Race to the Finish Line
Trabedersen, the most advanced antisense molecule in Antisense Pharma’s oncology pipeline, is a backbone-modified phosphorothioate-based oligodeoxynucleotide. “Synthesis of the phosphorothioates is one of the most evolved chemical processes, and we have reached near perfection in terms of efficiency and automation—an essential prerequisite for the entry into a pivotal Phase III trial,” says Klaus Lindner, Ph.D., head of manufacturing. He predicts that, in the future, continuing refinements derived from improved solid supports and materials will further increase yields and purity.
Trabedersen downregulates synthesis of the tumor-enhancing molecule transforming growth factor beta 2 (TGF-β2) and is being tested in a Phase III trial in Europe, the U.S., Canada, South America, and Asia to treat adults with high-grade glioma, an aggressive form of brain cancer. It is also in a Phase I/II trial in patients with metastatic pancreatic carcinoma, malignant melanoma, or advanced colorectal cancer.
Antisense Pharma is working with service provider Avecia OligoMedicines to optimize and characterize its manufacturing process by applying QbD principles. “A risk-assessment approach was used to identify the critical process parameters.”
“Subsequently, the design of an experiment tool was used to reach a high level of process understanding and to establish a design space for all the critical process parameters, which, in turn, delivered a robust manufacturing process.” In contrast to the advances in oligo synthesis, “analytical and purification techniques remain a big challenge. Improved HPLC methods will become necessary for adequate identification and quantification of all significant impurities.”
Santaris Pharma develops RNA-targeted therapies called LNA-antimiRs that target either messenger or microRNAs (mRNA or miRNA) and incorporate locked nucleic acid (LNA) chemistry to enhance the affinity of its antisense oligonucleotides. This increased affinity allows the company to design shorter molecules (12–16 nucleotides) than the 20-mers more typically used for antisense applications.
Santaris has developed two main drug-design formats: single-stranded DNA sequences that contain LNAs at the termini and that function via a traditional antisense mechanism, recruiting RNase H to destroy a target mRNA sequence; and single-stranded oligos containing LNAs dispersed throughout, called mixed-mers, which are capable of knocking out miRNAs. The company’s lead therapeutic compound, miravirsen, which has advanced into Phase II trials for the treatment of hepatitis C virus infection, is a mixed-mer that targets miR-122.
A third design format in development comprises tiny LNA-antimiRs, which are fully modified 8-mers containing only LNAs. These oligo drugs are short enough to target consensus sequences in families of miRNAs, offering the potential to knock out entire regulatory networks.
Santaris has entered into a strategic alliance with miRagen Therapeutics to develop miRNA-targeted drugs to treat cardiovascular disease; announced an expanded collaboration with Pfizer in which Pfizer will pay $14 million for access to Santaris’ LNA drug platform; and received an exclusive license from Mass General Hospital for IP related to the regulation of miR-33 to improve HDL levels in patients with cardiovascular disease.
As the length of an oligo drug decreases, manufacturing is simplified, explains Henrik Ørum, Ph.D., CSO at Santaris, as there is less opportunity for attenuated molecules to form during the iterative process in which nucleotides are added onto the growing DNA strand. This makes it easier to purify and characterize the full-length oligo product.
Dr. Ørum describes no substantial differences in the processes for manufacturing oligos with or without LNAs, or with LNAs at only the termini, dispersed throughout the strand, or comprising the entire molecule. The main difference is in the production of chemically active monomers containing LNA amidites.
Once these are in hand, oligomers are synthesized on automated instruments using standard solid-phase synthesis protocols for which the synthesis cycles may have to be modified slightly to optimize them for the incorporation of LNAs.
“We have gone from micromolar, laboratory-scale oligo synthesis to producing hundreds of grams of oligos seamlessly by moving from one automated synthesis platform to the next, using well-defined manufacturing processes across all scales.”
“The LNA chemistry has enabled the design of shorter-than-usual oligonucleotides that are pharmacologically active in many different tissues upon systemic administration of naked molecules. Several of these molecules are undergoing clinical trials that will eventually answer whether LNA is the chemistry that takes antisense to the finish line.”
If that is the case, he believes the focus in the antisense field will shift from developing therapeutically suitable chemistries to manipulating the biodistribution of the LNA oligonucleotides as desired to reach and affect target tissues. Additions or modifications to antisense oligos, or encapsulation or linkage strategies to facilitate targeting and delivery, could present new challenges for manufacturing and regulatory assessment.