Researchers recently enhanced the titer of a cell line still in development to produce adenovirus viral vectors by 7.5-fold, and without the risk of also producing replication-competent adenoviruses (RCA). If approved by regulatory bodies, it will join a handful of cGMP alternatives to the commonly used HEK293 (human embryonic kidney) cell line.

The proprietary cell line, SF-BMAdR, is derived from A-549 (adenocarcinomic human alveolar basal epithelial cells). It is being developed for the large-scale production of replication-defective adenovirus type 5 by researchers at the Biotechnology Research Institute at the National Research Council Canada (NRCC).

“By developing a novel serum-free suspension cell line (SF-BMAdR), we have expanded the possibilities for the production of an adenoviral vector devoid of RCA and demonstrated that titers can be improved through process development. This suggests that we have not reached the full potential of this platform,” Martin Loignon, PhD, senior author of the recent paper, and a member of the NRCC cell culture scale-up team, tells GEN.

Perfusion, batch, & fed-batch

Loignon’s team, including the paper’s first author and NRCC senior officer Chun Fang Shen, PhD, produced 6.3 × 10¹⁰ vp/mL in 3 L perfusion bioreactors. That’s a 7.5-fold increase in titer from the current SF-BMAdR culture process, making its titer comparable to that of PER.C6 (which typically achieves up to 8 × 10¹⁰ vp/mL) and some optimized HEK293 cell lines, but without their inherent risks. RCA, if present, were below the detection limit of 1 RCA in 5.3 × 10¹⁰ vp/mL.

“There is clear evidence that perfusion has the most significant impact on titers,” Loignon says. It may be more complex, but it more than doubles the cell titer. Neither batch nor fed-batch cultures achieved titers that were even remotely as high.

For batch culture, the scientists compared the growth profiles in four different commercial cultures. The highest titer achieved was 1.2 × 10⁶ cells/mL. Peak viable cell densities ranged from 2.2 × 10⁶ cells/mL to about 4.2 × 10⁶ cells/mL, depending on the media. Inoculation densities of 1, 2, or 3 × 10⁶ cells/mL E6 cells/mL were largely inconsequential in terms of adenovirus 5 production in the four media.

Further gains were achieved, though. For biomanufacturers choosing to use batch cultures, Shen and colleagues report that combining two particular media at a 1:1 ratio increased titer by more than 80%, to 2.6 × 10¹⁰ cells/mL. They also report that infecting the cultures at 7.5 × 10⁶ cells/mL and replacing the medium four times after virus infection achieved a virus titer of 7.3 × 10¹⁰ vp/mL. This is a 5.6-fold improvement from the culture infected at 1 × 10⁶ cells/mL with medium replacement, the team notes.

In fed batch cultures, neither increasing initial cell density nor altering the feeding strategy significantly increased virus productivity. Replacing media before virus infection helped increase process productivity somewhat.

Loignon and his team are working to further improve the productivity of the SF-BMAdR perfusion cell culture and thus make it more cost-effective.

In future research, “We will identify limiting nutrients and toxic metabolites,” he says, and analyze spent media “to identify and quantify the remaining concentrations of various metabolites and media components…apply[ing] a large-scale, targeted metabolomics method developed at the NRCC for the analysis and quantification of metabolites.

“Results will guide the customization of the cell media used for the production of adenoviral vectors using SF-BMAdR cells, as well as cell engineering using our CRISPR/Cas platform and other genomics and omics tools.”