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

1. “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.
2. “Onpattro Prescribing Information.” Alnylam Pharmaceuticals, 2018, https://www.alnylam.com/sites/default/files/pdfs/ONPATTRO-Prescrib­ing-Information.pdf. Accessed 31 October 2023.
3. 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
4. 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
5. 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
6. 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.

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.