You Say You Want a Revolution?
“Cells resistant to high osmolarity exhibit increased robustness and stability,” said Florence Wu, Ph.D., director, cell sciences, at the PD Direct Bioprocess Services division of Invitrogen (www.invitrogen.com).
To pursue this property, Dr. Wu investigated a gene-mutation protocol for increasing resistance to high levels of salt added to the culture medium. While high osmolality of the culture medium boosts specific productivity of recombinant proteins, the growth parameters of the cells deteriorate dramatically. So as salt concentrations rise to toxic levels, there will be a point of negative impact on cell performance.
Dr. Wu and her associates reasoned that if it were possible to engineer resistance to high osmolality in cells, then dramatic increases in protein production could be anticipated. They observed that when they cultured cells continuously in increasing levels of salt, up to 550 mOsm/kg, after 30 days the cells improved in viability and began to proliferate.
Initially it was not clear whether this was the result of gradual physiological adaptation or selection of a preexisting mutant population. Analysis of the growth kinetics of the cultures suggested, but did not prove, that a selection of genetic variants had occurred during this period of continuing growth in the high-salt medium.
Dr. Wu next applied the Invitrogen Revolution™ technology, which operates by interfering with mismatch repair of DNA, increasing mutation rates by as much as a 1,000-fold. Ordinarily, damage to the genetic apparatus is constantly being monitored and repaired, but the Revolution technology interrupts this process, and a torrent of genetic damage accumulates. The classic means of producing genetic variants in cultured cells has been through the use of compounds such as ethyl methane sulfonate, which cause errors during DNA replication.
The company argues that the Revolution system is much more effective than chemical mutagenesis, producing a more diverse and hardier spectrum of mutational variants. It is also more rapid, as Revolution-treated cultures produce high osmolality-resistant variants immediately, rather than after a longer lag period as seen in the untreated populations, the firm reports.
Dr. Wu then turned her attention to the CHO DG44 cell line, an important antibody producer. Not only was she able to select a CHO variant with 500 mOsm resistance, but it proved to be stable over at least 75 generations and retained its capacity to produce immunoglobulin.