Accelerating Cell Line Development for Vaccine Production

Mark Stockdale is amalgamator of business and biology at Solentim.

July 15, 2020

Around the world scientists are working to develop, test, and scale-up the production of a vaccine for the global COVID-19 pandemic. Working to unprecedented timeframes, by early July—only a few months after the initial acceleration of the pandemic—18 vaccine candidates were already in clinical trials with the WHO listing more than 129 candidates in preclinical development.¹

The standard timeframe to develop a novel vaccine, and establish its quality, safety, and efficacy, is 10–15 years,² making our search for a vaccine as an answer to the pandemic ambitious to say the least. However, the research speaks for itself: the timeframe to reach clinical vaccine trials is usually 5–10 years—we have achieved this in a matter of months. Moderna’s mRNA-based SARS-CoV-2 candidate entered a phase 1 clinical trial on March 16, less than 10 weeks after the first genetic sequences were released.³ So, how has this been possible?

Changing procedure and protocol

Vaccine developers usually follow a linear process, coupled with extensive data analysis and checks, to protect against traditionally high failure rates. Developing a vaccine during a pandemic has required a new paradigm, with a fast start and many steps executed in parallel without waiting for confirmation of success in the previous step. This accelerates the process but also elevates the financial risk.³

Advances in cell line development (CLD) technology and knowledge are also re-risking and accelerating research, including in silico methodologies and novel orthogonal methods like Oligomer Detection Assays and Quartz Crystal Microbalance (QCM) methods. Further integration of computational (predictive) methods with new analytical tools will provide detailed developability fingerprints in the discovery stages for many different therapeutic candidates.⁴ Optimizing the CLD phase of research not only increases the efficiency of drug development, and the chance of success, but also makes the transition to scale-up and manufacture more streamlined and successful.

The transition of host cell from embryonic chicken cells to mammalian cell lines (a transition which began over 50 years ago),⁵ such as Chinese Hamster Ovary (CHO) cells, has enabled developers to rapidly produce vaccine supplies with higher levels of stability and fewer concerns around sterility.⁶ CHO cells have emerged as the gold standard in the production of biologics and are considered by many to be the ideal cell line for protein production.⁷ Similarly, the Vero cell line is the most widely accepted continuous cell line by regulatory authorities and has been used for over 30 years for the production of polio and rabies virus vaccines.⁸

The perfect cell line

Not all cell lines are created equal, nor are all methods of cell line development equally successful.

Firstly, gene integration needs to be accurate, reliable, and stable to ensure that the sequence designed in the lab is exactly what ends up in the cell. Technologies such as ATUM’s Leap-In Transposase^® can increase the accuracy of this step, with 90% of clones retaining 100% copy number stability, and over 90% retaining expression stability. The more reliable the gene integration step, the higher the proportion of ‘high value cells’ (high-producing cells) in your pool. This will lead to greater pool homogeneity, which will reduce the level of screening required; the more uniform and stable the pool of clones, the less screening required as the majority of the clones are high-expression. The ability to reduce or eliminate the screening step can knock weeks off cell line development workflow timelines.

Growth media and supplements, either dispensed with the single cell or after single-cell seeding, should also be considered. There is no one-method-fits-all, and conditions need to be tried independently for the specific proteins to be expressed. Even with recommended media, modifications of some key ingredients and addition of supplements may be able to obtain excellent yields but keeping it simple usually seems to be a good bet.

For use in drug development, cell lines must be clonal—originating from a single cell. Traditionally this has been achieved using limited dilution (LD), a time-consuming and unpredictable method. The replacement of LD with single-cell seeding instruments can improve seeding efficiencies and shorten development timelines by months, providing users with highly stable, single clones for manufacturing purposes.

Crucially, when creating cell lines for vaccine development, proof of clonality must be assured. Securing this evidence has been made easier than ever due to new advances in imaging technology. The FDA has 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”.⁹ Ideally, a “double lock of assurance” will be secured: in-well assurance of the arrival of the single-cell into a well using a single cell seeder and day zero whole well imaging using a whole well imaging system, providing regulators with the confidence they need in cell line origin.

Scaling-up

By selecting or designing an optimized cell line for vaccine production and ensuring all stages of the CLD process have been de-bugged, researchers can maximize the likelihood of successful scale-up and manufacture. Cell lines suitable for scaling up vaccine production need to demonstrate high yield, appropriate antigenicity, and accurate product expression.

A significant challenge when considering the scale up of a COVID-19 vaccine is the range of vaccine platform technologies on trial. In the U.K., COVID-19 vaccines are expected from two main platforms, the RNA and the chimpanzee adenovirus (ChAd) vaccine vector. Each has a different formulation, specific upstream production, and downstream requirements. Without knowing which vaccine will move ahead to scale up, manufacturers will find it difficult to fully prepare their facilities for the task ahead.¹⁰

What’s in a vaccine?

As mentioned above, not only has the current pandemic pushed the global scientific community to accelerate its rate of drug discovery, it has also seen an increased focus on the development of lesser known types of vaccines. Until recently, most vaccine development has focused on live-attenuated vaccines (MMR, Chickenpox, Yellow Fever), inactivated vaccines (Hep A, Polio, Rabies) and subunit/recombinant vaccines (Heb B, HPV, Shingles).

Several of the most advanced vaccine candidates for COVID-19 make use of emerging technology platforms, including:¹¹

Nucleotide-based vaccines: Like traditional live-virus vaccines, these vaccines deliver a genetic sequence into a host cell and co-opt host machinery to express antigens of interest. However, rather than using a weakened SARS-CoV-2 to transport the code, Moderna's vaccine uses a synthetic lipid nanoparticle to carry mRNA templates.
Recombinant vaccines: The University of Oxford and AstraZeneca have embraced a recombinant vaccine called AZD1222 to achieve a similar effect, engineering a chimpanzee adenovirus to carry DNA for the spike antigen. Because adenoviruses are themselves immunogenic, such types of approach could generate robust memory B cell and T cell responses that might result in better prophylaxis with fewer doses.

But neither of these approaches have ever produced an approved vaccine in the United States or EU before, nor have they ever been produced or distributed at scale. It remains to be seen how these trials will play out, however it is possible that we will see an initial wave of clinical trials making use of RNA vaccines—fast to lab but untested and therefore possibly less likely to work—followed by a second wave of classical vaccines entering clinical trial.

The future of vaccine development

The global drug development community has come together against a common enemy, accelerating time frames and deploying new technologies to bring the world the COVID-19 vaccine we desperately need. Optimized CLD will be essential for this breakthrough. Long term, the lessons learned, and techniques used in the fight against this pandemic could enhance our capabilities for general biotherapeutic production and drug development.