The Essential Guide to Lipids for Successful LNP Therapy Development

Everything you need to know about lipids for developing LNP therapies Understanding the Foundation of LNP Formulation Success Table of Contents 03 Introduction 14 The importance of customization 17 Key characteristics of a lipid suitable for LNP therapies 18 The lipid sourcing journey: a strategic overview 20 Better understanding the building blocks of LNPs 20 References 14 Off-the shelf lipids: the promises and pitfalls 18 Early development 15 Putting down the catalogue: the case for customization 16 Common lipid customizations 04 Lipid types: the “what, why, and when” 04 Cationic lipids 06 Ionizable lipids 08 Phospholipids 10 Sterol lipids 12 PEGylated lipids 01. INTRODUCTION UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 03 Introduction Lipid nanoparticles (LNPs) — lipid-based spherical particles ranging from 50–200 nanometers in diameter — represent one of the most significant advances in drug delivery technology in recent decades. As drug delivery vehicles, LNPs offer numerous advantages that make them increasingly valuable in modern medicine — from safe and effective delivery of nucleic acid cargo to cell-free manufacturing processes and ease of production scale-up. The flexibility of LNP formulation also allows developers to optimize LNPs for specific applications — from gene therapies to next-generation vaccines. This comprehensive guide addresses these questions. After reading it, you’ll better understand: At the heart of every successful LNP formulation, though, lies its fundamental building blocks: lipids. The critical role that ultra-pure lipids play in determining LNP characteristics cannot be overstated, with specific types and combinations of lipids dramatically influencing numerous LNP characteristics and behaviors, including: · Size and structure · Surface charge · Stability · Cargo encapsulation · Cellular uptake · Targeting · Endosomal escape It is therefore critical that developers select the right lipids for their formulations. Indeed, suboptimal lipid selection could lead to the costly and unnecessary failure of an otherwise promising therapeutic candidate. However, navigating the vast landscape of lipid options and considerations can be overwhelming. Where should developers start? What specific knowledge about lipids is essential for developing successful LNP therapies? The types of lipids commonly used in LNP therapies, their function, and key considerations for their use The importance of lipid customization The key characteristics of an ideal lipid for LNP therapeutics What’s involved in sourcing the right lipids for your LNP therapy program UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 02. LIPID TYPES: THE “WHAT, WHY, AND WHEN” 04 Lipid types: the “what, why, and when” Understanding the different types of lipids used in LNP formulations is crucial for successful therapeutic development, since each lipid type serves specific functions in the final LNP complex. In this chapter, we explore the various lipid types commonly used in LNPs. Cationic lipids Cationic lipids are characterized by their head groups carrying a permanent positive charge. Common examples of these lipids include 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (Fig 1), which have been used in LNPs for mRNA delivery.1 DOTMA DOTAP Figure 1: Chemical structure of DOTMA and DOTAP molecules UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 02. LIPID TYPES: THE “WHAT, WHY, AND WHEN” 05 Cargo encapsulation: Cationic lipids strongly bind with negatively charged nucleic acid cargo, such as mRNA. This binding serves to both protect cargo from degradation, and aid compaction of the nucleic acid material into a more stable form. Toxicity The most pressing concern is toxicity. The permanent positive charge that makes these lipids effective at cargo binding can also lead to cytotoxicity through cell membrane disruption. This can then activate pro-apoptotic cascades and trigger pro-inflammatory responses, limiting the maximum safe dosage of the therapy. Non-specific interactions Non-specific interactions pose additional complications. Cationic lipids readily interact with negatively charged blood serum proteins, leading to the formation of protein corona, potential aggregation, and altered biodistribution patterns. Rapid clearance Rapid clearance presents another challenge. LNPs containing cationic lipids can be quickly recognized and cleared by the reticuloendothelial system (RES), resulting in shortened circulation times and reduced therapeutic efficacy. Immune activation Furthermore, cationic lipids can trigger unwanted immune responses, potentially causing inflammatory reactions and other adverse side effects. Due to these safety and toxicity concerns, the field has largely shifted toward using ionizable lipids, which are discussed below. Enhanced Cellular Uptake: The positively charged head groups interact with negatively charged cellular membranes, facilitating cellular internalization of the LNP complex. Donald Kelemen, Chief Scientific Officer, ABITEC Cationic lipids serve multiple critical functions in LNP formulations. Their primary roles include: While cationic lipids play an important role in the LNP complex, they present several significant challenges that developers must carefully consider: Function and Importance Key Considerations Fixed cationic lipids come with toxicity concerns that should make developers carefully consider whether this class of lipids is right for their LNPs. Ionizable lipids, on the other hand, play much the same functional role and are generally recognized to be safer. UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 02. LIPID TYPES: THE “WHAT, WHY, AND WHEN” 06 Ionizable lipids Ionizable lipids (Fig 2) are lipids that can change their charge based on the pH of their environment, being neutral at physiological pH, but positively charged in more acidic conditions, such as in endosomes. SM-102 Figure 2: Structure of SM-102, the ionizable lipid used in the formulation Moderna’s COVID-19 vaccine, Spikevax. Supporting nucleic acid complexation: In acidic conditions, ionizable lipids are positively charged and thus strongly bind negatively charged nucleic acids (such as mRNA), helping protect nucleic acid cargo and aiding compaction. Supporting endosomal release: Ionizable lipids are positively charged inside the acidic environment of endosomes, where the positive charge causes the LNP to fuse with the endosomal membrane to help facilitate endosomal release. Ionizable lipids have a critical multi-functional role in the LNP complex, similar to cationic fixed lipids: Functional Importance Reducing toxicity: Since ionizable lipids are neutral at physiological pH, they are less toxic than fixed cationic lipids, helping to create an LNP with a superior safety profile.2 UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 02. LIPID TYPES: THE “WHAT, WHY, AND WHEN” 07 Stability Ionizable lipids are particularly susceptible to degradation due to their reactive functional groups. For example, the presence of tertiary amines and other chemically reactive functional groups makes ionizable lipids vulnerable to oxidation, hydrolysis, and other chemical modifications, which can ultimately impact the structure and function of the LNP complex. As such, ionizable lipids require much more specialized storage and handling, as do the therapeutic LNP products that incorporate them. Intellectual property (IP) considerations The IP landscape for ionizable lipids is a minefield. There is a good chance that any novel or custom ionizable lipid that a developer creates is already covered by a patent. Patents are also fairly broad and vague when it comes to the design and synthesis of ionizable lipids, meaning that it is easy for companies to claim infringement. Large, well-funded organizations can easily tie up smaller organizations in legal costs — even if the supposedly infringing company’s ionizable lipid is truly novel. Developers looking to create their own customized ionizable lipids should therefore seek input from chemistry and legal experts deeply familiar with the space. While ionizable lipids play a crucial role in LNP complexes and are generally safer than fixed cationic lipids, LNP developers must be aware of several limitations. Key Considerations Customization concerns Given that most available ionizable lipids used in commercial therapeutics are already patented, these lipids are the most likely to require customization (assuming an organization doesn’t want to pay a licensing fee to use an existing lipid). But creating and using a custom lipid in a therapeutic LNP formulation demands adherence to strict regulatory requirements, which can be time- and resource-intensive. For example, developers will need to build out a data package to show the purpose, function, pharmacokinetics, and safety profile of the new lipid. Developers should be aware of (and plan for) this requirement as early as possible to minimize program delays and disruption. UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 02. LIPID TYPES: THE “WHAT, WHY, AND WHEN” 08 Phospholipids Phospholipids represent another crucial category of lipids used in LNP formulations. These molecules are characterized by their distinctive structure: a hydrophilic head containing a phosphate group and two hydrophobic fatty acid tails, all joined by an alcohol residue (typically glycerol). The most commonly used examples in LNP development are 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (Fig 3). DSPC DOPE Figure 3: Chemical structures of DSPC and DOPE. UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 02. LIPID TYPES: THE “WHAT, WHY, AND WHEN” 09 Promoting structural integrity: Phospholipids help create the basic structure of LNPs and maintain membrane stability. Modulating membrane fluidity and endosomal escape: The unsaturated tails of DOPE molecules, for example, can increase LNP membrane fluidity and promote non-lamellar structures that facilitate endosomal membrane fusion, which is critical for therapeutic cargo delivery. Often referred to as ‘helper lipids’ or ‘structural lipids,’ phospholipids play a fundamental role in both the structure and function of LNPs. The major challenge when working with phospholipids for LNP therapeutics is a lack of relevant phospholipid and phosphate chemistry expertise in the marketplace. While there are many researchers interested in phospholipids, the interest is typically from a basic science perspective (for example, understanding phospholipid biophysics or the role of phospholipids in biological membranes), rather than from a therapeutic application and cGMP synthesis perspective. Finding experts that can help synthesize and customize phospholipids for LNP applications can therefore be a bottleneck for LNP therapy programs. Function and Importance Key Considerations and Challenges UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 02. LIPID TYPES: THE “WHAT, WHY, AND WHEN” 10 Sterol lipids (such as cholesterol) Sterol lipids are characterized by a fused fourring core structure. The most commonly used sterol in LNPs is cholesterol (Fig 4), which is the most abundant naturally occurring sterol lipid in animals, where it helps maintain cell membrane structure and serves as a precursor for important hormones. Enhance membrane stability and integrity: Cholesterol intercalates between other LNP membrane components to facilitate proper lipid packing and drive membrane stability. Modulate membrane fluidity: Cholesterol can act as a fluidity buffer, increasing membrane fluidity at lower temperatures and decreasing it at higher temperatures by pulling lipids towards the liquid ordered phase. This results in better LNP particle stability, especially in the presence of temperature fluctuations. Sterols are another critical component of LNPs, helping to: Function and Importance Figure 4: Chemical structure of cholesterol Cholesterol UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 02. LIPID TYPES: THE “WHAT, WHY, AND WHEN” 11 Mammalian-derived cholesterol Being a product of animal origin, mammalian-derived cholesterol carries a risk of contamination with viruses and other adventitious agents such as prions (potentially leading to transmissible spongiform encephalopathies (TSEs)). As such, mammalianderived cholesterol is subject to more stringent and resource-intensive safety testing, and meticulous traceability documentation. When it comes to using cholesterol in LNP formulations, developers need to consider the source of the molecule. Several sources of cholesterol exist — mammalian-based, ‘plant-based’ (in the form of modified phytosterols), and synthetic — but not all are equal. Key Considerations and Challenges Synthetic cholesterol Opting for synthetic cholesterol alleviates many of the concerns associated with animaland plant-derived cholesterol. For example, synthetic cholesterol does not carry a risk of contamination with adventitious agents, and, unlike with plant-derived cholesterol, there is greater control over the chemical inputs of the synthesis pathway. Additionally, synthetic cholesterol offers increased flexibility for downstream chemical modification, thereby offering more opportunities for optimization for LNP applications. ‘Plant-derived’ cholesterol (modified phytosterols) Cholesterol can also be produced by chemically modifying purified phytosterols. Such modified phytosterols do not present a TSE risk and are thus more regulatory-friendly than animalderived cholesterol. That said, there is still a risk of batch-to-batch inconsistency with plantderived cholesterols, given that the starting material (plants) can be variable, and plants may be a source of other exogenous pollutants, such as pesticides. As such, there is still some traceability documentation burden. Phytosterols — a feasible alternative to cholesterol? Developers also have the option of using unmodified phytosterols purified from plants. While this approach would eliminate some of the safety risks associated with animal-derived cholesterol, phytosterols are chemically different to animal cholesterol. Using them in therapeutic LNP formulations would therefore require additional studies to evaluate their pharmacokinetics and safety profile. Dmytro Honcharenko, Principal Scientist, ABITEC. Not all cholesterols are equal. Synthetic cholesterol sidesteps many of the safety concerns associated with mammalian-derived cholesterol, which can add significant regulatory burden to an LNP program. UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 02. LIPID TYPES: THE “WHAT, WHY, AND WHEN” 12 Figure 5: Chemical structure of DSPE-PEG 2000, Na [PEGylated lipid molecule] Increase circulation time: The primary role of PEGylated lipids is controlling clearance. They do this by enabling LNPs to evade clearance by the mononuclear phagocyte system (MPS), increasing blood circulation time. Improve steric stability: PEGylated lipids form a hydrophilic surface layer around the LNP complex that prevents nanoparticle aggregation and improves colloidal stability. This helps ensure better stability both during preparation of the LNP formulation and during product storage. In the LNP complex, PEGylated lipids serve to: Function and Importance Modulate size: PEGylated lipid concentration impacts LNP particle size during formulation development, where lower concentrations lead to larger particle sizes. PEGylated lipids PEGylated lipids consist of a lipid anchor attached to a polyethylene glycol (PEG) polymer chain of varying lengths (Fig 5). Common types used in LNPs include DSPE-PEG 2000 and DMG-PEG 2000. Though comprising the smallest percentage of lipid components in LNPs, PEGylated lipids play an important role in LNP function. DSPE-PEG 2000 UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 02. LIPID TYPES: THE “WHAT, WHY, AND WHEN” 13 Immunogenicity One primary concern relates to potential adverse immunological responses. PEGylated lipids can trigger the body to produce anti-PEG antibodies, potentially leading to serious complications, including anaphylaxis. However, the extent of this immunogenicity can vary. To what extent it may pose a problem depends on the therapeutic context — namely the intended therapeutic effect (e.g., whether it’s a vaccine, where a certain amount of immunogenicity is desirable, or a gene therapy, where it is not), dosing levels, and the frequency of administration (with larger doses and more frequent administration increasing the risk of negative side effects). Despite their benefits, PEGylated lipids present a few important challenges that developers must address (or at least be aware of). Key Considerations and Challenges Reproducibility PEG molecules are incredibly amorphous. If synthesis is done poorly, you can end up producing a broad distribution of PEG molecules of varying chain lengths, which, as discussed above, can have implications for LNP structure and function. Ideally, therefore, companies need a synthesis process that produces a process that allows for control of the PEG dispersity. Achieving this level of consistency, however, requires considerable expertise and experience in lipid synthesis and characterization. A handful of different lipid types are used in therapeutic LNP formulations, each with different roles in the LNP complex, and associated with different challenges: Cationic lipids are important for nucleic acid complexation and cellular uptake of the LNP, although they are associated with considerable safety issues. Ionizable lipids largely replace fixed cationic lipids in LNP formulation today, supporting nucleic acid complexation, cellular uptake of the LNP, and endosomal release with reduced toxicity. But developers must be aware of a tricky patent landscape, and be prepared for lipid customization. Phospholipids promote structural integrity and stability of the LNP complex, as well as modulating membrane fluidity and endosomal escape. However, expertise in phospholipid synthesis and customization for therapeutic applications is hard to come by. Sterol lipids, primarily cholesterol, enhance LNP membrane stability and act as a membrane fluidity buffer. Opting for synthetic cholesterol may be best, owing to the reduced safety concerns and regulatory burden, and the greater control over chemical synthesis inputs relative to animal-and plant-derived alternatives. PEGylated lipids improve steric stability, modulate size, and prevent premature clearance of LNPs. However, immunogenicity concerns may preclude them from use in your therapy, and their amorphous structure may pose reproducible synthesis challenges. UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 03. THE IMPORTANCE OF CUSTOMIZATION 14 The importance of customization There are thousands of lipids for developers to explore when formulating LNPs. While off-theshelf tried-and-tested lipids offer benefits, they may not always be the best option. Off-the shelf lipids: the promises and pitfalls Opting for tried-and-tested lipids means you’ll be using well-understood molecules that may already have a wealth of safety data and are possibly already being used in approved therapies. As a result, you won’t have to do the extensive resource- and time-intensive work of creating a comprehensive data package to demonstrate function and safety. However, these benefits come at a cost; available off-the-shelf lipid chemistries may not be optimal for your specific therapy or target, reducing opportunities to create a safer, more efficacious therapy, and potentially even entailing more formulation development work than would otherwise be required. UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 03. THE IMPORTANCE OF CUSTOMIZATION 15 Putting down the catalogue: the case for customization Customized lipids allow developers to overcome the limitations of off-the-shelf lipids; with custom lipids, developers can create novel lipid chemistries optimally tailored to their specific therapy and therapeutic target. Moreover, custom synthesis can also lead to more exploratory opportunity; unlike selecting an offthe-shelf lipid, when creating a custom lipid, you’ll get a whole library of similar molecules that you can explore for potential application in your LNP formulation. Ultimately, with a greater chemical exploration space comes a greater chance of finding a better-fit molecule for your application. Aside from the inherent benefits of custom synthesis, in some cases there are limited other options. For example, most existing ionizable lipids are patented, and so unless developers are willing to pay for licensing of the lipid, they will have no choice but to create a custom molecule. As noted in the previous chapter, though, custom lipids do come with a higher development and regulatory burden owing to their novel, untested nature. Donald Kelemen, Chief Scientific Officer, ABITEC Taking an off-the-shelf approach to sourcing your lipids offers some good advantages, but it can ultimately stifle opportunity to create better formulations. It’s very one-dimensional. With an off-the-shelf approach, you won’t get any of the derivatives that could unlock greater LNP performance. UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 03. THE IMPORTANCE OF CUSTOMIZATION 16 Addition of bioactive molecules: Adding specific bioactive moieties — such as aptamers, antibodies, or antibody fragments — at certain points along the lipid molecule is an effective strategy for enhancing LNP function and performance. This approach is commonly used for enhancing specific tissue targeting, but it can also be used for enhancing the immunogenicity of vaccines, for example. Addition of biodegradable groups: Adding biodegradable elements to lipids can help improve the safety and efficacy of LNP formulations. With chemical structures that can be easily degraded by the body, lipids are less likely to aggregate in the body after the LNP has delivered its cargo. Common examples of biodegradable structures added to lipids include ester linkages and disulfide bonds. However, adding these structures is not an easy task. Location along the lipid molecule is key, and developers must also ensure that the degradation products do not accumulate or cause unwanted side effects — a particularly important consideration in therapies that require frequent repeated doses. pKa value changes: Modifying lipid pKa values is key for optimizing LNP performance, particularly in nucleic acid delivery. The ideal pKa value (typically 6.2–6.8) allows lipids to remain neutral at physiological pH while becoming positively charged in acidic endosomes, which is important for fine tuning endosome fusion and therapeutic cargo release (see ionizable lipids section of chapter 2). Structural modifications to adjust pKa include altering the chemical environment around ionizable amines,3 modifying the number and spacing of ionizable groups, and incorporating electron-withdrawing or electron-donating groups. However, these modifications must balance pKa optimization with other important properties such as biodegradability and LNP stability. Common lipid customizations Lipids can be customized in several different ways to optimize various LNP therapy characteristics — from stability and cargo encapsulation to tissue targeting, endosomal escape, and biocompatibility. Customized lipids offer developers the chance to optimize the safety and efficacy of their LNP therapies in ways that are not possible with off-the-shelf chemistries The novel nature of customized lipids entails greater testing and regulatory burden UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 04. KEY CHARACTERISTICS OF A LIPID SUITABLE FOR LNP THERAPIES 17 Purity It is critical that any lipid — customized or not — used in a therapeutic LNP formulation is pure. The presence of impurities doesn’t just present a direct safety risk to patients (through toxicity or immunogenicity); it can also affect LNP formation, stability, and biological activity. Because of these risks, undefined impurities in raw materials can, understandably, complicate regulatory approval processes and ultimately delay release to market. Reproducible synthesis pathways It is also critical that companies can create their lipids through reproducible synthesis pathways. Doing so helps deliver consistent quality across batches to ensure that the same product (and therefore the same LNP) is produced time and time again. Accordingly, reproducible synthesis will also need to be demonstrated for regulatory compliance. Scalable synthesis pathways Similarly, developers must be able to time-and cost-effectively produce large quantities of their lipid, as this will be critical for progressing an LNP formulation from the lab through to the clinic and commercial production. Biodegradability An LNP composed of biodegradable lipids can be rapidly cleared by the body to prevent accumulation and toxicity, leading to a safer and more tolerable therapy. As noted in the previous chapter, developers can increase the biodegradability of LNP lipids by introducing both ester and disulfide motifs, which accelerate their in vivo degradation. Flexible synthesis pathways There are many reasons why developers might need to alter lipid synthesis pathways — from further refining a lipid for superior LNP function to overcoming challenges in development (such as scaling production) and responding to changing regulations. A flexible synthesis pathway allows this to be done without having to completely redesign the synthesis pathway, which can be very challenging, costly, and time-consuming. Having to redesign a synthesis pathway from scratch could lead to significant program delays or even program failure. Key characteristics of a lipid suitable for LNP therapies Having a custom lipid tailored to your application is not enough — there are several other factors that you must consider when exploring lipids for LNP applications. Carl Arevång, Managing Director, Larodan Lipid purity is non-negotiable if you want to reliably create safe and effective LNP formulations. With the right QA/QC system and analytical tools, it’s possible to consistently create lipids with >99% purity. This is not an easy task, though. Lipids are notoriously hard to analyze for a variety of reasons, not least the fact that they are so structurally diverse and lack a chromophore. UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 05. THE LIPID SOURCING JOURNEY: A STRATEGIC OVERVIEW 18 The lipid sourcing journey: a strategic overview The journey from early-stage LNP formulation to commercial production requires companies to carefully plan their lipid sourcing, with requirements evolving significantly across development phases. Below we highlight some of the key requirements and considerations at each stage of an LNP therapy program: Early development Initial research and formulation development typically require small quantities (milligrams–grams) of lipids for screening and optimization. At this stage, Research Use Only (RUO)-grade materials from commercial suppliers are usually sufficient. Off-the-shelf lipids will typically work well for initial proof-of-concept studies, but a panel of custom lipids may be beneficial as developers look to refine and optimize their formulation. Custom lipids can be sourced from a suitable RUO partner or synthesized by experienced in-house organic chemists. Key challenges at this stage include managing costs when ordering small quantities (which can carry price premiums), establishing initial analytical methods to assess quality, coordinating multiple small batches for screening studies, and finding lipid suppliers with the technical expertise and capacity to collaboratively trouble-shoot, optimize, and service custom lipid requirements. Clinical development Clinical programs typically demand cGMPgrade materials in multiple grams to kilogram quantities accompanied by exhaustive regulatory documentation. The network of lipid providers who can address these requirements narrows significantly at this stage, leaving only those with cGMP-certified manufacturing facilities. Pre-clinical development As programs advance to pre-clinical studies, requirements shift to larger quantities (multiple grams) with potentially higher purity and more rigorous analytical documentation. During this phase, teams typically begin planning for eventual cGMP scale-up and production. The transition from research to cGMP-grade production can be complex and time-consuming, often requiring many months of lead time. Working with lipid providers who have expertise in lipid synthesis and analytical development across multiple development phases can significantly reduce the development timeline and minimize tech transfer risks. UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 05. THE LIPID SOURCING JOURNEY: A STRATEGIC OVERVIEW 19 At this stage, you’ll want to ensure that you have comprehensive supplier qualification and audit programs, detailed quality agreements covering specifications, testing, and change control, and supply chain risk assessment and mitigation strategies. Key challenges here are the lead times for GMP-grade lipids, which, without the right partner, can extend to several months. Fredrik Karlsson, Senior Scientist, ABITEC. cGMP manufacturing of lipids of any type is a key challenge for the market right now. Exceptionally few lipid providers have the knowledge and capability to deliver cGMP grade lipids at sufficient quantities, in part due to the difficulty of maintaining consistently high purity. Part of the purity challenge lies in the analytics; lipids are inherently difficult to analyze since they lack chromophores and have complex and diverse chemical structures. Key Recommendations: 1 Begin lipid provider identification and qualification early, particularly if custom lipid synthesis is a key requirement. 2 Develop scalable analytical methods and specifications from the start. Early investment in robust purification, chromatography, and analytical capabilities can prevent delays during development progression. 3 Implement a phase-appropriate quality system that can evolve with your program. Document key decisions and changes in specifications or suppliers. 4 Plan for increasing scale requirements by discussing long-term manufacturing capabilities with the lipid provider as early as possible. Partnering with lipid providers capable of supporting multiple development phases can offer significant benefits, including reduced supplier qualification burden, simplified tech transfer, consistent material quality across development stages, streamlined communication and project management, and the potential for faster development timelines. If working with multiple suppliers is unavoidable, be sure to establish robust analytical comparability protocols, plan adequate time and resources for tech transfer, maintain detailed documentation of manufacturing processes, consider the impact on regulatory filings, and budget for additional qualification and validation activities. 5 Consider regulatory requirements from the outset. Early attention to regulatory expectations can prevent costly reformulation or requalification later. 6 Consider your lipid provider as a partner in your LNP program. With the right partnership, you’ll be able to lean into their expertise and experience, which can help you better identify and sidestep roadblocks, and conceive novel and effective ways to tackle unforeseen challenges. 7 Develop a comprehensive supply chain strategy that includes safety stock requirements at each phase, backup supplier identification and qualification, risk assessment and mitigation plans, cost management strategies, and longterm supply agreements. UNDERSTANDING THE FOUNDATION OF LNP FORMULATION SUCCESS 06. BETTER UNDERSTANDING THE BUILDING BLOCKS OF LNPS 20 Better understanding the building blocks of LNPs LNPs hold remarkable promise as a drug delivery vehicle, offering safe, efficient delivery of a variety of therapeutic cargo to treat a myriad of medical conditions. But at the core of successful LNP formulations are their lipid building blocks. With even small changes in the lipidic composition of the LNP complex leading to structural and functional changes, LNP therapy developers need a solid understanding of lipids — from the types of lipids available, their role, and associated challenges, to the importance of customization, purity, and scalable, flexible, and reproducible synthesis pathways. With a firm grounding in these areas, developers will ultimately be better equipped to develop safer and more effective LNP formulations. Are you looking for a collaborative partner to develop and manufacture ultra-high purity custom lipids for your LNP program? At ABITEC, we have 60+ years of lipid chemistry experience, deep pharma industry expertise, and the team and facilities to synthesize and reliably deliver cGMP-quality lipids from milligram to kilogram scale. Discover how we can help you overcome your toughest lipid chemistry challenges: Download our brochure today References 1. Hou X, Zaks T, Langer R, Dong Y. Lipid nanoparticles for mRNA delivery. Nature Reviews Materials. 2021;6(12):1078–1094. https://www.nature.com/articles/s41578- 021-00358-0. https://doi.org/10.1038/ s41578-021-00358-0 2. Mrksich, K., Padilla, M.S., Mitchell, M.J. Breaking the final barrier: Evolution of cationic and ionizable lipid structure in lipid nanoparticles to escape the endosome. Advanced Drug Delivery Reviews. 2024; 214: 115446. https://doi. org/10.1016/j.addr.2024.115446 3. Jayaraman M, Ansell SM, Mui BL, Tam YK, Chen J, Du X, Butler D, Eltepu L, Matsuda S, Narayanannair JK, et al. Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo**. Angewandte Chemie International Edition. 2012;51(34):8529– 8533. https://doi.org/10.1002/ anie.201203263. 501 W 1st Ave Columbus, OH 43215 Toll Free (800) 555-1255 info@abiteccorp.com www.abiteccorp.com