Small Nucleic Acid Process and Cobetter Ultrafiltration Solution

Small Nucleic Acid Introduction

Among the many frontier areas of biopharmaceutical innovation, the field of small nucleic acids has demonstrated its huge potential and broad application prospects. Small nucleic acid drugs include antisense oligonucleotides (ASO), small interfering RNA (siRNA), micro RNA (miRNA) and nucleic acid aptamers, etc, which usually consist of 12 to 30 nucleotides to form a single-stranded or double-stranded structure. Each type of small nucleic acid drug has its own unique mechanism and advantages, and can achieve therapeutic effects on diseases by precisely regulating gene expression or repressing specific gene sequences (1).

Recently, the 2024 Nobel Prize in physiology and medicine was awarded to scientists Victor Ambrose and Gary Ruffkund for their discovery of microRNA (miRNA) and its role in post-transcriptional gene regulation. After many types of nucleic acid drugs such as ASO, siRNA, and mRNA have made breakthroughs, RNA therapy has once again attracted widespread attention.

This article mainly introduces the manufacturing process of small nucleic acid drugs and the application of membrane filtration technology to help everyone efficiently solve practical application problems encountered in the small nucleic acid process route.

Small Nucleic Acid Manufacturing Process - Synthesis

There are two main methods for synthesizing small nucleic acids: solid-phase synthesis and biosynthesis. The current mainstream chemical synthesis method is the solid-phase phosphoramidite method, which uses a synthesizer to precisely control the order of nucleotide addition on a solid-phase carrier, and synthesizes small nucleic acids of specific sequences through multiple cycles. Each cycle mainly includes four steps: deprotection, coupling, oxidation, and capping (Figure 1a). The key steps of the overall production process include synthesis, cleavage and deprotection, chromatographic purification, ultrafiltration, annealing (such as double-stranded oligonucleotides), and freeze-drying [3].

This solid-phase synthesis method is robust and versatile, and can efficiently and massively synthesize short to medium-length (5-80 nt) and heavily modified oligonucleotide sequences, but it is limited to the short length of modified oligonucleotide sequences. The nucleic acids prepared by polymerase-based biocatalysis (Figure 1b) can have no restrictions on their sequence length, but the control over the position, quantity, and complexity of the modified nucleotides is very limited.

Figure 1 a.Solid phase synthesis；b.Enzymatic synthesis

Small nucleic acid manufacturing Process - Impurity Removal

The main considerations for CMC of small nucleic acids include monomer supply, quality control, impurity characterization, separation and purification, etc. Among them, the analysis, characterization and control strategies of related impurities are particularly critical and more challenging.

For example, regarding the setting of impurity limits in phosphoramide, the accumulation caused by the number of times which is introduced in the synthesis needs to be considered based on the iterative attributes of solid-phase synthesis. The relevant ICH guidelines apply to oligonucleotides, including process-related impurities (ICH M7 genotoxic impurities), inorganic impurities (ICHQ3D) and residual solvents (ICHQ3C). For details, please refer to the relevant guidelines [4].

In addition, the Subcommittee of the Oligonucleotide Safety Working Group (OSWG) also discussed the process-related and product-related impurities of oligonucleotide drugs (s-6]). Process-related organic impurities include starting materials, ligands, activators, capping agents, sulfurizers and protecting groups, which can usually be removed during the synthesis, purification and ultrafiltration processes.

The ultrafiltration step can also effectively remove inorganic impurities in the API to a level which is below the detection limit. Product-related impurities are usually N-1, N-2, N-x (missing or short polymers), N+ impurities, long polymers, incompletely deprotected oligonucleotides, phosphodiester impurities (P=0), etc.

These impurities can be effectively removed or reduced to meet the limit during the chromatographic purification process. The accumulation of historical research data helps to prove the impurity removal ability during the development process, thereby achieving minimal detection.

Small nucleic acid manufacturing Process - Chemical Modification and Delivery

However, the drug development of small nucleic acids still faces challenges such as delivery difficulties, immunogenicity, and off-target effects. Over the years, through the exploration of various chemical structure modifications and delivery systems, the sites that can be chemically modified include the termini, backbone, ribose moiety, and bases (as shown in Figure 2).

For example, phosphorothioate (PS) modification of siRNA and 2'-F or 2'-OMe modification of ribose have been widely used in marketed siRNAs. These modifications can improve serum stability, prolong half-life, enhance RNAi ability, and reduce off-target effects [1].

However, on the basis of chemical modification, an excellent delivery system is also crucial for the intracellular delivery of small nucleic acids, among which LNP and GalNAc (N-acetylgalactosamine) are relatively mature delivery systems currently studied and applied (as shown in Figure 3). The GalNAc delivery system is particularly outstanding in liver-targeted delivery, and compared with LNP, it exhibits lower immunogenicity and better safety.

The LNP delivery system has a relatively high universality for the delivery of nucleic acid drugs and is currently the main delivery form for mRNA vaccine preparations. For details on the process application of LNP, please click here to view <mRNA-LNP Practical Understanding & Application Selection Part 1 Cobite helps LNP efficient process development.

Figure 2 Common Oligonucleotide Chemical Modifications

Figure 3. Common Nucleic Acid Delivery Systems [1]: (A) Viral Vectors; (B-E) Non-viral Vectors

Cobetter’s solution is on the above small nucleic acid manufacturing and LNP delivery system process applications, Cobetter can provide efficient and reliable solutions. Some application cases of its membrane filtration technology are shown below:

Membrane Cassettes – Pore Size Selection for Small Nucleic Acid Applications:

API Ultrafiltration

For nucleic acid substances, different nucleic acid sequence designs, buffer systems, pH and temperature conditions may cause differences in molecular spatial structures. Therefore, when selecting the retention pore sizes of ultrafiltration membrane cassettes, it is not recommended to simply rely on molecular weight for estimation and evaluation. In some cases, actual test of process flux and the presence or absence of permeation end loss is required to confirm the appropriate accuracy selection.

For small nucleic acid ultrafiltration, 3KD, 3KDH (3KD high retention product), 2KD, 1KD and other pore sizes can usually be used. Cobetter has the above regenerated cellulose RC membrane cassettes with various pore sizes and specifications (see Figure 4), which can take into account the needs of API ultrafiltration impurity removal, desalting and liquid exchange of multiple sequence lengths and volumes.

API Endotoxin Removal

For the control of endotoxin levels of small nucleic acid materials, on the one hand, it mainly depends on factory environment, raw materials, contact containers and other material’s sterility and endotoxin level.

On the other hand, it can also be ultrafiltered in the form of ultrafiltration with a 30KD or 50KD pore size membrane cassettes to achieve effective interception of some endotoxin aggregates, so the target small nucleic acid product can be collected at the permeation end, and the endotoxin level can be effectively controlled to a certain level. In addition, for the endotoxin control of related buffer solutions, a 6-10KD ultrafiltration membrane cassette can usually be used to achieve a retention and removal efficiency of more than 99.99%. Cobetter’s anion adsorption membrane Purcise Q can also be used to remove endotoxins with high efficiency and high load.

Figure 4. Cobetter RC Cassettes Option

Ultrafiltration Manufacturing Process and Case Analysis

Pore Size Selection

For small nucleic acid product selection and process data comparison, please refer to the following cases:

For small nucleic acids with a molecular weight of 6KD, the ultrafiltration selection was compared with Cobetter's 3KDH and 2KD membrane cassettes. The process data are as follows:

Table 1 siRNA Ultrafiltration -2KD Comparison with 3KDH

Figure 5. Concentration and Diafiltration Flux & TMP Curve Changes over Time

As shown in the above data, under the condition that the material yield can be guaranteed, the high-retention 3KDH membrane cassettes has a higher process flux in high-salt solution, and compared with the 2KD membrane cassettes, it has certain advantages in the membrane area or process time required for the same processing volume. Combined with our company's evaluation data, 3KDH or 2KD membrane cassettes have high versatility for different small nucleic acid sequences with conventional length or molecular weight of about 6-8KD, but in practice, comparative tests and combined with overall filtration flux, yield and process time and other indicators are required for pore size optimization.

Ultrafiltration Process

The small nucleic acid part usually contains organic solvents and salts after purification, which needs to be dialyzed and ultrafiltered. However, in the ultrafiltration process, due to the small pore size of the ultrafiltration membrane, even in the case of relatively high TMP, the permeation flux level is still relatively low, which also leads to the fact that the process time required for ultrafiltration is often very long. Therefore, the determination of the optimal concentration factor or liquid replacement time during the ultrafiltration process is particularly critical to the process duration.

The following case conducts ultrafiltration concentration of small nucleic acids in a salt system and a pure water system respectively, analyzes and evaluates the optimal concentration factor or liquid replacement time in different systems through the detection of real-time flux and corresponding filtered-particle concentration.

As shown in Figure 6, when the filtered-particle is ultrafiltered in a salt system, the membrane flux does not decay significantly with the increase of liquid concentration; while in a pure water system, the flux decreases significantly with the increase of liquid concentration. We mainly refer to the filtered-particle concentration range value at a higher flux level in a pure water system as the optimal concentration range value in the ultrafiltration process, and then perform the liquid replacement step, which can appropriately reduce the process time.

Figure 6. Confirmation of the Best Dialysis Point of Small Nucleic Acids in Salt Systems and Aqueous Systems

In summary, for the manufacturing process in the field of small nucleic acids, Cobetter's solutions can be applied to ultrafiltration concentration of liquid exchange, endotoxin removal, sterilization filtration, and membrane chromatography purification of API and LNP preparations. At the same time, for enzyme-sensitive materials such as nucleic acids, we can provide products such as nuclease-free storage bags, mixing bags, customized disposable components, and liquid preparation systems, as well as verification services.