The Problem That Most mRNA Gets Wrong

The appeal of mRNA therapeutics is straightforward: deliver the genetic instructions for a beneficial protein directly into cells, let the cell’s own machinery produce the protein, and achieve a therapeutic effect without permanently altering the genome. The challenge is equally clear: mRNA is a large, negatively charged, inherently unstable molecule that cannot cross cell membranes unaided, is degraded rapidly by extracellular RNases, and triggers innate immune responses that can abort expression before it begins.

Lipid nanoparticles have become the delivery system of choice for mRNA therapeutics precisely because they address these barriers. But not all LNPs deliver mRNA with equal efficiency. Understanding the intracellular journey from LNP uptake to protein expression, and the points at which that journey can fail, is essential for designing formulations that maximise therapeutic protein output.

The Intracellular Delivery Pathway

Step 1 — Cellular Uptake

LNPs enter cells primarily through endocytosis, a process in which the cell membrane engulfs the particle and internalises it within an endosome. The efficiency of uptake depends on the particle size, surface properties, and the extent to which the LNP has adsorbed apolipoprotein E (ApoE) from plasma, which facilitates receptor-mediated uptake in hepatocytes via the LDL receptor pathway. For non-hepatic targets, surface modification with targeting ligands can enhance uptake in specific cell types.

Step 2 — Endosomal Escape

This is the critical bottleneck in LNP-mediated mRNA delivery. After cellular uptake, the LNP is contained within an endosome, and the endosome matures progressively from early endosome through late endosome to lysosome, with decreasing pH at each stage. If the mRNA does not escape the endosome before lysosomal degradation occurs, it is destroyed and no protein expression results.

Ionisable lipids are the key functional component that drives endosomal escape. At physiological pH they are largely neutral, which reduces non-specific toxicity in circulation. At the acidic pH of the late endosome, they become protonated and positively charged, disrupting the endosomal membrane and enabling mRNA release into the cytoplasm. The efficiency of this process, often described as endosomal escape efficiency, is typically low: published estimates suggest that fewer than 2% of endocytosed LNPs successfully deliver their mRNA cargo to the cytoplasm.

Step 3 — Translation

Once mRNA reaches the cytoplasm, it is translated by ribosomes into the encoded protein. The efficiency of translation depends on the quality and integrity of the mRNA, including its 5-prime cap structure, the design of the untranslated regions (UTRs), and the codon optimisation of the coding sequence. mRNA degradation by cytoplasmic RNases competes with translation throughout the expression period.

Formulation Variables That Drive Delivery Efficiency

Formulation Variable	Effect on Delivery	Optimisation Approach
Ionisable lipid selection	Primary driver of endosomal escape efficiency; pKa affects pH-response profile	Screen ionisable lipids across a range of apparent pKa values; target pKa 6.2-6.8 for hepatic delivery
Lipid molar ratios	Balance between encapsulation, stability, and endosomal escape activity	Design of experiments to map formulation space; optimise N:P ratio for each mRNA
PEG-lipid content and PEG chain length	Modulates particle size, colloidal stability, and cellular uptake	Higher PEG content reduces uptake; shed-able PEG chains can improve delivery
mRNA : lipid ratio (N:P ratio)	Determines encapsulation efficiency and particle charge	Typically optimised to achieve greater than 85% encapsulation with near-neutral zeta potential
Microfluidics mixing parameters	Affects particle size and homogeneity during formulation	Optimise flow rate ratio and total flow rate for target particle size

Measuring Delivery Efficiency In Vitro

The standard cell-based assay for measuring LNP transfection efficiency uses a reporter system, typically luciferase or green fluorescent protein (GFP), where the mRNA encodes a readily detectable protein. Cells are incubated with the LNP at defined concentrations for a defined period, and protein expression is measured by luminescence or fluorescence. Comparing luminescence or fluorescence across formulations with identical mRNA doses gives a direct readout of relative delivery efficiency.

In vitro delivery efficiency is a useful screening tool for ranking formulations, but it does not always predict in vivo performance. Cell lines used for screening may have different uptake and endosomal biology to the target cell type in vivo, and the absence of serum proteins including ApoE in some in vitro assays can significantly underestimate hepatic delivery efficiency.

Ardena’s mRNA LNP Formulation Expertise at Oss

Ardena’s formulation scientists in Oss have expertise in ionisable lipid-based LNP formulation for mRNA and nucleic acid payloads. The site operates microfluidics and nanoparticle extrusion platforms for LNP manufacture at both screening and GMP scale, with integrated analytical capability for particle size, PDI, encapsulation efficiency, and zeta potential measurement.

Formulation development programmes at Oss are structured to move efficiently from initial lipid screening through to a robust formulation with defined critical quality attributes, supported by in vitro characterisation and stability data suitable for an IND or IMPD filing.

The Double Problem

The molecules that generate the most clinical excitement are often the ones that cause the most formulation headaches. Modern oncology APIs, for example, tend to be both highly potent (small doses, strict containment requirements) and poorly soluble (BCS Class II or IV, significant bioavailability challenges). Solving one problem in isolation is hard enough. Solving both simultaneously requires a formulation strategy that navigates containment requirements and bioavailability enhancement at the same time.

This article looks at how those two challenges interact and how development teams can avoid designing a solution for one that undermines the other.

Where the Challenges Collide

Spray Drying with HPAPIs

Spray drying is one of the most effective routes to an amorphous solid dispersion for a poorly soluble API. For a high-potency compound, it also generates an aerosol of fine particles during atomisation, exactly the exposure route that makes HPAPIs dangerous. The spray drying chamber needs engineering controls appropriate to the OEB classification of the API, and the secondary drying step, which typically involves fluid bed processing, must also be conducted within a contained environment.

This is not insurmountable, but it requires a facility where spray drying equipment is integrated with HPAPI containment infrastructure. A site that has spray drying and a site that has HPAPI containment are not the same thing as a site that has both in the same building.

Wet Milling with HPAPIs

Wet milling for nanosuspension manufacture is lower-risk from a containment perspective because the API is suspended in liquid throughout the milling process, reducing the risk of airborne exposure. However, the downstream steps, particularly spray drying or fluid bed granulation of the milled suspension, reintroduce aerosol risk. Closed transfer systems and contained fluid bed processing are standard mitigations.

HME with HPAPIs

Hot melt extrusion produces a solid extrudate rather than an aerosol, which gives it an inherent containment advantage over spray drying. The milling of the cooled extrudate to a usable particle size does generate dust, which requires containment. For many HPAPIs, HME followed by contained milling is a more practical manufacturing route than spray drying, provided the API is thermally stable at processing temperatures.

Formulation Technology Options for High Potency, Low Solubility APIs

Technology	Bioavailability Benefit	HPAPI Containment Considerations	Best Suited For
Hot melt extrusion (HME)	ASD formation; 2-10 fold AUC improvement	Contained milling of extrudate required; lower aerosol risk than spray drying	Thermally stable HPAPIs; OEB 3-4
Spray dried dispersion	ASD formation; similar bioavailability benefit to HME	Contained spray dryer and secondary drying required; aerosol generation in chamber	Thermally labile HPAPIs where HME is not feasible; requires dedicated HPAPI spray drying facility
Nanosuspension (wet milling)	2-5 fold dissolution rate improvement; crystalline API retained	Closed milling system; contained downstream processing	HPAPIs where crystalline form is required; OEB 3-4
Lipid-based formulation (SMEDDS)	Enhanced solubilisation via self-emulsification	Liquid handling; lower aerosol risk; containment simpler	Lipophilic HPAPIs; fill-in-capsule formulations
Cyclodextrin complexation	Solubility improvement via inclusion complex	Aqueous processing; low aerosol risk	Moderate potency; HPAPIs with suitable cavity fit for cyclodextrin complexation

Dose Accuracy: The Third Challenge

For HPAPIs dosed at microgram or sub-milligram levels, the analytical challenge of confirming content uniformity is significant. Standard HPLC-UV methods may lack the sensitivity needed at the concentrations involved. LC-MS/MS methods with lower limits of detection in the nanogram range are often required for content uniformity and dissolution testing of HPAPI drug products.

This analytical requirement must be built into the development plan early. A formulation team that spends six months developing an ASD strategy, then discovers the release method cannot measure the drug at the target dose, has a problem that is expensive to solve retrospectively.

Ardena’s HPAPI Formulation Capabilities at Pamplona

Ardena’s Pamplona (Idifarma) facility combines OEB 3, 4, and 5 containment capability with formulation development infrastructure for poorly soluble APIs, including HME, wet milling, and lipid-based formulation platforms. The analytical team at Pamplona develops high-sensitivity LC-MS/MS methods for content uniformity and dissolution testing of HPAPI drug products as an integrated part of the formulation development programme.

Two Mature Platforms, Two Different Profiles

Lipid nanoparticles and biodegradable polymeric nanoparticles are both well-established pharmaceutical delivery platforms with approved products and substantial clinical track records. LNPs are the platform behind the COVID-19 mRNA vaccines, liposomal doxorubicin, and a growing range of nucleic acid therapeutics. Polymeric nanoparticles, particularly those based on PLGA and PLA, have been approved in long-acting injectable formulations including the goserelin depot (Zoladex) and have a strong development pipeline across oncology, neurology, and infectious disease.

Choosing between them is not always straightforward. Both can encapsulate small molecule APIs and both can be engineered for sustained release. The decision depends on the physicochemical properties of the molecule, the intended route of administration, the target release profile, the regulatory pathway, and the manufacturing infrastructure available at the development partner.

Head-to-Head Platform Comparison

Factor	Lipid Nanoparticles (LNP)	PLGA Polymeric Nanoparticles
Primary payload types	Nucleic acids (mRNA, siRNA, DNA); small molecules; peptides	Small molecules; peptides; proteins; hydrophilic and hydrophobic drugs
Release profile	Typically rapid; burst release unless specifically engineered for sustained delivery	Tunable sustained release from days to months based on polymer MW and composition
Biodegradability	Lipid components metabolised via natural lipid pathways	Hydrolytic degradation to lactic acid and glycolic acid; natural metabolites
Stability in circulation	Ionisable LNPs designed for stability in blood; PEGylation extends circulation half-life	PLGA NPs stable in circulation; surface properties affect protein adsorption and clearance
Administration routes	IV (oncology, nucleic acid); IM (vaccines); potentially inhaled	IM/SC depot (sustained release); IV; potentially oral
Manufacturing technology	Microfluidics; extrusion; established at GMP scale	Nanoprecipitation; emulsion-solvent evaporation; established at GMP scale
Regulatory precedent	Strong: Onpattro (siRNA LNP), mRNA vaccines, Doxil (liposome)	Strong: Lupron Depot, Zoladex (PLGA microspheres); growing body for nanoparticles
Best suited for	Nucleic acid delivery; rapid onset injectables; vaccines	Sustained release injectables; small molecule payloads; depot formulations

When LNPs Win

If your payload is a nucleic acid, the case for an LNP platform is essentially settled. The ionisable lipid system is specifically designed to encapsulate negatively charged nucleic acids at high efficiency, protect them from enzymatic degradation in the extracellular environment, and facilitate endosomal escape after cellular uptake. No polymer nanoparticle system has demonstrated equivalent nucleic acid delivery efficiency in systemic applications.

For small molecule payloads that need rapid release, such as anti-cancer agents that need to achieve high intracellular concentrations quickly after uptake, LNPs also have an advantage over slowly degrading PLGA systems. The clinical success of liposomal doxorubicin (Doxil) in extending the circulation half-life of the drug while reducing cardiotoxicity relative to free doxorubicin is a demonstration of this principle.

When Polymeric Nanoparticles Win

For programmes where the clinical objective requires drug release over days, weeks, or months from a single injection, PLGA-based systems have an advantage that is difficult to replicate with LNPs. The tunable degradation rate of PLGA, combined with well-characterised manufacturing processes for microspheres and nanoparticles, makes it the platform of choice for depot injectable programmes where patient compliance with daily dosing is a limiting factor.

For small molecule payloads where stability in an aqueous lipid environment is a concern, encapsulation within a solid polymer matrix can offer better protection than an LNP core. Hydrophilic molecules that do not partition efficiently into the lipid phase of an LNP also tend to be better suited to polymer matrix systems that can encapsulate them within an aqueous core or adsorb them to a polymer surface.

Ardena’s Dual Platform Capability at Oss

Ardena’s nanomedicine team at Oss has development and GMP manufacturing capabilities for both LNP and polymeric nanoparticle platforms. The team can advise on platform selection based on the molecule’s physicochemical profile and the programme’s clinical and regulatory objectives, and can design feasibility studies that compare platform options before a full development programme is committed to a single technology.

Why Spray Drying Has a Thirty-Year Track Record in Pharma

Spray drying is not a new technology. It has been used in the food industry for over a century and in pharmaceutical manufacturing since the 1980s. What makes it particularly relevant to modern drug development is the growth of the BCS Class II molecule pipeline: drugs that are highly permeable but poorly soluble, and for which dissolution in the gastrointestinal fluid is the rate-limiting step in absorption.

For these molecules, converting the crystalline API into an amorphous form and dispersing it within a polymer matrix by spray drying provides a dissolution advantage that can increase oral bioavailability by two to tenfold or more, depending on the molecule and the formulation. The first spray-dried amorphous dispersion approved for a major indication was the HIV protease inhibitor ritonavir in Norvir capsules in the late 1990s. Since then, the technology has been applied to dozens of approved products across oncology, virology, and other therapeutic areas.

The Spray Drying Process: What Happens Inside the Dryer

In a spray drying process for amorphous solid dispersion (ASD) manufacture, the API and polymer are co-dissolved in an organic solvent, most commonly acetone, ethanol, methanol, dichloromethane, or a mixture of these. The solution is pumped to an atomiser, typically a two-fluid nozzle or a rotary atomiser, which breaks the solution into fine droplets that are sprayed into a heated drying chamber. Hot nitrogen or air flows through the chamber, evaporating the solvent from the droplets within milliseconds and leaving behind solid particles of the ASD.

The speed of solvent evaporation is what creates the amorphous state. The molecules do not have time to organise into a crystal lattice before the solvent is gone, so they are locked in a disordered, amorphous arrangement within the polymer matrix. The quality of that arrangement, specifically the degree of molecular dispersion of the drug within the polymer, depends on the polymer, the drug loading, and the processing conditions.

Key Process Parameters and Their Effects

Process Parameter	Effect on Spray-Dried Dispersion	Typical Optimisation Approach
Inlet temperature	Drives solvent evaporation rate; affects particle temperature and morphology	Optimise to achieve residual solvent below ICH Q3C limits without thermal degradation of API
Feed concentration	Higher concentration increases throughput; affects particle size and morphology	Balance between yield, particle size target, and solution viscosity
Atomisation rate and type	Controls droplet size and therefore particle size of the SDD	Nozzle type and pressure optimised for target particle size distribution
Outlet temperature	Indicates drying efficiency; must be above glass transition temperature of wet cake	Typically 10-15 degrees C above Tg of formulation; controlled by inlet temperature and feed rate
Nitrogen flow rate	Affects residence time in drying chamber; closed loop systems recover solvent	Matched to feed rate for consistent outlet temperature
Secondary drying	Removes residual solvent to below specification limits	Fluid bed drying or tray drying post-spray; temperature and time optimised by residual solvent measurement

Polymer Selection for Spray-Dried ASDs

The choice of polymer is the single most important formulation decision for a spray-dried ASD. The polymer must be miscible with the drug in the amorphous state, must dissolve in a common solvent with the drug, must form a rigid matrix with a sufficiently high Tg to prevent recrystallisation under storage conditions, and must release the drug appropriately when the dosage form contacts gastrointestinal fluid.

HPMC-AS (hypromellose acetate succinate) is the most widely used polymer for spray-dried ASDs in commercial products, offering good drug stabilisation, pH-dependent dissolution (releasing the drug in the upper small intestine at pH above approximately 5.5), and a well-established regulatory track record. PVPVA (polyvinylpyrrolidone-vinyl acetate) offers faster dissolution and is used where rapid drug release is needed. PVP-K grades and Eudragit systems are also used depending on the molecule and the target release profile.

From Spray Drying to Final Dosage Form

The spray-dried intermediate (the SDD powder) must be converted into a tablet or capsule for clinical and commercial use. The SDD typically has poor flow properties and low bulk density, which can make direct compression challenging. Granulation, either wet granulation or roller compaction, is often required to improve the powder properties before tabletting. The downstream processing step must preserve the particle size of the SDD and avoid conditions that would cause recrystallisation of the amorphous drug.

Ardena’s Spray Drying Capabilities

Ardena operates spray drying capability at its Somerset, New Jersey facility and at Pamplona (Idifarma) in Spain. Both sites provide spray drying for ASD development and GMP clinical manufacturing, with solvent handling systems, closed-loop nitrogen operation, and secondary drying capability. The formulation teams at these sites have developed spray-dried dispersions across a broad range of BCS Class II molecules and polymer systems.

Beyond the Simple Solution

The simplest injectable drug product is a solution: the API is fully dissolved in a compatible aqueous vehicle, the solution is sterile filtered, and it is filled into vials or prefilled syringes. A significant proportion of the injectable pipeline, however, cannot be formulated as simple solutions. Poorly water-soluble APIs, APIs that are unstable in solution, molecules that need to be targeted to specific tissues, and biologics that require depot-forming formulations for sustained release all require more complex formulation architectures.

Suspensions, emulsions, and liposomal systems are the three most clinically established complex injectable formulation platforms. Each offers distinct advantages for the right molecule, and each brings formulation challenges and manufacturing complexity that require specialist expertise to navigate.

Pharmaceutical Suspensions for Injection

A parenteral suspension consists of solid drug particles dispersed in an aqueous vehicle. Suspensions are used when the API has poor aqueous solubility, when a depot effect is desired (slowly dissolving drug particles at the injection site providing sustained release), or when the drug is more stable in solid form than in solution.

The critical quality attributes for injectable suspensions include particle size distribution (which affects both syringeability and dissolution rate at the injection site), resuspendability (the ability to achieve a uniform dispersion after settling with minimal shaking), viscosity (which must be low enough to allow injection through the intended needle gauge), and physical and chemical stability over shelf life.

Particle size control in injectable suspensions requires either micronisation of the API before suspension, or controlled crystallisation in the suspension medium. The final suspension must be sterilised, which for crystalline suspensions rules out sterile filtration and typically requires either terminal sterilisation (if the product is stable to autoclaving) or aseptic processing of each component followed by aseptic blending. ICH Q6A provides the quality standards framework for suspension characterisation.

Pharmaceutical Emulsions for Injection

Injectable emulsions are thermodynamically unstable two-phase systems in which one liquid is dispersed as droplets within another immiscible liquid, stabilised by an emulsifier. For pharmaceutical applications, oil-in-water (O/W) emulsions are most common, used to solubilise lipophilic APIs in the oil phase and deliver them via intravenous or intramuscular injection. Propofol, the widely used anaesthetic, is the most familiar example of an injectable O/W emulsion.

The critical attributes of injectable emulsions include droplet size (typically below 500 nanometres for intravenous emulsions, to prevent embolism risk), zeta potential (which governs colloidal stability), and drug distribution between the oil and aqueous phases (which affects dose uniformity). Physical stability, particularly resistance to droplet coalescence and Ostwald ripening over the product shelf life, is the primary formulation challenge and is addressed through emulsifier selection, oil phase composition, and processing conditions.

Liposomal Drug Products

Liposomes are spherical vesicles formed from phospholipid bilayers that enclose an aqueous core. The bilayer structure allows hydrophilic drugs to be encapsulated in the aqueous interior and lipophilic drugs to be incorporated into the bilayer membrane, making liposomes versatile carriers for a wide range of APIs. Commercially approved liposomal products include liposomal doxorubicin (Doxil/Caelyx), liposomal amphotericin B (AmBisome), and the COVID-19 mRNA vaccine lipid nanoparticles, which are closely related to classical liposomes.

Formulation Type	API Suitability	Key Stability Challenges	Primary Clinical Advantage
Injectable suspension	Poorly water-soluble crystalline APIs; sustained release depot	Particle size growth; physical instability; syringeability	Sustained release at injection site; suitable for low-solubility compounds
Injectable emulsion	Lipophilic APIs requiring IV or IM delivery; propofol-like molecules	Droplet coalescence; Ostwald ripening; API leakage from oil phase	Solubilisation of lipophilic APIs; IV administration possible
Conventional liposome	Hydrophilic APIs (aqueous core) or lipophilic APIs (bilayer)	Aggregation; drug leakage; phospholipid oxidation and hydrolysis	Extended circulation time; reduced systemic toxicity vs free drug
PEGylated liposome	As above; particularly cytotoxic oncology agents	As above; PEG shedding over time	Stealth effect; avoids rapid clearance by mononuclear phagocyte system
Targeted liposome	APIs requiring cell-specific delivery; ADC-like specificity	As above; ligand stability; targeting efficiency in vivo	Active targeting to tumour or specific cell type; improved therapeutic index

Manufacturing Considerations for Complex Injectables

Each of the formulation types described above requires a different manufacturing approach, and all share the requirement for aseptic processing to produce a sterile final product. Liposomal products are typically manufactured by thin-film hydration or microfluidics-based approaches, with size reduction by extrusion or high-pressure homogenisation and remote loading of the API after vesicle formation for ionisable drugs. Injectable emulsions are prepared by high-shear homogenisation followed by high-pressure homogenisation to achieve the target droplet size.

For all three formulation types, sterilisation is a particular challenge. The physical structures involved (crystals, droplets, vesicles) cannot withstand terminal steam sterilisation, so aseptic manufacturing, with the stringent environmental controls and validated processes that entails, is the standard approach.

Ardena’s Complex Injectable Capabilities at Ghent

Ardena’s development and manufacturing team in Ghent has formulation and manufacturing experience across injectable suspensions, emulsions, and liposomal products. The facility’s aseptic fill-finish capability supports GMP manufacturing of these complex dosage forms for clinical supply, with analytical support for the particle size, drug content, and stability testing required for regulatory filings.

Why Biologics Need Lyophilisation

Proteins, monoclonal antibodies, enzymes, and other biological molecules are inherently unstable in aqueous solution. In solution, they are subject to hydrolysis, oxidation, deamidation, aggregation, and physical denaturation, degradation pathways that can reduce potency, alter immunogenicity, and compromise the safety of the product. For biologics with an intended shelf life of two years or more, maintaining adequate chemical and physical stability in a liquid formulation is often not feasible without cold chain storage that is impractical for global distribution.

Lyophilisation, or freeze-drying, addresses this by removing water from the formulation while maintaining the structural integrity of the biologic molecule, converting the liquid drug product into a stable solid cake that can be stored at ambient or refrigerated temperature and reconstituted immediately before use. The process is technically demanding, time-consuming, and equipment-intensive, but for many biologics it is the only route to a commercially viable product.

The Three Phases of a Lyophilisation Cycle

Freezing

In the freezing phase, the liquid drug product in the vials is cooled to a temperature below the eutectic point or glass transition temperature of the formulation. The rate of freezing is critical. Slow freezing produces large ice crystals that create a coarser cake structure with good mass transfer properties during drying. Rapid freezing produces small ice crystals and a denser cake structure. Controlled nucleation, the induction of ice crystal formation at a defined temperature, is an emerging technique for improving freeze-drying cycle consistency and reducing vial-to-vial variability in cake structure.

Primary Drying

In primary drying, the chamber pressure is reduced and the shelf temperature is raised to supply the heat of sublimation, driving water directly from the frozen ice phase to vapour without passing through a liquid phase. The product temperature during primary drying must remain below the collapse temperature, which is typically close to the glass transition temperature of the maximally freeze-concentrated solution (Tg prime). Collapse, the loss of the porous cake structure, results in a product with reduced reconstitution performance and potentially altered potency.

Secondary Drying

After all ice has been sublimed, secondary drying removes the residual bound water from the amorphous matrix by desorption. The shelf temperature is raised to typically 20 to 40 degrees Celsius and held for several hours. The target residual moisture content for most lyophilised biologics is below 1%, as residual water acts as a plasticiser that reduces the Tg of the dried cake and increases molecular mobility, accelerating degradation.

Critical Formulation Components for Lyophilisation

Excipient Role	Common Examples	Function in the Lyophilisate
Bulking agent	Mannitol, glycine, sucrose	Provides physical structure to the cake; prevents collapse of the matrix during drying
Cryoprotectant	Sucrose, trehalose, sorbitol	Protects the protein during freezing by replacing water in the protein hydration shell; reduces denaturation
Lyoprotectant	Sucrose, trehalose	Protects the protein during drying; glass-forming excipients that immobilise the protein in a rigid amorphous matrix
Buffer	Histidine, citrate, phosphate	Maintains pH during reconstitution; some buffers crystallise during freezing and can cause pH shift; histidine preferred for many biologics
Surfactant	Polysorbate 20 or 80, poloxamer 188	Prevents protein aggregation at interfaces during freezing, drying, and reconstitution
Tonicity modifier	NaCl, mannitol, sucrose	Ensures reconstituted product is isotonic for injection

Cycle Development and Scale-Up Considerations

Lyophilisation cycle development begins at laboratory scale using a small-scale freeze-dryer with full data logging capability. The critical parameters to establish are the collapse temperature or Tg prime of the formulation, the primary drying shelf temperature and chamber pressure that keep the product below collapse temperature while maximising sublimation rate, and the secondary drying conditions that achieve the target residual moisture.

Scale-up from laboratory to GMP manufacturing scale is not straightforward. Heat and mass transfer characteristics differ between dryers of different sizes and designs, and the cycle parameters optimised at lab scale rarely transfer directly to a larger dryer without modification. Computational modelling approaches and process analytical technology including temperature probes (thermocouples), pressure rise testing, and near-infrared spectroscopy for real-time endpoint detection are increasingly used to facilitate scale-up and cycle transfer between facilities.

Ardena’s Lyophilisation Capabilities at Ghent

Ardena’s sterile manufacturing facility in Ghent operates lyophilisation capacity for both development and GMP clinical batch manufacture. The lyophilisation team has experience with small molecule, peptide, and biologic lyophilised products, and can support cycle development from initial formulation characterisation through to GMP-validated cycle execution.

Why Particle Size Matters for Oral Drug Absorption

The Noyes-Whitney equation, a cornerstone of pharmaceutical dissolution science, tells us that the rate of dissolution of a solid is proportional to its surface area. Reducing the particle size of a poorly soluble API increases the surface area exposed to the dissolution medium, which increases the dissolution rate, and for molecules where dissolution is the rate-limiting step in absorption, this translates directly into improved oral bioavailability.

Particle size reduction is one of the most widely used and well-understood strategies for addressing solubility challenges in oral drug products. It does not require a change in solid form, a new polymer system, or a significant change in formulation architecture. For many BCS Class II molecules, it can provide all the bioavailability improvement needed with a relatively straightforward manufacturing process.

The Three Primary Particle Size Reduction Technologies

Jet Milling (Micronisation)

Jet milling uses high-pressure streams of gas, typically nitrogen or compressed air, to accelerate drug particles to high velocities and drive them into collisions with each other and with the walls of the milling chamber. The result is a dry, crystalline powder with a particle size typically in the range of 1 to 20 micrometres.

Jet milling is well-established, scalable, and produces a product that is relatively easy to characterise and handle. It is the particle size reduction method of choice for potent compounds where containment during processing is critical, because the dry milling process can be conducted in a closed, contained system. However, it has limitations: it cannot reliably produce particles below about 1 micrometre, and it can cause polymorphic conversion or surface amorphisation if the API is thermally or mechanically sensitive.

Wet Milling (Media Milling)

Wet milling suspends the API in an aqueous medium with appropriate stabilisers and uses grinding media, typically ceramic or polymeric beads, to reduce particle size through attrition and impact. Wet milling can achieve smaller and more uniform particle sizes than jet milling, typically in the 200 nanometre to 5 micrometre range.

The stabiliser system used in wet milling is critical. The stabiliser prevents particle aggregation during milling and in the final suspension. Common stabilisers include HPMC, poloxamer, and polysorbate 20 or 80. The choice of stabiliser affects not only the physical stability of the suspension but also the dissolution performance of the final product.

Bottom-Up Precipitation (Nanosuspension by Anti-Solvent)

Rather than breaking down larger particles, bottom-up approaches start with a molecular solution of the API and induce controlled precipitation of nanoparticles by mixing with an anti-solvent. The particle size and distribution are controlled by mixing conditions, temperature, and the stabiliser system. Bottom-up approaches can achieve very small and uniform particle sizes but are more complex to scale and control than top-down methods.

Technology Comparison for Particle Size Reduction

Factor	Jet Milling	Wet Milling	Bottom-Up Precipitation
Achievable particle size	1-20 micrometres	200 nm – 5 micrometres	50-500 nm
Solid state of product	Crystalline (risk of surface amorphisation)	Crystalline	Variable; can be amorphous
Process type	Dry	Wet suspension	Wet suspension
Containment for HPAPIs	Excellent; closed system	Moderate	Moderate
Scale-up complexity	Established, well-understood	Established for pharmaceutical use	More complex; sensitive to mixing conditions
Regulatory precedent	Extensive	Extensive (Rapamune, Tricor precedents)	Growing
Key limitation	Cannot achieve sub-micron reliably	Stabiliser selection critical	Scale-up control challenging

Particle size ranges and characteristics above are representative of typical pharmaceutical processes and are drawn from published literature. Programme-specific outcomes depend on API properties and process conditions.

Choosing the Right Approach for Your Molecule

The selection of particle size reduction technology depends on several factors: the target particle size needed to achieve the desired dissolution enhancement, the physical and chemical stability of the API under milling conditions, the intended final dosage form, and the manufacturing scale at which the process will ultimately run.

For potent or cytotoxic compounds, the ability to conduct the entire process in a contained, dry environment makes jet milling the preferred first choice. For molecules where sub-micron particles are needed to achieve meaningful bioavailability improvement, wet milling or a combined approach may be required. A feasibility study comparing dissolution performance across particle size ranges is the most reliable way to establish the target specification and select the technology.

Ardena’s Particle Size Reduction Capabilities

Ardena’s Pamplona (Idifarma) facility has dedicated particle size reduction capabilities including jet milling for API micronisation, with containment suitable for high-potency compounds. The analytical teams at Ardena use laser diffraction and dynamic light scattering for particle size characterisation and dissolution testing in biorelevant media to assess the in vitro performance impact of particle size reduction.

Particle size reduction studies at Ardena are conducted alongside the broader formulation programme, ensuring that decisions about milling approach and target particle size are informed by dissolution data and downstream processability assessment rather than treated as a standalone analytical exercise.

The Stability Trade-Off at the Heart of ASD Formulation

The physicochemical advantage of an amorphous solid dispersion is real and well-documented. By eliminating the crystal lattice energy barrier, amorphous forms dissolve faster and achieve higher apparent solubility in biological fluids. For BCS Class II molecules with poor oral bioavailability, this translates directly into better clinical performance.

But amorphous forms pay for that advantage with thermodynamic instability. An amorphous solid is at a higher energy state than its crystalline counterpart. Given sufficient molecular mobility, the amorphous drug will tend to return to the crystalline state, losing the solubility advantage that made the formulation worthwhile. Managing this tendency, across the manufacturing process, the packaging, and the intended shelf life, is the central stability challenge of ASD development.

The Physics of Recrystallisation

Glass Transition Temperature (Tg)

The glass transition temperature is the single most important physical parameter for understanding the stability of an amorphous solid. Below the Tg, molecular mobility in the amorphous matrix is very low, and recrystallisation is kinetically inhibited. Above the Tg, mobility increases substantially, and the rate of crystallisation increases rapidly. For practical storage stability, the Tg of the amorphous drug or ASD should be significantly above the intended storage temperature. A rule of thumb used in the field is that the Tg should be at least 50 degrees Celsius above the storage temperature, though this is a guideline rather than a regulatory requirement.

The Effect of Moisture

Water acts as a plasticiser for amorphous solids, reducing the Tg and increasing molecular mobility. An amorphous drug that is stable under dry conditions may crystallise rapidly when exposed to elevated humidity. This makes moisture control during manufacturing and packaging critical. It also means that ICH stability studies at 75% relative humidity are a particularly challenging condition for amorphous formulations and an important test of the robustness of the stabilisation strategy.

Drug-Polymer Miscibility

In an amorphous solid dispersion, the drug must remain miscible with the polymer matrix throughout its shelf life. Phase separation, where the drug migrates out of the polymer matrix into drug-rich domains, accelerates recrystallisation. Drug-polymer miscibility is assessed using techniques including modulated DSC, solid-state NMR, and computational solubility parameter calculations during formulation development.

Stability Risk Assessment Framework

Risk Factor	Low Risk Indicator	High Risk Indicator	Mitigation
Tg of ASD	Greater than 80 degrees C at ICH storage conditions	Below 60 degrees C	Increase polymer content; use higher-Tg polymer
Moisture sensitivity	Tg reduction less than 10 degrees C at 75% RH	Tg reduction greater than 20 degrees C	Moisture-barrier packaging; desiccant; enteric coating
Drug-polymer miscibility	Single Tg observed; no phase separation by DSC	Two Tg events or crystalline peaks in XRPD	Reformulate with more compatible polymer
Recrystallisation tendency	No XRPD peaks after 6 months at 40 degrees C/75% RH	XRPD peaks within 1-3 months	Increase polymer ratio; add crystallisation inhibitor
Drug loading	Below 30% w/w	Above 50% w/w	Evaluate lower drug loading; consider alternative polymer

The thresholds above represent general guidance based on published ASD development literature and are not regulatory requirements. Programme-specific assessment is always required.

Analytical Tools for Monitoring Amorphous Stability

A stability monitoring programme for an ASD must be able to detect early-stage recrystallisation before it becomes analytically visible by standard release methods. The analytical toolkit for amorphous stability monitoring includes XRPD for detecting crystalline conversion, modulated DSC for measuring Tg and detecting phase separation, and dissolution testing using biorelevant media to capture changes in in vitro performance. Raman spectroscopy and solid-state NMR offer additional sensitivity for detecting early molecular-level changes.

How Ardena Manages ASD Stability

Ardena’s formulation teams at Somerset and Pamplona design ASD stability programmes that integrate physical stability monitoring with dissolution performance assessment from the earliest development batches. Stability studies are structured to generate data that supports regulatory filings under ICH Q1A(R2), with additional stress conditions designed to probe the specific failure modes relevant to the formulation.

The solid state research team in Ghent provides the advanced characterisation capabilities needed to understand drug-polymer interactions and phase behaviour, feeding data into the formulation decisions made at Somerset and Pamplona in a coordinated way.

The Solubility Problem in Modern Drug Development

Estimates from published literature suggest that more than 70% of new chemical entities in development pipelines have poor aqueous solubility. Many of these fall into BCS Class II (low solubility, high permeability) or Class IV (low solubility, low permeability) under the Biopharmaceutics Classification System. For these molecules, getting the drug into solution in the gastrointestinal tract fast enough and in sufficient concentration to achieve meaningful absorption is the central formulation challenge.

Amorphous solid dispersions (ASDs) address this challenge by converting the crystalline API into an amorphous form and dispersing it within a polymer matrix. The result is a solid that dissolves faster and achieves higher apparent solubility in the gut than the crystalline starting material.

The Science Behind ASDs

Why Amorphous Forms Dissolve Faster

Crystalline solids require energy to break down the lattice structure before molecules can enter solution. Amorphous forms lack this ordered lattice, meaning the activation energy for dissolution is lower. An amorphous API can achieve supersaturation concentrations in the gut, providing a higher driving force for absorption, provided the supersaturated state is maintained long enough for absorption to occur.

The Role of the Polymer

Without stabilisation, amorphous APIs will tend to recrystallise over time, losing their bioavailability advantage. The polymer matrix in an ASD serves two functions: it provides a physical barrier that inhibits molecular mobility and recrystallisation, and it can interact with drug molecules through hydrogen bonding or other non-covalent interactions that further stabilise the amorphous state. Common polymers used in ASD manufacture include HPMC-AS, PVPVA, and Eudragit systems, each with different dissolution and stabilisation profiles.

Supersaturation and Precipitation Inhibition

In the gastrointestinal environment, a well-designed ASD generates a supersaturated drug solution. Maintaining that supersaturation requires the polymer to act as a precipitation inhibitor, preventing the drug from recrystallising in the gut fluid before it can be absorbed. The selection of polymer type and drug-to-polymer ratio is therefore critical not just for solid-state stability during storage but for the in vivo performance of the dosage form.

ASD Technology Platforms

Technology	Principle	Best Suited For	Key Consideration
Spray drying	API and polymer dissolved in solvent, spray atomised and dried	High-throughput screening; scale-up to commercial	Solvent selection and residual solvent control
Hot melt extrusion (HME)	API and polymer melt-blended under heat and shear	Thermally stable APIs; solvent-free processing	API must be thermally stable at processing temperatures
Coprecipitation	Anti-solvent addition causes simultaneous precipitation	Research scale; proof of concept	Scale-up can be challenging
Electrospinning	High-voltage electric field creates nanofibre matrices	Very low dose, highly potent APIs	Complex scale-up; niche application

Development Considerations for ASD Programmes

Drug Loading

Higher drug loading reduces the tablet size needed to deliver the target dose, which is important for patient acceptability. However, higher drug loading typically increases the risk of recrystallisation during storage. The optimal drug loading is a balance between these competing requirements, established through screening studies that evaluate physical stability at relevant storage conditions.

Stability Testing

An ASD formulation must demonstrate physical stability over its intended shelf life. Stability studies following ICH Q1 guidelines, combined with accelerated stress testing and XRPD monitoring to detect any crystalline conversion, form the basis of the stability data package. The glass transition temperature of the dispersion is a key parameter: a high Tg relative to storage temperature provides a greater kinetic barrier to recrystallisation.

Downstream Processing

The ASD intermediate, whether produced by spray drying or HME, needs to be converted into a final dosage form. The hygroscopicity, flowability, and compactibility of the ASD intermediate determine what downstream processing approach is feasible. Some ASDs require granulation before tabletting; others can be directly compressed.

Ardena’s ASD Capabilities

Ardena has dedicated ASD manufacturing capabilities at its Somerset, New Jersey facility and at the Pamplona (Idifarma) site in Spain. Both sites operate spray drying and hot melt extrusion platforms with clinical and commercial-scale capability. The development teams at these sites work in close coordination with Ardena’s solid state research group, ensuring that the polymer and drug loading decisions made in screening translate directly into the development programme.

For BCS Class II or IV molecules that have shown limited bioavailability in early animal studies, a conversation with Ardena’s formulation scientists about ASD feasibility is a worthwhile investment before committing to a clinical formulation strategy.

Two Strategies for the Same Problem

Poor aqueous solubility is one of the most common challenges in small molecule drug development. A molecule that does not dissolve adequately in the gastrointestinal fluid cannot be absorbed at a rate sufficient to achieve the intended therapeutic effect, regardless of how potent it is in a biochemical assay. Two of the most widely used solid form strategies for addressing this problem are pharmaceutical salts and co-crystals. Both involve creating a multicomponent solid that modifies the physical properties of the API, but they do so through different mechanisms, and each has a different regulatory footprint.

What Is a Pharmaceutical Salt?

A pharmaceutical salt is formed when an ionisable API reacts with a pharmaceutically acceptable acid or base to form an ionic compound. The positive and negative charges of the salt components are held together by electrostatic interactions. Salt formation is only possible when the API has an ionisable group with a suitable pKa, and where the difference in pKa between the API and the counterion is sufficient to drive proton transfer. As a general rule, a pKa difference of at least two units is required for reliable salt formation.

Salts are regulatory well-established. They are classified as the same active ingredient as the free form in most jurisdictions, which simplifies the regulatory pathway. The FDA has published guidance on the pharmaceutical salts framework, and the EMA’s ICH Q6A guideline addresses solid state characterisation requirements.

What Is a Pharmaceutical Co-Crystal?

A co-crystal is a multicomponent crystalline solid in which the API and a coformer are held together in the crystal lattice by non-covalent interactions, typically hydrogen bonds, rather than ionic bonds. Because no proton transfer occurs, co-crystal formation is not limited to ionisable molecules. This makes co-crystals particularly valuable for neutral or weakly ionisable APIs where salt formation is not feasible.

The regulatory status of co-crystals has evolved significantly over the past decade. The FDA’s 2018 guidance on co-crystals classifies them as drug products containing a drug substance, meaning they follow a standard NDA or ANDA regulatory pathway rather than being treated as new chemical entities. The EMA has taken a broadly similar position.

Salt vs. Co-Crystal: A Practical Comparison

Factor	Salt	Co-Crystal
Applicable molecules	Ionisable APIs (pKa difference > 2 units)	Ionisable and non-ionisable APIs
Interaction type	Ionic (electrostatic)	Non-ionic (hydrogen bonds, pi-pi stacking)
Regulatory classification	Same active ingredient as free form	Drug product containing drug substance (FDA)
IP potential	Well-established, many precedents	Growing body of co-crystal patents
Solubility improvement	Can be significant; dependent on counterion	Variable; coformer selection is critical
Physical stability	Generally robust if polymorph is stable	Can be hygroscopic; humidity sensitivity must be assessed
Coformer options	Pharmaceutically acceptable acids/bases	GRAS substances, excipients, or other APIs

Choosing the Right Approach for Your Molecule

Start with Ionisability

The first question is whether the API is ionisable. If it has a basic nitrogen or an acidic group with a pKa in the right range, salt screening is typically the first strategy to evaluate. If the molecule is neutral or weakly ionisable, co-crystal screening becomes the primary option alongside other solubility-enhancing technologies.

Consider the Stability Requirements

Some salts are hygroscopic or unstable at elevated humidity, which creates challenges during manufacturing and storage. Co-crystals can also show humidity sensitivity depending on the coformer. Understanding the stability profile of candidate forms under conditions relevant to your intended market, using ICH storage conditions as a minimum, is essential before committing to a development form.

Factor in the IP Landscape

If the free form and common salts of your API are already in the prior art, a novel co-crystal form with demonstrated physicochemical advantages may offer a patentable differentiation. A solid state research team with experience in the IP dimensions of form screening can help structure the work to generate data that supports a patent filing.

Ardena’s Approach to Salt and Co-Crystal Screening

Ardena’s solid state research team in Ghent conducts both salt and co-crystal screening as part of pre-formulation development programmes. The screening process uses a design-of-experiments approach to evaluate a broad range of counterions and coformers, with XRPD, DSC, and solution NMR used to characterise and confirm the nature of each candidate form.

Screening results are evaluated against the full development context, including target dose, intended manufacturing process, and the regulatory and IP strategy, so that the form recommendation reflects not just the best physical chemistry but the best outcome for the programme as a whole.