Issue:September 2024
DRUG DELIVERY - Viewing Lipid Nanoparticle Delivery Technology Through the Lens of a CRDMO
LNP MARKET DYNAMICS
Lipid nanoparticle formulations have undergone remarkable progression in recent few years. To give you just one data point, according to our own internal market analysis, there are now approximately 150 active pipelines that incorporate LNP delivery technologies. What distinguishes this trend is not only its sheer number of pipelines, but also its rapid technological advancement.
It is also undeniable that the success of mRNA-LNP vaccines against the COVID-19 pandemic stands as a testament to the robustness of this delivery technology. Looking closer, a significant proportion of these pipelines — roughly three-quarters — are RNA-based therapeutics, specifically, LNP delivery supplementing to antisense oligonucleotide, siRNA, and mRNA modalities. Notably, LNP-based COVID-19 vaccines are only the tip of the iceberg of the “LNP club.”
So, if we look at advanced pipelines – defined as those that have progressed into late-stage clinical trials – we see targets for a diverse range of indications, such as cancer, endocrine disorders, and even viral infections. And, when we speak with innovators first-hand, we see real confidence in incorporating this delivery system within their newest drug designs. This confidence is anchored by the reliability of LNP formulation in terms of its developability with about 50% of LNP-formulated drugs advancing into the clinical trials.
OVERVIEW OF LNPS
LNPs share a similar concept to liposomes, as demonstrated in Figure 1, in that they involve the encapsulation of an active pharmaceutical ingredients (APIs) within lipid particles that circulate in the bloodstream, ensuring sustained release of the API. What is surprising perhaps is that the current market activity has taken so long to come to fruition. For example, although the inception of PEGylated liposome-based therapeutics dates back to 1995, with the commercialization of Doxil®, the approval of LNP-based therapeutics was delayed by over two decades, marking its breakthrough with Onpattro®’s approval by the FDA in 2018.1,2
What has certainly facilitated the recent emergence of LNP as a go-to delivery technology is the evolution of oligonucleotide-based therapeutics. Oligonucleotides used to encounter significant limitations due to their brief circulation time, which arose from serum degradation and limited cellular penetration capability. In this context, LNPs emerged as a robust formulation solution to this problem due to its ability to encapsulate and protect fragile cargoes, safeguarding their transport to target cells and enabling effective release after endocytosis.
Looking closer at their makeup, the fundamental components of LNP vehicles encompass functional lipids and stabilizing lipids.3 Functional lipids are cationic lipids or ionizable lipids. They play critical functions in cell penetration and release.4 Cationic lipids in early designs are permanently charged, and although able to effectively bind to the cell membrane and the oligonucleotide API, their cytotoxicity limits their application in LNP design. In contrast, new ionizable lipids exhibit pH sensitivity and maintain a neutral charge in serum. They then become protonated in endosomes, thereby promoting effective endosomal escape of the encapsulated cargo.
The other essential component of LNP vehicles is PEGylated lipids, characterized by their extended PEG polymer heads. These PEG lipids prevent aggregation of LNPs, which enhances their stability and prolongs circulation time within the bloodstream. The incorporation of PEGylated lipids in LNP self-assembly also increases encapsulation efficiency during manufacturing. However, the presence of PEG lipids can present some drawbacks, notably their bulkiness diminishes the affinity of LNPs to ApoE for cellular uptake. A further potential issue is that for patients with established immunity against PEG, the efficacy of drugs with LNP formulation may further decrease. This an ongoing dichotomy that developers face and underscores the intricate balance required when incorporating PEG lipids into LNP formulations.
In addition to the functional lipid and stabilizing PEG lipids, LNP design also incorporates structural lipids, such as cholesterol or phospholipids, and these critical components nowadays have long advanced from natural lipids. Consequently, in the process of new formulation development, fine-tuning of the lipid component selection is necessary. To accommodate the need for precise LNP formulation design, a large library, as demonstrated in Figure 2, with large selection of head groups, linkers, hydrophobic tails, and PEG polymers is a prerequisite for effective LNP development.
COMMON MANUFACTURING APPROACHES
When we turn to look at synthesis, the mainstream manufacturing approaches for LNPs all share the same concepts of mixing, followed by purification and filtration. These methods originate from a simple and quick lab-scale preparation known as ethanol injection, which involves the rapid mixing of a lipid-containing organic phase with an API-containing aqueous solution. However, it initially faced challenges in achieving optimal encapsulation efficiency while ensuring consistent particle size.
The answer was found after extensive research, which showed the crucial role fluidic dynamics and local ethanol concentration play in determining encapsulation efficiency during LNP manufacturing. For example, factors like flow speed, temperature, fluidic mechanics, and ethanol removal rate intricately influence the assembly process. Based on these findings, the modern landscape of LNP manufacturing engineering has diverged into two primary categories: T-mixers and microfluidic mixers.
T-mixers are large-scale mixing equipment featuring rapid flow speeds to achieve desirable particle sizes. This approach has remarkable advantages of simplicity, efficiency, scalability, and consistent quality. Notably, the T-mixers have been implemented in the manufacturing of prominent LNP-based drugs such as the mRNA vaccines.
In contrast, microfluidic mixers operate at the microscopic level, meticulously managing fluid dynamics to ensure precise and high-quality mixing. An illustrative example is WuXi STA’s multi-channel microfluidic mixer, equipped with multiple inlet valves. These valves supply solutions that are not only limited to conventional ethanol and aqueous solutions, but also other materials such as polymers, salts, and ligands for conjugation. It also offers adjustable parameters similar to flow chemistry reactors, enabling precise manipulation of reactions. Such a system is a powerful tool for advanced LNP formulation at a scalable level.
LNP & CONJUGATES – ADVANCED LNP MANUFACTURING
As previously mentioned, LNPs share physiological similarities to liposomes, with a natural affinity to ApoE; and unconjugated LNPs already have specificity for hepatic cells and facilitate efficient internalization. Therefore, for hepatic indications or those that do not require a specific target cell type, this intrinsic property eliminates the need for additional targeting agents in LNP formulations. In addition, studies hint at ApoE’s role in brain lipoprotein scavenging. The utilization of this pathway is therefore highly promising for siRNA-LNP therapeutics against brain indications, but no pipeline with this design has yet advanced to NDA submission. In contrast, for tissues that do not utilize the ApoE metabolism pathway, unconjugated LNP is futile, and in response, the conjugation of ligands, such as antibodies, has emerged as a potent strategy to enhance LNP targeting. By incorporating ligands onto the LNP surface, the ability to recognize and interact with specific target molecules is magnified. This approach introduces three methods: direct assembly, post-modification, and post-insertion.
Direct assembly involves integrating the ligands during the self-assembly mixing process, demanding specialized mixers and amphipathic ligands, often heavily modified. The tremendous size of common ligands severely impairs the efficiency of the self-assembly process. Therefore, small molecule (eg, shorter peptide) ligands are more favorable for this method.
Conversely, post-modification/insertion methods introduce the ligands after LNP vesicle assembly. This process involves the incorporation of the heterobifunctional PEG lipid anchors on the LNP surface during the initial assembly of the LNP. While ligands are clicked on these PEG lipids via azide-mediated ligation, after the assembly. Such design is already being tested on some siRNA-LNP pipelines.6
Despite remaining in preclinical phases, LNP-conjugate designs stand as a beacon of hope for future drug delivery strategies. Particularly promising for RNAi-related therapeutics, these innovations hold the potential to expand the indication selection by enhancing the efficacy and precision of LNP-mediated drug delivery.
LNP QUALITY ATTRIBUTES & CMC CONSIDERATIONS
Most LNP formulations are sterile injectables and thus are required to follow ICH Q8 R2 guidelines in the CMC process. However, for LNP formulations, there are still specific quality control considerations for effective development.
The balance of different lipid components significantly impacts the API encapsulation and nanoparticle assembly. And, this factor serves as the indicator of the quality of LNP products. Achieving the optimal ratio of lipid components is essential, not only for encapsulating the API effectively, but also for determining the LNP’s overall stability and performance.
Particle size distribution stands as another vital aspect of LNP quality control. The size distribution influences in-vivo release dynamics and ultimately the delivery efficiency. Also, in-vitro release testing is one of the key evaluations before preclinical evaluation. From this, developers gain a crucial understanding of the interaction between LNP and its cargo, before proceeding to animal PK studies. This insight provides a crucial opportunity for developers to make the most cost-efficient decisions.
In addition to the lipid component composition and LNP particle analysis, several other attributes should be considered and examined extensively for a successful formulation. Leachable and extractable studies are especially important for LNPs because of their pH sensitivity. The fill-to-delivered volume ratio reflects the osmolality and ultimately the delivery efficiency. Additionally, attributes such as sterility and endotoxin levels simply reflect the quality of the manufacturing process – therefore, good CMC practices with comprehensive analytical capabilities are fundamental for a successful LNP formulation.
THE IMPORTANCE OF INTEGRATED CAPABILITIES FOR LNP DEVELOPMENT
The complexity of LNP formulations demands diverse and integrated capabilities for development – incorporating lipids, oligonucleotides, small molecules, and sometimes antibodies or ligand, and thus it requires a cohesive approach. Additionally, a comprehensive analytical platform with specialized testing for LNPs is also a must for development and further regulatory applications. So, with this as the technology’s background, it is perhaps unsurprising that seeking partnership is the de facto approach for a majority of developers in LNP-based therapeutics.
Ideally, an established LNP platform by a CRDMO should cover the entire journey of the therapeutic’s development, including lipids/oligonucleotide research, LNP development, API and drug product manufacturing, and proper analytical work. This synergy ensures the seamless amalgamation of components, forming the bedrock of a successful formulation. With LNP development projects, each decision has consequences and shapes the trajectory of the final product. So developers more than perhaps in many other areas have to rely upon their partners to bring in the needed development expertise. Without prior experience, development will not be able to progress at the required rate and likelihood of achieving optimal results is reduced.
In fact, what sets successful CRDMOs apart is not only this experience but also access to cutting-edge manufacturing equipment coupled with specialized analytical supports. With an LNP, your development is only as strong as its weakest link – so it’s important that going into a partnership you find a CDMO that will not only offer services but is more of a strategic development enabler. With comprehensive capabilities, expert decision-making, and advanced infrastructure, CRDMOs play an indispensable role in translating LNP innovations into impactful therapeutics, benefitting patients around the world.
REFERENCES
- “Doxil Prescribing Information.” Pfizer, 1974, https://labeling.pfizer.com/showlabeling.aspx?id=530#:~:text=DOSAGE_AND_ADMINISTRATION,symptoms_of_cardiomyopathy_(2.2). Accessed 5 October 2023.
- “Onpattro Prescribing Information.” Alnylam Pharmaceuticals, 2018, https://www.alnylam.com/sites/default/files/pdfs/ONPATTRO-Prescribing-Information.pdf. Accessed 31 October 2023.
- Albertsen Hald C, Kulkarni JA, Witzigmann D, Lind M, Petersson K, Simonsen JB. “The role of lipid components in lipid nanoparticles for vaccines and gene therapy.” Adv Drug Deliv Rev 188 (2022): 114416. https://doi.org/10.1016/j.addr.2022.114416
- Sun D, Lu ZR. “Structure and Function of Cationic and Ionizable Lipids for Nucleic Acid Delivery.” Pharm Res 40.1 (2023): 27-46. https://doi.org/10.1007/s11095-022-03460-2
- Zhang Yuebao, Sun Changzhen, Wang Chang, Jankovic Katarina E, Dong Yizhou. “Lipids and Lipid Derivatives for RNA Delivery.” Chemical Reviews 121.20 (2021): 12181-12277. https://doi.org/10.1021/acs.chemrev.1c00244
- Swart LE, Koekman CA, Seinen CW, Issa H, Rasouli M, Schiffelers RM, Heidenreich O. “A robust post-insertion method for the preparation of targeted siRNA LNPs.” International Journal of Pharmaceutics 620 (2022): 121741. https://doi.org/10.1016/j.ijpharm.2022.121741.
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Dr. Lu Tian is Senior Director, LNP platform lead & CMC Project Management at WuXi STA. He earned his PhD in Chemistry from Rutgers University. He has extensive experience in novel drug development having spent over 4-years at STA and the preceding decade in roles at InnovForm Therapeutics, Merck, and Abbott Laboratories. At STA he leads a dedicated team on the development and implementation of WuX’s market leading suite of LNP technologies.
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