Freeze Drying vs. Spray Drying Analysis

Research will be limited to:
- Process: Freeze Drying
- Process: Spray Drying
- Product: Dry Powder Formulation
- Analyte: Protein Stability
- Metric: Scalability

Strategy: I will conduct a comparative analysis focusing on the trade-offs between freeze-drying (lyophilization) and spray-drying for protein therapeutics. The research will specifically target empirical data regarding secondary structure preservation (stability), particle engineering (morphology), throughput/energy consumption (scalability), and total cost of goods (COGs). I will prioritize recent reviews and comparative experimental studies that provide quantitative metrics for these parameters.
Queries:
- freeze drying vs spray drying protein stability secondary structure comparison
- spray drying vs lyophilization protein powder morphology particle size distribution
- scalability and cost analysis freeze drying vs spray drying biopharmaceuticals
- protein stability dry powder formulation spray drying vs freeze drying review 2020..2026
- techno-economic assessment spray drying vs lyophilization protein therapeutics

Executing 5 concurrent searches:
- freeze drying vs spray drying protein stability secondary structure comparison
- spray drying vs lyophilization protein powder morphology particle size distribution
- scalability and cost analysis freeze drying vs spray drying biopharmaceuticals
- protein stability dry powder formulation spray drying vs freeze drying review 2020..2026
- techno-economic assessment spray drying vs lyophilization protein therapeutics

Found 9 papers. Key excerpts:

Radio Frequency - Assisted Ultrasonic Spray Freeze Drying for Pharmaceutical Protein Solids. — Tarun Tejasvi Mutukuri et al., 2023

Biopharmaceutical products have an intrinsic instability in their solution state and are often subjected to drying using lyophilization or spray drying 1 Stability of the solid biopharmaceutical products often relies on the selection of excipients and the ratio of excipients along with the drying methods 1 Lyophilization is the most widely used drying process for biopharmaceutical products. 2 , 3 , 4 Despite the recent advancements in lyophilization, the process remains energy- and time-intensive due to the various steps involved 5 The lyophilization process also typically results in drug product formulations that are cake-like solids unsuitable for needle-free ballistic injections or inhalation therapies. 6 , 7 Recently, the use of microwave technology to reduce the drying duration of biopharmaceuticals has gained interest. 8 , 9 , 10 , 11 The shorter drying cycle time due to the increased heat transfer by microwaves makes it an attractive technology to incorporate into the lyophilization process.
There are other processes, such as spray drying, that can produce solid formulations with desired particle characteristics. 12 , 13 However, the high-temperature stresses generated during the spray drying process may have a negative impact on the stability of some proteins. 14 , 15 Spray freeze drying (SFD) is another technique that can be used to engineer particles without heat stress. 16 , 17 , 18 In the SFD process, the feed solution is atomized using a nozzle and the droplets are frozen using a cryogenic medium. These frozen droplets are then transferred into a lyophilizer where they are subjected to primary and secondary drying cycles to obtain the final powder product. The SFD process has been previously studied to produce several pharmaceutical products such as vaccines, 19 , 20 , 21 solid dispersions 22 and nanoparticles 23 A recent study by Adali et al. 24 discussed the impact of the processing parameters on the physico-chemical propertirs of spray freeze dried particles. In our previous study 17 we observed that spray freeze drying resulted in similar physical stability to freeze-drying for lysozyme and myoglobin when formulated with a stabilizing excipient such as sucrose.
In this study, we evaluate the application of the novel RF-assisted drying technology based on statistical electromagnetics 25 , 26 to spray freeze dry the protein formulation. The application of RF technology has shown to reduce the primary drying cycle times without effecting the structure of the protein 25 The

reduction in the primary drying cycle time is due to the conventional lyophilization heating coupled with the RF-assisted heating which distributes the energy volumetrically at a molecular level, targeting lossy dielectric materials, such as ice. The choice of the RF frequency has a large impact on the primary drying time since, for ice, higher frequency results in a higher loss factor and hence, higher absorbed power, resulting in a significant speed-up in the primary drying time. It is known that at higher frequencies, the loss factor of ice increases 27 The total absorbed power by ice is determined using: 28 (1) P a b s o r b e d = 2 π f ϵ 0 ϵ ″ ( f ) | E | 2 where f is the frequency of operation, ϵ 0 represents the free-space permittivity, ϵ ″ is the loss factor of the dielectric, and E is the electric field intensity due to the RF source.
A model protein, Bovine Serum Albumin (BSA), was formulated with varying levels of trehalose and mannitol to study the impact of RF-assisted drying on the physical stability of the protein solids. Various ratios of trehalose dihydrate and mannitol (with no excipient formulation as control) are chosen in this study because trehalose is a widely used stabilizing excipient and mannitol is widely used as an excipient in spray freeze drying to improve flowability. Different trehalose to mannitol ratios were selected to understand their impact on the stability of the proteins. The dried formulations were then characterized using powder X-ray diffraction (PXRD), BET surface area measurements, particle size distribution, particle morphology, and solid-state hydrogen/deuterium exchange (ssHDX-MS). Physical stability studies were performed by storing the samples at 40°C for 3 months and then determining the loss in monomer content using size exclusion chromatography (SEC).
A BSA formulation (containing 20 mg/ml of protein) with varying concentrations of trehalose dihydrate and/or mannitol as well as the excipient-free formulation with a potassium phosphate buffer (pH 6.8, 2.5 mM concentration) as shown in Table 1 was prepared and filtered using a 0.22 µm filter. Bovine Serum Albumin (BSA), trehalose

dihydrate, mannitol, potassium phosphate monobasic, and potassium phosphate dibasic were purchased from Millipore Sigma (St. Louis, MO, USA). To maintain the RH at 11% in the desiccator for ssHDX studies, a supersaturated solution of lithium chloride (Thermo Fisher Scientific, Waltham, MA, USA) in D 2 O (Cambridge Isotope Laboratories, Inc., Andover, MA, USA) was used.
An ultrasonic nozzle (Büchi, New Castle, DE) was used to spray the solution into a 250 ml borosilicate glass beaker containing liquid nitrogen at the flow rate of 3 ml/min . Upon evaporation of the liquid nitrogen, the frozen particles were transferred to 20 ml borosilicate vials each with a 50% fill volume. The frozen samples were then dried by placing the 20 ml 2R borosilicate glass vials in a Revo® laboratory-scale lyophilizer (MillRock Technology, Kingston, NY). The shelf temperature was maintained at -35 ° C until the vials were loaded onto the shelf. The vials were then equilibrated for 30 min at -35 ° C. The primary drying cycle was initiated by decreasing the chamber pressure to 100 mTorr for 1h and ramping the temperature to -20 ° C for another hour. The temperature was held at -20 ° C for 48h. The time of 48h was set as the end point for the primary drying cycle step for the SFD samples based on previous drying cycles developed for similar formulations. 29 The process data (Fig. S1) obtained during the cycle development supported the selection of 48h as the end point for the primary drying cycle. The pressure of 100 mTorr was maintained in the chamber and the temperature was raised at 0.15 ° C/min to 25 ° C and held for 10h during the secondary drying.
A Radio Frequency-assisted heating technique was used on the Revo® laboratory-scale lyophilizer (MillRock Technology, Kingston, NY) to perform RF-assisted drying of material prepared by ultrasonic spray freezing. The experimental setup, shown in Fig. 1 , comprises five main components: an auxiliary chamber (AC), a signal generator, a power amplifier, a transmitting antenna, and stirrers controlled by stepper motors. The AC is a metallic box that contains

The physicochemical properties, functionality, and digestibility of hempseed protein isolate as impacted by spray drying and freeze drying — Xuan Dong et al., 2024

Protein from hempseed ( Cannabis sativa L.) is considered a sustainable and nutritious alternative protein source. As a plant protein, the production of hempseed protein loads fewer environmental burdens than animal-derived proteins, particularly in reducing water and land resource demands and lessening greenhouse gas emissions ( Poore & Nemecek, 2018 ). From a nutritional standpoint, hempseed protein isolate (HPI) encompasses a complete profile of all the essential amino acids required by adults according to FAO/WHO standards and presents potential bioactive peptides after digestion ( Tang et al., 2006 , Mamone et al., 2019 ). In addition, HPI has been reported for its superior digestibility and lower allergenicity compared to widely accepted soy protein isolate ( Wang et al., 2008 , Mamone et al., 2019 ). Therefore, HPI is an excellent potential protein ingredient to be applied to food systems to add nutritional value and promote environmental sustainability.
Before incorporating protein into different food matrixes, it is essential to comprehend their physicochemical properties and functionalities. Previous studies have shed light on the impact of the production process on HPI, including dehulling, defatting, and extraction methods. Specifically, seed dehulling increased HPI extraction yield ( Shen et al., 2020 , Shen et al., 2020 ); Folch defatting ( Shen, Gao, Xu, Rao, et al., 2020 ), or micellisation extraction ( Hadnađev et al., 2018 ) improved the HPI thermal properties; alkaline extraction followed by acid precipitation, the most common HPI extraction method, resulted in a higher water holding capacity but a lower emulsifying activity than salt-extracted HPI ( Hadnađev et al., 2018 , Fang et al., 2023 ). In addition to protein extraction, dehydration is a necessary step to prolong the shelf life of HPI. Although one previous study investigated freeze, oven, and vacuum oven drying methods on the co-product during HPI extraction ( Lin et al., 2021 ), the influence of drying methods on HPI has not been thoroughly investigated.
Various drying methods are available in the food industry for drying different food ingredients, such as basic hot air drying, oven drying, vacuum drying, freeze drying, and spray drying. Although there is no standardised drying method for HPI production, freeze drying (FD) was the most selected method in previous HPI studies ( Hadnađev et al

., 2018 , Malomo and Aluko, 2015 , Potin et al., 2019 , Shen et al., 2020 , Shen et al., 2020 , Tang et al., 2006 , Fang et al., 2023 ). The benefit of using FD for HPI production is to minimise protein heat damage during dehydration. However, it is a time-consuming and expensive batch process. On the other hand, it is well known that spray drying (SD) is one of the most popular drying methods in the food industry, especially for milk and protein powder production. SD offers advantages over other drying techniques, particularly in rapidly producing uniform powders without an additional milling step. Nonetheless, the thermal energy involved in the SD process may potentially damage heat-sensitive nutrients during production. Given the unique advantages and considerations of both FD and SD, exploring the impact of these two drying methods on HPI is worthwhile.
Therefore, this study aimed to investigate the influence of freeze drying and spray drying on the physicochemical properties, functionality and digestibility of HPI. To the best of our knowledge, there has not been any comparative study investigating the impact of the drying method on the properties and digestibility of HPI. Hence, this study aimed to fill the research gaps and provide a better fundamental understanding of the above areas.
Dehulled hempseed was kindly provided by Hemp Farm New Zealand Limited (Tauranga, New Zealand). Petroleum ether was purchased from Avantor, Inc. (Radnor Township, Pennsylvania, United States). Bovine serum albumin was obtained from MP Biomedicals New Zealand Ltd. (Auckland, New Zealand). Amino acid standards mixture, Bradford reagent, β-mercaptoethanol, bile extract porcine, haemoglobin, isotope-labelled amino acid mix solution, l -methionine, o -phthalaldehyde, porcine pepsin, porcine pancreatin, p-toluene-sulfonyl- l -arginine methyl ester, and sodium dodecyl sulphate were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Sodium tetraborate was acquired from BDH Chemicals Ltd. (Poole, England). Ammonium carbonate, calcium chloride, magnesium chloride hexahydrate, potassium chloride, potassium dihydrogen phosphate, sodium chloride, sodium hydrogen carbonate, and sodium hydroxide were obtained

from ECP Ltd. (Auckland, New Zealand). Hydrochloric acid was purchased from J.T. Baker (Phillipsburg, New Jersey, USA). The following chemicals were obtained from Bio-Rad Laboratories, Inc. (Hercules, California, USA): Laemmli sample buffer, Tris/Glycine/SDS buffer, protein standards, and Coomassie brilliant blue R-250 staining and destaining solutions. Soybean oil was purchased from a local supermarket.
The HPI preparation process was based on method by Tang et al. (2006) with modifications. Dehulled hempseed was ground by an ultra-fast crush grinder (800Y, Huangcheng, Wuyi, China) before oil extraction. The oil was extracted twice from the ground hempseed using petroleum ether (PE) at a 1:10 (w/v) ratio for two hours with agitation. PE was chosen for defatting due to its low boiling point and ease of separation from the hempseed meal. The meal was separated from PE using vacuum filtration and thoroughly dried under nitrogen gas. After drying, the meal was ground as fine powders and stored at room temperature for further use. For protein extraction, hempseed meal was mixed with 20-fold (w/v) of pH 12 distilled water (pH pre-adjusted by 1 M NaOH) and stirred for 30 min at 35 °C. The final pH after the 30-min extraction was around 8.5. The samples were centrifuged at 8000×g for 20 min at 20 °C. The supernatant was adjusted to its isoelectric point (pH 5) with 1 M HCl. The isoelectric point was predetermined by measuring supernatant protein content after adjusting the pH of the alkaline extracted protein solution from 3.5 to 6 with a 0.5 increment. Then, the precipitated sample was collected by centrifuging at 8000×g for 20 min at 4 °C. The sample collected was labelled as the undried-HPI.
The hempseed protein isolate (from Section 2.2 ) was resuspended in water at a 1:4 (w/v) ratio, followed by homogenisation for 2 mins. The pH was adjusted to 7 with 1 M NaOH before drying. For freeze drying (FD

Effect of Methyl-β-Cyclodextrin and Trehalose on the Freeze-Drying and Spray-Drying of Sericin for Cosmetic Purposes. — Lorella Giovannelli et al., 2021

Proteins are considered to be suitable ingredients in the biomedical and cosmetic fields and are used in a variety of skin–care formulations. Thanks to their ability to bind water, proteins are characterized by film–forming properties that provide a smooth appearance and a feeling of softness upon the skin [ 1 , 2 , 3 ].
Proteins usually undergo drying via either freeze–drying (lyophilization) or spray–drying techniques, which are processes extensively employed to remove water from pharmaceuticals, biopharmaceuticals and foods, leading to an increase in their stability and shelf life. Both rhGH, which is the recombinant human growth hormone that is used to treat hypopituitary dwarfism, and insulin for inhalation have been converted into powder by lyophilization [ 4 ]. Spray–drying has been used to process immunoglobulin G (IgG) [ 5 ] and cosmetic proteins, such as hydrolyzed collagen [ 6 ].
During the removal of water, the complex protein structure is prone to collapse, causing denaturation. To preserve the original structure and therefore the properties of these molecules, it is necessary to avoid structural changes during drying. Carrier agents such as trehalose and sucrose allow the protein to be maintained by replacing the hydrogen bonds throughout spray–drying [ 7 ]. This “water replacement” theory gains even more importance in the freeze–drying process, in which lyoprotectants, such as polyols, mono–, di– and polysaccharides, are frequently used as functional process agents, due to their hydroxyl groups. Besides this theory, other hypotheses such as vitrification and water entrapment have been proposed to explain the stabilizing and preserving effect of those excipients. The vitrification theory states that during freeze–drying the protein is immobilized in a rigid, glassy sugar matrix and therefore the degradation drastically slows down, as the protein is unable to unfold thanks to its reduced molecular mobility. On the other hand, the water entrapment is based on the concept that excipients are able to form a cage around the protein, entrapping water molecules. All these theories result in stabilizing and preserving the structure of the protein [ 8 , 9 , 10 , 11 , 12 , 13 ]. Moreover, some of these substances, such as trehalose, perform the additional function of moisturizers in skin and hair–care formulations [ 14

].
The freeze–drying and spray–drying techniques have also been used to dry sericin, a globular glycoprotein characterized by low stability during storage in the liquid state [ 8 , 15 ]. Sericin is a natural hydrophilic and gelatinous protein produced by the silkworm Bombyx mori . Its main role is binding the two fibroin filaments together, granting structural integrity to the cocoon. In this way, sericin can effectively protect the silkworm from the external environment during its pupal stage [ 16 ]. Sericin’s molecular weight varies from 10–20 kDa to 310–400 kDa; it consists of three different polypeptides (sericin A, B and C), which are distinguished by their position in the cocoon shell (from the outermost to the innermost layer, respectively) and their composition and solubility in hot water. Upon cooling, sericin shows sol–gel properties due to its conversion from the random coil to the β–sheet form [ 17 ].
Fibroin and sericin are the two main components that form raw silk fibers. In the textile industry, the fibroin fibers are extracted by detaching sericin via degumming procedures [ 16 , 18 ]. The removed sericin has been considered a wastewater product for many years [ 19 ]. In recent decades, sustainability and the circular–economy concept have acquired an increasingly important role in manufacturing processes and consumer culture [ 20 ]. A great deal of effort has been made to recover this protein, and numerous studies have demonstrated the intrinsic biological properties of sericin [ 21 , 22 , 23 ]. Its biocompatibility, biodegradability and the absence of skin–sensitizing or skin–irritating potential have meant that sericin is used in the pharmaceutical, cosmetic and food fields. Indeed, numerous studies have demonstrated its antioxidant, anti–aging, photoprotective, antibacterial, antiproliferative and immunomodulant properties [ 24 , 25 , 26 , 27 ]. In cosmetics, sericin is a high–value component in a number of skin–care products and make–up formulations, such as mascara and nail cosmetics, in association with silk fibroin. Moreover, sericin is present in foundation creams and eyeliners as a coating agent for talc, mica, titanium dioxide, iron oxide

and nylon. This protein has also been included in sunscreen products containing triazines and cinnamic esters as UV filters, leading to the enhancement of the light–screening effect [ 28 , 29 ]. Furthermore, silk sericin resembles the natural moisturizing factor (NMF) [ 30 ] thanks to the moisture retention capacity of its hydroxyl groups. Sericin–containing products can thus be used to prevent trans–epidermal water loss (TEWL) and increase skin hydration [ 25 ]. Sericin has also been shown to provide an increase in skin elasticity, anti–wrinkle and anti–aging effects in creams and ointments [ 28 ].
Sericin shows strong affinity towards keratin, and it is therefore particularly suitable for hair–care products [ 31 ]. In fact, sericin hydrolysates are present in many hair–conditioning and straightening formulations. The proteins natural film–forming ability means that sericin can protect hair against damage, protein loss, roughness, dryness and swelling, as well as improving fiber flexibility [ 30 ].
In addition to polyols and linear carbohydrates, cyclodextrins (CDs) are becoming increasingly established as a new class of thermal stabilizers of liquid–protein formulations thanks to their anti–aggregation activity [ 32 ]. CDs are macrocyclic oligosaccharides that are composed of a different number of glycosidic units: 6, 7 and 8, respectively named α, β and γ [ 33 , 34 ]. Their main characterizing feature is their ability to form water–soluble complexes with lipophilic molecules that can be completely or partially included in CD cavities, which are a hydrophobic environment. The size of the CD cavity is particularly relevant for their complexation ability, indeed α–CDs are principally used to host aliphatic chains, whereas β–CDs have been demonstrated to effectively accommodate aromatic rings. These characteristics allow some amino acids, such as Phe, Tyr, His and Trp, to be hosted [ 35 , 36 ]. In particular, β–CD derivatives are generally singled out for their aggregation inhibitory properties towards protein molecules. Branched β–CDs and dimethyl–β–CD are able to inhibit the thermally and chemically induced aggregation of egg lysozyme and fibroblast growth factor [ 37 ]. Hydroxypropyl–β–CD can stop the interfacial aggregation

Physicochemical properties and enzymatic activity of wheat germ extract microencapsulated with spray and freeze drying. — Fahimeh Jamdar et al., 2021

Since ancient times, cereals have been one of the first known human foods that have always played a critical role in the economy and nutrition of people worldwide, particularly in developing countries. Cereal germ is the richest sources of amino acids, vitamins, and minerals and also contains proper amounts of fiber. They contain a variety of vitamins (A, B, C, D, E, K, and folic acid) and are excellent sources of iron, potassium, calcium, phosphorus, magnesium, and zinc (Almansouri et al., 2001 ). Among the cereals, wheat ( Triticum aestivum ) represents a foremost cereal product as the main human food crop and livestock feed (Zhang et al., 2019 ). With the current increasing population growth and raising the knowledge of nutrition quality, wheat germ can be used as a food ingredient. Germ is one of the most attractive and promising source of vegetable functional compounds (Rizzello et al., 2010 ).
Wheat germ is produced as a by‐product during wheat milling operations and is a relatively inexpensive protein source that, in spite of its exclusive nutritional properties, is mostly used for animal feed formulation and has limited use in the food industry (Rizzello et al., 2010 ). Wheat germ is a rich source of phytosterols, policosanols, unsaturated fatty acids, protein, lipase, acid phosphatase, flavonoids, B vitamins, dietary fiber, and minerals (Zhu et al., 2006 ). Wheat germ is the richest known source of plant‐derived vitamin E. Tocopherols are powerful fat‐soluble antioxidants that are effective in preventing cancer, diabetes, hypertension, and Alzheimer's disease. The acceptable percentage of unsaturated fatty acids in wheat germ oil plays an important role in lowering blood cholesterol and treatment of atherosclerosis and heart disease (Dunford, 2009 ). Antioxidants found in wheat germ include carotenoids, tocopherols, flavonoids, and phenolic acids. Few studies are available on the antioxidants and phenolic compounds of wheat germ. Phenolic compounds are the main antioxidant compounds, and their content is directly proportional to antioxidant activity (Zhu et al., 2011 ). Enzymes are considerably found in wheat germ, the most important of which include lipase and acid phosphatase. Lipase is widely used in various industries, particularly the food industry, and is commonly applied in the processing of oils

and fats, dairy, bakery, etc. The maximum amount of lipase is found in the aleurone and germ cells. (Cara et al., 1992 ; Elwira et al., 2010 ). Addition of wheat germ containing valuable compounds and enzymes can be one of the most desirable and simplest methods used to produce products with unique properties.
Recently, plant‐derived bioactive compounds have been widely investigated for their beneficial health outcomes. These metabolites have been receiving a great interest in the last decades for applications in food and pharmaceutical industries due to their antioxidant, antimicrobial, anticancer, antidiabetic, and anti‐inflammatory activities (Shahidi et al., 2020 ).
Many active compounds, such as antioxidants that are lipophilic, need to be protected from environmental effects (Gibbs et al., 1999 ). Microencapsulation is a technique by which the sensitive ingredients are packed within a coating or wall material. Microencapsulation technology launched in 1950 and is widely developed today, with wide applications in a variety of pharmaceutical, chemical, food, and printing industries (Augustin et al., 2001 ). It can envelop a solid, liquid, or gaseous substance within another substance in a very small sealed capsule (Fang & Bhandari, 2010 ). Microencapsulation can be defined as a process of building a functional barrier between the core and the wall material to avoid chemical and physical reactions and to maintain the functional properties of the core materials (Bakry et al., 2016 ). Oils and fats, flavoring compounds, oleoresins, vitamins, minerals, color compounds, and enzymes are among the microencapsulated substances in food products (Minemoto et al., 1999 ). Numerous wall materials or encapsulating agents are available for food application. Gum arabic, maltodextrins of different dextrose equivalent (Bakowska‐Barczaka & Kolodziejczyk, 2011 ), whey protein powder and mixtures of whey protein and maltodextrin (Bryant & McClements, 1998 ), pectin and guar gum (Ravichandran et al., 2014 ) are the most commonly used as wall materials.
The aim of this study was to investigate the effects of type and concentration of coating material and the microencapsulation method on the physicochemical properties and enzymatic activity of

microcapsules obtained from WGE.
Wheat germ was purchased from a local flour production factory (Zarrin Khoosheh, Karaj, Iran). Whey protein concentrate (WPC) with 80% protein and maltodextrin with a Dextrose Equivalent (DE) of 9–12 were obtained from Sigma Aldrich (Germany). All the required solutions were procured from Merck, Germany.
Wheat germ (30 g) was mixed with 450 ml of distilled water, heated in a waterbath (WNB22‐MEMMERT, Germany) at 60°C for 15 min, and centrifuged (Z36HK‐HERML, Germany) at 4500 rpm for 20 min. The supernatant was collected in dark vessels and stored at −18°C in a freezer (RR30 & RZ30‐SAMAUNG, South Korea) until further tests and the microencapsulation process (Mohamed et al., 2015 ).
According to the results of previous studies, maltodextrin and WPC solutions were used to prepare the wall at concentrations of 20% and then placed on a magnetic stirrer (LT108, V. 220, HZ. 50, Iran) at 4,500 rpm for 10 min. To produce a stable emulsion, equal amounts of maltodextrin and WPC (M‐W) were mixed with 1:3, 2:2, and 3:1 ratios (w/w) to reach a total solid content of 40% w/w. WGE was then added to form an optimum core (a core to wall ratio of 1 to 8) and placed on a magnetic stirrer at 4,500 rpm to be mixed for 10 min. Samples were transferred to a spray dryer (B 290, Buchi Laboratoriums‐Technik, Switzerland) at incoming and outgoing temperatures of 150 and 85°C, respectively, with an air flow rate of 35 m 3 /h. Dried samples were collected in dark vials and stored in a freezer (RR30 & RZ30‐ SAMAUNG, South Korea) (Simon‐Brown et al., 2015 ; Sing et al., 2015 ).
Maltodextrin and WPC solutions were prepared at 20% concentrations for wall preparation and placed on a magnetic stirrer at 90 rpm overnight.

Impact of Different Saccharides on the In-Process Stability of a Protein Drug During Evaporative Drying: From Sessile Droplet Drying to Lab-Scale Spray Drying. — Johanna Dieplinger et al., 2023

Dry powder of protein biopharmaceuticals is generally produced via lyophilization. In recent years, there has been increasing interest in using spray drying as an alternative process for developing dry powder biopharmaceuticals. This technique enables rapid drying of a protein-excipient solution atomized as droplets in a hot stream of air to produce dry powder particles. The benefit of spray drying is inherent to its single-step nature and rapid processing times, which could allow its application to the continuous manufacturing of biopharmaceuticals [ 1 – 3 ]. Compared to lyophilization, spray drying became a favoured alternative due to its lower energy consumption [ 4 , 5 ]. Furthermore, the particle size distribution (PSD) of dry powders produced via spray drying shows good reproducibility, and produced particles are generally small in size, providing additional advantages of this technique from a drug delivery standpoint such as for inhalation [ 5 , 6 ]. For the ProCept spray dryer used in this study, the size of particles generated using the different nozzle types can be between 1 – 350 µm [ 7 ]. Based on the authors experience with the bifluid nozzle, the average particle sizes obtained are between 1 µm (Division 10, Dv 10 ) and 90 µm (Division 90, Dv 90 ) [ 8 , 9 ]. A great variety of excipient classes can aid in the protection of the protein during drying, i.e., surfactants, amino acids, saccharides, polymers, or other proteins [ 10 – 14 ]. The excipients of main interest in this work are saccharides which have been shown to stabilize proteins during freeze-drying based on their size and steric hindrance [ 15 ]. During spray drying of protein-saccharide formulations, the protein is trapped in a glassy saccharide matrix, which leads to protein stabilization [ 15 – 17 ]. Two main mechanisms about how saccharides can stabilize proteins during drying have been widely discussed and reported elsewhere, namely the water replacement theory and the vitrification theory [ 1 , 16 , 18 – 22 ]. Additionally, in a recent study, different saccharides were analyzed according to their ability to successfully stabilize bovine serum albumin (BSA) after spray drying [ 23 ]. In the past 20 years, the production of biopharmaceuticals by spray drying has greatly increased [ 10 ], yielding the first

commercial products, e.g. inhalable insulin Exubera® in 2006. [ 24 ]. In 2015 the first aseptic biologic produced via spray drying, Raplixa®, was approved by the US-FDA [ 25 , 26 ]. However, the development of the spray drying process requires the use of a considerable amount of material to obtain an adequate good powder yield for analysis. Due to the limited availability and costs of biopharmaceutical materials, during the early phase of formulation development, miniaturized screening workflows can enable the generation of a considerable amount of data with a limited amount of material. Different miniaturized screening approaches are existing and are being used for predicting particle formation during spray drying. Generally, this can be divided into single droplet or film casting experiments. For single droplet drying, four main approaches have been described in greater detail elsewhere [ 27 – 29 ]: free-falling droplet, levitation of droplet (acoustic or air flow), droplet hanging on a thin glass filament, or dispensed on a hydrophobic surface. In thin film preparation, a liquid is dispensed on a hot surface and dried inside an adequate container [ 30 ]. Thin film drying experiments were reported to be complementary to drying experiments of single droplets [ 30 ]. The advantages and disadvantages of the different mentioned droplet drying techniques are presented in Table I . Table I Summary of Positive and Negative Aspects of Acoustic Levitation, Film Casting, and Other Single Droplet Techniques Miniature techniques Advantages Disadvantages Acoustic levitation - Monitoring of kinetics and droplet shape during the drying process [ 27 ] - Contactless drying minimizes unwanted change in droplet morphology - small amounts of material needed [ 31 ] - Evaporation rate indirect [ 27 ] - Acoustic waves influence the drying process and droplet position [ 27 ] - Production of large particles & long drying times [ 31 ] Film casting - Information on evaporation rate, kinetic and thermodynamic powder stability [ 32 ] - Solvent evaporation not representing droplet geometry [ 32 ] Free-falling droplet - Mimics the drying process as of a spray dryer [ 29 ] - Individual droplet analysis difficult [ 27 ] - Drying process cannot be tracked consistently [ 29 ] Pendant droplet drying - Different parameters of interest can be measured concurrently [ 29 ] - Development of particle morphology can be monitored [ 27 ] - Difficult to put droplet into

desired position [ 29 ] - Filament interferes with droplet morphology and transfer of heat [ 29 ] Sessile droplet drying - Possible to track size and temperature of the droplet as well as morphology and crust formation [ 28 ] - Interference of hydrophobic surface with droplet morphology and transfer of heat [ 28 ]
Summary of Positive and Negative Aspects of Acoustic Levitation, Film Casting, and Other Single Droplet Techniques
- Monitoring of kinetics and droplet shape during the drying process [ 27 ]
- Contactless drying minimizes unwanted change in droplet morphology
- small amounts of material needed [ 31 ]
- Evaporation rate indirect [ 27 ]
- Acoustic waves influence the drying process and droplet position [ 27 ]
- Production of large particles & long drying times [ 31 ]
- Individual droplet analysis difficult [ 27 ]
- Drying process cannot be tracked consistently [ 29 ]
- Different parameters of interest can be measured concurrently [ 29 ]
- Development of particle morphology can be monitored [ 27 ]
- Difficult to put droplet into desired position [ 29 ]
- Filament interferes with droplet morphology and transfer of heat [ 29 ]
So far, there is a lack of systematic screening approaches for selecting excipients for the spray drying of biopharmaceuticals. Likewise, aware of this issue, Morgan et al ., 2019, have developed a screening and selection of excipient approach for spray drying of viral vectors [ 31 ]. Their work aimed to confirm the conservation of particle morphology between screening (miniaturized drying approaches) and methods for production (spray drying) [ 31 ]. In our work, we aimed to develop a similar approach that would enable the simple and miniaturized screening of excipients by giving us valuable understanding of the aggregation behaviour of protein-saccharide formulations during spray drying at lab-scale. We based our work on the approach by Both et al ., 2019, where a sessile droplet apparatus was used to study single droplet drying by dispensing the sample on a hydrophobic membrane. [ 33 ]. Our miniaturized approach developed at the small scale has proven to successfully relate to the drying behaviour at the lab scale for the di- and oligosaccharides TD and DEX used, but not for the polysaccharide HPβCD. The simple setup does not require levitation, can be easily replicated

Drying Technologies for the Stability and Bioavailability of Biopharmaceuticals — Fakhrossadat Emami et al., 2018

1. IntroductionThe intrinsic instability of protein molecules is currently the predominant challenge for biopharmaceutical scientists [1,2,3]. Because of their higher molecular weights and diversity of composition, therapeutic proteins have much more complicated structures than conventional chemical drugs [3,4,5]. Exposure to some environmental stresses, such as pH extremes, high temperatures, freezing, light, agitation, sheer stress, and organic solvents, can cause protein instability [4,5]. Since proteins can be degraded easily during manufacturing and storage, some strategies are suggested to improve protein stability, including the addition of stabilizers, protein modification with biocompatible molecules, nanomedicine, and nano- or micro-particle technology [6,7,8,9,10,11,12,13].Drying strategies that process and dehydrate proteins to produce more stable protein formulations in the solid state are frequently used for biopharmaceuticals that are insufficiently stable in aqueous solutions [14,15,16]. Solid dosage forms of proteins are less prone to shear-related denaturation and precipitation during manufacturing and storage [1,15,17,18]. Because water molecules can induce mobilization of therapeutic proteins and other additives, liquid formulations of proteins are more susceptible to unfavorable physicochemical degradation. Consequently, water removal and embedding of proteins in a glassy matrix are good approaches for improved storage against physicochemical protein degradation [1,5,18].Dried therapeutic protein powders have shown good storage stability at room temperature (≤25 °C), and dehydration is an easy and economical approach [19,20]. Dehydration is not only a drying procedure for improving protein shelf-life, but may also be used for engineering protein particles for various routes of administration. Dried biopharmaceutical powders have gained popularity as inhalation preparations for pulmonary, nasal, and sustained drug-delivery systems [21,22]. Numerous reviews of drying strategies have been published [23,24]. However, most of these reviews focus on small molecules, and reviews of using drying methods to improve stability or pharmacokinetic properties of therapeutic proteins are relatively few [25,26]. Because proteins are sensitive to environmental stresses, the techniques available for producing dried biopharmaceuticals are limited by factors such as production time, temperature, and various process-related stresses [26]. The features and drawbacks of each drying procedure should be considered for rational selection of a drying method to improve the

stability of therapeutic proteins for different drug administration applications [27]. In this review, the drying techniques of biopharmaceuticals are discussed, with focus on the selection of appropriate drying methods for improving stability and desired pharmacokinetic properties of biopharmaceuticals. Stabilizers for protein formulations and applications of dried-powder formulations to local or systemic drug delivery are also highlighted. 2. Drying TechniquesGenerally, drying involves three steps, which may be operated simultaneously. First, energy is transferred from an external source to water or dispersion medium in the product. The second step is phase transformation of the liquid phase to a vapor or solid phase. Finally, the transfer of vapor generated away from the pharmaceutical product occurs. The characteristics of dried particles can be effectively influenced by process parameters, such as temperature, pressure, relative humidity, and gas feed rate, besides characteristics of protein formulations, such as composition and type of excipients, concentration of solutes, viscosity, and type of solvent [28] (Table 1). Drying based on the mechanism of removing water can be classified into subgroups. Drying can be performed using an evaporation mechanism, such as vacuum dying or foam drying; evaporation and atomization pathways such as spray drying (SD); sublimation mechanisms such as freeze drying (FD) and spray freeze drying; and supercritical fluid drying methods using a precipitation mechanism [27]. The most common drying techniques, namely freeze drying, spray drying, spray freeze drying, and supercritical fluid drying will be discussed in this review. 2.1. Freeze Drying (FD)The most common drying method for therapeutic proteins is FD [14,26,29], which has been used for many therapeutic proteins, including insulin dry powder for inhalation (Afrezza®, MannKind Corporation, Valencia, CA, USA) [14]. Since water molecules can induce mobilization of protein solution, protein stability can be improved by water removal and embedding of proteins in a glassy matrix through lyophilization [5]. FD is based on sublimation, where solid materials are directly transformed to the gaseous phase. The FD process involves the following three steps: freezing, primary drying, and secondary drying [1,5,18,30,31].Freeze-dried proteins have greater storage stability than proteins in liquid dosage forms; however, this process applies freezing and dehydration stresses to the proteins, which may result in the alteration of protein structure

[31,32,33]. Upon drying, the hydration shell surrounding the protein, which provides a protective effect, is removed. In addition, the protein solution becomes saturated because of ice crystal formation during the freezing process. The solute concentration, pH change, and ionic strength changes are formulation variables that should be considered for a stable protein formulation (Table 1) [1,33]. Recent infrared spectroscopic analyses have shown that acute freezing and dehydration stresses of lyophilization can induce protein unfolding [29,34]. To develop a successful protein formulation using an FD procedure, physical properties, such as glass transition temperature (Tg) and residual moisture content, and operational parameters, such as pH and cooling rate, should be considered [29].Furthermore, a hydrophilic molecule can be incorporated into the protein formulation as a lyoprotectant to overcome protein denaturation and preserve stability during lyophilization [1,17]. Stabilizers can protect proteins during freezing (cryoprotectants) and lyophilization (lyoprotectants) through water replacement and hydrogen bond formation (Table 2) [1,18,30,35]. Moreover, excipients have the potential to provide a glassy matrix to decrease protein-protein interactions and reduce protein mobility in a solid dosage form [36]. In summary, optimization of process variables and proper combinations of additives as stabilizers are requirements for stable freeze-dried products [35,36,37].Liao et al. investigated the effect of excipients, such as glycerol, sucrose, trehalose, and dextran, on the stability of freeze-dried lysozymes using second derivative Fourier transform infrared (FTIR) spectroscopy [17]. They showed that the combination of trehalose and sucrose could raise the Tg of freeze-dried lysozymes, leading to the stabilization of lysozyme in freeze-dried formulations. This study indicated that the Tg of freeze-dried formulations and the protein stability during lyophilization were dependent on the excipient type and excipient to enzyme mass ratio. A recent study by Tonnis et al. [38] showed the influence of size and molecular flexibility of sugars on the stability of freeze-dried proteins, including insulin, hepatitis B surface antigen, lactate dehydrogenase, and β-galactosidase. Among freeze-dried

Quality of a powdered grapefruit product formulated with biopolymers obtained by freeze-drying and spray-drying. — Luis A Egas-Astudillo et al., 2021

Freeze-drying and spray-drying are two techniques used to produce dehydrated food products. Both techniques are easy to use and offer high sensory, nutritive value, and functional quality to foods. However, both processes become difficult for foods with high sugar and acid content, such as fruits. This is because these products, once dehydrated, moisten quickly, causing a change in their physical properties, mainly in the mechanical aspects related to the start of a caking phenomenon. Therefore, incorporating high molecular weight biopolymers that act as facilitators or processors, prevent the structural collapse of the product. The aim of this study was to select the best process, between freeze-drying or spray-drying, to obtain a powdered grapefruit product with the higher quality. The impact of the biopolymers used to stabilize the powdered product was also tested. The properties analyzed were the solubility, wettability, hygroscopicity, porosity, and color of the powder together with the flow behavior, both in air and water. The results of this study show that using the freeze-drying technique, products have a better flow behavior, greater porosity, and a color more like fresh grapefruit. Biopolymers, especially when in combination, have a positive effect on the quality parameters studied.
Practical Application

           The results of this study allow freeze-drying to be proposed as a process to obtain a grapefruit product with better properties, both powdered and rehydrated, than that obtained by spray-drying. On the other hand, although the incorporation of biopolymers is necessary to facilitate the process and stabilize the product, no significant differences have been found between the different formulations tested, although it seems that their combination favours some of the properties of the powder, such as solubility, hygroscopicity, wetting time and dispersibility.

1 INTRODUCTION

     Grapefruit (Citrus paradisi) of the variety Star Ruby is a citrus well studied for being a source of bioactive molecules such as vitamin C (Vanderslice et al., 1990), eriocitrin, and naringin among the phenolics (Uckoo

et al., 2012; Zhang et al., 2011) or alpha and beta carotenoids (Holden et al., 1999; Hung et al., 2017; Peterson et al., 2006), compounds that seem to confer this fruit's biomedical properties (Cristóbal-Luna et al., 2018). Previous studies report that grapefruit juice has several biomedical activities, in relation to the cardiovascular system (Díaz-Juárez et al., 2009), metabolic syndrome (Fujioka et al., 2006), cholesterol, and low-density lipoprotein levels (Dow et al., 2012). The juice inhibits DNA damage (Alvarez-Gonzalez et al., 2011) while decreasing gastric lesions and onset of diarrhea. The grapefruit also promotes the benefits of glutathione in the body because of its antioxidant and anti-inflammatory properties (Cristóbal-Luna et al., 2018; Khan et al. Feroz, 2016).

     Despite the goodness of fruit in general and grapefruit in particular, there is a problem related to the consumption of these foods. Fruit intake is below the RDA, which may be due to its perishable nature in relation to new lifestyles. From this point of view, the design of more stable and easy-to-use fruit products could stimulate their consumption among the population. Both freeze-drying and spray-drying are two techniques easy to manage and are used to produce powdered products with high sensory, nutritive, value, and functional quality. Fruit powder, dehydrated or previously rehydrated, allows for incorporation in juice formulations, infusions, desserts, dairy products, salads, ice cream, among other products. It has the advantages of product storage stability and logistical improvements, such as increased product packing density and transportation. However, both processes are difficult when applied to foods with a high sugar and acid content such as fruits. Main grapefruit composition is about 89 g water/100 g and 10°Brix, the main soluble solids in this fruit being sucrose, fructose, glucose, and citric acid, in mass ratios of 45.5, 21.2, 18.0 and 15.3, respectively (Fabra et al., 2009). The normal water content of dehydrated powdered foods is in the range

3% to 5%. So, the soluble solids in the powdered grapefruit increase from 88% to 86%, which in fact is a very high sugar and acid content. The parameter that defines the loss of quality of these products when dehydrated, a consequence of their wetting, is the change in their mechanical properties, related to the start of the caking phenomena. These sample changes occur from the moment the glass transition begins and are previous to the color changes, associated with their non-enzymatic browning. Changes also occur before chemical and microbiological reactions take place, responsible for their deterioration. The glass transition supposes the change from the stable glassy to the more instable rubbery state of material and occurs above the so-called glass transition temperature (Tg), dependent on the water content and solutes composition. Therefore, keeping the powder products in a glassy state is essential to make sure their quality and stability. The critical viscosity that determines the start of caking phenomena in mixtures of sucrose and fructose, sugars present in fruits, occurs at approximately 20°C above the Tg (Roos, 1995).

     The low values of critical water content and water activity required for the glass transition of powdered fruit products make it necessary to incorporate high molecular weight biopolymers. For example, according to the data published by Silva-Espinoza et al. (2020), the Tg of the orange puree freeze-dried to 3 g water/100 g product is 11.4°C and increases to 20.32°C when gum Arabic (GA) and bamboo fiber (BF) (ratio 100:5:1) were added. This increase is enough to ensure the product stability during storage at room temperature. Examples of different biopolymers to be added to increase the Tg including gum Arabic, maltodextrins, starches, gelatine, methylcellulose, alginates, pectin, and mixtures of them (Telis & Martínez-Navarrete, 2009; Silva-Espinoza et al., 2020). Certain other biopolymers, such as proteins, insoluble fiber or inorganic compounds as silicon dioxide or tricalcium phosphate, delay the products’ caking phenomena through steric activity (Barbosa-Canov

Progress in spray-drying of protein pharmaceuticals: Literature analysis of trends in formulation and process attributes — Joana T. Pinto et al., 2021

1. IntroductionProtein pharmaceuticals are, normally, formulated as aqueous dosage forms. However, liquid dosage forms are often unstable, presenting limited shelf life that frequently requires storage and transport under refrigerated conditions. To overcome these limitations, protein pharmaceuticals can be formulated as a dry powder. Ideally, when in the powder form, the biomolecule, will remain stable and retain its activity for the intended periods (3 years or more) under ambient storage conditions. Traditionally, freeze-drying is the process of choice, when drying protein pharmaceuticals. In freeze-drying, the liquid formulation is first frozen and then the ice is removed by sublimation and desorption.[Citation1McAndrew, P. T.; Hostetler, D.; DeGrazio, F. Container and Reconstitution Systems for Lyophilized Drug Products. In Lyophilization of Pharmaceuticals and Biologicals: New Technologies and Approaches; Ward, K. R., Matejtschuk, P., Eds.; Humana Press: Totowa, NJ, 2019; pp 193–214. [Crossref], [Google Scholar]] As of 2014, there are over 400 approved freeze-dried products by the United States Food and Drug Administration (U.S. FDA). The estimated annual growth of freeze-dried products for 2018 was 13.5%.[Citation2DiFranco, N. Lyophilization of Pharmaceuticals: An Overview. https://lubrizolcdmo.com/blog/lyophilization-of-pharmaceuticals-an-overview/ (accessed Sep 16, 2020). [Google Scholar]] Many approved freeze-dried products are protein formulations (i.e. vaccines, antibodies, enzymes, peptide hormones, etc.) that emerged rapidly over the last years.[Citation3Comission, E. Study on the Competitiveness of the European Biotechnology Industry – The Financing of Biopharmaceutical Product Development in Europe, 2010. DOI: 10.2769/33524. [Crossref], [Google Scholar]] Likewise, the expansion of alternative drying processes able to sustainably support the rapid development of biopharmaceutical technologies, are a central need of this industry in years to come.While freeze-drying presents numerous advantages, there are some inherent challenges. Namely, the difficulty to control the particle/cake

properties and microstructures, inter-vial variability, challenge to process large quantities of material, enormous power, time, and resource consumption are some of the major hurdles. These have led many to the quest of finding alternative drying technologies, spray-drying being one of them.[Citation4Emami, F.; Vatanara, A.; Park, E. J.; Na, D. H. Drying Technologies for the Stability and Bioavailability of Biopharmaceuticals. Pharmaceutics. 2018, 10, 131. DOI: 10.3390/pharmaceutics10030131. [Crossref], [Web of Science ®], [Google Scholar]] Unlike spray-drying, other exploratory drying technologies reported for the processing of protein therapeutics, tend to be costly, time consuming, and not mature enough for industrial implementation. Moreover, one of the main advantages of spray-drying, and a critical driver in generating the interest of the industry in this technique, is the possibility for “continuous processing.”[Citation4Emami, F.; Vatanara, A.; Park, E. J.; Na, D. H. Drying Technologies for the Stability and Bioavailability of Biopharmaceuticals. Pharmaceutics. 2018, 10, 131. DOI: 10.3390/pharmaceutics10030131. [Crossref], [Web of Science ®], [Google Scholar]] This provides a promising and rapid solution in terms of large volume manufacturing in particular cases like the recent crisis caused by the coronavirus disease 2019 (COVID-19).In the last 15 years, spray-drying has, in fact, been successfully applied in the production of a few protein pharmaceuticals (Table 1). In 2006, the inhaled insulin powder, Exubera® (Pfizer), became the first commercial spray-dried protein hormone (later withdrawn from the market). Spray-dried alternatives of Poly(lactic-co-glycolic acid) (PLGA) microspheres for depot liquid crystal formulation of triptorelin pamoate and lanreotide acetate were approved in 2010 and 2013, respectively. More recently, in 2015, Raplixa® (ProFibrix BV) became the first approved protein drug manufactured via aseptic spray-drying. Beyond these, other protein pharmaceuticals produced via spray

-drying in a wide array of dosage forms are, presently under clinical development.[Citation5Vehring, R.; Snyder, H.; Lechuga-Ballesteros, D. Spray Drying. In Drying Technologies for Biotechnology and Pharmaceutical Applications; Ohtake, S., Izutsu, K., Lechuga-Ballesteros, D., Eds.; Wiley‐VCH Verlag GmbH & Co. KGaA: Weinheim, 2020; pp 179–166. DOI: 10.1002/9783527802104.ch7. [Crossref], [Google Scholar]]Progress in spray-drying of protein pharmaceuticals: Literature analysis of trends in formulation and process attributesAll authorsJoana T. Pinto, Eva Faulhammer, Johanna Dieplinger, Michael Dekner, Christian Makert, Marco Nieder & Amrit Paudelhttps://doi.org/10.1080/07373937.2021.1903032Published online:15 April 2021Table 1. Commercially approved protein pharmaceuticals produced via spray-drying.
Download CSVDisplay TableIn general, drying of protein pharmaceuticals can pose a risk to their chemical and physical stability.[Citation6Cicerone, M. T.; Pikal, M. J.; Qian, K. K. Stabilization of Proteins in Solid Form. Adv. Drug Deliv. Rev. 2015, 93, 14–24. DOI: 10.1016/j.addr.2015.05.006. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]] Thus, it is of utmost importance to understand how spray-drying’s formulation and process parameters (and their interactions) impact the quality of protein pharmaceuticals. To that end, this review will extensively discuss the learnings achieved over the last 30 years of research conducted in the field of solid protein drug formulations produced by one-step spray-drying process. The following subtopics will be covered: classes of protein pharmaceuticals and excipients used in spray-drying; solvent systems applicable to these types of formulations; spray-dryer equipment used and process parameters applicable to protein formulations; quality aspects of spray-dried protein pharmaceuticals. At the end, we concisely discuss the perspectives on the protein spray-drying based on the identified trends,

Spray Drying Is a Viable Technology for the Preservation of Recombinant Proteins in Microalgae. — Anaëlle Vilatte et al., 2023

To meet global food demand, the aquaculture industry had an average growth rate of 5.3% per year between 2001 and 2018 [ 1 ]. Of the 114.5 million tonnes of live weight produced in 2018, finfish accounted for more than 47%. The intensification of fish farming raises new challenges in disease management and the delivery of protection at scale. Of particular concern are salmonid alphaviruses (SAVs), which are serious pathogens affecting farmed Atlantic salmon and rainbow trout in Europe [ 2 ]. The resulting pancreas (PD) and sleeping diseases can be associated with high mortality rates, e.g., up to 63% [ 3 ] in the case of PD, and severe economic losses. Vaccination is an effective strategy to control SAV transmission in fish farming. Conventional vaccination methods involve the intraperitoneal injection [ 4 ] of anaesthetized fish with a commercial vaccine such as the multivalent vaccine AquaVac ® PD3, which contains inactivated SAV [ 2 ].
Vaccination by injection can be expensive, labour-intensive and stressful to the fish. An attractive alternative is to administer the vaccine via an oral route [ 4 ]. Typically, the production of edible vaccines would involve the microencapsulation of a recombinant subunit antigen and formulation into a feed or supplement [ 4 ]. There have been a number of potential oral vaccine candidates for veterinary applications expressed in plants (e.g., lettuce [ 5 ]), yeasts [ 6 ], and bacterial hosts such as Bacillus subtilis [ 7 ]. As natural and beneficial components of the aquaculture diet, whole cell microalgae are highly promising systems for such applications [ 8 ]. The advantages of microalgae as a production platform for therapeutic proteins have been extensively reviewed elsewhere [ 9 ]. Microalgae are photosynthetic microorganisms which can be cost-effectively grown in simple media, with water and sunlight. They can synthesize and correctly assemble a wide range of complex therapeutic proteins [ 9 ] such as monoclonal antibodies (mAbs) against the glycoprotein D of the herpes complex virus [ 10 ]. In addition, microalgae can be grown at large scale in contained and controlled environments, allowing the implementation of good manufacturing practices (GMP) [ 9 ].
The unicellular microalga Chlamydomonas reinhardtii is a model organism with well-established genetic engineering tools for both chloroplast and nuclear transformation [

11 , 12 ]. With its generally regarded as safe (GRAS) status, C. reinhardtii represents a promising platform for the production and delivery of edible vaccines [ 13 ]. Kiataramgul et al. reported the successful vaccination of shrimp against white spot disease, with a survival rate of 87%, using a transgenic line of C. reinhardtii expressing the VP28 viral envelope protein from white spot syndrome virus [ 14 ].
Several challenges in process development exist to make microalgae-based oral vaccination feasible and cost-effective at the industrial scale. A crucial step in the manufacturing process is the drying stage, where the dehydration of the algal cells is used both to kill the transgenic algae and to provide a natural method for the protective bioencapsulation of the vaccine [ 15 ]. Among the available drying technologies, freeze drying is a gentle technique which is conventionally used at the laboratory scale. Despite its efficiency, freeze drying is considered an expensive technology owing to its high energy requirements, potentially limiting its use at a larger scale of production [ 16 , 17 ]. Several studies have already demonstrated the potential of spray drying to preserve natural compounds, such as β-carotene [ 18 ], lipids [ 19 , 20 ], fatty acids [ 19 , 20 ], carbohydrates [ 19 ] and proteins, [ 19 ] in various microalgal species. However, to our knowledge, no study has investigated the potential of spray drying to preserve microalgae expressing recombinant proteins and, more specifically, to produce microalgae-based edible vaccines for aquaculture applications. For spray drying to be a viable option, the tolerance of the protein to elevated temperatures in the process must be established. In addition, the effect of formulation should be investigated: the microstructure of spray-dried powder has a high surface area [ 21 ], which may impact the protein stability.
The aim of this study was to investigate the suitability of spray drying for the manufacture of microalgae-based edible vaccines at the industrial scale. For this purpose, we created a transgenic line of C. reinhardtii expressing a SAV vaccine. The vaccine integrity after spray drying and its stability over time were evaluated. A techno-economic analysis (TEA) was performed using pilot data to forecast the process economics of spray drying compared to freeze drying in a scale-up scenario.
The C. reinhardtii strain used as the transformation recipient in this study was the photosynthetic mutant TN

72 (CC-5168: cw15 , psbH :: aadA , mt+), which was previously described in Wannathong et al. [ 22 ]. TN72, together with the transformant lines (TN72:E2-ecto) and control lines (TN72:empty and TN72:ptxD), were cultured in tris-acetate-phosphate (TAP) medium [ 23 ], which was modified as follows: (NH 4 Cl: 0.400 g/L, K 2 HPO 4 : 0.113 g/L, KH 2 PO 4 : 0.048 g/L, MgSO 4 .7H 2 O: 0.100 g/L, CaCl 2 .2H 2 O: 0.050 g/L, Trizma ® base: 2.42 g/L, glacial acetic acid: 1.13 g/L) with a revised trace element recipe (Na 2 EDTA.2H 2 O: 21.5 mg/L, ZnSO 4 .7H 2 O: 0.720 mg/L, MnCl 2 .4H 2 O: 1.19 mg/L, CuCl 2 .2H 2 O: 0.340 mg/L, (NH 4 ) 6 Mo 7 O 24 .4H 2 O: 0.035 mg/L, FeCl 3 .6H 2 O: 5.40 mg/L, Na 2 SeO 3 : 0.017 mg/L, Na 2 CO 3 : 2.32 mg/L) [ 24 ]. The strains were maintained at 18 °C on 2% TAP agar plates, with TN72 kept under dim light conditions (5–10 µmol.m −2 .s −1 ) and transformants under moderate light (50–100 µmol.m −2 .s −1 ) provided by fluorescent tubes. Liquid cultures were cultivated in Erlenmeyer flasks in an illuminated shaking incubator (Innova 4340, New Brunswick Scientific, USA) at 25 °C, 120 rpm, and under constant illumination provided by fluorescent tubes with an average light intensity of 100 µmol.m −2 .s −1 . Cultures were routinely tested for contamination on 2% Lysogeny agar (LA) plates incubated in the dark at 37 °C for 2–3 days.
The sequence of the