Clarity in cell line development

Conference Report on latest trends in Cell Line Development and Cell Culture

A summary of the Informa Cell Line Development & Engineering conference, held in Amsterdam in April 2018.

April 23rd-25th, 2018 Amsterdam: I recently attended the annual Informa Cell Line Development & Engineering conference, held in Amsterdam over the course of three days. I went in eager anticipation of listening to a wide range of pharmaceutical companies and solution providers telling us their stories about the continuing search for new and improved ways to develop and isolate clonal cell lines, their thoughts on existing technologies, and the trends moving forwards.

I’m pleased to say that the event didn’t disappoint! On the first day, the topic of clonality still remained on the agenda and provoked many interesting discussions.  Despite no presentation this year from the FDA, the emphasis shifted from purely regulatory requirements to how people have actually been ensuring clonality in new cell lines or trying to address it for legacy cell lines.  This wasn’t really surprising as our interest for “real-life” examples, case studies and user experiences will always be high – for me, this is always the highlight of any good conference or seminar.

Specific content for the rest of the day included presentations from pharmaceutical companies and solution vendors describing a range of novel single cell isolation technologies.  There seemed to be more focus on trying to select for expression combined with clonality, plus recovery in low volumes of media. Several manufacturers continue to focus on traditional techniques such as plating into semi-solid media; and determining the protein titre of a clone at the static phase.  We found this approach interesting as many cell line development groups have moved away from these approaches for a variety of reasons.  Some point out that semi-solid media selects for certain cell attributes, and titre at static phase does not correlate well with fed-batch.  Some of these novel approaches may provide enrichment for potentially higher producers, but equally pre-selection by FACS or vector optimisation can achieve the same sorts of results.

Two of the presentations promoted the benefits of using next generation sequencing (NGS) for proving the clonality of legacy production cell lines.  They discussed the use of whole genome NGS to improve clone selection by mapping transgene integration sites.  This process also takes into account transgene copy number, different integration sites, genomic mutations for clonal fitness, and possible transgene mutations.  Whilst this approach is great for determining clonality after the fact, there seemed to be a consensus that this can be quite labour intensive and so ensuring clonally up front maybe the easier way for companies to go.

In addition to the presentations mentioned above, we listened in on several fascinating discussions including: why ‘in-sourcing’ of cell line development is more productive for small biotech companies when dealing with complex molecules; and challenges around the production of recombinant proteins such as immunocytokines (antibody linked to a cytokine).

We also attended a talk where MedImmune discussed the strategies they are putting into place to reduce protein aggregates in bispecifics during cell line development, and Philochem presented some clinical data showing how the combined effect of certain therapeutics were producing dramatic results for cancer patients.

The last, but perhaps the most interesting, talk of the day explained the challenges of editing HEK 293T cell lines to produce lentiviral vectors.  We also heard about their increasing number of applications such as CAR-T therapy. Conrad Vink from GSKshowed us how traditional methods of transfecting several plasmids containing the individual viral components only resulted in transient expression.  Because of this, their efforts have focused on the development of methods to create a single plasmid (containing all the components required to make a viral vector) and transfect it into the cell.  Using this method they can then carry out selection and induction to produce a stable packaging cell line which will produce the viral vector.  The transgene can then be transfected as and when required. This is quite exciting for the progress of lentirviral production as the stable cell lines generated in this process will be easier to edit for future targets, hopefully making the entire process faster for patient treatments and enabling the large amounts of materials needed for clinical trials to be manufactured more easily and cost effectively.

The second day of the conference centred around the use of synthetic biology to help understanding of success and failure in cell line development. Prof David James from Sheffield University opened the day with a discussion around why companies should change their focus from ‘blind’ cell line development – without really understanding what’s happening to the cells – to the introduction of genetic analysis and synthetic biology techniques.  According to David, this will help us all reach a greater understanding of cell line development success. He also emphasised how design should be considered “up front” of cell line development in order to reduce unknowns and “tune cell factories for the production of any protein”.

Lin Zhang from Pfizer then echoed David’s views by outlining their approach to targeted integration with the use of recombinase mediated cassette exchanged (RMCE) to switch a ‘landing pad’ with whatever monoclonal antibody (mAb) they want to integrate. They used CRIPSR to generate the cell line with a ‘landing pad’ that can easily be switched out ensuring the integration site remains the same and is no longer random. Practically speaking, the landing pad means they can reduce the number of clones to be screened and the impact is that one person can now do three projects instead of just one.

In line with the themes above, Holger Laux from Novartis explained how they are increasing recombinant antibody expression using transcriptomics and engineering. As an example, they established that the loss of the telomeric region on chromosome 8 resulted in a higher expression of the antibody, and so they designed a knock-out cell line that eliminated this region.  As a result, the cell line is now predisposed to produce the protein at high levels (35x increase). This is another example of how understanding the cell’s genetics better can aid the production of stable cell line and future developments of cell lines.

Hannah Byrne from Valitacell introduced the idea of coated plates for measuring real-time IgG productivity in situ of static growing clones. Scientists are definitely looking for better alternatives to Octet and it will be interesting to see how the regulator responds to having detection proteins in the cloning plate.

There were talks by Arna Andrews from CSL and Jean-Marc Bielser from Merck in Vevey about the trend towards perfusion compared with fed-batch. Bielser talked about screening methods early on for clonal phenotypes in perfusion cell media. This really led to a recurring theme of screening for critical quality attributes earlier in the CLD process with best fit for intended process and product quality

I came away from the conference with a renewed sense of optimism about the exciting developments taking place in our industry overall, and affirmation that the further products we are launching and developing at our own company will make a significant contribution to scientists in this field.

Andrea Gough, Product Manager, Solentim