Clarity in cell line development

Collaboration: changing CLD practices

In January 2019 Solentim, a global leader in cell line development (CLD) instrumentation, and bioengineering company ATUM, announced a technology partnering and collaboration agreement to enable rapid, high expression, stable cell line development. But what exactly are the tangible benefits gained from both parties and for customers, and why does this fusion of technology offer more advantageous methods than other, traditional practices?

The Deal
The agreement demonstrates the benefits of integrating Solentim’s VIPS™ (verified in-situ plate seeding) hardware for isolating single cells with ATUM’s efficient Leap-In Transposase® expression technology. For potential customers, by applying both technologies to their CLD workflow, the need to pre-screen or select in-line for high producing clones is eliminated, saving time and money, and enhancing workflow efficiency.

The Science
But how exactly does this work?

The main limitation in creating cell lines from multi-ORF (open reading frame) constructs is that when the DNA enters the cell it tends to be randomly fragmented and rearranged, and those fragments are integrated into the genome, rather than the desired sequence. Due to this, the majority of cells may not contain the desired construct, and therefore pre-screening is necessary to identify those suitable for CLD.

The Leap-In Transposase technology provides a solution: it integrates the entire transposon (GOI) into the expression host genome, maintaining the structural integrity of the construct, linkage of all genes to the selectable marker, and the desired expression balance. The ratio balance is particularly valuable when dealing with antibody heavy/light chains and their different combinations for bispecifics and biosimilars. Consequentially, the majority of transfected cells are high producing clones, and therefore no screening is needed prior to seeding. The sites of insertion are in open regions of the chromatin with high resultant transcriptional activity. Additionally, the approach allows for low stringency selection which means better clone recovery and very high clone stability – both of these further reduce the timelines.

For the stable pools, Solentim’s VIPS technology comes into play for single cell seeding – with optimal use of the VIPS, seeding efficiencies of 70-90 % can be achieved, with single cell identity in each droplet on the bottom of the well confirmed by Z-stack imaging. The VIPS continues to add cell droplets to the well until a single cell has been confirmed. At this point, the well is filled with media.

Through licensing the transposases, customers are now able to optimise the stable integration of genes into a target genome, creating a population of clones for which the distribution is skewed for high-producers, and then using the high seeding efficiency of VIPS, the seeding process only requires a handful of plates to be seeded per project. Solentim is also able to assist with improvements in clone recovery and outgrowth which results in even better cloning efficiencies.

The Alternatives
Prior to this innovative combination, more “random integration” methods were used, necessitating a vital pre-screening step to identify high producing clones from a much larger population of clones. Traditionally, this has been achieved by using fluorescence activated cell sorting (FACS) to enrich for clones expressing a fluorescent marker for antibody or to even select and sort in-line for the highest producers. This is not only an expensive and technically challenging approach, it also only achieves separation of producers from non-producers. Typically, the ranking of clones from these methods is meaningless until you start growing the clones in their optimal media and in shaking culture.

Using ATUM’s technology, this enrichment or selection step is no longer necessary. The VIPS also uses much lower pressure than FACS systems when dispensing cells, resulting in higher cell survival rates, again increasing the outgrowth and cloning efficiency.

Future-proofing customers for the best enabling technologies in CLD
This synergy of technologies between two different vendors is a good example of the benefits of an open technology platform, where in this case improvements in vector design and transfection can be combined together and optimised for an innovative hardware platform which does both single cell seeding and whole well imaging for assurance of clonality.

These types of synergies are arguably lost with some of the novel closed platforms which are provided by a single vendor and restricts the customers to methods and consumables only provided by that vendor. Synergies are also often lost when customers are evaluating and purchasing pieces of equipment from a vendor to do just one part of the process e.g. the seeding step. An instrument may appear to be the best at that step when viewed in isolation but may be an orphan system in terms of its relationships to the best biology available and the other related instruments in the workflow.