High-titre Vector Producing Cells

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Therapeutic biologics are thriving, according to Zhou Jiang, director Cell Culture Services at Cytiva. “As we work to advance the next generation of therapeutics, we must address these challenges to deliver the medicines patients need. The increasing diversity introduces new challenges and mandates new solutions in upstream cell culture,” says Jiang.

Gary McAuslan, CEO aCGT Vector, outlines some central challenges, stating, “cell therapy manufacturing today is unsophisticated and in the main, extremely manual and people dependent, shocking from a microbiological contamination potential, and impossible to scale in a controlled manner from a mass uptake of precision medicines perspective. Batch failures, low viability, and time to patient exceeding the patient’s life expectancy are the stark realities of today’s pharmaceutical manufacturing response that attempts to bulk manufacture what are truly individual modern medicines at a scale of N=1."

“Only one of the CAR-T innovators ship fresh patient T-Cells for good reason,” he says. McAuslan concludes. The current default, for example, for the first CAR [chimeric antigen receptors]-T Kymriah from Novartis is to use frozen T-cells that are collected at the hospital from a patient, sent across the world frozen, thawed, cell engineered, frozen again, shipped again and thawed again, before being administered inside the same hospital over a month later, taking just 11 minutes to infuse into the same patient. The current logistical complexity is restricting cancer patient access to the just 15,000 treated since the first approval in 2017 versus the projected annual 2 million patients who might benefit from CAR-T treatment. Pharma can absolutely do better than this. Both of these therapies are manufactured and shipped back frozen, a number of weeks to an individual patient for administration inside a hospital center of excellence approved to do so. Being able to eliminate these complex logistics, and time especially (in the region of 27% of patients die waiting on their own CAR-T therapy), as a complementary manufacturing supply chain will increase access substantially.”

This need for speed comes in parallel, but case by case, when compared with the warp speed efforts during COVID-19 vaccine development and deployment programs. Jiang reflects on this phase, saying, “There is also the drive to produce the highest possible titers with the best product quality, which has led to efforts to produce more robust cell lines and media formulations suitable for a wide variety of cell lines and biopharmaceutical molecules. Insights from the cell biology of CHO [Chinese hamster ovary] cells, and understanding of the relationships between specific metabolites and cell culture outputs such as titer and quality are highly valued and sought after for the realization of these goals.” This is why so much research, investment, and time has been focused on advanced therapy medicinal products (ATMPs) in the past few years.

Clinical challenges

From a drug-development and clinical perspective, analytical characterization of material is important at every step, and making sure the critical quality attributes (CQA) stay within a threshold, within specifications and guidance, are chemistry, manufacturing, and controls (CMC) issues, which need to be tackled during the early manufacturing process development. Vibha Jawa, head of Biotherapeutics Bioanalysis in Nonclinical Disposition and Bioanalysis (NDB) organization, at Bristol Myers Squibb, points to this saying, “further, we are always quantitating transduction efficiency and target response ... titer quantitation helps us to visualize the transgene expressions in a much more reliable and specific manner whether we are looking to quantitate what that transgene expression is, or whether we are looking to characterize and optimize the amount of the vector we are going to administer. This is because a lot [of] safety and toxicity challenges turn out to be proportional to the amount of the virus used, and the content of that virus from an empty versus full capsids perspective. The best case option for the patient is to have the highest does load presented via the least virus delivery method possible.” Sometimes poor results or failure here even precludes proceeding with the originally intended ATMP plan. Sometimes caregivers revert to antibody drug conjugates or other well-understood treatments, as the novel medicines are sometimes simply not the best choice option for that patient.


The closer one gets to the situation in the clinic, the more these challenges stand out in stark relief. Jawa relates that groups have been struggling to get sufficient well-characterized material for clinical studies. “Even if you have plans to file an IND [investigational new drug application], unless you have a good contract development and manufacturing organization (CDMO) ready with that kind of capacity waiting for you, you have to be able to synchronize with their timelines, because they are busy making someone else’s viral vector. So you have to put yourself into that que,” she states, referencing the need to always be cognizant of many other people’s timelines. Jawa goes on to say, “because there are very few of them [CDMOs] that are good and reliable. And you really need material that is the least toxic in terms of residual or process-derived contaminants, but is effective to get your transgene in, and is a good drug product within that viral vector.”

Another logistics and business challenge, according to Jawa, is which patient cohort one is working with. “It’s a highly competitive situation, especially if you are targeting diseases which affect large numbers of patients. For rare disease states, there is no such resource pressure, and the landscape is not as [potentially] crowded with competitors ahead of you making these vectors.”

Sharing knowledge

Near patient manufacturing small footprint systems combined with increased automation seem to be where much of the current progress has been made. Baley Reeves, interim director at the National Center for Therapeutics Manufacturing, points out that, especially for small footprint systems, “with scaled-down/scale-out operations, quality control and regulatory [issues] are more difficult hurdles to manage. Automation and inline analytics become even more critical pieces of the process. This mainly impacts autologous cell therapies at the moment but could have future implications as personalized medicine takes off,” she suggests.

Common issues to achieving high titer levels are manufacturing plasmids that contain difficult genetic payloads (e.g., gene-of-interest size, base composition, toxicity, use of selection markers). When pursuing large plasmid sizes to navigate those challenges, one is then often confronted by leaky promoters that lead to pre-mature protein expression upstream, losses during alkaline lysis or repeated sequences such as inverted terminal repeats (ITR) and polyA tails that are more prone to deletion and recombination events. Some successful ways to navigate these obstacles are to optimize and balance buffer volumes, be more precise in terms of buffer contact times. By using more gentle mixing methods, one can reduce sheer impacts. And of course, it always helps to start with well-characterized pHelper and pREPCAP plasmids. Viral vector synthesis network Helper plasmid (pHelper) activates expression of the rep/cap gene on the packaging plasmid (pPackaging) and synthesis (k Cap_syn and k Rep_syn ) of viral protein (VP) and Rep protein (RepProtein). “Capsid proteins are assembled (k assembly) into empty capsids in the nucleus (EmptyCapNuc), and each capsid particle consists of 60 protein subunits. With helper functions from the helper plasmid, the Rep protein replicates (k DNA_rep) viral DNA (vDNA) from the transgene vector
plasmid (pVector)” (1).

A key to knowledge transfer may depend on reimbursement model modifications, and regulators embracing the idea of “equivalent centers,” leading to standardizing some equipment and protocols. aCGT Vector, for example, has concentrated on “right-size pharma—standardized, automated, and closed-system technology and expertise ... truly end-to-end services offering, that can increase access for innovators and patients alike. For example, Janssen with their CARTITUDE front-line defense for the most common blood cancer, multiple myeloma. Novartis also recently secured reimbursement by the National Health Service (NHS) in the United Kingdom for earlier treatment of their cancer treatment. In time as these precise targeted but individual treatments are demonstrated as a better use of healthcare budgets, the predictions point to locally distributed centers complementing large centralized facilities, where a patient can walk into their hospital, receive CAR-T treatment, and go home days later, with the hope of potentially surviving their own cancer. Excitingly, the rare disease landscape, where many of the 7000 untreatable diseases such as blood cancer, is changing and bringing hope to patients and their families,” says McAuslan.


Nguyen, T. et al. Mechanistic Model for Production of Recombinant Adeno-associated Virus via Triple Transfection of HEK293 Cells. 2021. Mol Ther Methods Clin Dev., CellPress.

About the author

Chris Spivey is editorial director for BioPharm International.

Article details

Pharmaceutical Technology Europe
Vol. 35, No. 4
April 2023
Page: 20-21


When referring to this article, please cite it as C. Spivey. High-titer Vector Producing Cells. Pharmaceutical Technology Europe 2023 35 (4) 20-21.