April 15, 2011 (Vol. 31, No. 8)

Vicki Glaser Writer GEN

Facilities Scramble to Meet Expected Deluge as Drugs Rapidly Advance through Pipelines

Oligonucleotide-based drugs continue to flow into developmental pipelines, and the relatively large numbers now being tested in humans and progressing through late-stage clinical studies have buoyed optimism that commercialization of oligo therapeutics may finally come sooner rather than later. In parallel to these advances is a high level of confidence in the industry’s ability to produce GMP oligos at large scale reliably, reproducibly, and cost effectively, and to meet the analytical and process-validation strategies needed to satisfy evolving regulatory requirements.

Stating that most of the challenges associated with scale-up of GMP oligos, particularly DNA sequences, have been managed, G. Susan Srivatsa, Ph.D., president of the independent consulting firm Elixin concludes, “I don’t believe our ability to manufacture is a factor in the industry’s success.”

The sole exception may be the chemistry, design, and engineering advances that will likely play an increasingly important role in oligo drug delivery strategies in development. Reproducible production, scale-up, and quality analysis of emerging delivery mechanisms—such as self-assembling nanoparticles or liposome-encapsulated oligos—and of complex dosage forms will present manufacturing challenges.

Large-scale oligonucleotide synthesizers and associated technologies to meet commercial needs are readily accessible. However, due to the relatively high cost of raw materials and to manage risk, companies continue to be prudent and manufacture at large enough scale to meet their commercial demands, while protecting their investment in raw materials and labor-intensive downstream processing in the event of a failed synthesis.

Dr. Srivatsa expresses some surprise at the buzz among CMOs at the recent “AsiaTIDES” conference in which they talked about building out additional capacity in anticipation of increased demand. Not so long ago, there appeared to be overcapacity for oligo manufacturing, and Dr. Srivatsa attributes the change in momentum and renewed energy to the critical mass of oligo drugs now in later stages of development.

“I think the CMOs realize that there are now enough balls in the air that even with a moderate success rate” enough drug candidates will make it through to regulatory approval and manufacturing demand will rise, she says.

Agilent Technologies’ Nucleic Acid Solutions Division has crunched the numbers on the current oligo therapeutic market—which has resurged following the 2009 economic downturn—and reports 2010 deals totaling $5.2 billion, with $529 million in direct investments. This investment drove up the number of oligo therapeutic development programs to a record 235 in 2011 (including both research and clinical stage).

Contributing to this increase has been a more than doubling of miRNA programs in 2011, making it the fastest-growing segment of the therapeutic oligos market. Furthermore, despite some upheaval in the RNAi sector and dire predictions regarding its future, Gary Carter, director of strategy and marketing at Agilent, reports that siRNA remains “the largest segment of the market with 81 programs in development, followed by antisense. We also saw record levels of deal activity in the siRNA segment, with $2.2 billion in total deal value in 2010.”

Operators at Agilent Technologies’ commercial oligonucleotide API manufacturing plant in Boulder, CO: The GMP plant expansion, which came on-stream in 2010, is producing multikilo lots of late-stage clinical materials and is ramping up for commercial supply.

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.

According to Santaris Pharma, its LNA chemistry can help make the promise of RNA-targeted therapies a reality. The increase in affinity that LNA chemistry reportedly brings to oligonucleotides means that LNA-based drugs can be made much shorter than previous antisense drugs based on other chemistries, while displaying strong affinity for their RNA targets.

Tackling Delivery

From a manufacturing standpoint, Agilent continues to see many early-stage programs and new customers, according to James Powell, GM, with a growing emphasis on more exotic, complex oligo conjugates to enhance delivery. “I think the industry has embraced the idea that there will not be any one delivery technology—no one-size-fits-all solution. Instead, delivery strategies are likely to be tissue-specific, which has resulted in a broad range of compounds we’re being asked to manufacture,” as well as demanding novel analytical solutions for characterizing conjugated oligos.

Girindus America (member of the Solvay Group), a CMO that specializes not only in oligo manufacturing but also produces small molecules, is leveraging this expertise to manufacture components for the increasingly complex oligo delivery systems in development and the conjugated oligo-based compounds being evaluated and scaled up for preclinical and clinical evaluation, according to Kathryn Ackley, Ph.D., director of project management.

She asserts that Girindus has a broad customer base and sees continued interest in RNA production, particularly for siRNA applications, in single-stranded antisense oligos, LNA-based oligo therapeutics, and oligos used as adjuvants in vaccine production.

The company recently hired additional chemists to enhance its oligo conjugation expertise and has invested in advanced analytical technology, such as UPLC. Girindus draws on the expertise of its sister company within the Solvay Group, Peptisyntha, for access to peptide sequences that are being conjugated to therapeutic oligos to improve their delivery and targeting.

At the upcoming “TIDES” meeting, Dr. Ackley will participate in a panel discussion of a white paper in development intended to provide a consensus view on the issues related to impurities in oligonucleotide-based active pharmaceutical ingredients. There is no formal regulatory guidance on this topic. This work represents an industry-wide effort in which multidisciplinary, informal working groups have formed to discuss best practices in the area of oligonucleotide production, including chemistry, manufacturing, and control and oligo drug safety, for example.

For Hüseyin Aygün, Ph.D., CSO at BioSpring, a GMP-certified producer of therapeutic oligos, the take-home message from the recent “AsiaTIDES” meeting is the focus on novel delivery methods, with talks on topics such as nanotechnology and the move toward formulated antisense compounds instead of naked oligos to improve uptake and reduce therapeutic doses.

Girindus America has a number of automated flow-through oligonucleotide synthesizers with dynamic axial compression columns in its cGMP manufacturing facility.

Process and Product Validation

At BioSpring, the number of projects in the corporate pipeline continues to increase. In particular, the past year has brought a growing demand for aptamers both with and without pegylation, notes Dr. Aygün.

Expressing the need for “new analytical techniques to characterize final and intermediate aptamer products,” he describes the company’s addition of differential scanning calorimetry (DSC) to its analytical toolbox and the positive feedback from customers, who are requesting DSC measurements to help characterize oligo and apatamer formulations and assess their stability.

A key advantage of DSC compared to chromatography and other analytical techniques, according to Dr. Aygün, is its ability to characterize the structure and dynamics of oligos at high concentrations—concentrations that would be present during manufacturing. As an example, he describes the use of DSC to determine the propensity for single-stranded oligos present at high concentrations to aggregate in different formulation buffers.

In addition to rising demand from BioSpring’s existing customer base, the company is seeing an influx of new customers entering the oligo drug development arena and ordering GMP oligos to support toxicology studies and clinical-development projects. Dr. Aygün notes a growing number of inquiries in recent months from Japanese biotech and pharma companies.

Trends in downstream processing include moving quality control points earlier in the process, according to Dr. Srivatsa. She points to two key analytical techniques that “have made a meaningful difference” in overall quality assessment: UPLC, yielding a complete impurity profile in a shorter amount of time; and LC/MS, giving manufacturers a “tighter handle on their processes and helping with process optimization.” Mass spectrometry is “probably not too far off from being a routine manufacturing tool.”

At Agilent, the company’s commercial-scale manufacturing facility in Boulder, CO, is up and running and producing large-scale batches for late-stage clinical development programs, which, according to Powell, “are looking very promising. We’re looking to support multiple NDA’s over the next few years.”

Paul Metz, director of operations at Agilent, who oversees the commercial facility, describes some of the challenges and opportunities the industry is managing with the adoption of the most recent GMP regulations and subsequent guidances for regulatory compliance and filings—ICH Q8 covering pharmaceutical development data, ICH Q9 related to quality risk management, and ICH Q10 regarding quality systems.

These guidances further clarify what regulatory authorities expect in terms of the design of processes, with an emphasis on process understanding and building quality into the process rather than demonstrating quality by testing a finished product against specifications.

Agilent has developed an internal framework for a risk-based approach to oligo development and manufacturing that integrates QbD and process analytical technology (PAT) concepts into its scale-up and process validation programs.

“These support our clients’ commercialization strategies and worldwide regulatory filings,” says Metz. Based on this QbD approach, “we have developed rugged, small-scale models for every unit operation, giving us the ability to project at small scale what we will see at large scale,” and to use the models to optimize process parameters before scale-up, he adds.

The PAT approach incorporates increasingly rapid inline and online process analytical technologies to yield real-time information on the synthetic and downstream purification processes. Agilent utilizes the analytical power of mass spectrometry to identify and track raw material and process-related impurities throughout the process and gauge their impact on product quality attributes.

Access to these types of data throughout the production of oligos from early- to late-stage clinical development may provide clients more flexibility in their product registrations with options such as continuous verification (CV). Implementation of CV involves continuous monitoring, evaluation, and adjustment, as needed, of the process. It represents a move away from validation as a discrete exercise and toward a life cycle approach to process validation.

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