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17 July 2019 by Nick Whitelock

Viral Vector Processing

The development of gene and cell therapy is a burgeoning area of research. With ever-improving understanding, methodologies and applications, it provides opportunities to treat and modulate diseases untouchable by conventional therapies.

Applicable to a range of maladies, from chronic genetic conditions to cancer, this class of therapeutics allows one to finely control the human genome, to knock-out pathogenic genes, to induce overexpression of required proteins, or to leverage CRISPR technology to replace defective genes with functional copies.

This and other developments mean the march of scientific progress is evolving into a sprint; humankind is ever closer to becoming master of his own domain, the species described in Yuval Noah Harari’s eponymous book, Homo Deus1.

Even with these exciting new applications of molecular biology towards improving public health, we must still balance the age-old issues of finance, ethics and chemistry to enable manufacture of these novel entities safely, robustly, and if possible, cheaply.


The Historical Context

It is easy to forget that pharmaceuticals themselves are a relatively new phenomenon. It is only a few generations since trepanning, blood-letting and exorcism (dashed with a healthy dose of mercury poisoning) were the peak of medical technology.

Germ theory was formulated only 250 years ago, with vaccines, aseptic technique and even the eradication of some diseases stemming from this simple thought.

Penicillin was discovered when Oxford University had already existed for eight centuries; and though perhaps the most therapeutically important molecule ever discovered, it had to be manufactured in repurposed hospital bedpans in an attempt to meet early demand2.

Whilst not quite as primitive, biopharmaceuticals such as monoclonal antibodies have had to undergo a baptism of fire.

In a few short decades, beginning in the mid-1970s, mAbs have evolved from obscure research toys expressed in murine cell lines at a concentration of a few milligrams of IgG per litre of cell culture fluid, into becoming a multibillion dollar industry treating millions of patients.

This industry can now produce fully humanized variants expressed at a concentration of up to 10 g/L in cell culture, a marked improvement in productivity, product quality and cost of goods.

This has been enabled by cell line, upstream and downstream process development, and ever improving technology and knowledge3.

These advancements have, in turn, enabled treatment of a plethora of diseases and have facilitated significant improvements to the quality of life for millions of otherwise helpless patients, the very dream that keeps us medicinal and bioprocess scientists slaving over our pipettes.


Difficulties to Address

It is certainly early days in gene therapy bioprocessing, where the analytics, process and product understanding are not as mature as with other biopharmaceutical processes.

Viruses, although the most abundant organisms on earth, with some estimates claiming each human harbours 1015particles4, are surprisingly troublesome to manufacture in a clinical setting, though it is also famously difficult to prevent their production in a biological setting.

This difficulty in clinical processing is not just the penalty of novelty: there are a number of difficulties that viruses present compared with other typical biopharmaceuticals at all stages of their lifecycle.

Cell line development must tackle the fact that some, often necessary, viral genes are cytotoxic, inhibiting stable cell line selection.

Often there is no known pathway for translocation of the expressed vector into the cell culture supernatant, or in cases where there is, this is usually to the detriment of cell viability. The stability of viruses is also problematic, requiring gentle but rapid processing, which is a paradox in principle.

The innate complexity of viruses means there are likely a myriad of product-related impurities that may be difficult to detect or remove.

The concentration of product in the initial stream is significantly lower than even in the early days of mAb production, by several orders of magnitude, though the product purity requirements may be just as strict.

Whilst the therapeutic potential of viral vectors are immense, they also have several limitations and hazards unique to their format.

These include host immune systems that are primed against this class of agent, the mutagenesis and oncogenesis activity inherent to viruses, and the nightmarish (but slim) possibility of replication-competent viruses, risking not only the patients’ health but that of their loved ones (and in the worst case scenario, another George A. Romero film).


Reasons to be Hopeful

That said, numerous technologies may be applied to viral vector processing from mAb manufacture. Single-use technologies allow one to intensify a process and remove the burden of cleaning and the associated validation, allowing lean, flexible processes and plants.

High-throughput techniques are increasingly employed at every stage of the cycle, from screening of drug candidates to screening cell lines and process methodologies, increasing robustness, consistency and safety.

Continuous processing, applied to increasingly complex products including mAbs, may alleviate the issues associated with poor stability of viral vectors by ensuring a rapid transition from fermenter to drug substance.

It may be that every step of viral vector production will owe a debt of gratitude to their mAb counterparts, where much of the equipment, methods and employees have been trialled and tested.

It is optimistic to hope viral vector manufacturing can reach comparable levels of robustness, quality and efficiency to mAb processes (though this same optimism contributed to mAbs reaching this level of quality and efficiency), considering the clear advantages mAbs have from a bioprocess viewpoint such as stability, simplicity and relative ease of upstream production.

However, by employing old techniques and developing new methods at all stages of development, from cell line screening to purification development, and collaborating at all levels, from suppliers to academic institutions, progress will continue to be made.

Whilst the ultimate goal of medicine, the eradication of disease, is less of a crackpot idea with every passing decade, the next few hurdles will take time, a lot more data, almost certainly some luck, but may enable yet another therapeutic revolution in the footsteps of antibiotics and mAbs.



1. Harari, Y. N. (2016). Homo deus: A brief history of tomorrow. Harvill Secker,ISBN: 9781910701874

2. Bud, R. (2013). Penicillin. Oxford University Press. ISBN 9780199541614

3. Liu, J. (2014). The history of monoclonal antibody development – Progress, remaining challenges and future innovations. Annals Of Medicine And Surgery, 3(4), 113-116. doi: 10.1016/j.amsu.2014.09.001

4. Elbehery, A., Feichtmayer, J., Singh, D., Griebler, C., & Deng, L. (2018). The Human Virome Protein Cluster Database (HVPC): A Human Viral Metagenomic Database for Diversity and Function Annotation. Frontiers In Microbiology, 9. doi: 10.3389/fmicb.2018.01110