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.
The PER.C6 Cell Line
“Percivia’s PER.C6® Development Center investigates the use of PER.C6 cells as a technology platform for recombinant therapeutic proteins,” said Marco Cacciuttolo, Ph.D., CEO of Percivia. The firm was formed in 2006 to provide technology transfer and technical support to the users of the PER.C6 platform.
Dr. Cacciuttolo stressed that although PER.C6 has only existed since 2000, it has already shown impressive performance, especially in terms of expression of IgGs and IgMs. “Our titers in fed batch have gone up from 5.8 to 7.8 g/L,” he continued. “This achievement for PER.C6 demonstrates the power and robustness of its manufacturing platform. We are confident that we will be able to drive productivity even further.”
In fact, PER.C6 researchers designed an ultrahigh-performance production process, named XD™, characterized by a short burst of exponential growth to densities of 150 million cells/mL, resulting in product titers in the range of 15 g/L for an IgG at harvest. “This is unprecedented and it represents a substantial increase over typical yields,” added Dr. Cacciuttolo.
Counting on Disposables
“The next breakthrough in disposable processing will come from old and boring enabling technologies,” explained Uwe Gottschalk, Ph.D., vp for purification technologies at Sartorius Stedim Biotech (www.sartorius-stedim.com). Challenges in today’s bioprocessing industry are formidable—complex and technically demanding, with ever-increasing regulatory scrutiny. Disposable components are looked upon as a means of resolving many of these issues. In his discussion, Dr. Gottschalk looked at the various steps in the downstream process including major separation, aggregate removal, polishing, and viral removal.
Sartorius Stedim Biotech and other companies involved in filtration technology are focusing on disposable chromatography, that is, products that enable efficient and cost-effective use of resources over a short time frame. Such single-use modules require lower capital investment per device and allow reduced exposure of the separation media to the environment. Examples of disposable chromatographic products include membrane absorbers, monoliths, and prepacked columns.
Although Protein A is the affinity separation medium of choice, selectivity comes at a high price. Protein A ligands for attachment to columns represent a cost contribution of about $16/g of antibody purified, while the much less specific approach of ion exchange will only add $2 in cost per gram of antibody purified. Two-column processing is becoming widespread in the industry, including membrane chromatography.