crystallography and biotinylation for assay and SPR.

Evotec also offers labelling of biologically relevant molecules and reagents for use in ELISA, FACS, FCS, plate readers and other fluorescence based detection systems. Our service helps clients to improve their research results in all areas of biological and pharmacological assays, as well as clinical diagnostics, analytics and agrochemicals.

Our expertise covers the labelling of the following species:

• Peptides and chemokines (as enzyme substrates, GPCR ligands, etc.)
• Proteins including antibodies
• Site-specific mutagenesis of proteins (for site-specific labelling)
• Glycoproteins
• Carbohydrates

For labelling purposes we use the optimally suited dyes and tags for your application, such as:

• Commercially available fluorescent dyes and the in-licensed MR121 dye
• Non-fluorescent markers and probes like affinity tags, biotin, reporter enzymes, etc.

High-Throughput parallelised protein production

For fast and efficient production of proteins we offer a high-throughput platform for protein expression scouting and purification.  Robust statistical models are employed to triage constructs. Informed decision-making to maximise throughput and minimise waste. The platform has been successfully employed over more than 1,500 unique constructs. The platform is suitable for E. coli, baculovirus infected insect cells and mammalian cell lines.

Standardised high-throughput protein production work flow:

• Expression Scouting of constructs in parallel
• In-house statistical model for review and prioritisation
• Large Scale Expression (1-3 liters)
• Parallel Purification and QC
• Blocks of 96 constructs from gene-to-protein within 2-3 months, ready for multiple downstream experiments