Spray-freeze-drying in the manufacture of pharmaceuticals

Over the last decade, the development of new drug delivery methods and devices for dry powder inhalation1, needle-free intradermal powder injection2 or sustained parenteral drug delivery3 has led to an increasing demand for powder formulations incorporating an active pharmaceutical ingredient (API)4,5.

In contrast to the production and handling of powders for oral dosage forms, methods to prepare stable biopharmaceutical powders are limited due to the sensitivity of peptides and proteins to powder processing conditions4,6. Furthermore, bulk properties such as size distribution or density of the final particles are different depending on the application4,5,7. Particles for dry powder inhalation, for example, should have particles of less than 5 µm in diameter and a narrow size distribution5, whereas particles for needle-free ballistic injection must be 30-60µm with a density greater than 0.7 g/ml7 for a successful intradermal delivery. One of the most commonly used methods of drying protein formulations is freeze-drying8-11 but, because it does not involve droplet formulation, the final dry cake can only be reduced to particles by subsequent mechanical milling or grinding4. Some reported disadvantages associated with this method of powder manufacturing include the following:

  1. Production of particles with diameters above 1 mm
  2. Broad particle size distributions
  3. Changes of solid state and degradation of the peptide or protein due to heat generation during inter-particle collision12,13

Spray-drying, i.e. atomisation of a liquid solution or suspension into a cloud of fine droplets to rapidly evaporate the solvent and form particles, is now an established method for producing and processing stable protein powders14-16; even though sensitive peptides and proteins can become inactivated during the process, due to adsorption and denaturation at the air-liquid interface as well as exposure to heat during evaporation13. Further available production techniques are the supercritical fluid technology that has already demonstrated its utility in practice1,8,17.

Spray-freeze-drying is a relatively new biopharmaceutical powder production method with the ability to enhance the solubility of poorly soluble chemical compounds; it is a method which has been attracting increasing interest in the last seven years.

Spray-freeze-drying systems

Spray-freeze-drying generally involves:

  1. The atomisation of a liquid solution or suspension using one-fluid, two-fluid or ultrasonic nozzles to form droplets
  2. Rapid freezing of these droplets in cryogenic gas or liquid
  3. Sublimation of the frozen water to obtain the final dry particles.

The term Spray-freeze-drying covers different production methods:

  1. Spray-freezing into vapour over a liquid cryogen (SFV/L)
  2. Spray-freezing into liquid cryogen (SFL). Both techniques can be performed with subsequent vacuum freeze-drying (FD) or freeze-drying at atmospheric or sub-atmospheric pressure (ATMFD).

One of the first reports on spray-freeze-drying is dated 1948 and was performed by Benson and Ellis24 to investigate the surface area of protein particles spray-frozen in the vapour over liquid nitrogen with subsequent vacuum freeze-drying. This method, today referred to as spray-freezing into vapour over liquid cryogen (SFV/L), is one of the most commonly used spray-freeze-drying techniques. The liquid feed is atomised into the vapour over a cryogenic liquid, such as liquid nitrogen3,5,6 or liquid propane25 using either two-fluid or ultrasound nozzles. The droplets begin to freeze during the time of flight through the cold vapour phase and completely freeze upon contact with the cryogenic liquid phase itself3,6,19,26. The suspended frozen droplets can be collected by sieve separation or after allowing the cryogen to boil off. Different setups and container geometries have been described in literature to manage the collection of the frozen droplets during SFV/L3,5,18,25. Maa et al.6 used a three litre two-neck, round bottom flask full of liquid nitrogen and agitated with a magnetic stirrer. To ensure the system’s low temperature, the flask was submerged into a quenching bath filled with liquid nitrogen. The spray of high pressure air from the two-fluid nozzle resulted in a lowering of the liquid nitrogen level due to evaporation which was compensated by continuous addition of Liquid nitrogen to the flask. For the subsequent freeze-drying the contents of the flask were poured into metal trays6. Sonner et al.5 used a self-constructed laboratory SFV/L rig consisting of an ultrasound nozzle at a height of 100 mm above a circular metal bowl filled with liquid nitrogen. The bowl itself was placed onto a magnetic stirrer and the complete apparatus was covered to minimise loss of liquid nitrogen. After the spray-freezing process the bowl was topped up with liquid nitrogen and placed onto pre-chilled freeze dryer shelves. Costantino et al.19 used a two-fluid nozzle with gaseous nitrogen for atomisation into a stainless steel chamber. The liquid nitrogen for freezing of the droplets was delivered via four one-fluid nozzles at a pressure of 22 psig. The frozen slurry was collected in stainless steel beakers and for freeze-drying poured into glass dishes afterwards. To use standard freeze-drying vials in the freeze-drying step, Webb et al.26 transferred the liquid nitrogen suspended frozen droplets into a cooler filled with dry ice to allow the residual liquid nitrogen to boil off. The frozen droplets were then filled into pre-cooled freeze-drying vials using a pre-cooled plastic spoon and tongs. An improved laboratory scale construction for SFV/L with transfer of the suspended frozen droplets into 20 ml standard freeze-drying vials was introduced by Gieseler25 in 2004. He used a separating funnel type bowl with a diameter of 220 mm that was filled with the cryogenic liquid via a curved copper tube at the bottom of the funnel. This ensured a constant flux of liquid nitrogen and movement of the frozen droplets avoiding the shear stress of a magnetic stirrer bar. The vials were connected tightly at the end of the bowl and immersed into a liquid nitrogen chilling bath. Transfer of the frozen droplets into standard freeze-drying vials enabled him to monitor product temperature and sublimation rate during the subsequent freeze-drying cycle by placing thermocouples in the vials26 or using a freeze-drying microbalance25. After the freezing and collection procedure, the respective containers containing the frozen droplets were placed on pre-chilled shelves between -45°C and -50°C and lyophilized by an empirical freeze-drying cycle. The two shortest cycles found in the literature were 16 hours 27 and 50 hours 5 in total, whereas the typical total lyophilization cycle time was 7-10 days19.

As an alternative to vacuum freeze-drying to sublime the frozen water, equipment for SFV/L with subsequent freeze-drying at atmospheric or sub-atmospheric pressures was developed28-31. Leuenberger et al.29 constructed a spray-freeze fluidised bed dryer and compared the drying characteristics of the spray-frozen droplets in the new process equipment to classical freeze-drying. The fluidised-bed drying showed short drying times and allowed advanced product particle shape and uniformity. Identified problems were the low yield in the primary drying phase and strong electrostatic effects during secondary drying29. Wang et al.31 developed a setup combining the spray-freezing step and fluidisation conveying using co-current flow to convey the frozen droplet powder to the exit filter. This modification overcame the difficulty of fluidising and elutriating cohesive frozen powder from a substrate and allowed completion of the drying process in 1-2 hours31.

Spray-freezing into liquid (SFL) is a fairly new atomisation and particle engineering technology in which the liquid feed, either an aqueous or an aqueous-organic cosolvent solution containing an active pharmaceutical ingredient (API) and formulation excipients, is atomised beneath the surface of a compressed liquid such as compressed fluid CO2, helium, propane, ethane, or the cryogenic liquids nitrogen, argon, or hydrofluoroethers32,33. Nitrogen is the cryogen of choice, because of its inexpensive, environmentally friendly and inert properties32. Two different SFL set-ups have been described by Rogers et al.21,33:

  1. A laboratory scale construction (solution volume < 50 ml) using an insulated one-fluid PEEK (polyether-ether ketone) tubing nozzle with an inner diameter of 63.5 µm and a length of 100 mm at 34.5 MPa constant pressure achieved by using a high pressure syringe pump and a solution cell (2) A pilot scale setup (solution volume > 50 ml) with an insulated one-fluid PEEK nozzle of 127 µm inner diameter with a length of 150 mm at 13.4 MPa constant pressure created by a HPLC pump for liquid delivery

The liquid-liquid impingement occurring as the two fluids collide, resulted in intense atomisation into fine microdroplets that freeze immediately32. The frozen suspended droplets were then transferred into 300 ml beakers and placed onto pre-cooled shelves (-80°C) to allow the liquid nitrogen to boil off34, or collected by sieve separation using a 150-mesh sieve21,33. The subsequent freeze-drying process took 52 hours34,35 to 72° hours22 to obtain the final powder product. Rogers et al.22 successfully used atmospheric freeze-drying (ATMFD) to increase mass transfer rates in sublimation in combination with SFL.

SFD product characteristics

One of the first studies by Maa et al.6 postulated the superior aerosol performance of the light and porous SFV/L particles in comparison to powder produced by spray-drying. Under the same atomisation conditions, SFV/L particles were larger, less dense and had an approximately 40 times larger surface area than the particles obtained from spray-drying6. The fine particle fraction of the spray-freeze-dried powder was better than that of spray-dried powder, attributed to better aerodynamic properties6. Effects of atomisation conditions and formulation variable on the particle size and stability were investigate by Costantino et al.3,19, delineating that the particle size was inversely related to the specific surface area and the amount of bovine serum albumin (BSA) aggregates formed. Trypsinogen was found to lose approximately 15% of its initial enzyme activity upon SFV/L with only marginal aggregation of 1.4%5. The origin of the trypsinogen inactivation could not be related to adsorption of the protein at air / liquid interfaces or the quench-freezing of the liquid droplets and must therefore have been the subsequent freeze-drying step5. In contrast to this result, Webb et al.26 showed adsorption of recombinant human interferon-g (rhIFN-g) at the air/liquid and ice liquid interfaces. The adsorption of rhIFN-g at the air/liquid interface was in the order of 4.5 mg/m2 with a surface area of 0.12 m2/g for the solution (50 µm droplets) and four times larger than at the ice/liquid interface26. The loss of protein was approximately 12% for the 5 mg/ml initial solution which was consistent with the measured amount or protein at the surface of the rhIFN-g powder26,34. Addition of 0.12% polysorbate20 reduced protein surface adsorption and decreased but could not completely prevent rhIFN-g aggregation26. Yu et al.34 extensively investigated the process stability of lysozyme in SFV/L compared to SFL. Both processes produced highly porous aggregates of protein particles34. Powders produced via the SFL process had a smaller degree of protein aggregation and smaller losses in enzyme activity than powders manufactured by SFV/L, primarily due to the smaller surface excess of lysozyme in SFL than in SFV/L during the atomisation34. Droplets in SFV/L start freezing during the time of flight through the cryogenic vapour phase and completely freeze upon contact with liquid phase of the cryogenic substance32. The particle morphology is not fixed until the droplets are fully solidified and therefore two important factors can have an unfavourable influence on the size distribution as the atomised droplets pass through the vapour gap over the cryogenic liquid: (1) collision and coalescence of the droplets and (2) solutes may precipitate and grow in the unfrozen regions of the droplets32. Based on previous reports, the fastest cooling rates can be achieved by immersing the liquid feed directly into the cryogen36. The maximum cooling rates achievable with liquid nitrogen are in the order of 103 K/s at a droplets size of about 10 µm37. Rapid cooling rates have the potential to minimise the formation of ice nuclei and therefore inhibit ice crystal growth12 as well as leading to the formation of a glassy structure before the protein undergoes aggregation in the freeze-concentrated solution or has time to adsorb to an interface with ice or glassy water34,38. Formation of an amorphous structure might also retard relaxation processes that could lead to the denaturation of the processed peptide or protein39,40. Independent of instabilities caused by interface adsorption, residual stress in the glass during the lyophilization cycle may have a negative influence on the peptide or protein stability41. SFL technology benefits from the liquid-liquid impingement that occurs as the two fluids collide – high Reynolds and Weber numbers lead to intense atomisation into micronised droplets directly inside the liquid cryogen, avoiding mechanisms of droplet / particle growth in the vapour phase as for SFV/L32,33.

Numerous recent investigations have demonstrated that SFV/L20 and particularly SFL21,22,32 may be the processes of choice if you want to produce powders of poorly water soluble pharmaceutical compounds with superior wetting and enhance dissolution properties owing to their amorphous character32,33. Rogers et al.21 demonstrated that SFL generated inclusion complexes of danazol / hydroxypropyl-b-cyclodextrin that dissolved faster and to a higher extent compared to those formed by conventional techniques, such as co-grinding or slow freezing. Similar results were presented by Hu et al.32 showing that their Danazol / Poloxamer 407 complex prepared by SFL dissolved to 99% within ten minutes, whereas the dissolution of the physical mixture was only 48% in one hour. More recently, Hu et al.42 developed an organic system to further enhance the dissolution properties of particles produced by SFL. The final high potency SFL powders from the danazol / PVP K-15 mixture in acetonitrile showed 95% danazol dissolution in only two minutes43. Pharmacokinetic in vivo analysis of these SFL danazol formulations showed an increased oral bioavailability and higher area under curve (AUC) values than the commercial product or crystalline bulk danazol44. Nebulised formulations of itraconazol produced by SFL also demonstrated high bioavailability and high efficacy against fungal infection in vivo, leading to extended survival rates of the animals tested44. At least Rogers et al.23 applied an o/w-emulsion system to produce enhanced dissolving SFL powders of danazol. They found that the micronised powders produced from emulsions had similar dissolution enhancement as the powders produced from solutions, but higher contents of API could be processed by SFL from emulsions.

Application of SFD

According to Leuenberger20, the spray-freeze-drying process is the method of choice if the following product properties are required:

  1. A porous product structure with a high specific surface area
  2. A free-flowing powder for use as final or intermediate product
  3. Improvement of bioavailability of extremely low water soluble compounds

The enhancement of dissolution rates and, thus, bioavailability of poorly water soluble APIs via SFL has been discussed in detail above. In one of the first studies Maa et al.6 successfully used SFV/L to produce protein powders containing recombinant human desoxribonuclease (rhDNase) and anti-IgG monoclonal antibody (anti-IgG Mab) for dry powder inhalation. SFV/L produced larger (~8-10 µm) and more porous particles compared to the small (~3 µm), dense particles obtained from spray-drying using the same atomisation conditions. The significantly better fine particle fraction (FPF) of the powder produced by SFV/L compared to spray-drying attributed to better aerodynamic properties6 and confirmed the concept of porous particles giving low aerodynamic size1. To develop a dry powder formulation for the inhalation of cetrorelix, Zijlstra et al.45 compared different particle engineering techniques and their influence on the powder properties for inhalation. They managed to produce particles similar to Maa et al.6 and Costantino et al.19 in terms of size, surface area and porosity. However, the FPF and the performance of the SFV/L particles were lowest compared to spray-drying and milling45. Zijlstra et al.45 ascribed these results to the porous and fragile particle structure that cannot withstand the production process of an adhesive mixture when spray-freeze-dried cetrorelix particles are exposed to impact forces from colliding lactose. The fragments from the crushed cetrorelix particles attached to the carrier and, once attached, were very difficult to separate again. The result was a relatively low separation explaining the low FPF of 25% during cascade impactor analysis compared to 66% for spray-drying45. Van Drooge et al46 discovered SFV/L from a water-tertial butanol mixture as a suitable process to include lipophilic drugs such as tetrahydrocannabinol (THC) in inulin glass matrixes to produce powder suitable for inhalation. THC was more effectively stabilised by SFV/L than by slow freeze-drying and FPF of up to 50% were generated by SFV/L indicating suitability for inhalation46. Costantino et al.3 and Wang et al.47 used SFV/L particles as intermediate porous product with high surface area for subsequent microencapsulation into biodegradable poly(lactide-co-glycolide) (PLGA) microspheres. Wang et al.47 managed to retain over 90% of the IgG integrity after SFV/L when the formulation was stabilised with mannitol and trehalose. The produced IgG microparticles were afterwards successfully microencapsulated into PLGA using a solid-in-oil-in-water encapsulation procedure47. In 2004, Maa et al.7 published a study developing a SFV/L process for preparing an influenza vaccine dry powder formulation for epidermal powder immunisation using needle-free ballistic delivery. They managed to reformulate a commercial flu vaccine by spray-freeze-drying a high concentrated liquid solution (35%wt) containing trehalose, mannitol and dextran (10kDa) to obtain particles containing 45µg vaccine per mg powder for successful ballistic delivery. In contrast to the small and highly porous particles with tap densities as low as 0.01 g/ml – ideal for application such as the already discussed dissolution enhancement of low water soluble drugs or pulmonary delivery6,7 – the particles for a successful needle-free ballistic injection had a median volume diameter between 30-60 µm and a tap density of approximately 0.7 g/ml7. The mentioned SFV/L powder formulation was furthermore successfully tested preclinically in Phase I human clinical trials7. Recent studies27 used trehalose, mannitol and hydroxyethyl starch as non-animal substitute for dextran to formulate suitable powders for needle-free ballistic injection of catalase. In the area of nanoparticle techniques, Leach et al.48 prepared nanostructured protein microparticles using SFL procedure with subsequent brake-up of these highly porous and friable protein particle aggregates into submicron particles. The obtained nanoparticles were then encapsulated into PLGA by an anhydrous solid-in-oil-in-oil (s/o/o) technique. Size exclusion chromatography indicated only a slight loss in BSA monomer of 3.9% in the encapsulated protein.


The suitability and selection of a specific particle and powder formation process relies on the need for specific applications1,4. The criteria for evaluation are particle size and size distribution, powder flowability, process efficiency and yield, scalability, long-term powder physical stability and long-term powder biochemical stability4. Spray-freeze drying has shown to be a feasible method if good particle size control, spherical particle shape and a high product yield are essential4,5. Furthermore, it may be the process of choice for improving the bioavailability of low water soluble pharmaceutical compounds20.


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