Increasing speed and efficiency of biotherapeutic drug development with stable pools
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Pressing Industry Challenges
I began the interview by asking Dr. Porter if she could tell listeners a little about herself and what she feels are the most pressing challenges for drug developers. She described how she’s been in the industry for 25 years and has held several different roles, including many areas of the drug development pathway with specialization in mammalian expression systems and mammalian cell line development. She went on to say that with respect to challenges today, she is still hearing the same common refrane – faster and higher: a desire for more product in the shortest time possible, so as to be the first in clinic. More complex proteins, which are becoming more common, can often be more difficult to express and harder to achieve the desired characteristics – however, they still come with the desire for higher and faster!Stable Pool Expression vs. Transient Expression and Stable Transfection
Next, I asked Alison if she could tell listeners about stable pool expression and its use in the industry. She explained that stable pools are a mixed population of stably transfected cells. She clarified that while in transient transfection, recombinant DNA does not integrate into the host genome, therefore it does not replicate, and it’s eventually lost to cell divide. This approach is typically used in discovery when assessing multiple variants. It is a good method if relatively small amounts of material are required quickly. At the other end of the spectrum is stable transfections. They begin transiently but are followed by the infrequent but critical event where recombinant DNA integrates into the host genome. Integration simply means the product gene can be replicated and descendants of those transfected cells also express the product. This is important if long term gene expression and large amounts of material are required. Typically, stable transfection also requires a cloning step to meet the current regulatory requirement of a cell line used for the manufacture of biotherapeutic originating from a single cell. A stable pool could be described as a halfway house between a transient transfection and a clonal cell line from a stable transfection. In fact, most people would recognize the pool as the start point from which they clone. So, a pool takes longer to create and obtain material from then a transient transfection, but it takes less time than a clonal cell line. It can generate more material than transient transfection, but is thought of as generating less than a stable cell line, although that is becoming blurred these days with higher levels of material produced in stable pools. She went on to say that Lonza first started looking at and using pools for various reasons in the early 2000s, then for use with customers in the mid-2000s. Initially they were used to supply material early in the development process, but soon they became a main part of the cell line construction workflow itself, helping to reduce timelines and subsequently their use has increased substantially.Stable Pool Expression Use
Reducing cell line development time
Next, I asked Alison about the best use for stable pools. She explained that using pools to reduce cell line development timelines had a significant impact early on. Now, with the advent of more complex proteins, pools can be used in other ways to further reduce timelines for those molecules. For example, in parallel to cloning, one could assess multiple pools using product concentration and/or product characteristics to identify the best pools to select to clone from. In addition, one could begin developing or optimizing the upstream process at this early stage if a particular molecule has special requirements.Generating early material supply
Another major area for pool use is material supply. Since a pool can generate more material than transient transfection, pools started to be used early on for this. And they continue to be used in the late stages of discovery where there are only a small number of variants with larger amounts of material required.Increased reproducibility
One challenge with transients is generating material with the same product characteristics that will be seen in the stable clonal cell line. This is especially true if the cell line being used is different between transient and stable production, for instance, HEK for transient and CHO for stable. Thus, pools can overcome the challenge of generating material quickly and generating material that is consistent with what will be seen in stable production.Assessing the production process
Lastly, development pools can be used to assess the selected production process and produce material to feed other development stages, which can happen in parallel to the remainder of cell line development and thereby further shorten timelines.Stable Pool Productivity
We then discussed titer expectations with stable pools and how this can be useful for drug developers. Dr. Porter stated that for antibodies, Lonza typically sees on average around 2.4 grams per liter in pools with their standard GS system® and platform upstream processes. However, they have seen over 6.5 grams per liter. It is not unusual to see slightly lower product concentrations from pools compared to final clonal cell lines where the range is 2 to 6 grams per liter with an average of 4 grams per liter for antibodies. When Lonza uses their transposon-based technology, GS piggyBac®, pools recover faster post transfection, and higher titers can be achieved. Lonza has seen substantial increases in titer for hard to express antibodies, complex antibodies, and bispecific antibodies when using GS piggyBac®. More product means drug developers can undertake the studies they need and speed towards being first in clinic.Predictiveness of Pools
We moved on to product concentration of the pools, how predictive the product concentration of the pools to a clonal cell line is at later stages and how representative the stable pool material is. Dr. Porter explained that the pools aren’t completely predictive of product concentration, but they are good enough for ranking purposes when doing early process development work. In Lonza’s Ibex® Design service, they use stable pool material for process evaluation and formulation activities, which helps them deliver shorter timelines for antibodies. This approach is only possible due to the stable pool material being sufficiently representative of that from the final clonal cell line. They have generated substantial data from multiple programs demonstrating that the process performance between the pooled and clonal material is comparable – with good comparability in growth profiles. Additionally, and most importantly, they have also demonstrated that the product characteristics are compatible.Future Applications for Stable Pools
I asked what other areas or applications pools might be used for in the future. Dr. Porter responded that for the last 10 years there has been conversations within the industry on whether material generated from pools could be used for toxicology material or even for Phase I clinical trials. If this is accepted, questions around material comparability material are going to be critical. She went on to describe how the current COVID-19 pandemic has pushed this concept front and center due to the big reduction in timelines they can enable. Dr. Porter presented with Professor Trent Monroe from the University of Queensland last year. She said that Dr. Monroe spoke about their rapid development of a potential COVID-19 vaccine and they did use material generated from pools when they first went into the clinic. Thus, there is certainly more consideration for the use of pools and precedents for the use of pools more broadly.Implementing the Use of Stable Pools
I closed the interview by asking how someone could get stared with incorporating stable pool expression into their workflow. Alison said that if listeners have no experience, consider using Lonza to generate material from pools using their CHO-based GS platform. Alternatively, if listeners want to do work in their own labs, they can take out a GS Gene Expression System® research evaluation license (REA), which would give users access to Lonza host cell lines, vectors and protocols for generating stable pools. This ensures that end users are working with materials that have demonstrated success in generating stable pools as well as detailed protocols. End users would also have access to GS piggyBac® and technical support from subject matter experts within Lonza who can assist with implementing the process. For more information, please visit https://pharma.lonza.com/This post is sponsored by Lonza.