Ion-exchange membrane technology targets Antisense production challenge - In the field: pharmaceutical science & technology news

An emerging family of biotech drugs promises to revolutionize the treatment of life-threatening diseases. "Antisense" drugs work at the molecular level by binding to messenger RNA--interrupting the process by which disease-related proteins are produced. Antisense drugs have been shown to inhibit the production of faulty proteins responsible for cancer, AIDS-related afflictions and cardiovascular diseases. And because they are highly-targeted therapies, antisense drugs should minimize patient side effects.

However, as companies now work to move many of these drugs forward from development and clinical trials into production, they face a number of challenges, one of which is the need to quickly and effectively purify large quantities of the antisense oligonucleotide active ingredient from very chemically similar production byproducts. Already, there are more than 25 antisense drugs in clinical trials, and significant bottlenecks may occur in full-scale manufacturing if traditional purification methods are relied upon.

To help address this challenge, Pall Corporation (East Hills, NY) is proposing a novel new purification technology. According to Ajay Lajmi, PhD, senior research scientist with Pall Corp., the company's "Mustang Q" ion-exchange membrane chromatography technology can provide a tenfold increase in antisense drug production speeds compared to conventional column chromatography.

The first step in the preparation of an antisense drug is the construction of the oligonucleotide active ingredient using automated DNA synthesizers. As explained by Lajmi, this process poses a special challenge since, as the antisense molecule is synthesized, a number of impurities are also created that are difficult to separate from it due to their molecular similarity.

Antisense oligonucleotides are synthesized by linking nucleotides, one at a time, to build a complementary sequence to the targeted messenger RNA (mRNA) strand. However, since all of the individual synthetic reactions don't go to 100% completion, a fraction of the building block molecules don't become attached, resulting in incomplete oligonucleotide molecules. These are called "failure sequences." So, for example, a process building a molecule composed of twenty nucleotides (20-mer), would result in 19 different short sequences remaining as impurities. As a result, a significant amount of the material produced by the solid-phase synthesizer consists of oligonucleotide impurities which must be separated from the desired molecule.

To accomplish this, the mixture must be passed through a purification system. First, ammonia is applied to "cleave" the molecular mix from the synthesizer. The ammonia is then removed by evaporation at reduced pressure, and the resultant mixture precipitated out and lyophilized.

Currently, the next step is to redissolve the compound in a mixture of water and acetonitrile or methanol solvent and load it into a reversed-phase chromatography column. The concentration of organic solvent is then gradually increased to eventually elute the desired molecule from the column. There are a number of limitations associated with this approach, however, including the cost and handling issues associated with the use of an organic solvent. The overriding concern for those planning for scale-up to large-scale production, however, is the slow throughput rate of the process.

As an alternative to this reverse-phased chromatography step, Pall is proposing the use of its Mustang Q ion-exchange membrane chromatography. The relatively large pore size (0.8 [micro]m) and chemical composition of the Mustang membrane significantly improves throughput rates.

The Mustang Q membrane chromatography process makes use of the fact that each of the failure sequence impurities produced during synthesis differ from each other by a single negative charge, with the largest negative charge associated with the complete antisense molecule. As a result, when a high salt (typically 1 molar NaCl) buffer is applied to the Mustang module, the antisense product molecules (which have the strongest attraction to the positive charge of the membrane) will "self-displace" the weaker-charged, shorter-sequence impurities. The NaCl gradient is then adjusted to elute the full-length oligonucleotide. According to Lajmi, "Mustang chromatography has been shown to achieve purity levels of up to 95% while eliminating the use of an organic solvent."

In addition to throughput and purity, scaleability is clearly a critical consideration for antisense product development. According to Lajmi, several companies are evaluating the Mustang Q technology for use in their antisense production processes, including a product now in Phase III clinical trials. Currently the company has 10 mL ([approximately equal to] 0.3 g purification capacity), 100 mL ([approximately equal to] 3.0 g) and 1 L ([approximately equal to] 30 g) bed volume purification modules and is in development with a purification system that can handle from 0.5 to 1kg/injection volumes.

One antisense drug already on the market is Isis Pharmaceuticals' (Carlsbad, CA), "Vitravene," targeted for the treatment of cytomegalovirus retinitis (CMV) in AIDS patients. Vitravene is marketed by Novartis Pharmaceuticals (Basel, Switzerland).

Isis currently purifies Vitravene using a combination of both reversed-phase chromatography and ion-exchange methods (chromatography followed by ion exchange). As explained by, Doug Cole, PhD, Isis vice-president, Technical Development, the relative efficiencies of the techniques differ according to the class of impurity. "Where there are distinct charge differences between molecules, ion-exchange will work well, while in other cases, you'll get better selectivity through reversed-phase chromatography. The chromatographic 'Holy Grail' will be one that combines the best features of both."

Isis has nine additional antisense products in the pipeline ranging from Phase I to Phase III clinical trials. Among these is a second-generation antisense drug recently announced by Isis and Eli Lilly (Indianapolis, IN). In preclinical tests, the antisense drug "LY2181308" has been shown to successfully inhibit tumor growth in animal models. LY2181308 targets Survivin, a molecule that promotes cell survival. Survivin is expressed in the vast majority of cancers, where it interrupts the natural cell death cycle, but not in normal tissue. Antitumor activity has been associated with significant reduction of Survivin expression in tumors, evidence that the drug was working through an antisense mechanism.

Isis has been working with Pall since the beginning of the Mustang Q project and has used the modules to purify a number of their developmental products. They haven't yet implemented the technology in a production setting, however.

Cole notes that ISIS may be in a unique position relative to comparing the relative benefits of reversed-phase and ion-exchange chromatography for antisense production. "The know-how relative to working up a reversed-phase system efficiently is not widespread, and we've developed our own proprietary techniques that are very fast." In fact, Cole says that their reversed-phase process is actually faster than ion exchange. He adds, however, that "you need the whole suite of technologies to make it work, and if you haven't got them, then you'll go with ion exchange."

Cole also points out another trade off between the two techniques. Though he acknowledges the drawbacks associated with the use of organic solvents, he adds that "on the other hand, given the fact that many of these drugs are parenterals, and we have excellent controls over microbiological contaminants, we like the fact that (with reversed-phase chromatography) our product (nucleic acids) is always in a medium that naturally inhibits bacterial growth. When you're doing ion exchange, you're sort of putting your product in a pure growth medium."

Regardless of the approach that companies choose for their full-scale antisense drug production, Cole observes that it's not too soon to be concerned about manufacturing capacity. "Given the number of people now in advanced development with nucleotide products, you have to look at the total available capacity and who has access to it." Three antisense drugs are now in Phase III clinical trials, one of which, the anticancer therapy "Genasense" from Genta Incorporated (Berkeley Heights, NJ), is now in NDA review.