In a recent interview with Cell Culture Dish, Dr Ian Taylor discusses the evolution of cell line development, new advances in technologies and workflows, and future advances. The discussion includes improved imaging techniques, manual limiting dilution versus single cell seeding, and achieving clonal outgrowth.
Going back to the 1980’s, it evolved largely from pioneers at Genentech in South San Francisco who had the idea of engineering a CHO cell line to produce recombinant therapeutic proteins. Cell line development has continued to evolve and now we have many CHO cell lines, mostly derived from those originally created by Genentech.
When it comes to optimization, high titre for the protein of interest is the most important. The current benchmark these days is around 10 g/L. It is also important that productivity can be scaled up for bioreactors of several thousand litres. Second priority is ensuring high protein quality.
There are three areas of importance. The first is educating researchers to understand that screening thousands of clones to find the “needle in the haystack” or best clone is unnecessary. There are far more efficient ways to pull out these high value clones, such as using transposases. The second is to make people realize that looking at protein productivity too early and for single cells is meaningless. Third, lots of single cells don’t grow and these could be top clones, but you wouldn’t know it. Using supplements to make sure you get a high recovery and the maximum number of clones to grow into colonies is very important.
Historically, customers needed two instruments, one for single cell isolation and one for whole well imaging. Both these steps are now combined into a single instrument, the VIPS. The VIPS system is also able to confirm a single cell in a droplet on the bottom of a dry well, presenting a new “microcosm” for clonality. This is a novel application that we are keen to get in front of regulators.
The VIPS enables single cell plate seeding at very high seeding efficiency, 80-95%, whilst retaining maximum viability. When combined with non-random integration methods (e.g. transposases or landing pads) a complete cell line development project can be completed in <200 clones, compared to the 5,000 clones typically used. Consequently, you can reduce the overall number of plates compared to your competitors, who may be running as many as 100 x 96 well plates, a process that requires automation. Another recent important example is one our customers, Biotheus, who have notified us that they had achieved a successful IND outcome with the regulator for a cell line developed using the VIPS.
We chose the HD-BIOP3 cell line from Horizon because it is one of the main commercial cell lines used by our customers. However, its outgrowth can be highly variable. To get more consistent and high-quality output our study tested conditioned media, commercial base media and the InstiGRO CHO supplement to improve clonal outgrowth and consistency. This study can be viewed in detail through our webinar and our application note.
On the face of it, single cell seeding generates far more wells per plate than LD. This is supported by more in-depth analysis using whole well imaging, revealing that a high proportion of LD wells actually contain more than a single cell. Hence the VIPS results are even more compelling because you have a high seeding density of 80-95% and you have more wells with single cells. This improvement is nicely articulated by Janssen in this Case Study.
Clonal outgrowth is the ability of a single cell to recover and actually grow into a viable colony. Many of the best clones can be lost at the single cell level because of poor clonal outgrowth. Solentim’s optimization study showed good recovery and growth for Horizon and Sigma cell lines, for host cells, expressing cell lines and different pools. This data was further validated by several customers, including Celgene, in our recent webinar.
Solentim is expanding its capacity to offer customers a complete A-Z holistic approach in the near-future. This would encompass instruments, reagents and reporting for the whole cell line development process, removing the need for customers to independently determine which elements they require to build out a cell line development lab. Another growing area is gene therapy and the production of viral vectors. There are lots of companies conducting clinical trials in this area that will need to make the move from transient to stable cell line production. They will require a similar set up with single cell isolation, assurance of clonality, and clonal outgrowth.
Please follow the link for further details about the VIPS.
Dr Ian Taylor, Chief Business Officer, Solentim
Dr. Taylor has a PhD in Biochemistry. He has more than 25 years working in the conceptualisation and commercialisation of novel high value life-science instrumentation. Prior to joining Solentim when it was founded 9 years ago, Dr Taylor was Commercial Director at Genetix PLC where he was responsible for the development and sales of the ClonePix FL for selection and isolation of high value mAb producing clones and a range of other products for cell culture. Prior to that, Dr Taylor was part of the senior leadership team for PerkinElmer Life Sciences.