The approval of Macugen for the treatment of age-related macular degeneration by the FDA in December 2004 marked the introduction of the first commercially successful oligonucleotide therapeutic to the marketplace.
Macugen contains a substituted RNA that specifically binds a protein that plays a critical role in the disease process. Researchers at Eyetech Pharmaceuticals (www.eyetech.com), the company that developed Macugen, worked closely with Prime Synthesis (www.primeesynthesis.com) to work out specific test methods and specifications for the solid support.
Founded in 1989 by Marc Rothstein and the late David Rothstein, Prime Synthesis specializes in developing economical consumable products for oligonucleotide synthesis.
David Rothstein, an electrical engineer who worked with early DNA synthesizers, recognized that the cost of making oligonucleotides was limiting the growth of this new market. He teamed up with his brother Marc, a chemist, to provide lower cost alternatives to the disposable columns then in use.
Now Prime Synthesis offers a growing line of products to large-scale manufacturers of therapeutic and diagnostic oligonucleotides, researchers, and core laboratories involved in oligonucleotide synthesis, including an assortment of cost-effective, bulk solid-phase supports and columns that are compatible with the most popular brands of synthesizers.
Controlled Porosity Glass
A key tool for oligonucleotide synthesis is a chemically inert glass powder called controlled porosity glass (CPG), to which the nucleoside attaches and grows. Wolfgang Haller, Ph.D., invented CPG in the 1970s as a packing material for size-exclusion chromatography columns, but it saw limited commercial success. Instead, it was adopted, fortuitously, as a solid support for oligonucleotide synthesis in the 1990s.
"We recognized that CPG was not perfect and finetuned the product to give higher yields and purity," says Marc Rothstein, president of Prime Synthesis.
The process took two years, and the resulting product is a special glass with a precisely controlled pore structure that is ideal for synthesizing oligonucleotides. The interconnected pores provide fast washout times and easy access for reagents to reach the interior of each particle. Prime Synthesis contracts with major glass manufacturers to supply custom formulations of melted glass.
"From that point on, we do all the processing to make CPG powder," says Rothstein. Customers can buy the company’s CPG, sold as Prime Performance Controlled Porous Glass, as a bulk powder for large-scale therapeutic synthesis or in smaller amounts as prefilled plastic columns for research projects.
Next, researchers at Prime Synthesis improved the chemistry that attaches nucleotides to CPG particles. They created novel "linkers" that anchor the chemical groups and allow DNA or RNA to grow one base at a time. In addition to tethering nucleotides to the glass, linkers are designed to prevent the glass from crowding the nucleotide as it grows. The reagents for synthesis must freely access the growing chain from all sides to produce a long chain molecule. Linkers that are too long can fold back on themselves and press against the glass.
"The molecular structure is the key to our linker," says Rothstein. The rigid design is just long enough to provide a space between the glass and nucleotide, yet it prevents folding and bending.
Fortuitous First Customer
The linker was developed in collaboration with Ribozyme Pharmaceuticals (now Sirna Therapeutics; www.sirna.com). The technology is exclusively licensed to Prime Synthesis. Starting in 1999, Sirna Therapeutics worked closely with Prime Synthesis to further develop CPG for the synthesis of larger quantities of RNA oligonucleotides for clinical trials.
The collaboration with Ribozyme also helped Prime Synthesis to establish a GMP-manufacturing facility to meet the needs of the pharma industry. The company recently completed construction of a 20,000-sq-ft GMP-compliant production area with a suite of organic synthesis work areas and state-of-the-art quality control laboratory.
Because of its early collaborations, Prime Synthesis focused on the large-scale therapeutic oligonucleotide market. Nearly every CRO that makes oligonucleotides uses Prime Synthesis CPG for some clients, according to Rothstein. The company is known for customized CPGs that produce the best results for a particular product, synthesis equipment design, and pricing objective, he says. For instance, a collaboration with Avecia Biotechnology (www.avecia.com) led to a chemical refinement that reduced the amount of an impurity and greatly lowered purification costs of the final product.
Prime Synthesis’ linker was developed to synthesize RNA-based oligonucleotides. As a result of recent advances in RNA interference, RNA-based drug development is becoming more popular.
RNA-based oligonucleotides, however, are more difficult to synthesize, but "this linker is exceptionally well suited for RNA synthesis," says Rothstein.
"The RNA area is starting to boom," says Fran Wincott, Ph.D., a synthetic organic chemist and consultant who advises companies developing RNA oligonucleotides. She recommends that her clients work with Prime Synthesis because of its track record. "Quality assurance people like Prime Synthesis," says Dr. Wincott.
Despite the advantages of glass for oligonucleotide synthesis, some researchers prefer polystyrene as a solid support, even though polystyrene may swell when exposed to solvents. Increasing the amount of cross-linking reduces swelling, but at the expense of nucleotide loading. Prime Synthesis responded to this market preference by introducing a new line of Prime Performance Polystyrene supports in 2006. "We developed one that has the best chance of success in the therapeutic oligonucleotide area," says Rothstein.
For more than a decade, many attempts have been made to create a universal support that does not contain one of four specific bases to initiate synthesis. Although there have been some successes, universal linkers give lower yields of fully cleaved and dephosphorylated oligonucleotides than do dedicated nucleoside supports.
Recently, a group at the University of Kuopio in Finland developed a universal linker that gives nearly 100% yield. "It uses a neutral functional chemistry that eliminates the need for base-specific supports," says Rothstein. Prime Performance offers the universal linker on both its CPG and polystyrene product lines, under license from Metkinen (www.metkinen.com) and Glen Research (www.glenres.com). A universal linker simplifies inventory and forecasting needs. "You only need one product instead of many," says Rothstein.
Prime Synthesis also launched a new division called 3-Prime (www.3prime.com) to make it easier for research customers to buy small quantities of its products online.