Freeze‐drying is usually an essential procedure when the ice‐templating technique is used for the preparation of porous and nanostructured materials. However, this technique has so far rarely been used to produce materials in industrial sectors. However, as a drying technique, the freeze‐drying method has been widely used in pharmaceutical, biopharmaceutical, and food industries. The objectives are usually to prepare well‐defined cakes with interconnected macroporous structures (to ensure easy and high‐standard reconstitution) and improved stability for storage and transport. Different types of excipients and/or control of pH have been investigated to maintain the activity or native state of the active ingredients in these materials whilst still producing the fine macroporous structures [1, 2]. Although the focuses are not on the porosity and properties of these materials, the freeze‐dried products in these industrial applications can be linked with or can provide insights into the preparation of porous materials by ice templating. Indeed, many of the ice‐templated materials, particularly polymeric materials, show similar pore structures or characteristics as the freeze‐dried pharmaceutical cakes [3, 4]. In this chapter, the purposes are twofold: (i) to give readers a general idea on how freeze‐drying processes have been employed in relevant industries, focusing on excipients, stability, and processing parameters and design and (ii) to provide thoughts/ideas for the preparation of ice‐templating materials, particularly polymeric materials and nanomedicine.
3.2 Excipients in Pharmaceutical Formulations
In brief, pharmaceutical excipients are components in a pharmaceutical dosage formulation other than the active pharmaceutical ingredients (APIs). The role of the pharmaceutical excipients is to guarantee the dosage, stability and bioavailability of the APIs [5, 6]. The excipients can be any component added intentionally to the medicinal formulations and the impurities in the formulations. More specifically, the excipients can act as fillers, binders, diluents, bulking agents, lubricants, wetting agents, solvents, preservatives, flavours, colouring agents, antiadherents, sorbents, coating agents, surfactants, etc. . Excipients can be obtained from different sources, including animals (e.g. gelatin, stearic acid, lactose, natural polymers), plants (e.g. starches, sugars, flavours, cellulose, polymers), minerals (e.g. silica, buffer solutions, inorganic salts, oxides) as well as synthesized polymers and stabilizers [5, 6].
In a pharmaceutical formulation, the percentage of excipients is usually very high. These excipients have their own properties and, similarly to APIs, may participate in adsorption and biodistribution. Therefore, the safety of the excipients is equally important. The excipients should be subjected to the same toxicity studies as demanded by APIs. When choosing certain compounds as excipients, it is always suggested to choose from those being used previously in pharmaceutical products or those originating from food industry and generally recognized as safe (GRAS). The second choice may be compounds chemically modified from those being approved and utilized in the pharmaceutical or food industry. The use of new compounds as excipients will need to be assessed to meet the requirements of the regulation bodies. The safety assessments comprise: (i) toxicity of the excipients; (ii) interactions of APIs–excipients at different stages; and (iii) impacts from production, distribution, and use.
Polymers have been widely used as pharmaceutical excipients for various roles including as diluents, bulking agents, stabilizers, in protective coating, osmotic pumps, and contribute to controlled drug release . These polymers may be either natural or synthesized. Some frequently used polymers include hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), poly(vinyl alcohol) (PVA), polyvinylpyrrolidone (PVP), alginate, dextran, poly(ethylene glycol) (PEG), starch, and so on. PVA, a synthetic polymer, is not only widely used as an excipient in food and pharmaceutical applications but also as a stabilizer for emulsion formulations and drug nanoparticles [3, 7, 8]. This may be attributed to its excellent safety properties. Based on a comprehensive evaluation of the scientific literature, it is inferred that PVA is not mutagenic or clastogenic, does not accumulate in the body when administered orally, poorly absorbed from the gastrointestinal tract (GI), and has very low acute oral toxicity (LD50 in the range of 15–20 g kg−1) .
Sugars are a type of excipients that have been employed for a variety of solid dosage formulation, either via freeze‐drying or other techniques [10–12]. As a filler/binder, mannitol has been widely used, and has even been compared to the conventionally low‐priced excipients such as lactose, microcrystalline cellulose, and calcium hydrogen phosphate. This is because of its very unique properties: low hygroscopicity, inertness towards the APIs and patient body, good taste, good compactibility, and the ability to produce stable tablets . Mannitol can exist in four polymorphic forms (α, β, γ, δ), of which the β form is the stable polymorph and dominantly available in the market.
The above‐mentioned polymers and sugars are mainly used as hydrophilic matrix to stabilize hydrophilic pharmaceuticals or biopharmaceuticals. There are also excipients that are more effective in stabilizing and delivering lipophilic drugs. Two excellent examples are cyclodextrin and lipids. Cyclodextrins are a family of cyclic oligosaccharides composed of α‐(1,4)‐ linked glucopyranose subunits. There are three types of cyclodextrins: α‐, β‐ and γ‐cyclodextrins, composed of 6‐, 7‐, 8‐glucopyranose units, respectively. The unique properties of the cyclodextrins are their hydrophobic internal cavity and hydrophilic external surface. As molecular carriers or chelating agents, via molecular complexation or encapsulation of hydrophobic compounds, cyclodextrins have been extensively investigated and utilized in a wide range of applications such as pharmaceutical excipients, drug delivery, separation, food, and catalysis . Lipid‐based pharmaceutical formulations have drawn considerable interest from the pharmaceutical industry and from researchers. Most of the lipid‐based formulations use lipid vesicles, emulsions or excipients to solubilize lipophilic drugs that are poorly water soluble, and thereby improve drug absorption in the body. An important question is whether the drug remains in solubilized form inside the GI after being administered .
A recent development is on excipient foods, i.e. designing food matrices to improve oral bioavailability of pharmaceuticals and nutraceuticals . This is different from medical food, which contains one or more APIs. By analogy to a pharmaceutical excipient, excipient food does not exhibit any therapeutic properties or bioactivity itself. But when consuming with pharmaceuticals, it can help increase the efficacy of any pharmaceuticals co‐digested with it. This may be achieved through bioactive liberation, increase in membrane permeability, and inhibition of efflux mechanism. Excipient food ingredients may include lipids, carbohydrates, proteins, minerals, surfactants, chelating agents, and phytochemicals .
There are additional requirements when excipients are used in freeze‐dried formulations. Because the freeze‐drying process has been mostly used in protein‐based solid formulations, the excipients must play the role of maintaining the integrity and stability of the proteins at different stages of the freeze‐drying process and during storage and reconstitution. Specifically, excipients that act as a cryoprotectant, lyoprotectant, or stabilizer in solid form should be added into the formulations. Fortunately, some excipients, e.g. sugars, can have all the protecting properties during freeze‐drying and act as a stabilizer in solid protein formulations. As it has been well documented, non‐reducing disaccharides trehalose and sucrose have been mostly used as stabilizers [1, 10]. Mannitol and glycine are good choices as tonicity modifiers . Bulking agents in freeze‐dried formulations usually include mannitol, glycine, lactose, dextran, povidone, and disaccharides [1, 18]. It is possible that one single sugar excipient can act in differing capacities such as a freeze‐drying protectant, stabilizer, bulking agent, and disintegrating agent.
When the excipients are present in or added to parenteral formulations, they can enhance or maintain API solubility and/or stability. The excipients also play important roles in assuring safety, minimizing pain and irritation, and controlling drug delivery . However, these excipients can have significant effects on the distribution and elimination of the co‐administered APIs. In a review focusing on the effects of pharmaceutical excipients on drug disposition, Buggins et al. summarized the effects of co‐solvents (e.g. DMSO, ethanol, propylene glycol, PEGs, cyclodextrins, and surfactants (e.g. Cremophor, Tween, Solutol) on distribution, metabolism, renal elimination, oral adsorption, hepatic elimination, etc. .
Physicochemical properties of excipients, such as solubility, ionization, hydrogen bonding tendency, molecular size and shape, have significant impact on their fate in the body. When assessing safety and therapeutic efficacy of pharmaceutical formulations, it is thus very important to obtain the data about the excipient pharmacokinetics and profiling . Pharmacokinetics is the kinetics of drug absorption, distribution, metabolism, and excretion (ADME). The parameters used to characterize ADME usually include volume of distribution (VD), elimination half‐life (t1/2), clearance (Cl), and bioavailability (F). The pharmacokinetic processes usually follow first‐order kinetics. VD describes how the compound is distributed within the body. A larger VD indicates that the API is in the blood plasma and is also highly tissue bound. Elimination t1/2 of the API is the time it takes to reduce its blood plasma concentration by 50%. Cl indicates how rapidly the API is eliminated from the body. It can be divided into hepatic clearance (ClH) and renal clearance (ClR). Bioavailability is the fraction (hence the symbol F) of an API absorbed from the GI into the general blood circulation . Whilst the excipient pharmacokinetics data should be reliably measured, some simple methods may be used to predict the ADME properties. The rule‐of‐five approach developed by Lipinski et al. is widely used to predict absorption from the GI and overall drug‐like properties of biologically active compounds . The Biopharmaceutics Classification System (BCS)  and The Biopharmaceutics Drug Disposition Classification System (BDDCS)  are also used to provide useful information on solubility, permeability and the extent of API metabolism. The physicochemical properties (Mw, solubility, Log P, H‐bond donors, and H‐bond acceptors) of 26 common pharmaceutical excipients and their pharmacokinetic parameters (t1/2, VD, F) have been summarized by Loftsson . The size/hydration diameter and the surface properties of the excipients are very important for clearance from the body. For example, PVP with Mw < 10 kDa is readily excreted via kidney by glomerular filtration, PVP of Mw up to 25 kDa is completely excreted within one day, while it can take 8 days to excrete PVP with Mw = 37 kDa . Polymer nanoparticles less than 8 nm are mainly excreted by renal clearance while the larger particles must disassemble into smaller particles before being excreted from the body . Although the safety and the pharmacokinetic parameters of pharmaceutical excipients have to be rigorously examined for pharmaceutical products, some insights may be gained from the pharmacokinetic data of the known excipients. For example, the size‐related effects are less likely to have an impact when the excipients can be readily excreted from the body. Hydrophilic low‐Mw polymers with small VD fall into this category, with t1/2 of about 2 h .
3.3 Improving the Freeze‐drying Process
Freeze‐drying processes are only industrially viable for pharmaceutical solid formulations or other high value‐added products. This is due to the long, energy‐intensive and complex freeze‐drying process. A freeze‐drying process may take 2–4 days, with the majority time used in the primary drying process. There are other important factors to consider – the activity and stability of the active ingredients in the formulations, e.g. proteins, vaccines, and food bacteria. Therefore, the efforts for improving freeze‐drying processes usually focus on reducing the freeze‐drying time and producing well‐defined cakes with high stability of APIs.
3.3.1 The Types of Freeze‐drying Processes
188.8.131.52 Spray Freeze‐drying Process
In a conventional freeze‐drying process, the vials containing solutions or suspensions are frozen on the shelf in a freeze‐dryer and then subjected to vacuum to allow the sublimation of the frozen solvent (usually water). The exposed surface area of the frozen body is directly proportional to the rate of sublimation. In other words, in order to increase the sublimation rate and reduce the freeze‐drying time, the size of the frozen sample should be reduced. This can be achieved by a spray freeze‐drying process, where a solution or suspension is either atomized into cold vapour over a cold liquid (usually liquid nitrogen) (Figure 3.1) or directly atomized into liquid nitrogen. The frozen powders (with the sizes of the particles in the micron range) can be subjected to a conventional freeze‐drying process . By analogy, in a spray drying process, the liquid or suspension is atomized into a chamber. Hot air or nitrogen flows inside the chamber in a co‐current or counter‐current direction, evaporating water from the sprayed droplets and producing a dry powder product [27, 28]. The spray drying process is not suitable for temperature‐sensitive compounds while there will be no such limit for a spray freeze‐drying process. The spray freeze‐drying processes are mainly used in drying of pharmaceutical products and high value foods, and in the encapsulation of active but sensitive compounds . Avoiding the blocking of the atomizing nozzles and preventing aggregation of sprayed droplets before they are fully frozen are the main operational difficulties [29–31].
184.108.40.206 Spin Freezing
Another way to reduce the size of the frozen body is by spin freezing. This is achieved by rotating the vials filled with liquid solution or suspension along their longitudinal axis. The liquid phase forms a thin layer coating the inner wall of the vial, which is cooled and frozen by using a flow of sterile gas with a controllable low temperature around the rotating vial (Figure 3.2) . The frozen sample is thin and spread over a larger surface. This can lead to faster freeze‐drying. It also has the great potential to realize a continuous freeze‐drying process. In a study to evaluate spin freezing versus conventional freezing as part of a continuous freeze‐drying concept (Figure 3.2), De Meyer et al. estimated that the total process time could be reduced by a factor of 10–40, depending on the specific formulation properties and vial dimensions .
220.127.116.11 Atmospheric Freeze‐drying
In a conventional freeze‐drying process, the frozen samples are subjected to high vacuum to allow the sublimation of the frozen solvent. This can significantly add costs to energy usage and limit the scale‐up of the freeze‐drying process. However, high vacuum is not necessary for a freeze‐drying process. It has been observed for long that a frozen sample can be dried naturally in dry and cold winter. As such, atmospheric freeze‐drying has been introduced [33, 34]. This process is viable provided the partial pressure of water vapour in the drying chamber is lower than the water vapour pressure of the frozen sample. This is usually achieved by circulating a cold and dry gas through the frozen sample . As expected, this method is very slow for bulky frozen samples. It is thus reasonable to combine a spray freeze‐drying process with atmospheric freeze‐drying to produce poorly water‐soluble drug formulations . Contacting the frozen microparticles with cold dry gas in a fluidized bed is proposed for a faster atmospheric freeze‐drying process . This allows better mixing between the frozen particles and the cold gas, enhancing mass transfer between the two phases and thereby a faster drying process. A problem with atmospheric freeze‐drying is the use of huge quantity of cold gas (albeit re‐circulation). The temperature of the cold gas should be lower than the collapse temperature of the frozen samples. The need for such a large quantity of cold gas supply is highly demanding . This problem has been partially addressed by sub‐atmospheric pressure freeze‐drying as it allows for shorter drying time and reduced use of cold gas [35, 36]. Owing to these issues with atmospheric freeze‐drying, conventional freeze‐drying process is still mostly used in research and industrial applications.
3.3.2 Process Development and Design
There are several stability issues during the freeze‐drying process for solid protein formulations. These issues include denaturation stresses from cold temperature, pH change, solute concentration as well as phase separation during freezing and critical temperatures during freeze‐drying . Various formulations have been investigated in order to achieve specific optimal conditions . In an effort to facilitate the adoption of modern techniques and move the investigations of freeze‐drying processes from the empirical and data‐driven approach to a more knowledge‐based route, the Food and Drug Administration (FDA) introduced ‘Pharmaceutical cGMP for the 21st Century: A Risk‐based Approach’ and ‘Guidance for Industry: Process Analytical Technology – A framework for Innovative Pharmaceutical Development, Manufacturing and Quality Assurance’, in 2004 . For a freeze‐drying process, this includes implementing quality by design (QbD) for proposing experiments, identifying quality target product profile and critical quality attributes, performing risk assessments, and defining the design space and process control strategy .
Based on the QbD approach, a fractional factorial design has been applied to investigate the effects of buffer type, pH, and excipients on glass transition temperature; monoclonal antibody concentration; unfolding transition temperature and particle size of the reconstitute solutions . The Pareto ranking analyses showed that pH, NaCl, and polysorbate 20 were the most important factors .
The design space can be regarded as the combination and interaction of input variables and process parameters that can generate the products with the desirable quality. For a freeze‐drying process, the product temperature at the sublimation interface is crucial in determining cake structure and residual moisture content. The other important parameters include shelf temperature, chamber pressure, collapse temperature, glass transition temperature (for amorphous products) or eutectic temperature (for crystalline products) . Mathematical modelling can be used to build the design space for a pharmaceutical freeze‐drying process. One such study was carried out on the primary drying stage to investigate the effect of shelf temperature and chamber pressure on product temperature and sublimation flux . In addition to shelf temperature and chamber pressure, the dried layer thickness was used as the third coordinate to construct the diagram.
The process control is then required to ensure that the freeze‐drying process can be performed within the variation of process parameters as defined in the design space. Usually, non‐invasive and real‐time monitoring and control of the process are preferred. This relies on the use of the state‐of‐the‐art process analytical technologies (PAT). PAT is used to design, analyse, and control manufacture via timely measurement of critical objectives of the final products [39, 42]. For the lyophilization of biopharmaceutics, the PAT tools can be used in the freezing step to monitor the degree of supercooling, in the primary drying step to probe product temperature and end point of primary drying, and in the secondary drying to monitor and control the residual moisture content . Table 3.1 summarizes the main analytic methods used as PAT during freeze‐drying processes [39, 42]. Various techniques that can be used to characterize the freeze‐dried products, include scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), powder X‐ray diffraction (PXRD), differential scanning calorimetry (DSC), solid nuclear magnetic resonance (solid NMR), and circular dichroism (CD) spectroscopy. These characterization data can provide the feedback on experimental design and process control. The application of PAT is expected to facilitate process transfer from laboratory to production or between manufacturers because the process is less equipment dependent but relies more on sound scientific and engineering knowledge.
Table 3.1 Summary of PAT tools used in pharmaceutical freeze‐drying.
|Analytic technique||Measuring attributes||Probe position|
|Resistance temperature detectors (RTD)||Product temperature||Inside sample vials, primary drying|
|Manometric temperature measurements (MTM)||Product temperature||Non‐invasively at the sublimation interface|
|Near‐infra red (NIR) spectroscopy||Moisture content, sublimation rate||Bottom of the vials|
|Raman spectroscopy||Water to ice conversion, purity and polymorphs of the drug||Top surface of the frozen body|
|Tuneable diode laser absorption spectroscopy (TDLAS)||Water vapour concentration, sublimation rate, predict the end of primary drying||Laser beam attached to the freeze‐dryer spool|
3.4 Applications of Freeze‐drying in Pharmaceutics
Pharmaceutics can be classified into three categories based on their molecular weights . As schematically shown in Figure 3.3, small molecule drugs with molecular weights of <500 Da are conventionally termed as pharmaceutics. Proteins, or biologics/biopharmaceutics, usually have molecular weights of >5000 Da. Between them, researches are emerging on peptide‐based drugs. Peptide‐based drugs, proteins, and vaccines are often classified together as biopharmaceutics, which are the focus of Section 3.5. In this section, the discussion is mainly on pharmaceutics or small molecular drug.
With the use of modern high‐throughput synthesis and screening techniques, a large number of potentially active compounds have been identified. However, a high percentage of them are poorly soluble in water, leading to poor bioavailability and therapeutic efficacy . These drug compounds may be classified into different categories, based on certain criteria: e.g. BCS classification based on solubility and permeability  and BDDCS classification based on solubility and metabolism . These classifications offer clarity and benefits for pharmaceutical research and regulatory bodies . Corrections and additional drugs may be continuously added to the list of the classifications . A simple and effective ‘rule‐of‐five’ was developed by Lipinski et al. to predict the likely poor absorption or permeation if the drug compounds have more than five H‐bond donors, molecular weights over 500 Da, Log P over 5, and more than 10 H‐bond acceptors. The rule is not applicable for compounds that can be used as substrates for biological transporters and biopharmaceutics .
Solid pharmaceutical formulation aims to improve apparent solubility and thereby permeability and bioavailability for poorly water‐soluble drugs . This usually involves the reduction in size of drug particles and the use of excipients, particularly polymers, as amorphous matrices to stabilize the drug particles [5, 44]. The dissolution rate of a compound is proportional to the exposed surface area. Therefore, smaller drug particles can increase the dissolution rate, supersaturation, or apparent solubility in the GI. This, however, does not necessarily transfer to equally higher permeability. The selection of excipients in drug formulations is very important because it can influence the solubility–permeability interplay . The lipophilic drugs in amorphous solid formulations can enhance both apparent solubility and flux across the intestinal membrane . However, as the amorphous glass state is not thermodynamically stable, crystallization can occur with time. This may bring out the stability and toxicity issues of the pharmaceutical products, which will have to be carefully examined .
Different solid formulation methods have been reported . The use of freeze‐drying in pharmaceutical formulations can usually offer fast dissolution of the solid formulation or provide high porosity for light and/or floating drug delivery system (FDDS). Of course, it is a necessary option for processing temperature‐sensitive drug compounds. Because the processed drugs are generally poorly soluble in water, non‐aqueous co‐solvents can be added to facilitate initial liquid formulation . This may also help to increase sublimation rate, due to the usually high vapour pressure of the non‐aqueous solvent. However, with the use of the co‐solvents, additional storage and processing safety need to be considered. The contents of residual solvent in the solid formulations should be assessed and, within the required limits, enforced by the regulatory bodies.
3.4.1 Spray Freeze‐drying
As described in Section 3.3.1, the spray freeze‐drying process can be used to produce pharmaceutical powder formulations. The produced formulations can enhance dissolution rate and apparent solubility of poorly water‐soluble drugs [29, 30]. When comparing spray freeze‐drying and spray drying, the powders produced by spray freeze‐drying may exhibit superior aerosolization efficiency and hence finer particles . Sugars and/or polymers are usually added as protectants or stabilizers to produce stable powder formulations. As a result of the spray freeze‐drying process, spherical, light, and porous micron‐sized powders are produced. This offers fast dissolution and good aerodynamics. In addition to spray freeze‐drying in vapour over liquid and spray freeze‐drying in liquid, it is possible to directly spray into a cold atmosphere in a chamber, which is like a direct opposite version of spray drying (cold atmospheres versus hot atmosphere). Another option is to spray onto a solid surface, where the liquid droplets form a thin film which is frozen ultrarapidly. Therefore, this process is sometimes called thin film freezing . To realize the thin film freezing process, the droplets of a liquid formulation can simply fall on a cold surface or spray onto a rotating steel drum containing a cryogen such as dry ice or liquid nitrogen [53–55]. To minimize water vapour condensation on the cold surface, the whole apparatus can be placed in a dry box with controlled humidity. The spray freeze‐drying process has been widely used for both poorly water‐soluble drugs and a variety of proteins and vaccines [52–56]. The powder formulations have been used for pulmonary, nasal, and needle‐free ballistic intradermal applications .
3.4.2 Orally Disintegrating Tablets (ODTs)
The orally disintegrating tablets (ODTs) are also known as orally dispersible drug delivery systems. The feature of the ODTs is that they can release drugs immediately when coming into contact with saliva . This type of drug formulations can be highly beneficial for patients who have difficulty in administering drugs via the oral route. Paediatrics, geriatrics, psychiatrics, nauseated or unconscious patients fall into this category of patients. Upon administration into mouth, the saliva penetrates into the highly porous ODTs that rapidly disintegrate to form a suspension of fine particles. The ODTs usually contain flavours and sweeteners (for taste marking) to enhance patient compliance. Hydrophilic polymers are usually used as porous matrices to stabilize drug particles, and with good wettability for fast disintegration . Unit size and disintegration time are the two main parameters to consider when assessing ODTs. The disintegration time is usually expected to be less than 1 min . ODTs can be categorized into three delivery groups: sublingual route, buccal route, and localized drug delivery .
Various methods have been used to fabricate the ODTs, e.g. spray drying, moulding, compression, and the cotton candy process, to name a few . The hot‐melt extrusion method may be of more industrial importance because the process can be operated continuously and is easy to scale up. The use of freeze‐drying is unique because highly interconnected porous and soluble polymeric structures can always be produced . This can lead to fast dissolution of the freeze‐dried products and instant formation of stable aqueous nanoparticle dispersions [8, 31, 59]. By using the freeze‐drying method, ODTs with ibuprofen have been produced . A factorial design approach was further used to optimize the conditions for the performance of the ODTs . Taste‐marking ingredients could be added into the freeze‐drying formulations and evaluated . Some examples of commercial ODTs available in the market include Zydis® tablets, Lyoc®, and Quicksolv® . The disadvantage for the ODTs produced by freeze‐drying is the weak mechanical stability and the potential to adsorb moisture. Thus, suitable seals and packaging are required for these ODTs.
3.4.3 Floating Drug Delivery System
The FDDS is one type of gastroretentive drug delivery system for improving gastric retention time [63, 64]. The prolonged presence or release of drugs can improve bioavailability and stable plasma concentration of the drugs, thereby reducing potential side effects and the amount of dosage. For orally administered drugs, their release and adsorption are directly related to gastrointestinal transit time. The stomach can be anatomically divided into three regions: fundus, body, and antrum pylorus. The fundus and body act as a reservoir for non‐digested mixtures while the antrum is the main site for mixing motion and pumping actions . The particle size of disintegrated drug dosages should be in the range of 1–2 mm to be pumped into the small intestine via the pyloric valve. The pH of the stomach in fasting state is 1.5–2.0, and 2.0–6.0 in fed state. A large volume of water or other liquids may raise the pH to 6.0–9.0 in the stomach. The resting volume of the stomach is approximately 25–50 ml .
There are different mechanisms for gastroretentive drug delivery, including mucoadhesion, sedimentation, and expansion . For the FDDS, once administered into the stomach, the density of the wetted dosage is lower than that of the gastric liquid, thereby floating and preventing it from pumping into the small intestine rapidly. This can be achieved by effervescent dosages and non‐effervescent dosages. In an effervescent dosage, swellable polymer and effervescent compounds (e.g. sodium bicarbonate) are included. When in contact with the acidic gastric fluid, CO2 is formed and released, which provides the buoyancy for the dosage to float. For the non‐effervescent dosage, a highly porous structure with good wetting and/or swellable property is required. When absorbing liquid in the stomach, the swollen gel‐like material with the trapped air can float and provide sustained release of the drug . The freeze‐drying is well placed to produce highly porous materials with tuneable pore structures  and hence is an effective technique to prepare the FDDS. For example, highly porous HPMC‐based tablets were prepared by freeze‐drying for the gastroretentive delivery of ecabet sodium (a locally acting antigastric ulcer drug). The porous structure lowered the tablet density and kept the tablets floating without any lag time, until the tablets were disintegrated and released . As expected, the FDDS is not suitable for drugs that may cause gastric lesions or are unstable under acid conditions.
3.4.4 Emulsion Freeze‐drying
Owing to the hydrophobic nature of many drug compounds, oil‐in‐water (O/W) emulsions have been employed for delivery and enhanced bioadsorption. Emulsion is a mixture of two immiscible liquid phases with one phase in the form of droplets dispersed in the other continuous liquid phase, usually stabilized by amphiphilic polymers or surfactants [66, 67]. Colloids or nanoparticles may be also used as stabilizers, producing Pickering emulsions. As per the requirement of immiscible liquid phases, emulsions are always composed of water and one immiscible organic solvent. Depending on which phase is the continuous phase, the emulsions can be classified as O/W emulsion (where water is the continuous phase) or water‐in‐oil (W/O) emulsion (where oil is the continuous phase) or double emulsions O/W/O and W/O/W (basically the pre‐formed emulsion disperses into another phase). Based on the size of the droplets, emulsions can be categorized into emulsions (droplet sizes from sub‐micron to 100 microns, thermodynamically unstable), miniemulsions or nanoemulsions (droplet sizes approximately 50–500 nm, better kinetic stability), and microemulsions (clear and thermodynamically stable, droplets <100 nm in diameter) .
O/W emulsions are usually used for lipophilic drug formulations, where the lipophilic drugs are dissolved in the oil droplet phase. The oils usually used include lecithin, soyabean oil, lipid, triglycerides, etc. Freeze‐drying is employed to improve the formulation stability and reconstitution properties. Because the oils may be liquid‐like but exhibit very low vapour pressure, they remain in the formulation after freeze‐drying. Cryoprotectants and bulking agents are added to generate stable lyophilized formulations. A submicron emulsion of antitumour drug Cheliensisin A with soyabean lecithin and medium chain triglycerides as the oil phase and sucrose (10 w/v%) added as cryoprotectant was freeze‐dried. The lyophilized formulation showed improved stability, lower IC50 values, and enhanced antitumour activity . Dry emulsion tablets produced by the emulsion‐freeze‐drying technique were studied for the delivery of a poorly water‐soluble drug hydrochlorothiazide . Medium chain triglyceride was the oil phase while the aqueous phase contained the bulking agent maltodextrin. The tablet strength increased significantly with the concentration of maltodextrin and decreasing pore sizes. Methylcellulose as an emulsifier–tablet binder also showed significant influence on tablet strength and disintegration time . Bufadienolides‐containing sub‐microemulsions and nanoemulsions were formed and freeze‐dried. The optimum cryoprotectants were found to be 20% maltose for nanoemulsions and 20% trehalose for sub‐micron emulsions. The freeze‐dried powders showed stability up to 3 months with no change in visual appearance, reconstitution stability, and particle aggregation . O/W microemulsions containing Amphotericin B were freeze‐dried, resulting in oily cakes, which could be easily reconstituted and stablized under the conditions studied. The microemulsion was prepared by adding polysorbate 80‐water solution to a lecithin/isopropyl myristate mixture under stirring. Amphotericin B solution in sodium hydroxide solution (1 M) was then added to the formed microemulsion at a temperature of 80 °C .
The emulsion‐freeze‐drying approach has been further developed by Zhang et al. to produce aqueous poorly water‐soluble drug nanoparticle suspensions [8, 31]. Unlike the use of non‐volatile oil phase in the emulsions and producing oily cakes [68–71], volatile organic solvents such as cyclohexane or o‐xylene are used to dissolve hydrophobic drugs. The formed organic solutions are then emulsified into aqueous phase containing polymer and surfactant to form O/W emulsions. Upon freeze‐drying, both water and the organic solvent are removed to generate dry porous nanocomposites. The highly interconnected porous structure allows the dry materials to dissolve in water in seconds to form stable aqueous drug nanoparticle suspensions. This will be the focus of Chapter 8 on freeze‐drying for nanomedicine.
3.5 Applications of Freeze‐drying in Biopharmaceutics
Based on World Preview 2016 Outlook to 2022 by EvaluatePharma, biologics will contribute 50% of the Top 100 product sales by 2022. Among the top 20 most valuable R&D projects, the majority of them deal with biologics, with monoclonal antibodies leading the way. Currently, in clinical applications or industrial research, the majority of the biologics/biopharmaceutics are proteins. Proteins function when they remain in the native secondary and/or tertiary structures. Many factors can lead to protein denaturation and aggregation. Indeed, this is one important reason that protein pharmaceuticals are traditionally administered by injection. This is the main barrier for biopharmaceutics during bioprocessing .
Because protein pharmaceutics are usually administered in parenteral formulations, liquid formulations would be preferred. However, due to relatively fast molecule motion and water‐facilitated reactions, the stability of proteins is a major issue during transport and long‐term storage. The stability issues may be addressed by suitable solid formulations, mostly prepared by freeze‐drying . This is because the degradation reactions can be avoided or slowed down sufficiently in the dry solid state. However, during a freeze‐drying process, the proteins experience freezing (cold) stress and dehydration stress, followed by the stability issue during solid‐state storage. When designing a freeze‐drying formulation and processing, protein stability is the ultimate target [1, 17]. All the formulation parameters (e.g. protein concentration and type and amount of excipients) and processing conditions should be optimized based on protein stability.
In this section, we describe first the different types of protein degradation mechanisms and procedures that can be taken to avoid or minimize such degradation routes. Then we will discuss the freeze‐drying of different biopharmaceutics and the important parameters investigated.
3.5.1 The Freeze‐drying Process for Biopharmaceutics
18.104.22.168 During Freezing
The dominant protein conformational motions are controlled by the hydration shell and the bulk solvent . The degree of hydration h can be defined as the weight ratio of water to protein. Dehydrated proteins do not function (some proteins begin to function at h ≥ 0.2) while full functions may require h > 1. Large‐scale motions are significantly affected by variation in the bulk solvent and controlled by solvent viscosity . When freezing a liquid protein formulation, solute concentration, increasing viscosity, and formation of ice crystals can all contribute to protein denaturation.
- Cold denaturation. The majority of proteins show cold denaturation well below the freezing point of water. This suggests that it may not have a significant impact on protein denaturation. However, with ice formation and protein concentration, the influence can be huge.
- Protein concentration. At a high concentration (whether because of initial preparation or freezing induced), the number of proteins in a certain volume is high. This can lead to stronger interaction between proteins and greater potential for aggregation and precipitation . Also, the surrounding bulky solvent may have higher viscosity, exerting impact on protein dynamics . However, there is potential advantage with higher concentrations: the percentage of proteins exposed to freezing stress may be lower . Since the percentage degradation during protein formulation is a critical parameter, suitable protein concentrations should be selected.
- pH change. Proteins are usually stable in a narrow pH range and many of them are only stable at physiological pH. During freezing, because of the solute or ion concentration effect and possible crystallization of certain salts, the pH can vary considerably and cause protein denaturation. Buffering species are usually added to stabilize the pH change but some buffering salts should be avoided due to the crystallization during freezing. Na2HPO4 crystallizes more easily than NaH2PO4, which can result in a significant pH drop . With potassium phosphate, the crystallization of dihydrogen salt may give a final pH near 9 . Therefore, the sodium phosphate and potassium phosphate buffers should be avoided. Buffers with minimal pH change upon freezing may be selected from citrate, histidine, and Tris .
- Ice–water interface. Adsorption of proteins at the interface can lead to protein instability. This is because the interaction with interface is favoured when a protein is partially unfolded to give greater exposure of hydrophobic amino acid side chains (usually in the core of protein) to the interface . Air–water interface exists in the liquid formulation whilst the ice–water interface develops during freezing. To minimize the interface damage, surfactants can be added to the formulations. Owing to their amphiphilic nature, surfactants tend to adsorb at the interface thereby preventing or reducing the adsorption of proteins at the interfaces.
To start with a stable formulation, buffers, ions, non‐aqueous organic solvents, and surfactants may be included [1, 11, 17]. In order to protect the freezing stresses as outlined earlier, excipients such as sugars, polymers, surfactants, and amino acids may be added depending on the specific proteins. The protection of proteins by sugars (used very often) during freezing (and also freeze‐drying) may be attributed to the universal thermodynamic mechanism developed by Timasheff for control of protein stability and reactions with weakly interacting co‐solvents . It has been established that, nearly any sugar or polyol, acting as excluded salts, can provide increased conformational stability of proteins. Furthermore, addition of amino acids, salts, and many polymers may also fall into this category .
22.214.171.124 During Freeze‐drying
Dehydration stresses are experienced by proteins through the partial removal of water molecules from the hydration shell. Generally, the water content in freeze‐dried solids is less than 10%. This can denature proteins and prevent their functioning . During a dehydrating process, the proteins may transfer protons to ionized carboxy groups and reduce the charges in the structural proteins. The resulting low charge density can lead to stronger protein–protein hydrophobic interaction and cause protein aggregation . Another stress is the increased surface (interface of solid/air) generated by sublimation of ice. In addition to the instability related with surface adsorption, this may have a pronounced effect during storage of the solid formulations.
FTIR has been mostly used to monitor protein denaturation during freeze‐drying, with the focus in the amide I, II or III region . Moreover, hydrogen bonds are disrupted during freeze‐drying, leading to an increase in frequency and a decrease in the intensity of hydroxyl bond stretching. As observed in protein solid formulations, β‐sheet contents are increased while the contents of α‐helix are decreased. An increase in β‐sheet content usually indicates protein aggregation and/or intermolecular interaction. This can be monitored by two major IR bands at about 1617 and 1697 cm−1 .
Because freezing and freeze‐drying are two separate processes with different stresses induced, both cryoprotectants and lyoprotectants are required for solid protein formulations generated by freeze‐drying. Fortunately, the widely used excipients such as sugars or disaccharides function in both processes to stabilize the proteins. Sugars such as trehalose and sucrose are the mostly used excipients. A ‘water replacement’ mechanism is generally utilized to emphasize how sugars can stabilize proteins during freeze‐drying. That is, sugar replaces water molecules from the hydration shell and then interacts/stabilizes the proteins through hydrogen bonding [11, 76].
126.96.36.199 During Solid State Storage
The instability of solid protein formulations during storage has been reviewed from the perspective of chemical and physical factors and also from the angle of thermodynamic and kinetic influences [10, 11, 77, 78]. In this section, we describe how chemical reactions and physical interactions contribute to protein denaturation/aggregation, what excipients may be added to stabilize the proteins, and then explain stabilizing mechanisms proposed in literature.
There are several typical reactions involved in the instability of solid protein formulations.
- Deamidation. This is a common degradation route for peptides and proteins containing Asn and Gln [10, 11]. For the Asn deamidation, it generates two degradation products (Asp and isoAsp) at the site of the original Asn residual. The mechanism and more detailed information may be found in the reviews [[10, 11], and references therein] and the website on this topic (www.deamidation.org).
- Hydrolysis. The degradation by hydrolysis at As/Asp residues and Asp‐associated hydrolysis of the peptide backbone. This type of reaction shows the sensitivity to pH and buffer species . The hydrolysis of peptide backbone is also observed in antibodies containing no Asp.
- Protein glycation(Maillard reaction). This type of reaction occurs between the carbonyl groups of reducing sugars and the lysine and arginine residues (the base) in proteins. A reducing sugar exhibits a free aldehyde group or ketone group and can act as a reducing agent. All monosaccharides (e.g. galactose, glucose, and fructose) are reducing sugars. Disaccharides can be either reducing or non‐reducing. Non‐reducing disaccharides include sucrose and trehalose. Their glycosidic bonds between anomeric carbons cannot convert to the open‐chain form with aldehyde group at the end. The examples of reducing disaccharides include lactose and maltose. The Maillard reaction is named after French chemist Louis Maillard and has been widely investigated in food industry. It is related to aroma, taste, and colour and is involved in cooking processes such as roasting, baking, and grilling. It is also known as non‐enzymatic browning reactions because of the colour developed during the reaction. The Maillard reaction is very complicated but the initial basic reaction is the condensation of a reducing sugar with a compound containing an amino group (e.g. lysine or lysine residuals in peptides and proteins). The condensation product is N‐substituted glycosilamine, which rearranges via Amadori rearrangement and then follows different mechanisms under different pHs to finally form Melanoidins (brown nitrogenous polymers) .
Oxidation. Proteins containing His, Met, Cys, Tyr, and Trp amino acids can be denatured by potential oxidation reactions on these side chains. The reactions may be easily initiated by atmospheric oxygen or the presence of other oxidation agents. The reactions may be photocatalysed or metal‐catalysed. Apart from the intrinsic nature of the amino acids, surface area, moisture content, pH, and the presence of metal ions or other impurities in the solid protein formulations can have significant impact on the rate of oxidation. To avoid or limit the oxidation reactions, the solid formulations should be sealed properly (to reduce moisture or other solvent adsorption), exposure to oxygen (air) and potential UV irradiation should be minimized, and excipients should be added to reduce oxidation rate (e.g. mannitol as free radical scavengers, ethylenediaminetetraacetic acid (EDTA) to chelate metal ions) .
- Disulfide formation. The Cys residues can form disulfide bonds, which may cause aggregation and affect the overall protein conformation. This process may be retarded by the removal of free Cys residues .
Physical instability mainly involves thermal denaturation (particularly at high storage temperature) and denaturation from surface adsorption. Freeze‐drying usually leads to a highly porous structure with high surface areas, which may contribute significantly to surface adsorption. Thermally induced denaturation, characterized by DSC, is usually irreversible because the unfolded protein molecules form aggregation. There are different mechanisms of protein aggregation induced by various factors [11, 80]. For a freeze‐drying process, protein aggregation can occur from freezing and lyophilization stresses. During storage of the solid formulations, the aggregation is likely to result mainly from chemical modifications, motion through the amorphous matrix, and surface‐induced aggregation. Moisture content, pH, and other impurities can considerably influence the protein stability. The interaction of excipients with proteins and glass transition temperature (Tg) of the amorphous matrix are two important factors for storage of protein formulations. Generally, amorphous matrices with higher Tg reduce protein motion ability and contribute to higher stability. The presence of moisture can considerably reduce the overall Tg of the matrix and thereby the stability of the protein. In addition to the common stabilizing excipients such as sugars and surfactants, bulking agents (e.g. mannitol) can help produce an elegant cake structure and stabilize the proteins. However, the crystallization of the excipients or bulking agents can have detrimental effects on protein stability.
Upon reconstitution, small aggregates may be soluble (reversible aggregates) or well dispersible (for sizes 1–100 nm). The larger particles may be suspended or precipitated from the liquid formulations. Protein aggregates may show significant cytotoxic effects and immune response. The relationship between protein aggregates and immunogenicity has been examined and a mixed picture has been shown . This may depend on the specific formulations and specific proteins. The important thing is that the protein aggregates in the formulations should be properly characterized and provided as a pharmaceutical product.
Improving Protein Stability
Depending on the proteins involved, suitable excipients should be selected for the formulations. Some aspects of choosing excipients have already been discussed earlier, for example, the use of non‐reducing sugars, surfactants, suitable buffers, etc. The pH of the formulations should be considered both in the freeze‐drying process and in the reconstitution stage. The solution pH of the liquid solution after reconstitution can be regarded as a measure of the solid state microenvironment pH. This can help monitor the possible ‘pH’ of the solid formulation and its effect on protein stability and improve on the general protein formulation.
As mentioned earlier, the moisture content plays an important role. Maintaining a low moisture content is favourable for protein stability. Moisture transfer from the stopper to the solid formulation should be avoided. The proper seal on the vials should be kept all the time. The vials containing solid protein formulations may be stored in a dry and inert atmosphere. This will also decrease the chance of oxygen exposure.
Storage temperature is another important parameter. The degradation kinetics is faster at higher temperature. A low storage temperature is always preferred although this is not always possible particularly during shipping. Another potential problem is the crystallization of amorphous matrix. The rate of crystallization generally increases with increasing temperature and moisture content. Crystallization of the matrix can denature a protein because of the loss of closer excipient interaction with proteins and the possible decrease in Tg of the amorphous matrix. With the crystallization, water molecules can be excluded from the crystalline phase and thereby increase the moisture content of the amorphous phase. The increasing moisture can result in the decrease of Tg. For example, with the widely used excipient/bulking agent mannitol , a metastable phase of mannitol can crystallize when storage/shipping temperature is higher than 45 °C, leading to disastrous destabilization of proteins . For mannitol, there are three anhydrous crystalline forms (α, β, δ forms) and two metastable phases (hemihydrate and amorphous). Mannitol tends to form crystalline forms during freeze‐drying but the addition of an excipient such as sucrose can facilitate the formation and stabilize the metastable forms .
Stabilization Mechanisms in Solid Protein Formulations
- ‘Water replacement’ mechanism. This falls into the category of thermodynamic consideration. As mentioned in the stabilization measures during freeze‐drying, excipients such as sugars or polyols replace water in the hydration shell. This involves the interaction (mostly hydrogen bond formation) between the excipients and the proteins to maintain the native conformations of the proteins. Amorphous states of proteins and excipients allow closer contact between them and greater H‐bonding opportunities. The crystallization of excipients (e.g. mannitol) during storage can cause increasing instability due to less hydrogen bonding . The residual water in the matrix residing at the protein–sugar interface (so‐called ‘water entrapment’) is also able to provide thermodynamic stabilization of the proteins via hydrogen bonding .
Matrix vitrification. This is dynamic (kinetic) control of protein degradation in amorphous matrix. The very high viscosity in the glass state can significantly slow down the motion, conformation relaxation, and chemical denaturation of the protein. As the storage temperature may vary, amorphous matrix with higher Tg is usually thought to offer higher stabilization, although this may not always be the case. The vitrification mechanism is related to the slow α relaxation of the sugar matrix. A longer α relaxation time τα may indicate slower degradation . In the pharmaceutical field, it is common to assume that log (τα) scales approximately with (Tg − T) . τα may be also determined by positron annihilation spectroscopy or by enthalpy relaxation techniques .
- β‐Relaxation of the matrix. Although there are plenty of experimental observations that can be explained by the above two mechanisms, they cannot adequately account for the observation of protein stability in amorphous sugar matrix with antiplasticizing additives. Instead, the observed protein stability is directly linked to high‐frequency β relaxation processes . Amorphous solids exhibit three characteristic dynamic processes over a wide range of timescales: βfast relaxation (picoseconds, temperature independent), Johari–Goldstein β (βJG) relaxation (microsecond to millisecond), and the slow α relaxation (seconds to months) . In the study of protein in sugar matrix with antiplasticizing additives, a linear relationship between degradation rates and β relaxation was observed. It was proposed that the high frequency β relaxation process impacts protein degradation through coupling of the β relaxation to local protein motions and diffusion of small molecular reactive species in the amorphous matrix .
3.5.2 Biopharmaceutical Formulations
In Section 3.5.1, the routes to protein degradation and the ways of improving protein stability have been described. Selection and addition of suitable excipients are key to stable protein formulations. Figure 3.4 shows the molecular structures of the common excipients employed in protein solid formulations. In this section, we discuss, through some examples (without attempting to cover the literature comprehensively), how freeze‐drying processes have been applied in biopharmaceutical solid formulations and how the processing conditions and the selected use of excipients improve the stability of the biopharmaceutics.
188.8.131.52 Peptide Formulations
In the classification spectrum of drugs based on molecular weight (Mw) (Figure 3.3), peptides can be positioned in the Mw region of 500–5000. Compared to small molecular drugs and proteins, pharmaceutical peptides are under‐investigated, although certainly emerging. There are more than 60 US FDA‐approved peptide medicines and around 140 peptide drugs are in clinical trials . But there are only approximately 20 new chemical entities (NCEs) for peptides and this number has not changed too much over the years . Generally, small molecule drugs exhibit good solubility, membrane permeability, oral bioavailability, and metabolic stability, but poor target selectivity. Biopharmaceuticals such as proteins show target specificity and high potency but low stability and permeability. It is thought that the peptide drugs may combine the advantages of small drugs and proteins . It should be pointed out that the classification based on Mw of 500–5000 is quite arbitrary. There are no distinctly different properties or scientific meaning for peptides and proteins around Mw 5000. For example, some researchers put insulin in the category of peptides and some others have reported it as a protein. Peptides may be defined as molecules containing fewer than 50 amino acids .
Native peptides in general do not cross cell membranes. Therefore, membrane permeability elements are inserted into the peptide molecules to form ‘cell penetrating peptides’ . Via rational design and chemical modifications, e.g. introducing α‐helixes, forming salt bridge, lactam bridge, the peptides can show better pharmaceutical properties . Peptides with cyclic backbone are better resistant to proteolytic degradation . Currently, most of the peptide drugs have less than 20 amino acids, representing 75% of existing marketed peptides . Greater effort may be made to discover peptide drugs with the size up to 50 amino acids.
To improve the stability of peptide drugs, suitable excipients may be added into the formulations. Micro‐ and nanoencapsulations are also effective and have been investigated . Freeze‐drying is an effective method to prepare stable dry peptide formulations. For example, glucagon was dissolved in three different buffer solutions and then mixed with the excipient solutions. Polysorbate 20 was added as a polymeric surfactant. Three carbohydrate excipients, trehalose, hydroxyethyl starch, and β‐cyclodextrin, were investigated. The freeze‐dried formulations were characterized by MS, HPLC, FTIR, DSC, and turbidity . Contrary to proteins, it was thought that stabilizing the secondary structure of glucagon was not a prerequisite for its stability. The study showed that stable freeze‐dried glucagon formulations could be produced. The presence of polysorbate 20 facilitated the decrease of glucagon aggregation and chemical degradation .
In a freeze‐drying study with a model peptide hormone‐human secretin (0.002 w/v%), excipients of NaCl (0.9 w/v%), cysteine‐HCl (0.15 w/v%), and mannitol (2 wt%) were investigated . After reconstitution, the amount of secretin was measured by HPLC while the aggregated particles were examined by dynamic laser scattering (DLS). The storage of the solid formulations was studied at different temperatures (−20, 4, 25 and 25 °C/60% RH) and different periods (0, 1, 4, and 8 weeks). The study demonstrated that the increased crystallinity of mannitol occurred with time. The aggregated particles and increased reconstitution time were observed with longer storage period and higher temperature/humidity .
184.108.40.206 Protein Formulations
Freeze‐drying has been mostly employed in solid protein formulations [1, 10, 11, 17, 56]. Some of the examples include monoclonal antibody (e.g. IgG) [88, 89], insulin [90, 91], cytokine , and enzymes [76, 90, 93]. Ó'Fágáin and Collition described the practical procedures of freeze‐drying protein formulations, aiming to achieve protein stability during freeze‐drying and storage . Prevention of bacterial contamination, selection of suitable excipients, low temperature storage, and the operational procedures of the freeze‐drying were all covered, with useful notes on best practice and common pitfalls . The research and development on solid protein formulations have focused on optimizing the excipients, formulations, and freeze‐drying process controls.
For example, alkaline phosphatase (a protein enzyme) was stabilized with inulin or trehalose and also ammediol as a plasticizer. The enzymatic activity of the protein remained stable when the Tg was well above the storage temperature . It was concluded that the vitrification mechanism played a dominant role in stabilization when the storage temperature was up to 10–20 °C higher than Tg whilst the water replacement mechanism was the main factor for stabilization at higher Tg . For the same protein, the freeze‐drying process was also performed with water‐binding substrates (sucrose, lactose) or non‐water‐binding substrates (mannitol, PVP). Not all the water‐binding substrates (the reducing sugar, lactose) could help to maintain the protein activity . In the freeze‐dried insulin formulation with trehalose or dextran, the two types of formulations showed different stability behaviours during storage with regard to storage temperature and relative humidity. β‐Relaxation mechanism seemed to be responsible for the degradation of insulin in the matrix of trehalose and dextran .
There are many studies focusing on the use of different excipients and newly designed excipients. It is important to find out what exactly affects the stability of the target proteins and what alternative methods may be taken to improve the stability. Oligosaccharides dextran and inulin of approximately the same Mw was combined with four proteins of different Mws (6–540 kDa). It showed that molecular flexibility played a significant role in stabilizing the proteins. As long as the matrices are amorphous, smaller and more flexible sugar molecules can provide better stabilization due to less steric hindrance . New excipients may be designed and synthesized to give better stabilization of protein pharmaceutics. For example, a series of polyether‐modified N‐acy amino acids were synthesized and they were used as non‐ionic surfactants in protein formulations. The surfactant containing a phenylalanine moiety has shown slow thermal degradation and aggregation of IgG and IgG‐derived pharmaceutics, compared to the commonly used polysorbate surfactants .
Different freezing techniques, including freeze‐drier shelf , spray freeze‐drying , and thin film freezing , have been used for the preparation of solid protein formulations. An inhibition of mannitol crystallization was observed by addition of sucrose and NaCl during freezing. However, the annealing step promoted the formation of mannitol hydrate, which is known to undergo conversion into the anhydrous polymorph upon storage . It was found that the drying step was necessary to cause aggregation of the recombinant human interferon‐γ in a freeze‐dried sucrose formulation, not the adsorption to the ice/liquid interface. However, an additional annealing step resulted in more native‐like protein structures and suppressing the aggregation upon reconstitution .
It is favourable to perform the freeze‐drying step at a higher temperature as this will considerably shorten the freeze‐drying cycle and it is economically beneficial. It is widely accepted that the product temperature during freeze‐drying should be below the Tg in order to get an elegant cake structure. Higher temperature freeze‐drying may be achieved by selecting suitable excipients that have high Tg. Recently, it was shown that it was possible to freeze‐dry above the Tg in amorphous IgG formulations while maintaining the product quality. This was achieved by using high‐concentration protein formulations . However, this should be carefully examined, depending on specific protein formulations. This is because high protein concentration and the drying procedure could promote the transformation of excipient polymorphs .
Proteins have been encapsulated to protect their stability (either in the storage or in the GI) and enhance the chance of oral administration with better patient compliance . Spray drying and spray freeze‐drying are some of the widely used methods [56, 100]. Dry micron‐sized particle powders are usually produced. Precipitation and emulsion evaporation are the mostly used methods to produce protein‐encapsulated nanospheres [85, 100]. As these nanospheres are prepared as aqueous suspension, freeze‐drying of the nanoparticle suspensions is usually required to produce dry formulations with improved stability for shipping and storage . Similarly to freeze‐drying of liquid protein formulation, the proteins in nanoparticle suspensions during freeze‐drying are highly likely to experience the same freezing stress, freeze‐drying stress, and the stability issues during solid storage. This indicates that suitable excipients should be also included in the protein‐encapsulated nanoparticle suspensions.
Insulin‐loaded poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles were prepared by an emulsion evaporation method based on W/O/W double emulsions and were freeze‐dried with different cryoprotectants (trehalose, glucose, sucrose, fructose, and sorbitol) at a concentration of 10 w/v% . In general, the addition of cryoprotectants improved the protein stability. Compared to the formulations without cryoprotectants, the content of insulin native structure in the freeze‐dried formulations was enhanced from 71% to 79% on average. Among these formulations, the sorbitol‐based formulation showed better stabilization capability . Effects of different carbohydrates and polymeric cryoprotectants (Microcelax®‐mixture of lactose and Avicel, AvicelPH102‐microcrystalline cellulose, mannitol, sucrose, Avicel RC591, maltodextrin, Aerosil, and PEG4000) on freeze‐drying of nanostructured lipid carriers were investigated. Among the tested excipients, Avicel RC591 was found to be most effective in maintaining the particle sizes . Different sugar excipients at the concentrations of 1–3 w/v% were applied to the freeze‐drying of non‐loaded and doxorubicin‐loaded PEGylated human serum albumin (HSA) nanoparticle suspensions. It was found that 3 w/v% concentration generated the best stabilization result and sucrose and trehalose displayed better long‐term storage stability than mannitol . Later, a similar finding for freeze‐drying of HI‐6‐loaded recombinant HSA nanoparticles was reported . Instead of adding sugar excipients into the nanoparticle suspensions, the co‐encapsulation of cryoprotectants (trehalose, sucrose or sorbitol) into insulin‐loaded PLGA nanoparticles could also mitigate the freezing stresses to maintain protein activity . Similar to freeze‐drying protein formulations, annealing of frozen nanoparticle samples could affect the drying process. This was shown by the accelerated sublimation rate but without affecting the nanostructure when annealing a nanocapsule sample .
220.127.116.11 Vaccine Formulations
Vaccines save millions of people over the years by immunization against tuberculosis, polio, diphtheria, tetanus, measles, hepatitis B, and pertussis. It is estimated that these vaccines can save about 2.5 million lives each year. However, the significant issue of vaccine shortage remains and vaccines are not accessible to every person in developing countries. It is the goal of the Global Vaccine Action Plan (GVAP), to deliver full access to immunization by 2020. Vaccine shortage has been enhanced by stability problems during storage and shipping as well as by the lack of medical workers (because vaccines are usually available as injection formulations). Vaccines can be classified into different types: live attenuated vaccines, inactivated vaccines, subunit vaccines, toxoid vaccines, conjugate vaccines, DNA vaccines, and recombinant vector vaccines . Live attenuated vaccines consist of viruses that have lost their virulence but are still able to provide a protective immunity against a virulent virus. These types of vaccines are easier to produce (than inactivated vaccines) and are the most successful of the human vaccines because they can offer long‐term immunity .
Adjuvants are materials added to vaccine formulations in order to improve immunological response. The criteria for an adjuvant are safety, stability, and the cost. Adjuvants are usually introduced to subunit and recombinant protein vaccines . These vaccines are highly purified antigens, contain only a part of the pathogen to generate immune response, and are safer because they have no ability to revert to a virulent form. The often used adjuvants include aluminium salts, emulsions, and liposomes, which have been proven to be safe and cheaper [109, 110]. There are newly developed adjuvants that combine the usual adjuvants and immune potentiators, e.g. AS04 [109, 110]. For vaccine formulations, the interaction of vaccine and adjuvant and the stability of both vaccine and adjuvant should be considered .
Vaccines are conventionally produced in liquid forms. The stability of vaccines may be maintained in a cold medium with suitable cryoprotectants such as non‐reducing sugars, polymer surfactants, and buffers. The storage temperatures may go down to −20 °C or even −60 °C. A storage temperature above 8 °C is always harmful for vaccines . Whenever the cold temperature cannot be maintained during storage, shipping, or delivering, the outcome of the accelerated degradation in aqueous medium can be disastrous.
Like solid protein formulations, the freeze‐drying of vaccine can also provide higher stability and the potential for oral administration [108, 111]. The latter development can be highly beneficial because a trained medical worker may not be required to deliver the vaccine to patients. Similar stabilizing mechanisms and approaches from solid protein formulations may be applied to vaccine freeze‐drying [110, 112]. However, for live attenuated vaccines, additional freezing stabilization may be required, like what has been required in cell cryopreservation [108, 113]. The control of ice nucleation and crystal growth is critical in cryopreservation where the dehydration stress during freezing and mechanical damage of ice crystal growth should be minimized. The effects of change in pH, osmolarity, and solute concentration can be more significant. In the vaccine freeze‐drying applications, similar excipients as those used in solid protein formulations have also been employed, e.g. potassium phosphate (pH 6–8, in the range of virus activity, despite small pH change during freezing), sucrose/trehalose/sorbitol . Usually, the vaccine and adjuvant are mixed together before using. This can generate financial and technological barriers. By freeze‐drying a nanoemulsion containing both tuberculosis antigen and adjuvant, one single vial containing the dry formulation of both components can be produced, reducing the cold‐chain dependence of the vaccine product . Although freeze‐drying is very promising, other drying methods such as foam drying should still be considered, depending on the type of vaccines. In a recent study, it was found that the dry formulation of live attenuated influenza vaccines prepared by foam drying was an order of magnitude more stable than those prepared by freeze‐drying and spray drying .
18.104.22.168 Nucleic Acid‐based Formulations
Nucleic acids are biopolymers composed of monomers called nucleotides. A nucleotide is formed from the condensation reaction of phosphoric acid, 2‐deoxyribose (or ribose), and N‐containing organic bases. The polynucleotides formed from 2‐deoxyribose are called deoxyribonucleic acids, abbreviated as DNA, while the ones from ribose are called ribonucleic acids, abbreviated as RNA. DNA are double strands formed from pairing bases. Three kinds of RNAs are identified: ribosomal RNA (rRNA, the majority), messenger RNA (mRNA), and transfer RNA (tRNA). In both RNA and DNA, the acidity and negative charges result from the phosphoric acid in the polymer chains.
Nucleic acids are used as biopharmaceutics to treat infectious diseases and incurable diseases such as cancer and genetic disorders. The therapeutic treatments depend on either replacing a modified gene with the healthy one or completing a missing one to express the required proteins . This means that the nucleic acid pharmaceutics need to transport through cell membrane (for RNAs) and further move into the nucleus (for DNAs). It is very difficult for the naked RNAs or DNAs to survive the transporting process. Viruses are initially used as carriers to transport RNA/DNA into the cell or cell nucleus. However, the risk of triggering immune response by using viruses as delivering vectors can be disastrous. Furthermore, the costs of preparation and storage of virus formulations are very high. Non‐virus gene carriers have thus been widely investigated and used .
The nucleic acid‐based therapeutics include plasmid DNAs (pDNAs, introduce transgenes into cells), oligonucleotides (short single‐stranded segments of DNA, duplex with mRNA for antisense applications, triplex with DNA for antigene applications), ribozymes (RNA molecules able to sequence‐specifically cleave mRNA), DNAzymes (analogues of ribozymes with greater stability), aptamers (small single‐stranded or double‐stranded segments, directly interact with proteins), and small interfering RNAs (siRNAs, short double‐stranded RNA segments, downregulate disease‐causing genes via RNA interference) . pDNAs typically contain 5000 base pairs or more while siRNAs usually have only 20–25 base pairs . In order to deliver DNAs/RNAs, the commonly used non‐virus carriers are cationic, forming complex with DNAs/RNAs via electrostatic interactions. These carriers include lipids (forming lipoplex), polymers (forming polyplex), and nanoparticles with positive surface charges . Different lipids and polymers such as polyethyleneimine (PEI), poly(lysine), chitosan, and dendrimer polyamidoamine have been widely used. The complexation of polycations and pDNAs can condense the large pDNA molecules into very small structures that can enhance its transport across cellular barriers. But this condensation effect may be limited to polynucleotides greater than 400 bases .
Liquid formulations can be readily prepared and are convenient to use particularly in parenteral administration. However, polyplexes and lipoplexes in aqueous media are not favoured for shipping and long‐term storage . Freeze‐drying the liquid formulations is an effective route to enhancing storage stability. Linear PEI (Mw 800 Da–800 kDa) was complexed with oligodeoxynucleotide (ODN) and ribozyme. The freeze‐dried products showed no loss of activity for ODN–PEI and ribozyme–PEI but a decrease of the transfection efficiency was observed for plasmid–PEI. However, this could be addressed by adding trehalose, mannitol or sucrose as lyoprotectants . In another study, the freeze‐dried plasmid/PEI (Mw 22 kDa) polyplex showed long‐term stability. The products from isotonic formulations with 14% lactosucrose, 10% hydroxypropylbetadex/6.5% sucrose or 10% povidone/6.3% sucrose showed no particle size change after storage for 6 weeks at 40 °C. Polyplexes with lactosucrose or hydroxyproplybetadex/sucrose showed high transfection efficiencies and cellular metabolic activities . Mesoporous silica nanoparticles were coated with PEI and then complexed with DNA. The freeze‐dried formulations showed enhanced gene expression and higher efficiency. The use of trehalose as lyoprotectant facilitated the formulation stability – maintaining the activity for at least 4 months storage at room temperature . Freeze‐dried lipid/DNA complexes with glucose, sucrose, or trehalose as lyoprotectants were investigated for longer term storage (up to 2 years) at temperatures of −20 to −60 °C. Degradations were observed in terms of transfection rates, particle size, dye accessibility and supercoil content in all the temperatures. It was found that preventing the formation of reactive oxygen species in the dry state storage was very important to maintain the stability .
It was observed that freeze‐drying siRNA–liposome in ionic solutions led to the functionality loss of 65–75% while the addition of sugars (trehalose, sucrose, lactose, glucose) could maintain transfection efficiency with no loss . The freeze‐dried siRNA with PEGylated lipids showed the mucoadhesive property and could be rehydrated by body fluid to form a hydrogel and achieve the sustained release of siRNA . Trehalose‐based block copolycations were synthesized and could form stable complex with pDNA. These polymers could act both as stabilizer and lyoprotectant. Without adding sugar as lyoprotectant, the freeze‐dried formulation showed good stability and high gene expression after reconstitution. From the study of the in vivo pDNA delivery, these polymers exhibited favourable properties in cytotoxicity, cellular uptake, and transfection ability .
Spray freeze‐drying is an alternative technique for lyophilization of nucleic acid‐based formulations, particularly for lipid nanoparticle [127, 128]. In addition to the enhanced stability, spray freeze‐drying is highly effective in producing porous powders for pulmonary delivery via inhalation devices . Particularly, due to its poor pharmacokinetics, it is preferred to deliver siRNA for local administration, e.g. inhalation for respiratory diseases . Sugars are usually added as lyoprotectants to enhance the formulation stability. This has been demonstrated by lipid/pDNA  and PEI/DNA with sugar additives (sucrose, trehalose, mannitol) . Biodegradable polycations were synthesized and formed complex with pDNA. Porous particle powders (5–10 μm in diameter) were produced by the spray freeze‐drying process, with the integrity of pDNA maintained . It was also noted that the addition of L‐Leucine in the formulation could improve the inhalation performance . pH responsive peptides were complexed with DNA. Mannitol was added as lyoprotectant for the preparation of inhalable dry powders . This study also compared the performance of the formulations produced both by spray freeze‐drying and spray drying. The spray drying powders showed some advantages . pH responsive peptides were also used in the inhalable siRNA formulations, which showed antiviral activity against H1N1 influenza virus, although prepared by a spray drying method .
3.6 Freeze‐drying in Food Applications
3.6.1 Simple Freeze‐drying
Drying is a process used to preserve food. Industrial production of foods is generally achieved by convective drying or hot air drying. However, these drying methods often lead to significant shrinkage and quality loss of the foods. Freeze‐drying has been widely used for food preservation to improve the long‐term storage, but mainly for high‐value foods. The high‐value foods may include but are not limited to: (i) seasonal and perishable commodities; (ii) baby foods with maximum quality and nutrition; (iii) nutraceutical foods; (iv) organoleptic foods (e.g. flavours, aromatic herbs, coffee); and (v) specially designed foods for outdoor activities such as military foods, space foods, etc. . The drying process may cause changes in the physical aspects including colour (although this may be a result of chemical degradation/reaction), structure (e.g. hard casing, shrinkage), and chemical deterioration of aroma compounds and degradation of nutritional components. Generally, freeze‐drying can lead to minimal shrinkage, maintain the porous structure and texture, and offer the best potential for rehydration. For example, when drying berries, the volume shrinkage during freeze‐drying is around 5–15% while it is approximately 80% for air drying . Owing to the greater costs than air drying, freeze‐drying can only be economical for the production of high‐value foods. But it has been widely used in the production of dry meat, fruits, vegetables, and functional foods [135–138].
Although freeze‐drying is a better drying technique in preserving food quality, the freeze‐dried food may be not as nutritious or delicious as fresh food. For example, the content of phenolics is lower in the freeze‐dried extract . After freeze‐drying fruit pulps, the activity, texture, and browning index should be evaluated against the fresh ones . Other techniques may be combined with freeze‐drying to reduce the drying time and/or improve shelf time. For example, during atmospheric freeze‐drying of shrimps, pre‐treatment by vacuum thermal drying and osmotic dehydration were used to reduce the drying time and the effects were investigated . Broiler chicken meat was treated by a flow of ozone before freeze‐drying. This process could extend the shelf‐life up to 8 months .
For sensitive and/or volatile food additives, simple freeze‐drying would not be able to preserve these additives. Encapsulation of these additives into matrix or nanospheres/microspheres has been an effective approach to addressing this problem. The encapsulation can protect the active/volatile ingredients from the surrounding environment, prevent or reduce the loss, and control the release of these ingredients in a timely manner at the right place. This method can transfer the liquid form to an easily handled solid form, also offering longer‐term storage [28, 143]. The encapsulation procedure involves the core materials that are incorporated into particles or matrix and the wall materials that encapsulate or surround the active core materials. A variety of food components, protein powders, oils, flavours, cells, and enzymes have been used as core materials. The common wall materials include maltodextrin, chitosan, modified starch, gums, whey protein, skimmed milk powder, soya protein isolate, gelatin, cyclodextrin, lecithin, and modified cellulose [28, 143–145].
Different methods have been used for the encapsulation, including spray drying, extrusion, coacervation, emulsification, molecular inclusion, and fluidized‐bed coating [28, 143–145]. Freeze‐drying is unique in that it can result in minimal shrinkage, has a good structure and is suitable for heat‐sensitive actives. The encapsulation can be carried by directly freeze‐drying aqueous solutions containing the wall material and food ingredients (e.g. fruit extract) . The prerequisite is that these food actives should be soluble in water although it is possible to suspend powders in the aqueous medium. For the hydrophobic compounds or oils, an emulsion may be formed first and then subjected to freeze‐drying [147, 148]. However, the mostly used method is spray freeze‐drying. Different liquid formulations, including aqueous solutions, suspensions, and emulsions, can be spray freeze‐dried to produce dry encapsulated powders [28, 36, 143–145, 149]. These powders can be easily handled for packaging or mixing with other food ingredients. The type of the wall materials and the porosity and the size of the microparticles may be tuned for controlled release or fast rehydration.
3.6.3 Probiotic Foods
Probiotics can be defined as live microorganisms that benefit the host when administered. Probiotic foods can benefit the immune system, strengthen the mucosal barrier and suppress intestinal infection. High levels of viable microorganisms (in excess of 109 cfu day−1) may be required in probiotic foods for efficacy . For probiotics to be used in foods successfully, several criteria must be met: good viability and activity, stability during processing and storage, being alive during passage in the upper GI tract and arriving at the action site, and functioning in the gut environment . The dairy fermentation industries, including production of cheese, yoghurt, and sour cream, are ranked second only to the production of alcoholic beverages. It has been well established that some lactic acid bacteria play a very important role in the fermentation processes . In fermented probiotic processes, it is common to culture probiotic bacteria with other types of bacteria (starters) in order to generate good sensory properties.
Freeze‐drying is a preferred method to make dry formulations of microorganisms, which offer the benefit of easy handling, transport, and long‐term storage . Again, the low shrinkage and porosity associated with freeze‐drying contribute to fast rehydration with the maximal activity of microorganisms. Like cell cryopreservation  or freeze‐drying of live vaccines, the control of the freezing process is very important for maintaining the probiotic bacteria activity. Cryoprotectants should be added. Fast freezing with small ice crystals may be beneficial for the bacteria .
Similar to what has been described for freeze‐drying of biopharmaceutics, a variety of sugars and polymers can act as cryoprotectants and lyoprotectants for the freeze‐drying process as well as stabilizers for storage. The protectants that are usually used include trehalose, sorbital sucrose, glucose, lactose, maltodextrin, skim milk powder, whey protein, betaine, dextran, and PEG [150, 152]. The protecting compounds may be added to the probiotic bacteria suspensions before freeze‐drying. For example, glucose, lactose, trehalose, and skim milk were added either alone or combined before freeze‐drying Lactobacillus rhamnosus and Lactobacillus casei/paracasei. Survival rates greater than 94% were observed immediately after freeze‐drying. When stored under refrigeration, the freeze‐dried formula with skim milk alone or supplemented with trehalose or lactose showed the best performance after 39 weeks of storage. The lowest survival was found with glucose and glucose plus milk for non‐refrigerated storage; no viable cells were left at the end . Conventional freeze‐drying and spray freeze‐drying were compared for the microencapsulation of L. paracasei. The spray freeze‐drying method was found to be better, with >60% of probiotic cell viability. In the freezing stage, high concentrations of trehalose helped preserve the cell viability. Overall, higher concentrations of protectants (maltodextrin, trehalose) contributed to high probiotic viability during freeze‐drying and storage . In addition to sugars and disaccharides, polymer‐based formulations could also support the viability of probiotic cells effectively .
Sugar protectants may be also added to the culture media prior to fermentation to facilitate the adaptation of probiotic cells to the environment. When grown in batch culture, the growth of bacterial culture may be divided into four stages: log, lag, stationary, and death phases. At the stationary phase, the cells develop a general stress resistance and are thus more resistant to the surrounding stresses. Therefore, freeze‐drying the probiotic cells at the stationary phase is the optimal stage to maintain cell viability . When adding sugar protectants in the culture media, they can accumulate within the cells. The osmotic difference between the internal and external environment is reduced. This can help mitigate the stresses induced by freezing. For example, the presence of sugars such as lactose, sucrose, and trehalose during the growth of probiotic cells was found to help adapt to freezing and thawing stresses . The lower decrease in viability after freeze‐drying was observed when Lactobacillus bulgaricus was grown with mannose (the best), fructose, and sorbital . Metabolites such as mannitol, sorbitol, and glutamate, which in most cases remain inside the cells, may contribute to the improved survival rate during freeze‐drying .
The selection of protectants for the freeze‐drying process can have significant impact on the storage. Non‐reducing sugars may be a better choice due to the potential Maillard reactions of reducing sugars with cell proteins . Storage temperature, relative humidity, powder composition, and exposure to oxygen and light are the important factors on the viability of probiotic cells in the dry powders during storage. Rehydration is the final critical step for the revival of probiotic cells after storage. It has been suggested that freeze‐drying the cells at the stationary phase and employing a slow rehydrating procedure may produce the best results .
Freeze‐drying has been widely used in solid pharmaceutical and biopharmaceutical formulations, with the main objective being high stability during long‐term storage and reconstitution. Excipients have been added to the formulations to maintain the functions of APIs, reduce the stresses during freezing and freeze‐drying, and enhance the stability during storage. Different types of excipients are described and some of the common excipients are given as examples. From the industrial point of view, reducing the freeze‐drying costs, by reducing freeze‐drying time or raising the freeze‐drying temperatures is essential; this has been addressed in this chapter. This may be achieved by the use of different freezing techniques (e.g. spray freeze‐drying, spin freezing) and various freeze‐drying options.
This chapter has also focused on the applications of freeze‐drying in pharmaceutics, biopharmaceutics, and foods (especially probiotic foods). Special attention is paid to solid protein formulations, with stabilizing mechanisms, degradation routes, and stabilizing options being discussed extensively. A variety of solid formulations in each category are described in detail, emphasizing the advantages resulting from freeze‐drying, providing examples for freeze‐dried formulations, and comparing with other formulation approaches. This demonstrates the importance and uniqueness of the freeze‐drying technique in industrial applications. The principles and formulations that are used to produce high‐quality products may be adopted to fabricate ice‐templated materials as will be discussed in the following chapters.
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