Cell line development is an important part of the process development for recombinant proteins or mAbs. Even if not the only key for success of your biotherapeutic development programme, the selection of a good CHO clone is essential to ensure the subsequent development of an efficient and robust process.

The last few years have seen major evolutions in production processes for therapeutic antibodies, allowing expression yields to drastically increase and leading to the introduction of purification platforms that only need minor adaptations from one antibody to the next. This progress is the result of sizeable efforts in development on several levels:

• The development of optimised expression vectors
• Platforms allowing for the selection of CHO clones with high specific productivity
• The development of high performance culture media and feeding solutions
• The development of systems to optimise culture conditions in parallel at small scale
• The development of purification platforms (including new affinity supports), as well as new supports for ion exchange – or mixed mode – for intermediate and polishing chromatography steps

These improvements have spawned from various market players: pharmaceutical companies, CMOs, reagent suppliers and service providers, who have conducted joint and parallel efforts in order to respond to significant market expansion. However, the expression yield for an antibody remains strongly linked to the selection of the production clone and to its specific productivity (the quantity of antibody produced per cell per day). Indeed, even if seeking optimised culture conditions and feeding strategies specific to each clone can lead to significantly improved expression yields, seeking a stable CHO clone with the best possible productivity is directly linked to the quantity of stable clones screened. This number often ranges from 1,000 to 5,000. So, independent of the elements of the expression vector used to improve the inherent productivity of stable clones (promoters, optimised 5’UTR and 3’UTR, introns, insulator sequences, gene optimisation, use of one single vector or two co-transfected vectors for heavy and light chains), there has been an array of research efforts aimed at maximising the chances of identifying a high productivity clone by screening a lower number of clones, i.e. by increasing the proportion of high expressing clones in the screening pool.

Among these strategies are approaches which aim to:

• Minimise the number of stable clones for which the transgene insertion locus would hinder the expression using specific genetic elements such as UCOE® (Merck Millipore) or SGE® (Selexis), or GPEx® (Catalent) lentiviral vectors
• Increase gene copy numbers through gene amplification by combining a toxic agent which induces gene amplification (MSX or MTX) and resistance to said toxic agent through the selection gene (GS and DHFR). This gives increased chances of identifying overproducing clones through promoting expression at the insertion locus.
• Select the best clones by using strategies that minimise the expression of the selection gene (for example, in the Lonza approach, GS) through weak promoters, modified 5’UTR sequences or mutated IRES.

Nowadays, the expected standard for any new antibody development project is to obtain a CHO cell line with a productivity above 3 grams per litres and a culture process length of about 14 days. In this context, and despite a broad offer for cell line development, the Lonza cell line development platform remains the gold standard. Indeed, this proven platform has enabled the development of numerous products which are currently on the market. Even though it has evolved through the years to allow for faster cell line selection, minimising the quantities of MSX and increasing the stability of selected clones, this platform remains based on a CHO cell line derived from CHO-K1 line, created by knocking out the glutamine synthetase (GS) alleles. Unlike other technologies (UCOE®, SGE®, GPEx® for example), this one is now in the public domain. It does, however, still involve contracts with high Lonza licensing costs.

So, despite the performance and robustness of this approach, it seems that it is now more and more difficult for young biotech companies to access it, partly because of:

• The costs associated with cell line development with Lonza
• The lack of accessibility of this platform: indeed, the timeframes to access available slots are not compatible with the speed of development of a young biotech company.
• Lonza has a marked interest for projects that have a high probability of getting to market, therefore it is perceived that it fails to properly consider projects brought on by young biotechs, which often bare more risk.

With these considerations in mind, the challenge for biotech companies is often to select an accessible but still performant CHO platform and cell line development partner.

It is a complicated task to compare the various available technology solutions. Often, that’s only possible on the basis of achieved productivity. However, reaching record productivity is not the only token of a platform’s performance. First of all, as well as productivity, the quality of the molecule produced (notably its glycan profile) is a key factor to consider. But, taken in isolation from this key criterion, productivity alone must be treated carefully. Indeed, it derives from the screening of a large number of clones, all with varying levels of productivity, and chance plays a non-negligible part in the performance levels obtained. A platform that allows to reach productivity levels above 3 grams per litre in the majority of cases appears to perform a lot better than one that has reached up to 8 grams per litre, but with an average between 1 and 2 grams per litre. In evaluating the potential of one approach, there is also added complexity brought on by the fact that productivity levels are obtained in different conditions depending on the level of effort put into development (optimising the culture medium and feeding conditions).

At GTP, we have spent time performing a broad inventory of the platforms available for cell line development and can help you select the one most adapted to your constraints. We have indeed chosen to offer, through our partnerships with different companies, various CHO technologies. Our main considerations when selecting these technologies are their potential to reach productivity levels in line with the current standards, a proven track record of transfer to industrial scale production (robustness / stability of the cell line, proper documentation and traceability in accordance with industry requirements) and a cost of access compatible with biotech company constraints. If you currently are in the process development stage of your biotherapeutic candidate, looking for a CHO cell line development solution, and wish to know what GTP can offer, please do not hesitate to contact us.