Oligonucleotide Formulation & Process Development

Process Development
Oligonucleotides have rapidly gained momentum as a new class of biotherapeutics, offering target specificity and biocompatibility across a diverse array of medical modalities. Oligonucleotide molecules possess special characteristics for API process development which are highly dependent on the unique chemistry and synthetic methods used to produce them. The manner of preparation for an individual oligo can affect the purity as well as the secondary structure of bulk material. These in turn can have important implications for solubility and stability. Establishing a robust process for oligo drug production is critical in enabling pre-clinical development and ultimately clinical trials. Wolfe Laboratories has developed a unique platform to rapidly propel oligonucleotides through the development process. We have developed specific strategies for initial characterization, duplex partner annealing, secondary structure assessment, sizing and oligomeric state of nucleic acid particles in solution, purity analysis and quantitative detection of primary components and degradation products in raw materials, process samples and final drug substances.

Oligonucleotide molecule types developed at Wolfe Laboratories:

• Native and modified DNA (single- and double-stranded)
• Small interfering RNA (siRNA)
• O-methyl/locked nucleic acids (LNA)
• Peptidic nucleic acids (PNA)
• Phosphorodiamidate morpholino oligos (PMOs)

Regulatory Requirements for Oligonucleotides: Currently, there are no FDA regulatory guidelines specific for oligonucleotide drugs. Despite their large size and biochemical properties, oligo-nucleotide drug substances are often treated as small molecules, due to the synthetic manufacturing processes used in their production. For most siRNA drugs, a single strand purity test is required, as well as a duplex purity method to assess completeness of annealing.

Long-term stability in aqueous buffer cannot always be maintained, due to hydrolysis and other mechanisms of degradation. For this reason, many oligonucleotide are lyophilized for increased shelf life and long-term potency. Lyophilization of oligos enables storage at ambient conditions and simplifies preparation of the administrable dosage form. Wolfe Laboratories optimizes the lyophilization process using the Design of Experiments (DoE) approach, based on the observations of preliminary lyophilization runs and the formulation optimization results. Lyophilization process development requires a thorough characterization of the oligonucleotide and the excipients, in order to remove the necessary solvent molecules without disrupting or degrading the target species. Typical factors that are investigated during the process optimization stage are solution temperature and treatment prior to lyophilization, time and temperature of lyophilization cycle steps, and the impact of moisture content on product stability.

Formulation Development
Development for oligonucleotides includes bulk drug product formulation, development of the final dosage form and fill-finish. The outputs generated during preformulation characterization and process development inform the boundary conditions within which the optimal drug product composition will be determined. Oligonucleotides are formulated in a range of vehicles, including liquid solution or suspension, lipid nanoparticles, and virus-like particles. Routes of administration include IV or subcutaneous delivery, for which dosage volume, syringability, and other factors must be addressed. At Wolfe Laboratories, a rational approach to formulation development is utilized, based upon the characteristics of the molecule and the target product profile. .

Vehicle components typically evaluated

Vehicle Component Purpose

Buffer pH control for long-term stability (minimize hydrolysis)

Ionic strength modifier Isotonicity and stability of the oligonucleotide

Surfactant Prevent aggregation and increase solubility of vehicle components

Oligonucleotides - RNA in particular - are extremely sensitive to hydrolysis and degradation at room temperature, since a single phosphodiester bond cleavage can render an oligonucleotide drug useless. To foster rapid formulation development, Wolfe Laboratories evaluates the performance of the formulation in response to processing, storage and administration conditions, as well as accelerated stability testing to identify and rank-order formulations, and thus define the optimal composition of the final drug product.