Clarity in cell line development

Webinar | Revolutionizing Cell Line Development Workflows with New Technology

Leaping Ahead in Cell Line Development with the Latest Technology

Last week over 300 participants tuned in from 33 countries to hear experts in cell line development (CLD) discuss technology that could revolutionize their workflows. Mark Stockdale and Oren Beske, Amalgamators of Business and Biology at Solentim and ATUM respectively, discussed the essential pillars of cell line development, the practices revolutionizing workflow efficacy, and the best technologies to streamline your lab. Focused on how we can accelerate cell line development (CLD) while maintaining the quality of the process, increasing efficiency and decreasing regulatory queries, we kicked off.

 

A full recording of the webinar can be accessed by clicking the button

The Three Pillars of CLD

Mark opened the discussion by outlining the three pillars of CLD: cell line, gene insertion and media. These three variables offer anyone working in CLD a wide variety of choice.

When considering which cell line, researchers must decide between founder cell lines, requiring significant investment in time and resource to optimize, wild-type suspension variants, which tend to be low cost and ready to work with, and highly optimized variant cell lines, tailored for use via engineering or directed evolution offering faster timelines and better productivity. Money, resources, expertise, desired outcome and end use are all factors that will impact this decision.

Most of those who dialed into the webinar work with CHO cells. These reliable, well characterized cell lines are a common favorite and, when asked whether CHO cells would always be used for biotherapeutic production, Oren confidently answered, “Yes! They have a huge capacity to produce a variety of different molecules, are flexible and stable. We may look at creating designer CHO cells going forward, but CHO will be here a for long time.”

When deciding upon which media to use, researchers must choose between using a trusted provider or developing custom media in-house. Custom media are often optimized for expression but may not suit CLD and therefore require supplements. It can be tempting, when developing the perfect supplement for your media, to over-complicate the problem. As Mark described, previously in his career, “during a lab move, our team had controlled for every parameter, but within a few weeks of setting up the new lab not all the limited dilution (LD) plates were recovering colonies. Due to the complex cocktail of supplements being used it was almost impossible to identify what had gone wrong. In the end, it was in fact a light in the cold room that was inactivating a component in the media.”

Then Mark turned his attention to gene insertion – “how do I get the DNA into the cell?” Random integration offers easy to access, robust vectors but requires time-consuming enrichment steps, and while the recently in-vogue landing pads offer the opportunity for strong productivity, there have been questions raised around how homogenous the pools really are. Another option is transposase integration of transposons.

Transposase Technology

Gene integration needs to be accurate, reliable and stable. This is why ATUM’s technology relies on systems evolved over billions of years to be just that. Transposons are DNA sequences that can change positions within in genome but ONLY in the presence of a transposase enzyme. This is the guarantee of stability – no enzyme, no movement of your inserted gene. The Leap-In Transposase® system acts as a cut-and-paste mechanism for transposons of any size, integrating a single copy of the desired gene at each site and achieving perfect integration of elements between ITR’s every time. The system integrates single copies of the entire synthetic transposon into multiple sites (5-60+) across the genome, maintaining structural integrity and achieving comparable expression, functionality and product quality between integration sites. “Each integration event is equally functional, ensuring that exactly what you have designed ends up in the genome”, said Oren.

“Over 90% of clones retain 100% copy number stability, and over 90% retain expression stability”, said Oren. “The system is also cell line agnostic, and to some extent species agnostic. It has been seen to work in all mammalian and yeast cells tested, and we would expect it to work in any ligase-active cell – which is virtually all of them!”

Controlling Expression Ratios

Within a vector, ATUM can alter and design regulatory elements to vary the expression of different open reading frames to vary relative expression and control the ratio of each product produced. This level of control, instead of randomness, can lead to a massive increase in the amount of the desired product (Shown in figure 1). The ability to fine-tune production of different reading frames within one vector is a key advantage in the production of bispecific antibodies, and has been used by Horizon Discovery.

Maintaining structural integrity of the sequence integrated into the host genome also ensures that regulatory elements remain associated with the appropriate open reading frames, and desired balances between multiple open reading frames are maintained.

 

Figure 1: Controlling ratios with construct design

Pool Homogeneity

The Leap-In Transposase system also uses drug-free selection, selecting instead the absence of glutamine. This allows quick and robust selection, and rapid recovery of the population (<2 weeks). “This is possible due to the uniformity and stability of our pools. The majority of the clones are high expression which reduces the amount of screening needed”, explained Oren.

 

To illustrate this, when using Solentim’s VIPS alongside the Leap-In Transposase system, 62% of clones are in the top quartile of expressers and 82% are in the top half (see figure 2).

 

 

Janssen has adopted this technology and has achieved data reiterating the quality and uniformity of this process (see figure 3).

“It is predictive; pool titers predict clone titers, and in turn this predicts product quantity and quality” said Oren.

 

Figure 2: Typical clonal distribution for Leap-In Transposase (ATUM)
Figure 3: Stable pools predict derivative clone titers, Janssen case study

Saving Time and Money

ATUM’s approach – in combination with Solentim’s instruments – has cut CLD workflow timelines from between 27-36 weeks, down to 10-12 weeks, from transfection to RCB. This significant time saving, coupled with the high uniformity of cell pools and no loss in productivity or transgene copy numbers after 90+ doublings, makes this an absolute must to enable next generation biologics.

When asked by the audience how much the workflow can be shortened using Solentim’s instruments alone, Mark responded, “The biggest impact comes from the Cell Metric®, which will definitely take at least 3-4 weeks off the process, but you will also see time savings from the clonality report.”

Double Lock Assurance of Clonality

Solentim has worked for 10 years to create instruments that facilitate integrated workflows with strong audit trails, providing regulators the evidence they need of derivation from a single cell”, said Mark. Together, the VIPS and Cell Metric provide a double lock of assurance. In-well assurance and day zero whole well imaging provide users and regulators with the confidence they need in cell line origin.

The VIPS, when compared with manual LD, achieves 40% higher well growth while maintaining high cloning efficiency, meaning more clones are produced that meet FDA requirements from a single round of clone isolation. The FDA has also spoken out in support of this methodology, stating that “imaging technology offers an attractive way of providing supportive data to assure clonal derivation of production cell lines in lieu of additional laboratory work” . “The final report produced by our instruments will provide all the key information needed to show that your cell line was derived from a single clone, including images, in an easy to transfer document”, said Mark.

The combined technologies of Solentim and ATUM were shown throughout the webinar to provide significant, tangible benefits to the user, but some participants were concerned as to how long the transition from LD to this system would take. “Other than an inevitable technology transfer and set up period, the transition is very smooth. The biology between the two processes is very similar, removing the need for any big changes to the lab, and there are support staff at both companies ready and willing to help” reassured Mark.

Keep the Conversation Going

Both Mark ([email protected]) and Oren ([email protected]) are available for questions via email, and would be delighted to hear from you.

A full recording of the webinar can be accessed by clicking the button

You can find more information about both Solentim and ATUM at their respective websites: www.solentim.com and www.atum.bio.

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