Had I Known Then What I Know Now

July 31, 2024 by Emilija Robinson

Early decisions can make or break manufacturing success

I have been a researcher for most of my professional life. After a decade of mostly pursuing basic research, entering the field of viral gene vectors has been exciting, because it was an opportunity for me to make a tangible real-world impact through applying my molecular biology skills and knowledge in developing therapeutic modalities.

From the beginning
Success is about more than scientific discovery. During my five-year journey from academia into the biotech world and working across a variety of viral vectors, I learned many valuable lessons through successes and failures. Those lessons are worth sharing to help early-stage advanced therapy medicinal product (ATMP) developers who find themselves in similar situations.

When it comes to the long-term success of ATMPs, groundbreaking science is nothing without a robust process for manufacturing the final therapeutic. However, establishing that process is arguably one of the most challenging steps in the development of therapies, and it is influenced by everyone involved.

Those who define the process, i.e. preclinical researchers working in the discovery phase, must understand the significant impact their decisions will have on future manufacturing success, or failure. So, what do I know now about making those decisions that I didn’t at the beginning?

Laboratory protocols do not equal manufacturing processes
From a purely scientific perspective, producing ATMP is a seemingly straightforward process. So I thought too, as I was starting to work with viral vectors. Having years of experience with plasmid DNA and transfecting cells, I was expecting that the key to success to be (a) an optimized and well-written laboratory protocol which yields good titers, and (b) technical expertise to master the downstream process. But without a CMC team to provide continuous feedback, relying solely on production protocols often leads to workflows that cannot be upscaled.

I originally approached upscaling of viral production by simply making more; bigger batches and more production cycles, which resulted in saturated systems and failed batches. For example, the extent to which the volume of production cells can be increased is limited by the capacity of the downstream process. In my case this was an ultracentrifuge. This then required a change of the downstream process from ultracentrifuge to chromatography.

However, not all upstream protocols are compatible with chromatography. This triggers a change in production protocols (change of process) which may no longer yield good titers and, if implemented later, will require comparability studies to show that the process change has not impacted the activity of the therapeutic modality.

How can this best be addressed? From early on, the production protocol should be developed and optimized with chromatography compatibility in mind. This can be achieved only by developing a thorough understanding of the individual steps and materials. Building that understanding allows the operator to not only control the process and reduce batch variation, but also make predictions on how it might respond when implementing changes, which are inevitable.

Work with high-quality RUO material, or risk adding time, cost and additional studies
The quality of the starting material is one factor that directly affects the consistency and reproducibility of the ATMP production processes. Take plasmid DNA, used in viral gene therapies, as an example: it is considered stable and easy-to-produce consumable. However, plasmid DNA can vary widely in quality, and this becomes evident once root cause analyses (RCAs) are introduced to identify sources of process inconsistencies.

What characteristics does “high-quality” plasmid DNA have?

• It contains minimal amounts of impurities such as endotoxins, host cell protein, host cell RNA and or DNA, and has high sterility. Both factors have a significant impact on ATMP production cells
• It is homogeneous, meaning it consists mostly of supercoiled forms and is not fragmented or degraded, which affects the potency of the molecule. For example, a plasmid DNA preparation consisting of 90% supercoiled form will have higher transfection efficiency compared to a preparation consisting of 20% supercoiled form with fragmented and degraded DNA
• It has the right identity. During the growth of the bacteria, some of the plasmids, i.e. ITR-bearing ones, can recombine and create hybrid forms whose biological properties have not been assessed and thus cannot be controlled.

In my experience, research laboratories often exclusively pay attention to the third factor. The other two are often overlooked, either because there is no awareness of the risk associated with them, or because there is no dedicated analytical team to establish and validate methods for testing.

Getting the right help early can save money long term
When needing plasmid DNA, researchers often turn to the broad range of commercially available plasmid DNA extraction kits. They come with short and easy-to-follow workflows which can be accommodated between experiments. They do come with limitations though, mainly that they are inherently unscalable. Additional disadvantages are the costs of the kits, as well as the need to screen and test several results to identify the optimal plasmid.

Further challenges associated with in-house production of plasmid DNA for ATMPs are caused by their intrinsic properties. ITR-encoding, large, or poly(A)-encoding plasmids tend to recombine during the growth phase, resulting in mixed plasmid populations, loss of ITRs, and reduced poly(A) tails. A mitigation strategy is to conduct a screen for identifying optimal bacterial strain for the plasmid reamplification and screen for optimal growth conditions, which reduce the risk of recombination events and increase product yield. However, performing such screenings requires operator time and increases the overall cost of the plasmid DNA.

An effective way to address these issues is to partner with a CDMO. The key point to keep in mind when making such a decision is that CDMOs have specifically developed capacities and capabilities to address plasmid DNA production. They also have expertise to help you set up your process from the start, to ensure you avoid problems later with manufacturing and scaling.

Considering the time and financial burden of ATMP development, it is critical to select a CDMO that will be a reliable partner throughout the process. They should be able to support your needs as you move your program through the individual phases of the drug discovery life cycle. Some offer support from discovery all the way through clinical trials and commercialization including regulatory support. Others do not and can support programs only to a certain stage, which requires a CDMO change at some point, leading to additional costs associated with technology transfer.

Traceability is another extremely important regulatory aspect for ATMPs and, unfortunately, this is not always a given as some CDMOs do not offer the service. Reliability regarding timelines and product quality is also essential. I personally have made the mistake of choosing a CDMO based on price over reliability – and have ended up performing extra material validation steps in-house, wholly defeating the purpose of outsourcing.

The Takeaway
Developing ATMPs is complex, and you need to invest your time and resources wisely. Focus on the aspects you know best and can control. Understanding your modality, its MOA, and intrinsic properties is where your time is best invested. Get a CMC team and professionals to help you build a robust manufacturing process from the very beginning, then choose a CDMO with the capacity to support you from discovery through clinical to commercial, and that can provide the expertise needed to understand the challenges of the cells and gene therapy industry. This will help you to avoid pitfalls that result in unnecessary delays and costs.

Download our whitepaper, Set Your Cell and Gene Therapy Program Up For Success From Day One

• Learn more about Aldevron’s custom manufacturing
• Visit our Plasmid DNA manufacturing page
• Have questions on this topic? Contact Emilija Robinson
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