Bicistornic Vectors + piggyBac = Improved Antibody Cell Line Production

To produce stable cell lines for monoclonal antibody production, manufacturers typically use the piggyBac transposon system. For larger, multi-specific antibodies that have payloads of three or four cistrons (sections of genes that express one complete, functional polypeptide), piggyBac is only the starting point.

For those larger, more complex antibodies, the challenges of multi-cistron vector architecture can lead to such upstream bottlenecks as unbalanced ratios of both heavy and light chains across cistrons, chain mispairing (which contributes to product heterogeneity), and genetic stability over time. Combined, they slow cell line development and hamper titer productivity.

Scientists at Sanofi’s Framingham, MA, site have developed a vector engineering strategy for multicistronic antibodies for coordinated transgene expression. It appears to improve promoter configuration and cassette topology, thus resolving those issues and improving titer productivity up to six-fold. It is, they suggest, “the first reported use of light-chain-selection marker-heavy chain topology for monoclonal antibody expression.”

Modified bicistronic vectors

Rather than use the traditional piggyBac monocistronic method and conventional vector designs, Jason Vitko, senior scientist, and colleagues modified bicistronic vectors and combined them with the piggyBac transposon system. Their work focused on altering promoter sequences, reporter placements, and cassette configurations.

As they report, “Double human cytomegalovirus promoter configurations driving both heavy and light chain genes significantly enhanced pool productivity (2 to 2.5-fold) and reporter expression compared to separate promoter designs.” More specifically, the light chain-glutamine synthetase-heavy chain configuration they used improved productivity from 1.5- to 6-fold, heavy chain RNA transcript ratios by 1.4- to 3.8-fold, and light chain RNA transcript ratios by 1.3- to 6.1-fold. The glutamine synthetase selection marker that was placed between the light and heavy chains improved the expression balance and productivity.

Improvements were most notable under fed-batch conditions, but negligible under unfed-batch conditions. The clones produced by the optimized piggyBac/bicistronic system “exhibited significantly high productivity, with the best clone producing 9.6 g/L.

“This approach simplified transfection workflows while providing enhanced control of gene expression and stability,” the scientists point out.

The monoclonal and multi-specific antibodies produced under this optimized piggyBac/bicistronic system are expected to be very stable, highly productive, correctly assembled, more homogeneous, and faster to develop than antibodies produced using standard piggyBac technologies. This work, Vitko and colleagues write, is foundational, establishing “a robust and scalable platform for the development of high-producing cell lines for next-generation therapeutic proteins.”

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