A View of the Future for Manufacturing Viral Vectors

Larry Forman, founder and chief executive officer, CHO Plus

Forman gave a unique perspective on the states of cell-line development and engineering for adenoassociated virus (AAV) as someone not trained in those fields. The viral-vector space is oversaturated with contract development and manufacturing organizations (CDMOs), he stated, with few technological differences to distinguish them from one another. Such CDMOs experience universal challenges with low productivity, low ratios of full-to-empty AAV capsids, and suboptimal product quality — not to mention the inability to keep up with demand efficiently. Viral-vector manufacturing requires 10-L production capacity to make enough material for the treatment of a single patient who suffers from a common monogenic disorder. To treat a million patients, manufacturers would need to run hundreds of thousands of such processes — or to scale production significantly in stainless-steel bioreactors.

Forman began CHO Plus to explore methods of improving therapeutic-protein production efficiency with Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. CHO Plus applies directed evolution to engineer HEK cell lines to manufacture AAVs. CHO Plus scientists fuse the desired host cells repeatedly to shuffle their genomes and amplify whole chromosomes randomly. From the resulting genome library, which contains ≥500,000 carrier types, engineers screen and select desired phenotypes. As an example, Forman compared preengineered CHO-K1 cells against engineered CHO cells with 10× more endoplasmic reticulum (ER) per cell. Screening revealed that the engineered CHO cells are not necessarily bigger than preengineered ones despite the increased amount of ER that they contain. Using those screening data, CHO Plus scientists can manufacture cells with 40× more ER per cell.

Some barriers to high-dose AAV manufacturing concern cost, safety, and productivity. Drawbacks in those areas could lead to undesired consequences for patients postdelivery, so it is imperative to start with materials with optimal critical quality attributes (CQAs). Packaging cell lines, such as the ones from CHO Plus, offer optimized feed and media while minimizing variability of downstream CQAs. The CHO Plus platform can achieve high AAV yields without compromising the percentage of full capsids. Specifically, engineered cell lines from CHO Plus have seen 12-fold increases of AAV titers across multiple serotypes, 15-fold increases of AAV produced per cell, and two fold increases of full-to-empty capsid ratios.

The aim of directed evolution is to exceed cellular limits in AAV production. Directed evolution also can isolate favorable cellular characteristics for improved virus manufacturing and CQAs. CHO Plus’s engineered cells are likely to be approved readily by regulatory agencies because they are not manufactured with foreign genes or viruses. Additionally, scale-up should be a straightforward process because the cells are engineered in shake flasks. Cell-line development at CHO Plus includes suspension adaptation, with cells cultured using commercially available, serum- and animal-free media.

An audience member asked about key criteria to improve full-to-empty capsid ratios in AAV manufacturing. Forman responded that the goal should be batches with 80% full capsids before purification steps. Certain mitochondrial attributes of the host HEK cells are associated with higher percentages of full capsids, so during cell engineering and screening, operators should identify and use those attributes to maximize cell productivity.

Although scientists are unsure why such characteristics result in increased cell efficiency, Forman explained, the reason why is not important for screening. “There are so many genes involved with all these processes. We can’t possibly know what all of them are, let alone what they do and how they interact,” he said, “so we look for the things that we know correlate, and we hope to learn more as we go. But that’s going to be a very long process.”

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