Mitra Mosharraf, PhD, M.B.A., CSO, HTD Biosystems will do a talk on "Strategies for Manufacturing and Analysis of Lipid-Based Nanomedicines and Vaccines" at PharmSci 360, on 10th November at 4pm. EPM spoke to Mosharraf about the talk.
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Mitra, can you start by giving us a quick overview of what lipid-based nanomedicines are?
Lipid-based nanomedicines are medicines that are formulated using a lipid nanocarrier to enhance delivery or bioavailability of an active payload. The lipid carrier is usually a nanoscale liposome (lipid vesicle) or lipid nanoparticle (LNP), depending on the payload. Liposomes are lipid vesicles with an aqueous core that have been used in oncology for delivery of small molecules like doxorubicin or in vaccine formulations for delivery of recombinant protein antigens. Lipid nanoparticles (LNPs) are a non-viral platform technology designed to deliver small interfering RNA (siRNA), messenger RNA (mRNA), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas components including the Cas enzyme, guide RNA (gRNA) and DNA templates into target cells. LNPs have applications in formulation of vaccines, personalised medicine and cancer immunotherapy.
Why have they become so popular over recent years?
Liposomal drug delivery systems are not a new concept and have been used in several marketed products (e.g. Doxil) for many years. LNPs on the other hand have emerged as a novel drug delivery system platform for delivering RNA. The first product that used LNPs for delivering siRNA was Onpattro, but it was not until the pandemic that LNPs received remarkable tractions across the industry, because several vaccines that saved us from COVID-19 pandemic (i.e. Comirnaty and Spikevax) were mRNA LNP based vaccines. These systems are relatively safe, easy to scale up and manufacture, while they are also cost-effective. A forecast for the LNP market between 2023-20234 shows that the LNP market is growing at a CAGR of 14% from 2023 to 2034 (and at a faster rate than the market for liposomes during the forecasted period). As of Oct 2025, 48% of mRNA-LNP related clinical trials are on vaccines, 33% on cancer immunotherapy and 19% are on therapeutic proteins/ enzyme replacement therapy. LNPs have become very popular in recent years because they are better suited for delivering nucleic acids. They are less toxic than cationic liposomes. They can be designed to transfect cells and be used for non-viral delivery of nucleic acids, a property that is very important for gene-delivery and personalized medicine. They are easier to scale up and manufacture due to innovations in controlled mixing (e.g. microfluidics) which provide better control over the particle size and poly dispersity of these systems, increasing reproducibility and reducing batch to batch variations. They are platform technology and can be applied to deliver different mRNAs for example. These properties are important because they can reduce the time for drug and vaccine approvals and cut drug development time and cost.
Your talk highlights the importance of composition and design. What are the key components of a lipid-based vaccine or nanomedicine, and why do those details matter so much for performance?
The main components of lipid-based vaccines or nanomedicines are the lipid-nanocarrier (e.g. LNPs), the pay load (e.g. mRNA), and stabilizers (e.g. sugar).
There are four major types of lipids that are used in lipid nanoparticles (LNPs):
- Ionizable lipid (for nucleic-acid complexation and endosomal escape). This lipid is a key component of LNPs. It is positively charged at the time of formulation and interacts with negatively charged mRNA payload, while it is uncharged under physiological condition. This property reduces its toxicity compared with traditional cationic lipids.
- Helper Phospholipids (to enhance fusogenicity and structure)
- Cholesterol (to improve packing and rigidity)
- PEG-lipid (to prevent aggregation and improve pharmacokinetics)
The payload could be nucleic acid (for example siRNA or mRNA).
Performance depends on the design, for example payload length/structure, N/P ratios, molar ratios of lipids, total lipid concentration, particle size/PDI, surface charge, encapsulation/association efficiency and potency. Buffers and pH are important for complexation and performance while stabilizers like sucrose/trehalose are important for stability, and lyophilisation feasibility.
For liposomes, depending on the nature of the payload and product design, payload can be entrapped in the hydrophilic core (hydrophilic) or in the lipid bilayer (for lipophilic molecules). It can also be associated with the liposome surface (ligands/antigens) to target specific cells.
In both systems (LNPs and liposomes), the product design and formulation are driven by target product profile, TPP (e.g. dose, route of administration, indication, shelf-life). It is also important to identify the critical quality attributes (CQAs) of the product to ensure product quality is acceptable throughout the product shelf-life and during the time the product is in-use. Regulatory agencies like FDA and EMA have issued regulatory guidelines for liposome drug product/ nanomedicines, biologics, vaccines and CMC guidelines for gene therapy products to create a standardised evaluation protocol and ensure quality by design across all stages of development and post-marketing. These regulatory requirements define the critical quality attributes (CQAs) that must be checked at release and during stability studies to ensure product quality. Required analytical assays are phase dependent, but in general characterisation of particle size (using orthogonal methods), polydispersity index (PDI), surface charge, morphology, encapsulation, integrity, purity, content, safety and activity are usually required.
You mention regulatory requirements for understanding things like payload amount, location, and particle size. What makes these factors so critical for safety and efficacy?
Particle size, encapsulation efficiency (payload content in the LNP) and charge are a few critical quality attributes that are important for product quality.
Particle size is important because particle size impacts biodistribution, cellular uptake, efficacy, solubility and stability. Smaller particles usually have longer circulation, and better tissue permeability. Particle size is also important for filterability of the product during manufacturing. It impacts filtration efficiency, manufacturing consistency and product quality. The particle size of LNPs is usually about 30 to 100 nm. An increase in particle size is often due to aggregation and instability and can lead to fusion and impact efficacy, immunogenicity and product safety.
For mRNA-LNPs premature release of payload leads to loss of bioactivity. Similarly, leakage of small molecules entrapped in liposomes can impact activity and safety. Payload amount is important because dose is delivered only if the associated/encapsulated fraction remains within specification limits, and functional potency is maintained.
To ensure product quality it is important to determine particle size, PDI, amount of free versus encapsulated RNA (or other payload) determine identity content, purity, impurities (e.g., dsRNA for mRNA) to prove these remain within specification limits through shelf life.
Are there common pitfalls or misunderstandings scientists face when trying to characterise or manufacture these nanoparticles?
Yes, there are a few pitfalls that I can mention when characterizing LNPs. For example, if scientists rely on a single assay for characterization of particle size, they might miss some sub populations that would impact particle size. For example, if the LNP solution is turbid, Dynamic Light Scattering (DLS) might not accurately determine its particle size. In these cases, it is better to also use other methods such as field-flow fractionation and cryo-TEM to get a better size assessment. Scientists might also assume that in vitro results would correlate with in vivo outcomes, but the in vivo environment is more complex. Efficacy and behavior of LNPs might change if they interact with immune cells and proteins in the plasma.
Another pitfall is in calculation of encapsulation efficiency (EE) of mRNA. It can be inaccurately overestimated if scientists calculate EE by comparing the amount of encapsulated RNA to total sample RNA instead of total RNA input into the formulation.
When it comes to manufacturing pitfalls, relying on supplier quality specifications for raw material can increase the risk of product failure. It is important to verify raw material quality. Similarly neglecting impurities and degradation in lipid raw materials can impact m-RNA stability and activity and should be avoided. Mishandling m-RNA and lipids during freeze-thaw cycles and storage can also lead to instability, leakage and loss of product activity and scientists should be careful when handling these temperature sensitive materials.
Stability is a big theme, especially the push for thermostable LNP-based vaccines. What are some of the most promising strategies to improve room-temperature stability?
Lyophilisation, and spray drying are some of the approaches that are utilized to enhance thermal stability, because biophysical and biochemical changes occur more rapidly in aqueous solutions than in solids, different drying techniques can be used to improve product stability by reducing degradation and aggregation rates. However, it is important to ensure that product quality attributes like particle size are unchanged before and after lyophilization. To achieve this, and to ensure a good, lyophilized product, addition of lyoprotectants like sucrose or trehalose in the formulation is crucial to prevent particle aggregation during/after freeze-drying. Selection of buffer and pH are other important formulation parameters. It is desired for a lyophilized cake to have a rapid reconstitution time (usually < 1 min), low moisture content (usually 1-2 %w/w). In addition to particle size, the payload is usually checked for integrity and purity before and after lyophilization. Control and optimisation of lyophilisation parameters such as freezing temperature, primary drying temperature/ length and secondary drying temperature/length and rates are important process parameters that should be studied.
Freeze-drying is one approach mentioned—how does that work to enhance stability, and what challenges does it introduce?
Freeze-drying is a method that is used to remove water from the product. It is known that instability is related to presence of water activity. As the product gets dried, it also becomes vitrified. In such dried solids all chemical degradation and biophysical instability (e.g. aggregation) occur more slowly than in an aqueous solution. To protect the product during lyophilisation, a lyoprotectant like sucrose is added to the formulation. As a result, when the water is removed by sublimation, aggregation of nanoparticles is prevented/reduced, as sugar replaces the water molecules and the lipid nanoparticles would be frozen and dried in a sugar matrix that protects them. The freeze-drying parameters should be also optimised to minimise aggregation while preserving bioactivity. A good cake structure has a moisture content < 1-2% and ideally can be reconstituted within seconds in water for injection/diluent prior to any injection.
In some cases, freeze-drying, or spray drying is used for manufacturing powder for nasal delivery or inhalation. In those cases, they should be free flowing and of specific particle size to enable optimised delivery.
Manufacturing these particles sounds complex. What are some of the biggest hurdles in scaling up production while maintaining quality?
When a typical mRNA-LNP product goes through different stages of development, the number of analyses, batch size and length of manufacturing time changes. The process time for mixing lipid-ethanol solution with mRNA solution could increase from 1-2 hours at small scale (2-60 mL) to 8.5 hours or more at large scale (400 L) using microfluidic mixers. Ensuring that mRNA is intact and stable throughout the process is important. Microfluidic mixers have helped improve control of particle size, PDI, reducing heterogeneities and batch to batch variations. However, translating the critical process parameters from small scale to large scale is essential for tech transfer. The process parameters like total flow rate, flow‑rate ratios, temperature, post‑mix dilution and hold times are usually tested in laboratory scale during pre-clinical studies and then they are re-tested and adjusted at larger scales during tech transfer to ensure the product quality is maintained.
There are also process parameters that are tested during purification (ethanol removal) and buffer exchange by tangential flow filtration (TFF). For example, trans‑membrane pressure, shear and diafiltration volumes affect size and integrity. It is important to verify residual solvents to ensure ethanol is removed and buffer is exchanged. Ensuring batch to batch consistency and process reproducibility as well as raw material sourcing for large scale manufacturing are additional challenges for scaling up production. Other hurdles are ensuring that aseptic strategies are adequate for achieving bioburden and endotoxin targets. Also, sterile filtration feasibility, and extractables/leachable from single‑use systems are tested. The final drug product is characterised for particle size, PDI, charge, activity and safety to ensure its quality is maintained throughout the process.
Finally, what do you hope attendees will take away from your PharmSci 360 talk?
I hope that the attendees will understand how lipid nanoparticles are made. How their quality is assessed and what are their critical quality attributes. The opportunities that they provide and challenges that scientists and manufacturers are facing today.