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GTP's Smart CHO cell line development

Published on 2019-10-15 Upstream Process Process development

Smart CHO cell line development

To provide an integrated offer for the preclinical development of biotherapeutics, GTP has added mammalian cell line development services to its portfolio. We can now supply a CHO stable cell line  ready for transfer to GMP manufacturing.

Though not the only key for the success of your biotherapeutic development programme, selecting a good CHO clone is essential to ensuring the subsequent development of an efficient and robust process.

A recognised and efficient CHO platform

We use CHOvolution™, a proven technology with 25+ cell lines already used in preclinical and clinical trials in the EU, the US and Asia.

This technology could be summed up in two words: simplicity and efficiency. Broadly speaking, it involves simultaneously transfecting a non-genetically modified CHO-K1 with two expression vectors, both encoding for the gene of interest but each carrying a different antibiotic resistance gene (neomycin and hygromycin). This ensures that at least two copies of the expression cassette are integrated into the genome. By optimising the coding sequence of the gene of interest - through an defficient and proven algorithm - and using a strong transcriptional promoter (pCMV), a strong expression of the gene of interest is then promoted.

CHO cell line development vectors


A robust cell line development workflow

What sets apart our two-step cell line generation process is that it ensures higher assurance of clonality than classic methods. A first dilution is performed post-transfection to allow the generation of parental clones. Even if we have no guarantee of clonality at this stage, our dilution process is made in such a way as to obtain isolated cells. Then, a second limiting dilution is conducted from 5 parental clones selected based on their bioproduction characteristics (doubling time, volume and specific productivity, and so on). At this stage, clonality is confirmed by a high-resolution imaging system.

This process provides reliable and predictive results for upcoming scale-up, as parental clones and final clones are selected based on a fed-batch assessment.

High volumetric productivities, up to 8g/L, have been achieved with the technology. The diagram below summarises the distribution of the productivities generally observed in the production of monoclonal antibodies.

Average antibody productivities


CHOvolution™ data also show that the generated cell lines go on to display high genetic and physical stability (growth, productivity), as seen in the linear scale-up from 250mL to 1,000L presented below.

CHOvolution scale up


Case study: Development of a CHO cell line expressing Trastuzumab

The results obtained in the case study presented below confirm the efficiency of the CHOvolution technology and workflow. This study consisted of establishing a cell line expressing the Trastuzumab model antibody.

First step: selection of parental clones

Following transfection and the first limiting dilution, over 4,000 parental clones were obtained. We called on two robots to help us sort through this huge population. The Cell Metric™ imaging robot (Solentim) set out to identify wells with sufficient cell growth while the Automated ELISA Workstation (ThermoFisher) was responsible for determining the productivities (FastELISA (R), Biotech RD). The distribution curve obtained is fairly conventional, similar to an inverse function, with some hyperproductive clones.

Parental clones repartition Parental clones productivities


To refine the screening, the 20 most productive parental clones were evaluated in fed-batch culture, a more predictive condition for bioproduction. At this stage, a parental clone already stood out with a volumetric productivity of 3 g/L!

Second step: selection of lead clones

The 5 top parental clones were then subjected to another limiting dilution step, this time with complete documentation of cellular clonality via our Cell Metric™ imaging robot (Solentim). As before, the most productive clones were identified after automated ELISA screening, then submitted to an evaluation of fed-batch culture. This second limiting dilution round proved to be beneficial, generating a top clone with a productivity close to 5 g/L!

As productivity serves no purpose without stability, the stability of the top clone has been evaluated and validated over more than 60 generations.

CHO clones productivities

CHO clones stability


SMART CHO Cell Line Development Service adapted to your project

As a science-driven CDMO, we are convinced that customising the process is of essence to ensure the success of biotherapeutic development projects. This is particularly true for challenging & non-standard molecules.

For cell line development as for the rest of our services, we have developed a smart approach that is very well adapted to biopharma companies with challenging molecules looking for a partner offering expertise, flexibility and guidance.

Benefit from integrated process development services

Performing cell line development and process development under the same roof is a great asset to ensure the success and timely completion of your project.  In addition to our SMART cell line development service, we offer an extensive experience in full process development, including upstream & downstream process development and the development of supporting analytical methods.

Our flexibility, broad scientific expertise and dedication to projects make us the ideal partner to take charge of your protein and mAb therapeutics from research up to transfer to our privileged cGMP CMO partners.