Nano spray drying technology for heat sensitive biopharmaceuticals

Faculty of Pharmaceutical Sciences & Pharmaceutical Industries
Accredited by NAQAAE
Graduation Project Thesis
Nano Spray Drying Technology for Heat Sensitive
Biopharmaceuticals & Biotherapeutics: Enzymes and
Proteins
Thesis presented by
Name: Madona Hana ID: 20141568
Name: Sawsan Monir ID:20142629
Name: Norhan Reda ID: 20143146
Submitted for partial fulfillment of graduation project
Under supervision of
(Prof. Dr.)
Dr. Heidi AbdelMageed, PhD
Assist. Prof. of Pharmaceutics
(Department)
Pharmaceutics & Pharmaceutical Technology Dept.
Future University in Egypt
Academic year: 2018 – 2019

Faculty of Pharmaceutical Sciences & Pharmaceutical Industries
Accredited by NAQAAE
APPROVAL SHEET
For graduation thesis entitled “Nano Spray Drying Technology
for Heat Sensitive Biopharmaceuticals & Biotherapeutics:
Enzymes and Proteins”
Committee in Charge
Prof. Dr. Hussein Ammar, Ph.D.
Professor Of Pharmaceutics And Industrial
Pharmacy
Pharmaceutics And Pharmaceutical
Technology Dept.
Assist. Prof. Heidi M. Abdel Mageed,
Ph.D.
Assistant Professor of Pharmaceutics and
Pharmaceutical Technology
Technology Dept.
Assist Prof Amira M. Ghoneim, Ph.D.
Assistant Professor Of Pharmaceutics And
Industrial Pharmacy
Technology Dept.

Acknowledgements
We wish to express our sincere gratitude to our supervisor Dr. Heidi
AbdelMageed for her continuous support, encouragement and guidance
throughout this thesis. Also we would like to thank her for her great belief in
us and our project. She has her own way, on being a tremendous source of
inspiration and given us invaluable advice throughout this semester on our
thesis. She has always taken time to discuss questions – big or small –
whenever we needed help. For this we wish to express our unbounded
gratitude.
A special thanks goes to our family. We are grateful beyond words for the
unceasing support of our family during our study.

Table of Content
List of figures i
List of tables ii
Abstract iii
2. Introduction: 1
3. Spray Drying 15
3.1. Mechanism of Action of Spray Drying:........................................... 15
3.1.1. Spray drying Layouts:.............................................15
3.1.2. Spray dryer major phases:.......................................20
Atomization:............................................................................................................... 20
Droplet-to-particle conversion..............................................................24
Particle collection ................................................................................................... 27
3.2. Nanospray dryer B-90............................................................................ 29
4. Optimizing of the Nano Spray Drying Process
parameters: 32
4.1. Atomization pressure and cap size.................................................... 32
4.2. Feed properties.......................................................................................... 32
4.3. Feed rate...................................................................................................... 33
4.4. Inlet and Outlet temperature................................................................ 33
4.5. Residence time inside drying chamber ............................................ 34
4.6. Drying gas flow rate................................................................................ 34
5. Stress factors on spray drying of proteins...............36

5.2. Influence of the Shear Stress................................................................ 38
5.3. Influence of the Temperature............................................................... 38
5.4. Influence of Dehydration....................................................................... 40
6. Stabilization of spray dried Proteins........................40
6.1. Excipients..................................................................................................... 41
6.2. Immobilization........................................................................................... 43
6.2.1. Adsorption...............................................................43
6.2.2. Disulphide bonding.................................................44
6.2.3. Entrapment..............................................................44
6.2.4. Encapsulation..........................................................44
6.2.5. Cross-linking...........................................................45
7. Applications of Spray Dried Enzymes and
Proteins 46
Conclusion 57
References: 59

i
List of Figures:
Figure :1 Secondary structure of protein, α-helix and β-pleated sheet (Kabir 2016) ......2
Figure 2: Schematic illustration of drying methods using freeze-drying (FD), spray
drying (SD), spray freeze-drying (SFD), and supercritical fluid drying
(SCFD) technologies..................................................................................7
Figure :3 Schematic illustration of traditional spray dryer and its components..........10
Figure :4 Schematic representation of spray-drying mechanism. (1) Atomization. (2)
droplet-to-particle conversion (3) Particle collection...............................16
Figure :5 Different morphology characteristics............................................................17
Figure 6 : Schematic outline of spray drying systems: open cycle (A), closed cycle (B)
and semi-closed cycle (C)......................................................................18
Figure 7 : Schematic illustration of vibrating and static mesh ......................................21
Figure :8 Simplified sketch of Rotatory atomizer and Two-fluid nozzle................23
Figure 9 : Schematic outline of Air-droplet flow patterns within the drying chamber.
(A) Co-current flow. (B) Counter-current flow. (C) Mixed flow. ...........25
Figure 10: Schematic outline of typical collectors used on spray drying. (A) Cyclone
separator (B) Bag filter (C) Electrostatic precipitator (D) Venturi wet
scrubber. ...................................................................................................28
Figure 11 : Nano Spray Dryer B-90 (left) and atomization by spray nozzle (right).......31
Figure :12 Schematic representation of possible stresses on a protein during spray
drying .......................................................................................................37
Figure 13 : Molecular structure of trehalose..................................................................41

ii
List of tables:
Table 1 : Relationships between spray- drying parameters.......................35
Table 2: Applications list of spray dried products using the laboratory
scale Nano Spray Dryer B-90 from (BÜCHI Labortechnik AG)
(derived from literature) ..............................................................49
Table 3 : Examples of spray- drying enzymes...........................................51
Table 4: Overview of protein drugs newly registered at the United States
Food and Drug Administration (FDA) in 2015 and their type....55
Table 5: Studies of solid protein formulations prepared by spray drying
methods in the presence of stabilizers and applications..............56

iii
1. Abstract
Numerous drying methods have been used for preparing dried protein
powders, although that choosing a suitable drying technique remains a
challenge. In this thesis, we will discuss spray drying as a drying method for
improving the stability and bioavailability of therapeutic proteins. Spray
drying is a simple, fast, continuous, and scalable drying technology that is
well established in biotechnological and pharmaceutical industry as for
excipient production, micro-encapsulation, or granulation also it plays a
crucial role in the processing of pharmaceutical products such as pills,
capsules, and tablets as it is used to convert drug-containing liquids into dried
powdered forms. Nano spray drying is in particular used to improve drug
formulation by encapsulating active ingredients in polymeric wall materials
for protection and delivering the drugs to the right place and time in the body.
The Nano spray dryer developed in the recent years extends the spectrum of
produced powder particles to the submicron- and Nano scale with very
narrow size distributions and sample quantities in the milligram scale at high
product yields. This enables the economical use of expensive active
pharmaceutical ingredients and pure drugs. Critical process parameters like
atomization pressure and cap size, feed rate, feed pressure, inlet and outlet
temperature, residence time inside drying chamber and drying gas flow rate
are optimized based on the required pharmacokinetics. In an optimized
drying procedure, the screening of formulations according to their protein
properties is performed to prepare a stable protein formulation for various
delivery systems, including pulmonary, nasal, and sustained-release
applications as they produce particles with different sizes and morphologies.

1
2. Introduction:
Drying is the final phase in many procedures and the product
from a dryer is usually ready for final packaging (Namaldi 2005). Many
materials must be dried to bring their moisture content to a prescribed value
before being sold. Others foodstuffs, biological materials, and
pharmaceuticals are dried to preserve them for storage and shipment without
the need for refrigeration (Baines 2003; Namaldi 2005).
Enzymes are one of the most important groups of biotechnological
products and they serve important functions in detergent, food,
pharmaceutical, and biochemical processes (Namaldi 2005). In order to
increase the shelf life, easy storage and transport, reduce transportation cost
and protect the biological activity of these molecules they are often preserved
in dry form (Kalisz 1988; Namaldi 2005).
To understand the effect of spray drying on the catalytic activity of the
enzymes it is important to first understand the structure of the enzymes and
how the enzymes work. Enzymes are proteins that may work as catalysts of
chemical reactions. They temporarily bind a number (or in some cases just
one) of the reactants of the reactions which they catalyze. The activation
energy needed for the reaction is thereby lowered and the reaction rate
increases. These proteins & polymers are formed by the linkage of α-amino
acids by a peptide bond. The specific properties of different enzymes are
determined by the sequence of α-amino acids (Sloth 2007).
Proteins have multiple levels of structure. The most basic is the primary
structure which simply is the order of the amino acids. The secondary
structure is the common repeating structures found in proteins. There are two
types of secondary structure, one being the α-helix, the other being the β-
sheet (Figure :1 ).

2
Figure :1 Secondary structure of protein, α-helix and β-pleated
sheet (Kabir 2016)

3
In α-helix, protein twists in a clockwise direction. Also, the carbonyl groups
of the peptide bonds form hydrogen bonds with amine groups which also are
a part of the peptide bonds. These hydrogen bonds are always formed by
amine and carbonyl groups which are four amino acids apart in the helix. The
hydrogen bonds are parallel to the axis of the helix and strengthen the
structure quite significantly. Unlike the α-helix, the β-sheet consists of pairs
of amino acid chains lying side-by-side. The structure is like a pleated sheet
held together by hydrogen bonds between the carbonyl group on one chain
and the amine group on the adjacent chain. Thus, like for the α-helix, the
existence of the hydrogen bonds is crucial to maintain the β-sheet structure.
Tertiary structure is the three-dimensional structure of the polypeptide chain.
Thus, the structure describes the folding of the secondary elements, including
α-helices, β-sheets and more undefined elements called random coils. Some
proteins contain multiple polypeptide chains and the quaternary structure is
their interconnections and organization (Sloth 2007).
Enzymes require the presence of a non-protein component called a cofactor
to have catalytic activity. These may be metal ions such as Zn2+
, Cu2+
, Mn2+
,
K+
or Na+
. The additional compound may also be a small organic molecule
in which case it is termed a coenzyme. Coenzymes may be covalently bonded
to the enzymes but some coenzymes are bonded more loosely and may, in
fact, bind only transiently to the enzyme as it performs the catalytic act (Sloth
2007).
During spray drying the rapid changes in droplet temperature and moisture
content influence the enzyme directly. An enzyme functions best at a certain
temperature. The activity of the enzyme is reduced below and especially
above this temperature. The former trend reflects the general effect of
decreasing the temperature in a chemical reaction, the latter trend reflects the
loss of catalytic activity as the enzyme becomes denaturated. The term

4
“denaturation” is explained below but it basically means that the enzyme
loses activity due to a structural change. Another factor that may denaturate
enzyme is when removing the aqueous medium surrounding the enzyme that
may lead to breakage of the hydrogen bonds. Further, the interactions
between the hydrophilic groups may change. These factors lead to enzyme
denaturation and inactivation as mentioned above. The enzyme suspension
which is subjected to spray drying may contain additional materials such as
salts, organic compounds, and buffers. The dehydration causes changes in
e.g. salt concentrations. A significant increase in salt concentration will alter
the electrostatic interaction between charged amino acids. The alteration
might give rise to enzyme denaturation. This also applies to changes in pH
which can occur because one buffer compound precipitates before the other
during the dehydration process. The function of a protein is absolutely
dependent on the three-dimensional structure. denaturation. However, none
of these breaks the peptide bond between the amino acids and thus, this part
of the primary structure remains intact. The denaturation process is not
necessarily irreversible. If an enzyme has been denaturated it might
spontaneously resume the native three-dimensional shape when it is returned
to the normal conditions (e.g. upon rehydration). Thereby the catalytic
activity is regained (Sloth 2007).
The adsorption to an interface is commonly assumed to be controlled by the
diffusion rate and the surface activity of the protein, making the adsorption a
three-step process. First, diffusion of the protein to the subsurface region
followed by adsorption to the air-liquid interface. The final step is the
rearrangement of the adsorbed molecule at the surface (Sloth 2007).
Millqvist-Fureby et al. explain that adsorption of trypsin to the droplet
surface drying may lead to two kinds of inactivation. First, thermal
inactivation is increased because of the droplet temperature. Secondly,

5
interactions between the surface and the trypsin account for some of the
inactivation (Millqvist-Fureby, Malmsten, and Bergenståhl 1999; Sloth
2007).
Other factors than those mentioned above may contribute to the inactivation
of enzymes during spray drying. Elversson and Millqvist-Fureby mention
that shear forces during droplet formation in a nozzle or rotary atomizer is a
source of loss of protein structure (Elversson and Millqvist-Fureby 2005;
Sloth 2007).
Any inactivation during the spray drying process represents a loss of valuable
enzyme and a reduction in the quality of the final product. Thus, a number of
measures are usually taken to avoid this inactivation. Process conditions may
be altered but also, different additives (usually called excipients) are added
to the enzyme-containing formulation prior to dehydration (Sloth 2007).
The intrinsic instability of protein molecules is currently the predominant
challenge for biopharmaceutical scientists. Therapeutic proteins have much
more complicated structures than conventional chemical drugs Because of
their higher molecular weights and diversity of composition. Protein
instability can be caused by exposure to some environmental stresses, such
as pH extremes, high temperatures, freezing, light, agitation, shear stress, and
organic solvents. Some strategies are suggested to improve protein stability
since proteins can be degraded easily during manufacturing and storage,
including the addition of stabilizers, protein modification with biocompatible
molecules, nanomedicine, and nano- or micro-particle technology (Emami
et al. 2018). 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. Solid
dosage forms of proteins are less prone to shear-related denaturation and
precipitation during manufacturing and storage. Because water molecules

6
can induce mobilization of therapeutic proteins and other additives, liquid
formulations of proteins are more susceptible to unfavorable
physicochemical degradation. Consequently, water removal are good
approaches for improved storage against physicochemical
protein degradation (Emami et al. 2018). The challenge of protein spray
drying is to avert a negative impact of the multiple processes related stress
factor on protein stability by finding suitable formulation compositions and
process conditions. Among the main causes for chemical and physical protein
instabilities, the influence of some stress factors while in case of the enzyme
by using spray dryer the rapid changes in droplet temperature and moisture
content influence it directly in its conformation.
Generally, 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 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, the concentration of solutes, viscosity, and type of solvent. Drying
based on the mechanism of removing water can be classified into subgroups.
Drying can be performed using an evaporation mechanism, such as vacuum
drying or foam drying; evaporation and atomization pathways (Figure 2)
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 (Emami et al. 2018).

7
Figure 2: Schematic illustration of drying methods using freeze-drying
(FD), spray drying (SD), spray freeze-drying (SFD), and supercritical
fluid drying (SCFD) technologies.

8
Freeze drying is the most common technique for obtaining a dry formulation
of enzymes which has been used for many therapeutic proteins, including
insulin dry powder for inhalation (Emami et al. 2018). As during freeze-
drying, materials are not subjected to high temperatures hence the freeze-
dried products maintain their high quality and initial nutritious character
(Johansen Crosby 1989). It is operated on the principle of sublimation under
reduced pressure to remove water from a frozen material where solid
materials are directly transformed to the gaseous phase (Namaldi 2005;
Emami et al. 2018; Ribeiro et al. 2016). The FD process involves the
following three steps: freezing, primary drying, and secondary drying.
Freeze-dried proteins show greater storage stability than proteins in liquid
dosage forms. It is usually considered to have a less thermal effect on the
enzyme when compared to other drying methods. However, the process has
several disadvantages; during the freezing step, enzyme and buffer
components tend to be concentrated in the phase between ice crystals. Such
a concentration effect can result in a dramatic change in the pH and ionic
strength of the enzyme environment and thus, lead to a change in biological
activity, i.e., denaturation. Therefore, the solute concentration, pH change,
and ionic strength changes are formulation variables that should be
considered for a stable protein formulation. To reduce the degree of
denaturation, stabilizing agents are added to a protein solution prior to freeze-
drying (Namaldi 2005; Emami et al. 2018). When compared with other
drying processes, freeze drying is an energy-intensive and time-consuming
process also raises the concern of high production cost and product produced
is porous and hygroscopic (Belghith, Ellouz Chaabouni, and Gargouri
2001; Mumenthaler, Hsu, and Pearlman 1994; Namaldi 2005; Johansen
Crosby 1989).

9
Spray drying is currently a well-established method for processing liquids
into powders. It is the most common particle engineering method that
generates solid proteins for pharmaceutical applications. And as a single-step
process, it may provide dried protein particles with the required size and
morphology. Spray drying technology comprises atomization, drying, and
separation of particles (Figure :3 ). Unlike freeze drying, spray drying-
utilizes heat from a hot gas stream to evaporate micro-dispersed droplets
created by atomization of a continuous liquid feed and is therefore a very fast
and cost-effective dehydration method as it is about six times cheaper, it also
increases the surface area to improve the heat transfer efficiency (Namaldi
2005; Emami et al. 2018; Johansen Crosby 1989).
The first detailed description of drying of a liquid system through a spray and
the hot gas system appears in an 1872 patent; however, spray drying started
to be widely used in the dairy and detergent industries in the 1920s
(Johansen Crosby 1989). Spray drying is defined as the transformation of a
fluid from a liquid state into a dried particulate form by spraying the fluid
into a hot drying medium (Arpagaus et al. 2017; masters 1987). It is a
suitable one-step process for the conversion of various liquid formulations
(Arpagaus et al. 2017). It is a continuous one-step process for the conversion
of various liquid formulations (e.g., aqueous and organic solutions,
emulsions, and suspensions) into dry powders that allow simultaneous drying
and particle formation (Johansen Crosby 1989; Sloth 2007). In addition,
spray drying can be considered a continuous process that provides scaling-
up benefits and better quality, therefore, it reduces time-to-market (Johansen
Crosby 1989). Spray drying is a simple, fast, and scalable drying technology
that is well-established in the chemical, food, and pharmaceutical industries
(Arpagaus et al. 2017). Some of the materials used in spray dryer in the field
of the pharmaceutical industry are antibiotics like penicillin, vitamins

10
Figure :3 Schematic illustration of traditional spray dryer and its components

11
as ascorbic acid and vitamin B12 and proteins including enzymes such as
amylase, protease, lipase, and trypsin (Johansen Crosby 1989). Spray
drying is a part of the production line for numerous products – among the
most well-known are instant coffee, laundry detergents and powdered milk
(Sloth 2007). The cooling effect of the evaporating solvent conserves the
droplet temperature relatively low; therefore heat-sensitive products can be
dried with negligible degradation (Arpagaus et al. 2017; masters 1987).
When we use this technique the number of unit operations is reduced,
improving production efficiency and reducing costs, especially that spray
drying is a technique that can be easily automated and equipped for in-line
product analysis (Johansen Crosby 1989). The powders produced are high
in quality and have low moisture content, resulting in high shelf stability
(Anandharamakrishnan and Ishwarya 2015; Arpagaus et al. 2017).
Spray drying can result in several types of particle shapes, such as smooth
and spherical, collapsed, dimpled, wrinkled, raisin-like, and highly crumpled
or folded particles (Arpagaus et al. 2017). Spray drying offers high
flexibility to control particle size and morphology by optimizing the process
parameters and feed formulation (Arpagaus et al. 2017). The dried solid
particles are separated from the gas stream by a cyclone and collected in a
collection vessel (Arpagaus et al. 2017).
Spray drying of enzymes is done for three main reasons: for drug
administration through inhalation; needle-free injection as a non-invasive
immunization technique; and as a possibility to produce bulk protein powders
as an alternative to a classical freeze-drying process (Yakes et al. 2017).
Another effective and versatile technique for transforming protein solution
into dried particles is spray freeze drying, which is the combination of
traditional FD and SD processes. Atomization, fast freezing, and drying by

12
ice sublimation are the three phases of SFD process. SFD is a method for
preparing lyophilized protein powders with spherical microparticles.
SFD has potential applications for thermo-labile active pharmaceutical
ingredients as it involves the atomization of protein solution via a nozzle at
extremely low temperatures. Because of this critically low temperature, the
atomized droplets are rapidly frozen. The frozen micronized droplets are
sublimated using a lyophilizer under vacuum to prepare a dried powder. In
SFD, the liquid solution is sprayed into a vapor via a nozzle using a cryogenic
fluid, such as liquid nitrogen (Emami et al. 2018).
Spray freeze-dried powders may be produced for different drug delivery
system applications. Specific physical characteristics such as particle size
distribution, density, surface area, and volume are required, depending on
their application. SFD typically produces highly porous particles with a high
percentage of fine particle fraction (FPF) and proper aerodynamic behavior
for pulmonary delivery.
In addition, spray freeze-dried particles have applications for needle-free
intradermal injection system, nasal, colonic, and ophthalmic drug delivery,
as well as in processing for microencapsulation platforms. Spray freeze-dried
particles with a geometric diameter of 7–42 µm and very low density could
be effective in pulmonary drug delivery systems. However, for nasal delivery
and intradermal injection systems, particles with geometric diameters of 25–
70 µm and 34–50 µm are required, respectively (Emami et al. 2018).
Supercritical fluid drying (SCFD) is an attractive alternative drying method
because dehydration can be rapidly accomplished in the absence of extreme
temperatures. SCFD may produce large amounts of dried biopharmaceuticals
with adjustable particle sizes and morphology. The critical
the temperature of a liquid is the temperature at which its vapor cannot be

13
liquefied, no matter how much pressure is applied. The pressure that is
needed to condense a gas at its critical temperature defines its critical
pressure. SCF exhibits the appropriate characteristics of gas and liquid,
including penetration of gas and solubility of liquids (Emami et al. 2018).

14
Table2:Comparisonofcharacteristicsofdifferentdryingtechnologies

15
3. Spray Drying
3.1. Mechanism of Action of Spray Drying:
Spray drying involves the spraying of liquid solutions, suspensions or
emulsions' feed into a hot drying inert gas or nitrogen. The droplets formed
by the atomization process are dried through solvent evaporation to form
particles which are collected as a dry powder (Figure :4 ). The process of
drying the spray continues until achieved the desired moisture content in the
dried particles, and the product is regained from the air. It is a unique drying
process that involves both particle formation and drying (Jain et al. 2011).
The physicochemical properties of the produced powders influenced by some
process parameters such as; inlet and outlet temperatures of the drying
medium and the atomization pressure. With the different designs of spray
dryers available, it is possible to select a dryer layout to produce fine or
coarse particle powders, agglomerates or granules (Jain et al. 2011).
Many spray dried particles are spherical or nearly spherical with a solid or
hollow interior. However, variations in the spherical form are often observed
as a number of quite different morphology characteristics exist. As shown in
Figure :5 particles may have collapsed or contracted while others have
blowholes or craters. Likewise, some particles have cavities, fractures, cracks
or expand during drying. Exceptional morphologies include mushroom-head
shape particles or large particles holding smaller particles.
3.1.1.Spray drying Layouts:
A great advantage of the spray drying process is that numerous different
products with specific properties may be produced. This is possible because
a large number of different process layouts have been designed. The different
spray drying layouts are the open cycle, closed cycle and semi-closed cycle
(Figure 6 ). The exhausted air is cleaned using combinations of cyclones,

16
Figure :4 Schematic representation of spray-drying mechanism. (1) Atomization.
(2) droplet-to-particle conversion (3) Particle collection.

17
Figure :5 Different morphology characteristics.

18
Figure6:Schematicoutlineofspraydryingsystems:opencycle(A),closedcycle(B)and
semi-closedcycle(C).

19
bag filters, electrostatic precipitators, and scrubbers before it discharged
(Jain et al. 2011). That means that the drying gas passes only once through
the drying chamber before it is emitted to the surroundings. This is the most
commonly used design of spray dryer system (Gohel, Patel, and Pandya
2009).
In the other hand, the closed cycle system is based upon recycling the drying
medium (Jain et al. 2011; Gohel, Patel, and Pandya 2009). Often nitrogen
is used as the drying gas and closed loop thereby allows for drying of many
products which cannot produced in a conventional open loop process. Drying
chambers are combined with a cyclone/bag filter, solvent vapor condenser,
exhaust drying medium particulate cleaning in wet scrubbers and indirect
drying medium heating (Jain et al. 2011). It is possible to manufacture
products which degrade in contact with oxygen, dust explosions are avoided
and release of hazardous compounds is prevented, allowing drying of
solutions based on organic solvents (Sloth 2007).
Semi-closed dryer design is a combination between open and closed cycle
dryers where the off-gas is divided into two flows where one is recycled and
mixed with air from the atmosphere. The recycled gas has very low oxygen
content, making it suitable for materials that cannot be exposed to oxygen. A
direct-fired heater is used and the air entering the system is limited to that
required for heating (Gohel, Patel, and Pandya 2009; Sloth 2007). Less
energy is required to heat this gas stream but the humidity is increased.
Consequently, a higher temperature is necessary for sufficient product
drying. However, Masters states that an energy utilization reduction of 20%
is attainable by using a semi-closed loop setup (Sloth 2007).
To produce agglomerated products, a fluid bed is often introduced
in the process layout. Also, a fluid bed may help reduce the residual moisture
content of the powder or improve the overall heat efficiency. The fluid bed

20
may be implemented externally or internally in the spray dryer and may be
either vibrating or stationary (Sloth 2007).
3.1.2.Spray dryer major phases:
The spray-drying mechanism is based on moisture elimination using for that
a heated atmosphere to which the feed product is subjected. Spray drying
process described by three major phases; atomization, droplet-to-particle
conversion, and particle collection (Santos et al. 2018).
The solution is atomized, breaking up the liquid feed into a spray of fine
droplets. After that, the droplets are ejected into a drying gas chamber where
the moisture vaporization and formation of dry particles occurs. Finally,
using an appropriate device, the dried particles are separated from the drying
medium, is then collected in a tank (Santos et al. 2018).
Atomization:
The design, as well as the operation of the atomizing device, is most
important both for the final product properties and the overall process
economy (Sloth 2007). Optimal evaporation from the formed droplets in the
drying chamber is highly dependent on the spray characteristics. the droplets
must have a large surface area compared to their mass, but it is equally
important that the droplets have a narrow size distribution. The broad size
distribution leads to the formation of large droplets which compared to
smaller droplets that require a prolonged residence time inside the drying
chamber which is particularly unfortunate when drying temperature-sensitive
materials (Sloth 2007; Walton 2000). The aim of atomizing the concentrate
is to provide a very large surface to let the evaporation to take place. The
smaller the droplets, the bigger the surface, the easier evaporation, and better
thermal efficiency of the dryer are obtained (Gohel, Patel, and Pandya
2009).

21
Figure7:Schematicillustrationofvibratingandstaticmesh

22
The atomization process into droplet form could be accomplished by
pressure, centrifugal, electrostatic or ultrasonic energy, by using specific
devices called atomizers. There are different atomizers, which are used
according to the desired product characteristics (shape, structure, and size) as
well as depending on the nature of the feed solution (Santos et al. 2018).
There are 3 basic designs of atomizers, defined by the source of energy
utilized in the droplet formation process: Rotary atomizer (atomization by
centrifugal energy), Pressure nozzle (atomization by pressure energy) and
Two-fluid nozzle (atomization by electrostatic energy) (Jain et al. 2011).
Rotary atomizer uses a high speed-rotating disc or cylinder which is located
horizontally at the top of the drying chamber that produces energy to divide
the bulk liquid into droplets. The feed is introduced at the center of the device
and, it flows over the surface to the periphery by a centrifugal force, broken
down and disintegrates into droplets (Figure :88 )a (Gohel, Patel, and
Pandya 2009; Sloth 2007). The feed may be designed differently to obtain
specific droplet properties. The channel design, the disc angular velocity, and
the feed flow rate control the mean droplet size. The exact velocity necessary
depends on the flow rate, composition, and viscosity of the feed (Sloth 2007).
rotary atomizers have many advantages and are used in many high
throughput industries. Examples are the production of dairy, colored pigment
and foodstuffs products as well as inorganic chemicals. The most important
disadvantage of the rotary atomizer is that it is mechanically complex and
requires regular maintenance of the moving parts. In two-fluid or pneumatic
nozzles, Liquid feed and compressed air (or steam) are combined in a two-
fluid nozzle. Droplet generation is based on contacting a liquid jet with a high
velocity compressed gas. Their design atomizes the liquid jet into droplets
(Gohel, Patel, and Pandya 2009; Sloth 2007). The size of the droplets
generated by a two-fluid nozzle is dependent on the specific nozzle design,

23
Figure :8 Simplified sketch of Rotatory atomizer and Two-fluid nozzle

24
It also depends on liquid properties, surface tension, density, and viscosity.
Further, gaseous flow properties such as velocity and density influence the
droplet size (Sloth 2007). The advantage of the use of a two-fluid nozzle is
their ability to atomize highly viscous feeds and to produce very fine
particles. However, two-fluid nozzles are expensive to operate because of the
high cost of compressed air (Gohel, Patel, and Pandya 2009).
Recently, ultrasonic energy has been used to form droplets instead of pressure
or centrifugal force. In this method, a liquid is placed on a rapidly vibrating
surface at ultrasonic frequencies. At sufficiently high force, the liquid spreads
becoming unstable and collapse, resulting in the formation of a very fine
droplet (Gohel, Patel, and Pandya 2009).
Droplet-to-particle conversion
After the spray formation, the droplets are dried which is the next important
stage of the spray drying process. The droplet drying takes place inside the
drying chamber where liquid evaporates from the droplets and is transferred
to the drying gas that involves the spray-air contact, mixing and
droplet/particle flow. The choice of flow mode is a design parameter which
characterizes a given spray dryer unit. The flow mode influences the course
of droplet drying and thereby the drying kinetics, morphology formation, and
enzyme inactivation when drying enzyme containing formulations. The
drying chamber flow field determines how a droplet “experiences” the stay
in the drying chamber (Jain et al. 2011; Santos et al. 2018; Sloth 2007).
There are different drying chamber configurations, in which the flow pattern
between the hot gas and the spray of droplets is separated: co-current flow,
counter-current flow and mixed flow (Jain et al. 2011; Santos et al. 2018;
Sloth 2007).

25
Figure9:SchematicoutlineofAir-dropletflowpatternswithinthedryingchamber.(A)Co-
currentflow.(B)Counter-currentflow.(C)Mixedflow.

26
In a co-current flow, the feed is atomized and sprayed through the drying
chamber in the same direction as the flow of the heated drying medium. The
dried particles are dropped at the bottom of the chamber, where they are
released together with the gas. The drying gas is hottest in the uppermost part
of the drying chamber and evaporation from the newly formed droplets is
fast. During evaporation, the drying gas is cooled while it is mixed well in
the drying chamber. In such configuration, there is no time for the drying gas
to exchange some of its heat with the surroundings, Thus, there is a low
temperature with a somewhat uniform distribution in most of the drying
chamber. However, this implies an instantaneous high rate of solvent
evaporation, allowing the dried particles to contact with moderate
temperatures that avoid unwanted thermal degradation leading
to optimal solvent evaporation for spray drying of heat-sensitive
materials like enzymes, peptides, and proteins (Santos et al. 2018; Sloth
2007).
Alternatively, In a counter-current flow design, atomization takes place in the
top of the dryer while the drying gas enters in the bottom of the dryer (Sloth
2007). A counter-current dryer offers more rapid evaporation and higher
energy efficiency than a co-current design. This usually results in improved
heat utilization, but the almost dried powder is subjected to a very high
temperature at the bottom of the dryer. Therefore, counter-current mode is
often used when drying thermostable materials which require elevated
temperatures in the later part of the drying process (Gohel, Patel, and
Pandya 2009; Sloth 2007). Also, it may be beneficial to combine counter-
current mode with nozzle atomization because the drying gas flow reduces
the downward velocity of the droplets (Sloth 2007).
Mixed flow dryer construction combines both co-current flow and counter-
current flow, that is, atomized droplets are fed from the bottom of the

27
chamber in counter-current relative to the downward streamline of the drying
gas which is supplied from the top. The dried particles and the drying gas are
then released at the bottom of the chamber. In mixed flow, dried particles are
subjected to intense heat (Santos et al. 2018; Sloth 2007). Mixed flow also
includes fountain mode where the atomizer is placed at the bottom of the
drying chamber and sprays upward. This mode is well-suited for the
production of large particles and coarse powders (Sloth 2007). Both counter-
current design and mixed flow dryer expose the driest particles to the hottest
air, so these designs are not used with heat-sensitive products (Gohel, Patel,
and Pandya 2009).
Particle collection
It is necessary to collect the dried particles Once the droplet-to-particle
conversion is completed. This suggests a separation procedure, in which the
dried particles are disassociated from the drying gas. The choice of separation
unit depends on e.g., the gas and particle flow rates and the level of product
loss accepted. Finally, the drying gas may be passed through a wet scrubber
before it is released to the surroundings to prevent the emission of harmful
particles. Such separation happens generally in two phases, called primary
and second separation. In the primary separation, the densest particles are
recovered at the conical bottom of the drying chamber. On the second
separation, the finest or smallest particles are transferred to external devices,
where they are separated from the humid air. The cyclone separator, the bag
filter and the electrostatic precipitator considered as the most common dry
collectors used (Figure 10) (Santos et al. 2018; Sloth 2007). The separation
mechanism of the cyclone separator relies on centrifugal force. This device
presents a cylindrical upper part, the barrel, and a conical part on its bottom,
the cone. The streamline of air comes from the drying chamber containing

28
Figure 10: Schematic outline of typical collectors used on spray drying. (A)
Cyclone separator (B) Bag filter (C) Electrostatic precipitator (D) Venturi wet
scrubber.

29
the dried particles and is supplied into the top of the cyclone, namely
tangentially to the barrel. Then, this streamline follows a downward flow,
creating an outer vortex. The high velocities of the outer vortex create a
centrifugal force on the particles, allowing the particles-gas stream
separation. An inner vortex is created in the opposite direction as soon as the
gas reaches the cone. Thus, the gas is expelled from the cyclone at its top,
while the particles settle into a collection chamber present on its bottom
(Santos et al. 2018). In Filtration based on bags, the air streamline containing
the dry particles enters the bag filter under pressure or suction by its hopper
and is passed through a fabric, which stops the path of the particles. Dry
particles are retained on the bag surface whereas the clean air passes through
it, being expelled from the device. The accumulated particles on the bag
surface are then collected due to the pulses of compressed air injected in
counter-current flow inside the bags (Santos et al. 2018). Electrostatic
precipitation is a method for collection of particles whose principle is based
on electrostatic forces. A high voltage is applied to discharged wires, forming
an electric field between them and the collecting plates that constitute the
precipitator (Santos et al. 2018).
3.2. Nanospray dryer B-90
The Nano Spray Dryer B-90 by Buchi (figure 11) comprises three
technological novelties concerning the spray drying process: a vibrating
mesh spray technology was implemented to generate fine aerosol droplets, a
laminar drying air flow in the spray chamber to provide instant drying of the
aerosol at mild conditions, and an electrostatic particle collector to effectively
separate finest particles from the drying air. Submicron particles with 0.5 µm
and 0.8 µm mean particle size were obtained at high yields for 50 mg powder
amounts (Schmid 2011). The Nano Spray Dryer B-90 uses the vibrating
mesh spray technology to generate the aerosol. The liquid stream is atomized

30
into fine droplets by a piezoelectric driven vibrating mesh atomizer, then
subjected to drying in a drying chamber in order to yield solid particles, and
finally, separated and collected by a suitable electrostatic dry powder
collector (Arpagaus 2011; Schmid 2011).
The piezoelectric actuator causes the vibration of a thin, perforated stainless
steel membrane with ultrasonic frequencies. The vibration of the membrane
(spray mesh) causes a ‘micro pumping action’ and the formation of droplets
with very narrow size distribution. Spray meshes are available with 4.0 µm,
5.5 µm, and 7.0 µm apertures. The drying gas passes through a compact
porous metal heater which provides optimum energy transfer and short
heating-up rates of up to 120°C inlet temperature. The heater enables a
laminar gas flow within the drying section and extremely droplet drying
times. Due to this, the instrument is suitable for spray drying, heat-sensitive
biopharmaceutical products.
Instead of using a cyclone to collect the dry particles, the Nano Spray Dryer
is equipped with an electrostatic particle collector consisting of a stainless-
steel cylinder (anode = particle collecting electrode) and a star-shaped
counter electrode (cathode) inside the cylinder. During the spray process,
high voltage is applied between the electrodes and spray-dried particles get
electrically charged and deposited on the inner wall of the cylinder electrode.
The collection mechanism is based on electrostatic charging of the particles,
which is independent of particle mass in contrast to cyclones. The
electrostatic precipitator collects even thin-walled particles without breaking
those. After completion of the spray drying process, the fine powder is gently
collected from the internal wall of the electrode cylinder using a particle
scraper. This particle separation principle enables the collection of powder
particles in the micron to submicron size range at high yields even for small
sample quantities in the milligrams range (Schmid 2011; Arpagaus 2011).

31
Figure 11 : Nano Spray Dryer B-90 (left) and atomization by spray nozzle (right)

32
4. Optimizing of the Nano Spray Drying Process parameters:
4.1. Atomization pressure and cap size
Atomization stage is done under pressure so as it is involved in this
process it has an impact on droplet size. For a certain atomizer device
and feed solution, droplet size decreases with increasing pressure (Cal,
Sollohub. 2010; Anandharamakrishnan 2015). In the case that the
feed solution is pumped into the atomizer at a controllable rate.
Keeping the atomization pressure constant, droplet size will increase
with increasing feed flow rates (Anandharamakrishnan 2015). Also
the statistical analysis showed that the hole size of the spray cap
membrane had a significant impact on the particle size of the spray
dried powder. Using a smaller cap size resulted in smaller particles. It
was also revealed that the ethanol content, as well as the interaction
between cap size and ethanol content had a significant influence.
4.2. Feed properties
In case that the feed viscosity increased, in order to overcome the large
viscous forces of the solution, a great percentage of atomization energy
supplied to the nozzle is used. According to that a small amount of
energy is left for the droplet fission, leading to larger droplet sizes
(Anandharamakrishnan 2015; Anandharamakrishnan 2017), also
due to the disruption of the feed surface tension Atomization occurs
which means that a feed solution with higher surface tension hinders
the atomization process. So before starting the spray-drying process,
emulsification and homogenization of the feed are usually made in
order to reduce their surface tension (Anandharamakrishnan 2015).

33
4.3. Feed rate
The feed rate is the amount of fluid sprayed per unit of time, also in
the Nano spray dryer it depends on the spray mesh size, the set value
of the relative spray rate intensity, the recirculation pump rate, and
the feed formulation The feed rate increases with mesh size, but also
depends on the application, the parameter settings, and the spray head
used (Büchi Labortechnik, 2010). The addition of organic solvents
to the water tends to increase the feed rates. This can be attributed to
the lower surface tension. Pretreating the mesh with surfactant
solutions increases the feed rate (Aquino et al. 2014).
4.4. Inlet and Outlet temperature
Inlet temperature refers to the heated drying gas temperature that was
measured right before its entry into the drying chamber. The thermal
charge of inlet drying gas show its capacity to dry the humid atomized
droplets, so the higher the inlet temperatures the higher the solvent
evaporation rates. To achieve better drying performances the inlet
temperature should not just be increased as it also has an impact in the
wet-bulb temperature of the surrounding air. In fact, the reason for
preventing thermal degradation of the final product is the lower inlet
temperatures which lead to lower surrounding air wet-bulb
temperature. So, a good choice of inlet temperature, based on these
factors, should be done according to the feed properties (Cal, Sollohub
2010; Anandharamakrishnan 2015). While the outlet temperature is
the temperature of the air containing the dried particles also it is the
highest temperature to which the dried powder can be heated, although
in the countercurrent dryers the final product may present a higher
temperature than the outlet air (Cal , Sollohub 2010;
Anandharamakrishnan 2015). While outlet temperature results from

34
all heat and mass exchanges inside the drying chamber. So that it is not
directly regulated by the operator (Cal, Sollohub 2010).
4.5. Residence time inside drying chamber
It refers to the exposition period of the atomized droplets inside the
drying chamber; it is an important factor with a direct influence on the
final product quality. To assure that the main goal of the drying stage
is accomplished this period should be long enough, that is, to obtain
dried particles. When the dried particles are subjected to longer
residence times, thermal degradation may occur, especially when
using heat-sensitive materials. It is hard to experimentally predict the
minimum residence time to be used so in general fine particles should
not stay more than 10–15 sec inside the drying chamber
(Anandharamakrishnan 2015; Schmitz-Schug I, Foerst, Kulozik
2013).
4.6. Drying gas flow rate
Drying gas flow rate means the volume of drying gas which is supplied
to the drying chamber per unit time. High gas flow rates will make the
particle movements inside the chamber increase and reducing air-
droplet interaction time. Besides, it is also reported that as the drying
gas flow rate increased, as the efficiency obtained during cyclone
separation increased which means that the drying gas flow rate should
be low enough to be sure of a complete particle moisture removal, but
on the other hand, it should be suitable for the subsequent separation
procedure (Cal, Sollohub 2010). (As shown in Table 1)

35
Table1:Relationshipsbetweenspray-dryingparameters.

36
4. Stress factors on spray drying of proteins
Protein naïve structure is a result of a fine balance between various
interactions including covalent linkages, hydrophobic interactions,
hydrogen bonding and van der Waals force (Diwaker et al. 2011).
This intra protein interaction together with protein-solvent interactions
maintains the structure of the protein and any change in the
surrounding environment can cause damage to these interactions and
may trigger denaturation or interaction. Protein may come across
several stress factors during spray dryer as illustrated in Figure :12 .
4.1. Influence of Surface Adsorption
The atomization of the protein solution during spray dryer negatively
affects the stability of most proteins due to the large expansion of the
air-liquid interface. Proteins are adsorbed at the droplet surface during
the drying process; they tend to the interfaces due to their amphiphilic
nature (Maa, Y.-F. and S.J. Prestrelski. 2000). This can result to the
orientation of hydrophobic amino acids residues toward the
nonaqueous environment which means that the Hydrophobic regions
which are normally directed to the core of the protein become exposed
and so that it may interact with chains of other molecules, which in the
end, will lead to protein unfolding and the formation of aggregates
(Maa, Y.-F. and S.J. Prestrelski. 2000; Tripp, B.C., J.J. Magda,
and J.D. Andrade 1995). These interfaces produced during the
Formulation of proteins, e.g. filling of vials, mixing, filtration
processes or during spray drying. Proteins can be distinguished in
“Hard” and “soft”. Hard proteins are considered to be resistant against
conformational changes due to their rigid structure. They have a high
resistance against denaturation which in some cases is explained by
intramolecular covalent disulfide bonds. While soft proteins are highly

37
Figure :12 Schematic representation of possible stresses on a protein during spray
drying

38
hydrophobic and show a high degree of flexibility which makes it easy
to rearrange their tertiary structure. Therefore, they tend to form a
greater foam on shaking due to their higher affinity to interfaces
(Tripp, B.C., J.J. Magda, and J.D. Andrade 1995).
Also, the extent of protein surface adsorption depends on the number
and the distribution of the hydrophobic amino acids on the protein
surface and the rigidity or the flexibility of the protein in solution. So,
in order to prevent protein adsorption and aggregation at the air-liquid
interface, surfactants are commonly added to the spray solution
(Millqvist-Fureby A, Malmsten M, Bergenstahl B. 1999). This
preventive action is mainly related to the displacement of protein
molecules from the air-liquid interface (Adler M, unger M, lee G.
2000). In addition, their binding to the hydroscopic sites of protein
molecules avoids intermolecular protein interactions and aggregation
(Abdul-fattah AM, Lechuga-ballesteros D, Kalonia DS, pikal.
2007).
4.2. Influence of the Shear Stress
During the spray drying process, proteins are exposed to several
shear stresses, for example, shaking, pumping and atomization
through the nozzle. Shear stress can cause structural changes and
inactivation of proteins; in some cases, shear stress alone does not
harm proteins during a typical spray drying process (Maa, Y.F. and
C.C. Hsu. 1996). However, Shear rates from atomization which
usually are in the range of 104 -105 sec can enhance the interaction
of proteins with interfaces (Banga, A.K. 2005). Finally, the studies
indicated that shearing stress occurring during pumping, flow and
atomization, do not appear to cause major damage to Proteins.

39
4.3. Influence of the Temperature
The structure of proteins depends on hydrogen bonds for the
maintenance of the secondary, tertiary and quaternary structures. It is
known that increasing temperature weakens hydrogen bonding, in
contrast to hydrophobic interactions (Volkin, and C.Middaugh.
1992). At some point, the temperature disrupts these non-covalent
forces, which leads to an increase in flexibility and therefore partial
unfolding. With higher temperatures the collision frequency increases,
resulting in aggregation, loss of biological activity or solubility. All
peptides lose their native structure when exposed to sufficiently high
temperatures (Chi, E., et al. 2003). This thermally-induced
denaturation process can be either reversible or irreversible depending
on the ability of the protein to return to its native structure when
returning to surrounding temperature (Brange, 2000). During a spray
drying process and after atomization, a protein may denature as it
comes into contact with the hot drying medium. Although the
temperatures in spray drying processes are high, the contact time
between droplets and the hot air is very small (Banga, A.K. 2005). So,
the solution of this problem may be through operating the spray dryer
at a lower inlet temperature, however, it may represent undesirable
manufacturing conditions. The residual moisture content would be
high, leading to poor storage stability (Luyben, K.C.A.M., J.K. Liou,
and S. Bruin. 1982; Samborska, K., D. Witrowa-Rajchert, and A.
Gonçalves. 2005). So finally, thermal protein denaturation during
spray dryer not only depends on the temperature level but also on the
time of exposure of the proteins to the hot drying air (Abdul-fattah
AM, Lechuga-ballesteros D, Kalonia DS, pikal MJ. 2007). As the
exposure time of drying droplets to the elevated temperature ranges in

40
the millisecond scale, thermal denaturation during spray drying is
often regarded as negligible.
4.4. Influence of Dehydration
Proteins need a certain amount of water to maintain their three-
dimensional, native conformation (Prestrelski, S.J., et al. 1993). So,
in course of the drying process, the protein molecules are deprived of
the surroundings, protective water and are thermodynamically
destabilized by losing their hydrogen bonding to water molecules. The
removal of the water might lead to structural changes and protein
denaturation (Lee, G. and M. 1996). Apart from the protein, the liquid
feed may contain excipients like salts, organic compounds or buffers
so removal of water causes changes in the composition, e.g. salt
concentration which may lead to an alteration in electrostatic
interactions between charged amino acids. A change in buffer
concentration may shift the pH of the solution. Both can cause or at
least promote denaturation (Wang, W. 2000).
5. Stabilization of spray dried Proteins
Any activity loss during spray drying means a reduction in the quality
of the final product. Although some proteins can successfully be spray
dried without excipients, they are necessary in most cases to improve
the process and storage stability. Because enzymatic inactivation can
be caused by a number of different mechanisms, the stabilizing
principle must be adapted to retain the entire activity after rehydration.
So as freeze drying is by far the most popular drying method for
proteins solutions in the pharmaceutical industry, spray drying has
been successfully employed as an alternative to ameliorate protein
storage stability. A lot of methods are used to improve protein and
enzymes stability as addition of excipients and immobilization.

41
5.1. Excipients
Water removal can lead to conformational changes in the protein
leading to loss of activity. Many proteins have been successfully dried
with minimal activity loss by the use of stabilizing additives. They can
protect proteins during the dehydration process by forming hydrogen
bonds with the proteins. Several monosaccharide and disaccharide,
and several polyols and amino acids are known stabilizers against
water removal stress. Examples include lactose, trehalose, sucrose,
mannitol, sorbitol, lysine, histidine or arginine (Arakawa et al., 1993).
Amongst these examples, non-reducing sugar as sucrose and trehalose
are the most commonly used. They are believed to stabilize proteins
during drying mostly by hydrogen bonding to the surface of the dried
protein in place of the removed water layer and hence inhibiting
unfolding. Trehalose (Figure 13) is known to be the best sugar for
stabilizing proteins during spray drying processes (Adler and Lee,
1999) as it's one of the most stable saccharides which has high
thermostability and a wide pH-stability range (simperler et al.2006).
It has been used previously as a protective agent in protein spray
drying applications in order to improve the stability of the proteins
(Adler and Lee, 1999). Also, trehalose was selected as an excipient as
it is generally considered to be the gold standard for protein
stabilization during the drying process (Balcão and Vila, 2015;
Manning et al. 2010).
Figure 13 : Molecular structure of trehalose

42
The protective effects of carbohydrates other than trehalose have been
investigated (Labrude et al. 1989; Tzannis et al. 1999) suggested that
sucrose has the ability to prevent from inactivation of oxyhemoglobin
and trypsinogen. The analogous result has been reported that the
addition of hydroxypropyl-β-cyclodextrin inhibits the aggregation and
inactivation of β-galactosidase during spray drying (S. Branchu et al.
1999). The addition of polyols such as mannitol is also effective
hydrogen bond formers and may thus protect proteins from
dehydration induced stress; however, their tendency to crystallize on
drying reduces suitability as stabilizing excipient in the formulation. It
was found that the ability of mannitol to provide the desired level of
protein stabilization for the spray dried powder of recombinant
humanized anti-IgE monoclonal antibody was limited its tendency
toward crystallization (coastantino et al. 1998).
The protective effects of carbohydrates other than trehalose have been
investigated (Labrude et al. 1989; Tzannis et al. 1999) suggested that
sucrose has the ability to prevent from inactivation of oxyhemoglobin
and trypsinogen. The analogous result has been reported that the
addition of hydroxypropyl-β-cyclodextrin inhibits the aggregation and
inactivation of β-galactosidase during spray drying (S. Branchu et al.
1999). The addition of polyols such as mannitol are also effective
hydrogen bond formers and may thus protect proteins from
dehydration induced stress; however, their tendency to crystalize on
drying reduces suitability as stabilizing excipient in the formulation. It
was found that the ability of mannitol to provide the desired level of
protein stabilization for the spray dried powder of recombinant
humanized anti-IgE monoclonal antibody was limited its tendency
toward crystallization (coastantino et al. 1998).

43
Surfactants are incorporated into several marketed protein solutions
and help to protect the protein from surface induced aggregation as
shaking or agitation. Polysorbates are commonly used in order to
prevent protein surface adsorption and aggregation. It was showed that
the addition of 0.1% (w/v) polysorbate 20 into the recombinant human
growth hormone formulation reduced the formation of soluble and
insoluble aggregates by approximately 90% on spray drying
(Mumenthaler et al. 1994). However, the use of surfactants,
especially polysorbates, is limited due to reduced long-term protein
stability due to enhanced protein oxidation by residual peroxidase
(Schmid 2011).
5.2. Immobilization
5.2.1.Adsorption
Adsorptions of the enzymes during spray dryer are a very old and
simple method which has wide application and high capability enzyme
loading relative to other immobilization methods. By simply mixing
the enzymes during spray drying with a suitable adsorbent, enzymes
can be immobilized under appropriate conditions of pH and ionic
strength. It is most commonly used for attachment of cells; however,
enzymes adsorbed on different carriers are also found in various
biotechnological processes. It is based on weak forces, however, still
enabling an efficient binding process. Usually, in bonds formation
several forces are involved: van der Waals forces, ionic and
hydrophobic interactions and hydrogen bonds (Guisan 2006;
Flickinger 1999; Kumar 2009). Sometimes also affinity binding is
included in this group (Guisan 2006). While the method is easy in
preparation, costs are low; it has many disadvantages and very few
applications. As a result to the weak interactions between the support

44
and the biocatalyst, there is a very high rate of leakage, binding is
unstable, there is no possibility to control the loading, so the
reproducibility is also low (Flickinger MC, Drew SW. 1999; Bucur,
Danet, Marty. 2004).
5.2.2.Disulphide bonding
It is generally applied for enzyme immobilization during spray dryer
and may be seen as a variation of covalent bonding, as there are stable
covalent bonds formed between activated support and free thiol group
(for example on cysteine) in the biocatalyst. However, those bonds can
be easily broken using a suitable agent under mild conditions, what
classifies this method as a reversible immobilization. Dithiothreitil
(DTT) is the most popular agent for disulphide bond decomposition.
5.2.3.Entrapment
Entrapment is an irreversible method, where immobilized particles or
cells are entrapped during spray dryer in a support matrix or inside
fibers. Drawbacks of this type of immobilization is usually connected
with the costs of immobilization, diffusion limitations, and
deactivation throughout the immobilization and abrasion of support
material during usage. Another disadvantage is low loading capacity
as biocatalysts have to be incorporated into the support matrix. This
aspect creates the problem of choosing the best support material.
5.2.4.Encapsulation
Encapsulation is another irreversible immobilization method similar to
entrapment. In this process, biocatalysts are restricted by the
membrane walls (Bickerstaff. 21997; Cao 2005). The factor
determining this phenomenon is the correct pore size of the membrane,
attuned to the size of the core material. This limited access to the
microcapsule interior is one in all the most benefits of

45
microencapsulation, for it protects the biocatalyst from the harsh
environmental conditions. As most immobilization method, it prevents
biocatalyst leakage, increasing the process efficiency as a result (Park
JK, Chang HN 2000). However, like every technology,
microencapsulation has some disadvantages. The most severe one is
the necessity for a very strict pore size control, which is especially
difficult in the case of small biocatalysts like some enzymes What is
more, this technique is not available for biocatalyst with a size similar
to their reaction product, as it would result in a leakage of both or burst
of the capsule. The encapsulation is also effective for the stabilization
of proteins during spray dryer against thermal and dehydration
stresses.
5.2.5.Cross-linking
It is an irreversible method of enzymes immobilization that does not
require support during spray dryer. This method decreases costs, at the
same increasing time specific. There are two main methods of cross-
linking. First one is Cross-Linking Enzyme Aggregates (CLEA) and
the other one is the Cross-Linking Enzyme Crystals (CLEC). This first
method had some very serious drawbacks, like low mechanical
stability, low reproducibility and low activity retention. While it was
proved that in CLEC method enzymes are much more resistant to
denaturation by heat or organic solvents and to proteolysis which gave
this method advantage over CLE (Guisan 2006; Sheldon 2007). The
main disadvantage of this method is the necessity for very high
purification of the enzyme and the need to crystallize it, what is
laborious and time-consuming. As a result, another improvement
emerged CLEA, which allowed working in aqueous solutions. Basics
of CLEA are that by addition of salts, organic solvents or nonionic
polymers the aggregates of enzymes are formed which maintain their

46
activity, but their stability is increased. It was found to be cheaper and
easier than CLEC and, what is more important, has a wider range of
applications. However, the Main disadvantage of both CLEC and
CLEA methods are constraints of diffusion when aggregate or crystal
size is increased. An important factor is to use a proper cross-linking
agent. Usually, it is glutaraldehyde, as it is inexpensive and readily
available in commercial quantities, nevertheless, it was found
unsuitable for immobilization of some enzymes, for example, nitriles,
where sometimes low retention or even no retention was observed. In
this case, dextran polysaccharide can be used as a cross-linking agent
(Sheldon 2007).
6. Applications of Spray Dried enzymes and Proteins
Spray drying medical applications are mainly focused on the production
of microparticles designed for encapsulation purposes and drug delivery
systems, which can be then administered orally, pulmonary, parenterally,
ophthalmologically, nasally or even vaginally it focused on dry heat-
sensitive components, like enzymes or proteins, without compromising
their biological activity makes the production of such systems possible.
As a result, within the biomedical field, spray drying is primarily used to
tune active pharmaceutical compounds and to produce dry powder
aerosols which making them suitable and useful for drug delivery (Jain
Manu S, Lohare Ganesh B, Chavan Randhir B, Barhate Shashikant
D, Shah CB. 2012). In that sense, different strategies have been used to
design the sprayed products according to the desired goals. With the
introduction of the Nano Spray Dryer B-90 from (Büchi Laboretechnik
AG (Switzerland) in 2009), the nano spray drying of protein
nanotherapeutics became reality on a laboratory-scale. It is expected that
the increased customer demand for the laboratory product, combined

47
with promising new applications, will promote and stimulate the
development of more industry-relevant models. In order to further
explore the potential of nanospray drying, by grenha et al. they developed
microencapsulated protein (insulin) loaded chitosan/tripolyphosphate
nanoparticles by spray drying (Grenha et al.2011). In specific, by taking
advantage of the ionotropic gelation of chitosan with the
tripolyphosphate, they prepared the nanoparticles and then incorporated
the protein content within such particles using aerosol excipients (lactose
and mannitol) at which release suitable microspheres for lung protein
delivery were produced. The reality, the microspheres presented a good
protein loading capacity, being released in a matter of a few minutes
toward the lung environment. However, the authors realized that the
mannitol/protein ratio influences the microspheres morphology,
specifically spherical shapes that are obtained in the existence of higher
amounts of nanoparticles.
In fact, the release profile of phosphodiesterase V (PDE-5) which is a
drug prescribed to treat severe pulmonary hypertension encapsulated
within spray-dried polymeric particles. In that manner, they produced
particles from organic PLGA solutions and as well as composite particles
obtained from aqueous PLGA nanosuspensions. They concluded that
both spray-dried products were aerodynamically appropriate for deep
lung deposition. However, the particles produced from the organic
solution are preferred over the composite ones to act as pulmonary drug
delivery vehicles as they showed an extended drug releasing profile
(Beck-Broichsitter et al.2012).
Until now, the new Nano Spray Dryer B-90 technology with the ability to
effectively formulate temperature-sensitive compounds (e.g. proteins (Lee
S.H., D. Heng, W.K. Ng, H.-K. Chan and R.B.H. Tan (2011) and

48
enzymes (Bürki, K., I. Jeon, C. Arpagaus and G. Betz. 2011) in the
submicron scale in (Table 2) has been successfully used for a variety of
drug delivery applications such as :
• Bovine serum albumin used as a model protein, (Lee, S.H.; Heng, D.;
Ng, W.K.; Chan, H.-K.; Tan, R.B.H. 2011).
• ß-galactosidase as a model enzyme with trehalose as a stabilizer
(Bu¨rki, K.; Jeon, I.; Arpagaus, C.; Betz, G.2011).
• Arabic gum, whey protein, polyvinyl alcohol, modified starch, and
maltodextrin as different polymeric wall materials for encapsulation ;(
Li, X.; Anton, N.; Arpagaus, C.; Belleteix, F.; Vandamme,
T.F.2011).
Jain encapsulated curcumin in cross-linked human serum albumin
particles. Curcumin is a highly active antioxidant; it is also a food
additive and preservative. The particles were smooth, spherical, and
submicron-sized in a range of 0.2–0.7 μm. Cross-linking of albumin
produced spray dried particles with slower curcumin release, which
followed a first-order release profile (Jain et al. 2011). Cross-linked
horseradish peroxidase enzyme with ethylcellulose particles formed
by nanospray drying. The enzyme catalyzes the removal of phenolic
compounds from wastewater. As expected, the residual activity of the
enzyme increased upon decreasing the spray-dried particle size, thanks
to the higher specific surface area. The immobilization also improved
the pH tolerance up to a range of 4–10, as well as the storage stability,
compared to free enzyme. The immobilized enzyme preserved over
50% of its activity after 4 weeks of storage at room temperature
(Dahili et al. 2015).

49
Table2:ApplicationslistofspraydriedproductsusingthelaboratoryscaleNanoSpray
DryerB-90from(BÜCHILabortechnikAG)(derivedfromliterature)

50
The Nano Spray Dryer B-90 can be used to evaluate spray drying
during the early stages of product development in a different of
applications including spray drying of solutions, nanoemulsions,
nanosuspensions as well as structural transformations or micro and
nanoencapsulation. The Nano Spray Dryer B-90 has been assessed in
previous studies for the preparation of submicron particles of
polymeric wall materials, the encapsulation of nano-emulsions (Li et
al., 2010) as well as the drying of pharmaceutical excipients and model
drugs (Schmid et al., 2010). Recently, Nano Spray Dryer B-90 has
been applied for manufacturing protein nanoparticles (Lee et al.,
2011). The aim of this study was to evaluate the Nano Spray Dryer B-
90 with regard to the drying of proteins, -galactosidase, together with
trehalose as a stabilizer. According to literature, trehalose is described
to prevent protein degradation by glassy immobilization and/or water
replacement (h-bonds) as possible mechanisms (Crowe et al., 1996;
Anhorn et al., 2008;Maltesen et al., 2008;Maury et al., 2005a,b;
Schüle et al., 2008).
The use of dehydrated enzymes for industrial applications has become
increasingly common, especially in formulations of food and
pharmaceutical (Samborska et al., 2005; Namaldi et al., 2006;
Kurozawa et al., 2009; de Jesus and Maciel Filho, 2011). In between
these enzymes most used mention α-amylase. Alpha-amylase which is
an endoenzyme that Applications list of spray dried products using the
laboratory scale Nano breaks down starch by hydrolysis to maltose
(Lévêque et al. 2000) and is widely used in the food and
pharmaceutical industries, laundry detergents and in “desizing” in
textiles (Sivaramakrishnan et al., 2006; Biazus et al., 2009; de Jesus
and Maciel Filho, 2011). Some spray dried enzymes and their
applications in industry are given in Table 3 .

51
Table 3 : Examples of spray- drying enzymes

52
Other enzymes and proteins used as encapsulation wall materials ( excipients
), binders, stabilizers and dispersing agents, are applied in drug formulation
studies, including water-soluble saccharides (e.g. Arabic gum ) ,and proteins
(i.e. whey protein, sodium alginate, leucine, silk fibroin, gelatin, and serum
albumin ) and Bovine Serum albumin ( BSA ) used as targeting lungs
passively after intravenous injection and to treatment of Asthma . There is
the ability to process small sample amounts makes a Nano spray dryer very
suitable for testing valuable biological materials such as for example
monoclonal antibodies, siRNA-based therapeutics or recombinant proteins
And, nanospray drying enables the encapsulation of drugs in polymers with
high efficiency of over 95% and adjustable drug loading. Intravenous (e.g.
simvastatin in PLGA as cancer chemotherapeutics, antipsychotic clozapine
and risperidone in PLGA, or small interfering RNAs loaded in human serum
albumin particles to treat genetic disorders). Also can be used in the food
industry, especially the dairy industry, produces large quantities of food
proteins such as whey protein concentrate and whey protein isolate (WPI).
Spray-dried food protein powders are subsequently used in baby formulae
and other food products. The understanding of drying of protein solutions in
the spray drying process is important in the drying of probiotic and non-
probiotic bacteria as well as the denaturation of cellular protein that is
responsible for the death viable cells (Ghandi et al., 2012a).
Spray-dried protein powders are also very commonly used as encapsulating
shell materials for food color, aroma, herbal extracts (Re, 1998; Rodrigues
and Grosso, 2008), and sticky food materials (Adhikari et al., 2009).
Other types of enzymes have been attractive in the detergent industry due to
they are derived from renewable sources, they reduce costs ,energy use and
they are highly space-efficient, since a little amount of enzyme can remove a
lot of different stains (Olsen, H.S. and Falholt, P. 1998).

53
Moreover, enzymes account for about 6% of the total raw material costs for
powder detergents (Edser, C. 2009) As Lipase and Savinase are two types
of enzymes which are used in detergents due to their cleaning properties. On
one hand, lipase is classified as hydrolase, since it catalyzes the hydrolysis of
water-insoluble long chain triglycerides (Esteban-Torres M., Reverón, I.,
Santamaría, L., Mancheño, J.M., de las Rivas, B. and Muñoz, R. (2016).
It can remove tomato, butter, oils, chocolate and cosmetic stains (Edser, C.
2009). On the other hand, savinase represents a subgroup of high-alkaline
proteases belonging to subtilase enzyme family, so they catalyze the cleavage
of peptide bonds in other proteins (Lipińska, A., Świerczyńska, D.,
Tymoszuk, Nowakowska, E. and Walusiak-Skorupa, J. 2013,
Mukherjee, A.K., Adhikari, H. and Rai, S.K. (2008). Specifically, it is a
serine protease (Olsen, H.S. and Falholt, P. 1998). Savinase enhances the
cleaning of protein-based soils, such as grass, blood, human sweat and egg
(Edser, C. 2009). In the past Using of protein therapeutics for the
pharmaceutical industry has grown from a nearly negligible role to being a
primary focus. As proteins are generally not stable for prolonged periods of
time, formulation scientists faced many challenges in achieving sufficient
shelf life for these proteins therapeutics (E.Y. Chi, S. Krishnan, T.W.
Randolph, J.F. Carpenter 2003, M.C. Manning, D.K. Chou, B.M.
Murphy, R.W. Payne, D.S. Katayama 2010). A lot of these challenges
have been overcome, as is illustrated by the fact that in 2015 nearly 30% of
drugs newly registered at the United States Food and Drug Administration
(FDA) were protein drugs (Centre of Drug Evaluation and Research,
Novel drugs 2015, 2011). However, all but 1 of these protein drugs are liquid
formulations which require refrigerated (2–8 (ϲ° remaining dry powder
formulation (mepolizumab, Nucala) must be stored and transported below 25
C, see in (Table 4). SD is the most common particle engineering method
that generates solid (particulate) proteins for pharmaceutical applications see

54
in (Table 5). Some of the solid proteins are used in spray dryer on pulmonary
drug delivery as ( Catalase, IgG, influenza vaccine and Alkaline phosphate )
and Erythropoietin used in drug delivery sustained release (Maa, Y.-F.;
Nguyen, P.-A. 1999, Ramezani, V.; Vatanara, A.; Najafabadi, A.R.;
Gilani, K.; Nabi-Meybodi, 2013 , Faghihi, H2014, Li, H.Y.; Song, X.;
Seville2010 , Bittner, B.; Morlock, M.; Koll, H1998) . But other of solid
proteins can be used to improve stability of proteins as (Trastuzumab, Anti-
IgE Mab, rhDNas) (Ajmera, A.; Scherliess, R2014, Ramezani, V.;
Vatanara, A.; Najafabadi, A.R.; Shokrgozar, M.A.; Khabiri, A.;
Seyedabadi, M. A2014) by using specific stabilizer for each protein and
mechanisms of these stabilizers in (Table 5) .

55
Table 4: Overview of protein drugs newly registered at the United States Food and Drug
Administration (FDA) in 2015 and their type

56
Table 5: Studies of solid protein formulations prepared by spray drying methods in the
presence of stabilizers and applications

57
Conclusion
Enzymes are one of the most important groups of biotechnological and
pharmaceutical products. And in order to improve their shelf life storage
stability, reduce transportation cost and preserve the biological activity of
these molecules, they are often preserved in dry form. Drying can be
performed using various techniques such as evaporation, evaporation and
atomization, sublimation and supercritical fluid drying. Recently, Spray
drying was introduced where it involves spraying of liquid solutions,
suspensions or emulsions' feed using open or closed system. The droplets
formed by the atomization process are dried through solvent evaporation to
create particles which are collected as dry powder in the electrostatic
collector or drying chamber. There are different drying chamber
configurations, in which the flow pattern between the hot gas and the spray
of droplets is separated: co-current flow, counter-current flow, and mixed
flow. Co-current flow considered as the most suitable for heat sensitive
materials such as enzymes as proteins. In a co-current flow, the feed is
atomized and sprayed through the drying chamber in the same direction as
the flow of the heated drying medium. The drying gas is hottest in the
uppermost part of the drying chamber and evaporation from the newly
formed droplets is fast allowing the dried particles to contact with moderate
temperatures so avoid unwanted thermal degradation of enzymes and
proteins. It is necessary to collect the dried particles once the droplet drying
is completed. The most common dry collectors include the cyclone separator,
the bag filter and the electrostatic precipitator. Nanospray drying has been
successfully applied in a wide range of pharmaceutical applications on a
laboratory scale, such as increasing the bioavailability of poorly soluble
drugs as well as the encapsulation of nanoparticles, nanoemulsions, and
nanosuspensions in biocompatible polymeric wall materials for sustained

58
drug release. This thesis shows that every different process step in the spray
drying of proteins may result in a loss of protein activity. However, the extent
to which the different steps add to the total process stability of the protein may
significantly differ for different process equipment used and for different
proteins. As fast removal of water is important for the most efficient
stabilization of proteins during spray drying where water has to be replaced
by an amorphous interaction-partner, e.g. trehalose. The drying process is
gentle and contributes to maintaining the stability and activity of heat
sensitive materials, such as peptides, proteins, and enzymes. The most
important adjustable process parameters during spray drying are the drying
gas temperature, the drying gas flow rate, the spray mesh size, solvent type,
the solids concentration in the feed, and the selection of the corresponding
excipients, stabilizers, and surfactants. The prepared spray dried drug-loaded
particles are administrated in various ways, including pulmonary, oral,
intravenous, topically, ophthalmic, intraperitoneal, intravesical or even
cerebral, which underlines the versatility of nanospray drying technology.
Moreover, the ability to use different feedstocks and the high productivity
and broad applications of this technique makes it more and more attractive to
the scientific community. In conclusion, with the introduction of the
nanospray dryer B- the nanospray drying of proteins as nanotherapeutics
became a reality on a laboratory scale. It is expected that the increased
customer demand for the laboratory product, combined with promising new
applications, will promote and stimulate the development of more industry
relevant models. In order to further explore the potential of nanospray dryer.

59
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