Assays for Quality Control in Small Nucleic Acid Drugs

Importance of Quality Control in Small Nucleic Acid Drugs Manufacturing

Quality standard control is an important aspect in the manufacturing of oligonucleotides. Like the acceptance criteria for any active pharmaceutical ingredient, the quality standards for oligonucleotide raw materials are based on preclinical and clinical data, variability in production processes, and analytical control strategies. Oligonucleotide raw materials can generally be categorized into two main classes. The first class comprises single-stranded molecules, including phosphorothioate antisense oligonucleotides and aptamers. The second class consists of double-stranded oligonucleotides used in RNA interference (RNAi) methods and other approaches. Oligonucleotide therapies can be further classified into non-conjugated molecules and those conjugated with non-oligonucleotide modifying structures such as polyethylene glycol, cholesterol, or lipids.

Diagram of a drug development laboratory that maintains strict quality control.Diagram of a drug development laboratory that maintains strict quality control.

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Assays for Quality Control in Single-Stranded Oligonucleotides

Currently, single-stranded oligonucleotides are being researched for various applications such as antisense therapeutics, immune stimulants, aptamers, and splice-modulating drugs, necessitating evaluations across various chemical structures and oligonucleotide lengths. Generally, the tests used to determine the identity, purity, and potency of oligonucleotide active pharmaceutical ingredients (APIs) are quite similar to those used for small molecule APIs. These include tests for residual solvents, heavy metals, bacterial endotoxins, and microbial limits. Similarly, standard analytical techniques used for small molecule raw materials, such as gas chromatography for residual solvent determination, are also fully applicable to the analysis of oligonucleotide raw materials.

Identification Test

Identification tests are a series of analytical methods and techniques aimed at confirming the identity and properties of drugs such as oligonucleotides. The purpose of these tests is to determine the molecular structure, sequence, and composition of the drug, as well as to detect possible chemical or biological variations, ensuring that the produced drugs comply with specified quality standards and regulatory requirements. The results of identification tests are crucial for ensuring the quality and safety of drugs. They typically involve multiple analytical techniques, including mass spectrometry analysis, high-performance liquid chromatography, nuclear magnetic resonance, etc., to determine the sequence, structure, and composition of the drug, and to assess its identity. Identification tests also include tests conducted after the release of raw materials to ensure that the identity of the raw materials matches that of the raw materials actually produced or processed.

Pair Ion Detection

Pair ion detection is a type of identification testing method targeted at small molecule drug substances that exist in salt form. In this approach, the identity of the drug is confirmed by detecting the paired ions within it. Typically, quantitative detection of ions is not necessary; instead, sensitivity to changes in ion content is achieved through assay-based detection methods. However, for polyatomic anions such as oligonucleotides, pair ion detection becomes more complex, as confirming the composition of paired ions is challenging with qualitative identification tests and assay-based detection. Therefore, it is recommended to include quantitative testing for paired ions in the specifications of drug substances. Common methods for quantifying paired ions in oligonucleotide drug substances include ICP-MS, ICP-OES, AA, or IC.

Content Test

Content testing is one of the key aspects of release testing, aimed at determining the content of various components in the raw material and ensuring their accurate measurement. For most oligonucleotide drug substances, content testing needs to consider various factors such as moisture content, residual solvents, and inorganic salts. This test typically employs multiple analytical methods such as UV spectroscopy, capillary gel electrophoresis, HPLC, etc., to ensure accurate measurement of the content of raw materials. Non-specific methods can also be used for content testing, but correction of the results using specific methods for the raw material is required. The purpose of content testing is to obtain accurate content of the raw material to ensure the quality and safety of the drug.

Moisture Testing

The moisture content of oligonucleotides fluctuates depending on their environment, reflecting their current surroundings rather than being a fixed attribute. While moisture content alone does not indicate quality or predict stability of pharmaceutical substances, its determination remains valuable. It can indicate the success of drying operations, container closure system integrity, and serves as a calibration parameter for content testing. Moisture and content testing can be conducted on the same sample after equilibration, with moisture content subtracted from the content test. Moisture detection aids in using DS in DP production. When setting acceptance criteria for moisture content, it's important to consider the hygroscopicity of most oligonucleotide APIs, especially for APIs with unpredictable stability.

Purity and Impurity Analysis

The quantity and complexity of impurities in oligonucleotides pose a challenging task for their control. Many impurities, such as n-1 impurities formed during solid-phase synthesis, are structurally related to the parent compound. While HPLC can typically separate n-1 impurities from the parent components, this becomes more difficult with longer nucleic acids. It's often impractical to separate individual components of n-1 impurities for most oligonucleotides. Similar challenges apply to other common impurities like n+1, depurination, and CNET impurities, which are mixtures of individual components. It's generally recommended to control individual components within impurity categories rather than separately. Grouping impurities based on retention time or mass may be preferred in cases where separation is not feasible.

3D Configurations

The activity of aptamers relies on their specific three-dimensional structure, making the assessment of this structure crucial. While conformational testing is essential for aptamers, it may not be critical for oligonucleotide therapies like antisense and siRNA. Aptamer conformation is often inferred from biological assay results, ensuring correctness if they meet criteria. Due to assay complexity, alternative methods like selective excitation HNMR and biophysical techniques such as Tm and CD spectroscopy are explored. The main challenge in developing direct conformation measurement methods lies in establishing the relationship between physiological data and physicochemical test results.

Assays for Quality Control in Double-Stranded Oligonucleotides

Double-stranded oligonucleotides are being used in siRNA and other methods for controlling protein expression. These therapies consist of two complementary single strands, each synthesized independently and then annealed to form the double strand. Assuming the annealing mixture is at chemical equilibrium, the distribution of double-stranded and single-stranded species in the mixture is entirely determined by various binding constants between the chemical species.

Identification Testing

It is essential to confirm the identity of each strand in the double strand. Typically, identification tests for single strands are completed before annealing at the single-strand intermediate stage. Once they are confirmed before annealing, there is no need to confirm the sequence of the single strands again after the formation of the double strand. In these cases, the focus of identification testing for the double strand is to determine whether the correct single strands have been mixed and whether the desired double strand has formed during the annealing step. To assess whether the correct single strands have been mixed, the quality of the intact double strand or the quality of the dissociated single strands can be measured. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS) techniques have been used to determine the quality of single and double-stranded oligonucleotides. Assuming the sequence of the single strands has been established, high-resolution mass spectrometry may not be necessary. The important thing is to demonstrate that the correct double strand has formed during annealing. Typically, determining the identity of the single strands before annealing, combined with measurements of the quality of single or double strands, along with Tm analysis of the double-stranded API, is usually sufficient for identification testing.

Purity and Impurity Characteristics

Determining the purity and impurity characteristics of double-stranded oligonucleotide therapeutic drugs is quite complex. Because the double strand is formed by annealing two single strands, there may be double-stranded variants closely related to impurities annealed from single-strand impurities. Therefore, assigning a purity value to the target double strand alone may be impractical. Impurities in double-stranded drug substances may include residual single strands, double-stranded variants, and aggregated chains. These types of impurities can only be measured by denaturing techniques, which typically do not allow detection and quantification of individual impurities due to inadequate chromatographic resolution. The complete definition of impurity characteristics may be captured by analyzing drug substances in both double-stranded and single-stranded states. The main analytical challenge is to develop methods capable of separating and quantifying residual single strands and aggregated components from the double strand without disrupting it. SEC is generally accepted for minimizing structural conformational effects on drugs but is typically limited in resolution compared to other techniques. Additionally, for some denaturing methods, double-stranded variants may be sufficiently separated from the API to evaluate their distribution. Besides SEC, other commonly used denaturing techniques include AEX, IPRP, and capillary electrophoresis.

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