![Ratio between the project area equivalent diameter (da) and the total solids weight [mg/ml] (a) and the ratio between the polymer and one of the selected carriers, mannitol or lactose, (r) (b). The scale bars for the images shown per each case are 500 mm and 50 mm for the low and high magnification pictures, respectively.](https://www.researchgate.net/profile/Alberto-Baldelli/publication/361553029/figure/fig2/AS:11431281079398201@1660683312580/Ratio-between-the-project-area-equivalent-diameter-da-and-the-total-solids-weight_Q320.jpg)
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Drying Technology
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Spray freeze drying for protein encapsulation:
Impact of the formulation to morphology and
stability
Alberto Baldelli, Aylin Cidem, Yigong Guo, Hui Xin Ong, Anika Singh, Daniela
Traini & Anubhav Pratap-Singh
To cite this article: Alberto Baldelli, Aylin Cidem, Yigong Guo, Hui Xin Ong, Anika Singh, Daniela
Traini & Anubhav Pratap-Singh (2022): Spray freeze drying for protein encapsulation: Impact of the
formulation to morphology and stability, Drying Technology, DOI: 10.1080/07373937.2022.2089162
To link to this article: https://doi.org/10.1080/07373937.2022.2089162
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Spray freeze drying for protein encapsulation: Impact of the formulation to
morphology and stability
Alberto Baldelli
a
, Aylin Cidem
b
, Yigong Guo
a
, Hui Xin Ong
b
, Anika Singh
a,c
, Daniela Traini
b
, and
Anubhav Pratap-Singh
a
a
Faculty of Land and Food Systems, The University of British Columbia, Vancouver, BC, Canada;
b
Woolcock Institute of Medical
Research, Faculty of Medicine & Health, University of Sydney, Glebe, NSW, Australia;
c
Natural Health and Food Products Research
Group, Centre for Applied Research & Innovation (CARI), British Columbia Institute of Technology, Burnaby, BC, Canada
ABSTRACT
Proteins, the building blocks of life, are increasingly being used as therapeutics for treating
several diseases. Yet, there are challenges in the delivery of highly labile materials like pro-
teins, which is often circumvented with the help of encapsulation for targeted delivery and
enhanced stability. Spray drying technology has recently been employed for encapsulation
due to its’low cost and scale-up capabilities, yet the high temperatures of drying air makes
the technology unsuitable for proteins. More recently, spray freeze drying has evolved as an
emerging technology that combines spray drying with freeze drying by using low tempera-
tures, and is thus suitable for maintaining the stability of proteins. This study investigates
the correlation between formulation parameters and the properties of protein encapsulated
microparticles prepared by spray freeze drying. Morphology was investigated using micro-
scopic methods, and protein stability was examined using infrared and mass spectrometry.
By using bovine serum albumin, we verify that increasing the total weight to 15 mg/ml
results in microencapsulates with a projected area equivalent diameter of 100 mm larger. We
demonstrate that some types of amino acids are essential for shell formation; however, glu-
tamine generates an increase in dimer areas in mass spectra of 5.5. D-Mannitol is the sug-
gested carrier for high encapsulation efficiency (above 90 %). The formulation containing
polyvinylpyrrolidone, mannitol, and leucine (at 6, 9, and 2 mg/ml, respectively) produced the
lowest reduction in the stability of a few types of proteins; deconvoluted infrared peaks
show a difference of less than 2% compared to the free protein. Understanding the spray
freeze drying phenomenon for protein encapsulation would allow the control over morpho-
logical and chemical properties of microparticles containing active proteins.
GRAPHICAL ABSTRACT
ARTICLE HISTORY
Received 29 November 2021
Revised 29 April 2022
Accepted 9 June 2022
KEYWORDS
Spray freeze drying;
encapsulation; proteins;
particle formation
CONTACT Anubhav Pratap-Singh anubhav.singh@ubc.ca Faculty of Land and Food Systems, The University of British Columbia, 2205 East Mall.,
Vancouver, BC V6T 1Z4, Canada.
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07373937.2022.2089162.
ß2022 Taylor & Francis Group, LLC
DRYING TECHNOLOGY
https://doi.org/10.1080/07373937.2022.2089162
1. Introduction
Novel approaches for enhancing shelf life and delivery of
protein therapeutics are increasingly being researched.
[1]
By improving the efficiency and efficacy of delivered
proteins, researchers have successfully found treatments
for, or alleviated the symptoms of, a broad range of
diseases.
[2,3]
Despitethese,moreeffortsareneededfor
stable and effective delivery of proteins. Challenges in
protein delivery relate to the high molecular weight
and complex structure of proteins, and weak non-cova-
lent interactions, such as van der Waals forces and
electrostatic interactions, which make proteins inher-
ently unstable.
[2]
In addition, for most proteins, any
stress due to factors such presence of metal ions, tem-
perature, pH, and adsorption etc. leads to the collapse
of their complex structure resulting in denaturation,
aggregation, or modification, indicating a loss of activ-
ity.
[1,4]
The instability and vulnerability of proteins to
many external factors generate a short shelf-life, mak-
ing therapies involving proteins less effective.
The idea of encapsulating proteins in order to preserve
their integrity and properties is derived by the nature, i.e.,
viruses. Despite the large number of attempts in the litera-
ture to develop innovative delivery approaches, the most
common method to deliver proteins is still intravenous
(IV), considered invasive and challenging for self-adminis-
tration.
[2]
Alternative delivery methods, such as nasal, lung,
or gastrointestinal, are possible by encapsulating a protein.
During the delivery process, the proteins are protected by
the ‘enclosure’shell. Once the target is reached, the pro-
teins are slowly released.
Spray drying is the most common technique used to
produce microencapsulation.
[3]
The advantage that makes
this technique so appealing is the high applicability. The
main limitations are high polymorphism and possible
damage to the drug from the high temperatures required
for the atomizing procedure.
[4]
The first limitation could
be overcome by carefully selecting each parameter
involved in the particle formation process.
[5,6]
High tem-
perature can denature; the proteins unfold and break.
Each protein can show sign of damage at a precise tem-
perature and exposure time.
[7]
The damage from the high
temperatures in the atomizing process could be avoided
by employing alternative techniques, such as spray freeze
drying (SFD).
[7]
The cold temperatures involved in SFD
allow the conservation of the protein chemical structure.
SFD usually includes using a cryogenic fluid to maintain
the temperatures below a certain level. The advantage of
this technique is the ease of use and the production of
microparticles with a high porous surface structure, which
could produce superior aerosol performance due to a
favorable aerodynamic diameter.
[8]
For efficient encapsulation through SFD, a polymer
and a carrier are preferable. The polymer is useful for
generating a shell during the particle formation pro-
cess.
[5]
Due to the very high molecular weight of most
polymers, during the evaporation of sprayed droplets,
they show a high Peclet number and, thus, distribute
on the surface. The number of available polymers for
spray drying is restricted; the atomizing process limits
viscosity. A polymer with a molecular weight higher
than the protein’s one is recommended for a more
probable encapsulation.
[5]
Polyvinylpyrrolidone (PVP)
is a common choice for many studies thanks to the
broad range of molecular weights and high water
solubility.
[9]
High solubility and low viscosity compo-
nents aid the spray drying process; these components
are usually named carrier agents. The most common
examples are mannitol and lactose.
[10]
These two car-
riers maintain their amorphous state if stored at low
humidity, increasing the stability and the shelf-life of
encapsulated proteins.
[10]
Lactose and mannitol can
seem not ideal as lyoprotectants in protein formula-
tions because of their propensity to crystallize. In
aqueous solutions, the crystallization tendency of both
mannitol and lactose is enhanced during flash-freezing
involved in the SFD process.
[11]
However, due to the
very short distance between the atomizer and the
liquid nitrogen container, mannitol or lactose is fun-
damental for promoting particle formation.
[11]
Some types of amino acids have been applied as
drugs in biopharmaceutical formulations since they posi-
tively affect the solubility and stability of proteins.
[12]
Increasing protein melting temperature, preferential
exclusion, direct protein binding, buffering capacity, and
antioxidant activity are the mechanisms supported by
the use of excipients, such as some amino acids, in pro-
tein spray drying . Leucine is possibly the most common
amino acid used for promoting particle formation.
[12]
Other amino acids have been used, though, as stabil-
izers, such as arginine,
[13]
tyrosine,
[14]
and glutamine.
[15]
However, a guideline for selecting the amino acid gener-
ating the highest stability of proteins is missing for spray
freeze drying. Bovine serum albumin has been chosen as
thetestingproteinasitiscommonlyused,haswell-
known properties, and low cost.
[16]
Alternative proteins
are proposed to validate the applicability of spray freeze
drying for protein encapsulation.
There are some attempts to generate a guideline on
the use of spray freeze drying.
[16,17]
A previous study
analyzes the influence of the parameters of the spray
dryer parameters, such as drying air and liquid flow
rates, on the morphology of spray freeze dried micro-
particles.
[18]
Here, we investigate the influence of the
2 A. BALDELLI ET AL.
formulation on the morphology of the microparticles
and on the stability of the encapsulated proteins. The
novelty of this study relates to describing for the spray
freeze drying enabled encapsulation of various pro-
teins, and studying the relationship between various
formulation parameters.
2. Methods and materials
2.1. Solution preparation
2.1.1. Materials
The main protein used for testing the encapsulation
efficiency of spray drying in a cryogenic liquid was
Bovine Serum Albumin (BSA, 9048-46-8, VWR
Chemicals). In the case of fluorescence analysis, fluor-
escent BSA was used (Albumin from Bovine Serum
(BSA), FITC conjugate, A23015, ThermoFisher).
Other proteins were used to verify the encapsulation
efficiency of this procedure: Pea and whey protein iso-
late (Canadian protein), soy protein (Red Mill),
Hemoglobin from bovine blood (H3760, Sigma
Aldrich), b-Lactoglobulin from bovine milk (L3908,
Sigma Aldrich). One polymer was involved in generat-
ing a shell during the particle formation process:
Polyvinylpyrrolidone (PVP 1300, Mw 1,300,000 Da,
437190, Sigma Aldrich). The same polymers with dif-
ferent molecular weights were used (PVP 360, Mw
Table 1. List of the formulations used to verify the impact of chemical compounds on the properties of SFD microparticles.
# PVP Lactose Mannitol Leucine Tri-leucine Protein Total Ratio Substitute
Change in total weight [mg/ml] 1 15 12 2 0.1 29.1 1.2
2 13 10.5 2 0.1 25.6 1.2
3 11 9 2 0.1 22.1 1.2
4 9 7.5 2 0.1 18.6 1.2
5 7 5.5 2 0.1 14.6 1.2
6 8 13 2 0.1 23.1 0.6
7 10 16 2 0.1 28.1 0.6
8 15 22.5 2 0.1 39.6 0.6
Change in ratio between polymer (PVP) and carrier (Mannitol) (r) 9 14 1 2 0.1 17.1 14
10 12.5 2.5 2 0.1 17.1 5
11 10 5 2 0.1 17.1 2
12 8.5 6.5 2 0.1 17.1 1.3
13 6 9 2 0.1 17.1 0.6
14 4 11 2 0.1 17.1 0.3
15 14 1 2 0.1 17.1 14
16 12.5 2.5 2 0.1 17.1 5
17 10 5 2 0.1 17.1 2
18 8.5 6.5 2 0.1 17.1 1.3
19 6 9 2 0.1 17.1 0.6
Change in Ratio between Leucine and mannitol (r
a
)206 110(r
a
¼0) 0.1 17.1 0.5
21 6 10 0.5 (r
a
¼0.05) 0.1 17.1 0.6
22 6 10.5 1 (r
a
¼0.09) 0.1 17.1 0.6
23 6 9.5 1.5 (r
a
¼0.15) 0.1 17.1 0.7
24 6 8 3 (r
a
¼0.37) 0.1 17.1 0.9
25 6 6.5 4.5 (r
a
¼0.97) 0.1 17.1 0.7
Change in Ratio carriers (Mannitol/ Lactose, r
c
)26618(r
c
¼8) 2 0.1 17.1 0.5
27 6 2.5 6.5 (r
c
¼2.7) 2 0.1 17.1 0.5
28 6 4.5 4.5 (r
c
¼1) 2 0.1 17.1 0.5
29 6 6.5 2.5 (r
c
¼0.4) 2 0.1 17.1 0.5
30 6 8 1 (r
c
¼0.1) 2 0.1 17.1 0.5
31 6 9 (r
c
¼0)2 0.1 17.1 0.5
Change of protein 32 6 9 2 0.1 17.1 0.5 Pea
33 6 9 2 0.1 17.1 0.5 Whey
34 6 9 2 0.1 17.1 0.5 Soy
35 6 9 2 0.1 17.1 0.5 Hemoglobulin
36 6 9 2 0.1 17.1 0.5 B-lactoglobulin
Change of polymer 37 6 9 2 0.1 17.1 0.5 PVP 360
38 6 9 2 0.1 17.1 0.5 PVP 55
Change of amino acid 39 6 9 0.5 1.5 0.1 17.1 0.5
40 6 9 1 1 0.1 17.1 0.5
41 6 9 1.5 0.5 0.1 17.1 0.5
42 6 9 2 0.1 17.1 0.5 Glutamine
43 6 9 2 0.1 17.1 0.5 Tyrosine
44 6 9 2 0.1 17.1 0.5 Arginine
Assumed to be 0.
For this case, the commonly used material is substituted with the one listed under the column "Substitute."
The protein mostly used is BSA; however, when indicated with "", the protein is substituted with pea, whey, or soy protein. The polymer used for most
formulations is Polyvinylpyrrolidone with a molecular weight of 1’300’000 Da (PVP 13000). The symbol "" indicates the substitution of one chemical
compound. The term "ratio" indicates the ratio between the quantity of polymer and carried contained in the formulation.
The term “total”indicates the total amount of solid content in the sprayed formulation.
PVP, Lactose, Mannitol, Leucine, Tri-leucine, Protein and Total contents are provided in mg/ml
DRYING TECHNOLOGY 3
360,000 Da and PVP 55, Mw 55,000 Da Sigma
Aldrich). The carrier employed D-Mannitol (M4125,
Sigma Aldrich) and Lactose (61345, Sigma Aldrich).
primary main amino acid used to produce different
formulations was Leucine (L-leucine, L80000, Sigma
Aldrich). Besides, trileucine (L0879, Sigma Aldrich),
L-Arginine (A5006, Sigma Aldrich), L-Glutamine
(G7513, Sigma Aldrich), L-Tyrosine (T3754, Sigma
Aldrich) were used.
2.1.2. Mixing procedure
PVP was added to water by weighting the dry powder
with a CP2245 Sartorious, microbalance. The solution
was stored at 20 C for about 24 hours. Later, the solu-
tion was stirred at 100 rpm for 2 hours. The other com-
ponents were added in 10 ml of PVP solution. The
obtained solution was shaken using a C25KC Incubator
shaker (New Brunswick Scientific) for 3 hours.
2.1.3. Formulation matrix
To verify the impact of each component on the phys-
ical and chemical properties of microparticles achieved
via SFD, a broad range of formulations was created,
as shown in Table 1. Due to a short window (only
10 cm) for the shell to be formed before entering into
the liquid nitrogen, highly concentrated (>14.6 mg/
ml) solutions were used. With the following matrix,
the impact of the total weight, the ratio between poly-
mer and carrier (r), the ratio between amino acid and
carrier (ra), the ratio between two carriers (rc), the
molecular weight of the polymer and the protein, and
a change in an amino acid is verified.
2.2. Drying procedure
2.2.1. Spray freeze drying
A Buchi 290 with a double-flow atomizer with a noz-
zle of 0.7 mm was used. The drying chamber was
removed and instead, a lab jack holding a Cylindrical
Form Borosilicate Glass Dewar Flask (250 ml,
StonyLab) filled with liquid nitrogen was placed. The
distance between the atomizer and the liquid nitrogen
container was set at 10 cm. The flow of the feeding
liquid and the air were 5 ml/min and 3 L/min, respect-
ively (Figure 1). The rationale governing the choice of
these parameters are shown in the Supplementary
Information (SI) section (Figure S1).
2.2.2. Freeze drying
After the collection into liquid nitrogen and after the
liquid nitrogen is fully evaporated, the slurry was col-
lected using a spatula and transferred to an aluminum
weighting dish (Fisherbrand). The aluminum dish was
enclosed using a Kim wipe tissue tightened with an elas-
tic band. The slurry was stored at 80 Cfor12hours.
Subsequently, it was placed in a freeze dryer (Labconco
Freezone 6) at 300 mTorr and 3±4C for 3 hours
and at 100 mTorr and 8 ± 4 Cfor16hours(tempera-
tures were recorded at the shelves).
[16,18]
Different con-
ditions have been shown to highly impact the freeze
spray-dried microparticles (Figure S2 in the SI).
2.3. Characterization techniques
2.3.1. Morphology
A Scanning Electron Microscope (Hitachi S4700 SEM,
Ultrahigh resolution SEM with field-emission gun) was
used to obtain the morphology of SFD microparticles.
The parameters of 10 Kv and 8 mA were used. About
1 mg of material was placed on an Isopore membrane fil-
ter (13 mm in diameter and 0.4 mm pore size), positioned
on an SEM stub. A layer of 16 nm of gold was coated
over each sample by using a Cressington Sputter Coater.
Image analysis of SEM pictures was used to derive
the size distribution of SFD powder. The projected
area equivalent diameter (da) of microparticles was
obtained using ImageJ.
[19]
2.3.2. Distribution of chemical compounds
SFD microparticles containing fluorescent BSA have
been investigated under a Confocal microscope
(Olympus FV1000 Laser Scanning/Two-Photon
Confocal Microscope). Samples were analyzed using a
laser with a wavelength of 527 nm. Four cases have
been analyzed: numbers 6, 13, 23, and 26 in Table 1.
Figure 1. Illustration of the parameters selected for the pro-
cedure of spray freeze drying (SFD).
4 A. BALDELLI ET AL.
X-ray photoelectron spectroscopy (XPS) analysis
was performed using a Kratos Analytical Axis ULTRA
spectrometer containing a DLD spectrometer using a
monochromatic aluminum source (AlKa, 1486.6 eV)
operating at 150 W (10 mA emission current and
15 kV HT). Analysis was conducted on a 700 300
mm2 area of the sample. High-resolution scans were
obtained at a 100 meV step size, pass energy of 20 eV,
and averaged over 3 scans. The calibration procedure
was performed following the ISO 15472 procedure.
2.3.3. Protein stability
The stability of the protein was analyzed using a
High-Performance Liquid Chromatography (HPLC)
(Agilent 1100 series, Agilent) composed of a quater-
nary pump, an autosampler, a column heater, a DAD
detector, and Fourier-transform infrared spectroscopy
(FTIR) (Spectrum 100, PerkinElmer). The process is
reported in a previous reference and in the SI.
[20]
Attenuated total reflectance (ATR) –FTIR was used.
The spectra were deconvoluted using OriginPro deriving
peak areas. The IR spectrum of BSA is well-known and
reported in several references.
[16]
However, we suggest a
summary in the SI.
2.4. Statistical analysis
Repetitions were performed for every type of test
described above. The da was obtained by measuring
from 300 to 600 microparticles deposited in five differ-
ent areas on the SEM stubs.
[5]
The stability of encapsu-
lated protein was derived by analyzing FTIR and HPLC
spectra. Three spectra were obtained per sample.
3. Results
3.1. Morphological analysis
The morphological properties can be controlled by
adjusting the total solids weight, the ratio between poly-
mer and carrier, the amino acid weight percentage, the
ratio between two different carriers, and the substitution
of one or more chemical compounds. Figures 2–5show
the effect of these parameters on the size distribution of
SFD microparticles.
3.2. Protein encapsulation
In the case of fluorescent proteins, a fluorescent con-
focal microscope can be used to image the position of
the protein within the encapsulating shell, as shown
in Figure 6.InFigure 6, the bright and white areas,
highlighted with a red circle, indicate the presence of
BSA. The bright gray areas mainly distributed on the
surface of SFD microparticles are derived from the
strong fluorescence of PVP 1300.
[21]
While the confocal microscope provides clear and
visual validation of the protein’s location within the
microparticles, an alternative technique is required for a
more accurate analysis. Table 2 shows the typical peaks
of oxygen, carbon, nitrogen, sulfur, and sodium of both
reference materials and some of the samples shown in
Table 1 obtained using an XPS. The atomic concentra-
tion shown in Table 2 relates only to the first 10 nm of
the surface of SFD microparticles. BSA shows a unique
signature containing atomic concentration for S2p and
Na1s of 0.59 and 0.37, respectively. BSA is present in
the first 10 nm of SFD microparticles when these two
types of atoms are detected.
Figure 2. Ratio between the project area equivalent diameter (da) and the total solids weight [mg/ml] (a) and the ratio between
the polymer and one of the selected carriers, mannitol or lactose, (r) (b). The scale bars for the images shown per each case are
500 mmand50mm for the low and high magnification pictures, respectively.
DRYING TECHNOLOGY 5
3.3. Protein physical stability
BSA and the alternative proteins shown in Table 1 have
a strong signal when exposed to an infrared light. The
IR peak located in the wavelength range 1600–1700 cm
1
contains important references to the chemical structures
of proteins. Figure 7a shows an example of deconvolu-
tion of this peak into four sub-peaks, b-sheet, a-helix,
b-turn, and b-antiparallel (explained in the material sec-
tion). The IR spectra of each case are shown in Figure
S4 in the SI. In general, shift of the amine I peak
(1650 cm
1
) to the right could be caused by the inter-
action of nitrogen ions, present in the liquid nitrogen,
with BSA, with the C-O and C-N groups. This would
occur in case of contact between BSA and liquid nitro-
gen and, thus, in case of an improper encapsulation. A
lower and broader peak is a sign of structural perturb-
ation.
[16]
For a facile interpretation, the percentage differ-
ences between SFD encapsulated BSA and free BSA
peaks are shown in Figure 7b–h.
FTIR is considered a semi-quantitative technique.
HPLC is known to show higher accuracy, and it can
be used to determine the quantity of protein con-
tained in SFD microparticles, the spraying yield, the
change in the chemical structure of proteins, and the
Figure 3. Trend in projected area equivalent diameter for an increase in the ratio between amino acid and carrier (mannitol) ra,
and an increase in the ratio between two carriers, mannitol, and lactose, rc. For plots a) and b), the total mass is 17.1 mg/ml, and
the ratio between the polymer and the sum of the carriers and amino acid is 1.8.
Figure 4. Impact of the molecular weight of different proteins, a), and of different polymers, b), on the size of SFD microparticles.
6 A. BALDELLI ET AL.
Figure 5. Impact of the ratio between trileucine and leucine, a), and of types of amino acid, b), on the size of SFD microparticles.
Figure 6. Fluorescence images of different layers along one axis of SFD microparticles. Cases 6, 13, 23, and 26 in Table 1 are
reported as a, b, c, and d.
Table 2. Atomic concentrations of the surface (first 10 nm layer) of SFD microparticles and reference materials.
Atomic concentration [%]
Type # Description O1s N1s C1s S2p Na1s
Reference materials PVP 1300 10.4 ± 2.22 10.8 ± 1.21 78.8 ± 1.55
Mannitol 43.1 ± 3.54 56.9 ± 1.47
Lactose 39.6 ± 4.75 60.3 ± 1.53
Leucine 18.8 ± 2.87 9.33± 1.73 71.9 ± 2.18
BSA 15.5 ± 3.56 13.4 ± 2.47 70.1 ± 2.42 0.59 ± 0.14 0.37 ± 0.16
Total weight [mg/ml] 7 28.1 (r ¼0.6) 26.3 ± 0.95 7.14 ± 2.54 66.5 ± 2.34
4 18.6 (r ¼1.2) 16.3 ± 1.34 8.75 ± 1.77 74.9 ± 0.45
5 14.6 (r ¼1.2) 16.5 ± 0.23 9.27 ± 2.65 74.3 ± 0.56
Ratio carrier/polymer (r) 10 5 18.3 ± 1.34 8.78 ± 1.87 73.9 ± 0.23 0.13 ± 0.10 0.34 ± 0.21
11 2 17.6 ± 1.65 8.66 ± 2.34 73.2 ± 1.48 0.30 ± 0.19
13 0.6 17.9 ± 0.97 9.86 ± 1.76 72.2 ± 1.23
14 0.3 19.3 ± 1.35 8.86 ± 1.98 71.6 ± 2.35
Ratio amino acid/carrier (r
a
) 21 0.05 20.9 ± 3.45 7.30 ± 2.22 71.8 ± 2.89
23 0.15 16.7 ± 2.33 8.67 ± 1.96 74.6 ± 4.41
25 0.97 17.2 ± 2.87 8.18 ± 2.36 74.6 ± 5.68
Ratio mannitol/lactose (r
c
) 26 8 17.2 ± 3.45 8.26 ± 1.88 74.5 ± 6.64
27 2.7 19.2 ± 5.82 8.62 ± 2.03 72.2 ± 5.93
29 0.4 18.4 ± 3.34 9.32 ± 3.23 72.3 ± 4.53
DRYING TECHNOLOGY 7
efficiency of encapsulation, as shown in Table 3. The
calibration curves necessary to calculate the quantity
of protein contained in SFD microparticles are shown
in Figure S5 in the SI. The difference in the area of
the BSA monomer and dimers shows a possible pro-
tein degradation.
[16]
4. Discussions
4.1. Total solids weight
In spray drying techniques, it is well-known that the
total solids weight percentage (tw) is proportional to
the projected area equivalent diameter da in spray
Figure 7. In a), the deconvolution of the amide I of BSA is shown; in b to h), the percentage difference between the areas of
b-sheet, a-helix, b-turn, and b-antiparallel of the BSA spectrum and the spectra of samples reported in Table 1 is shown.
8 A. BALDELLI ET AL.
dried powders.
[5]
For a ratio between polymer and
carrier (r) of 1.2, increasing the tw from 14.6 to
22.1 mg/ml generates a surge in da from 110 to
220 mm, Figure 2a. By dividing r by a half, higher tw
appears to stimulate an increase in diameter; a differ-
ence in tw of 16.5 mg/ml produces an increase in da
of 120 mm. A richer solution would create more vis-
cous droplets, which tend to produce spray-dried
microparticles with a larger diameter.
[22]
However, the
two-flow nozzle shows a limitation in viscosity, which
is reflected in the highest tw with an r of 1.2. In these
cases, the microparticles are not formed.
PVP shows an average solubility of 100 mg/ml. By
employing agitation, the solubility could be further
increased, boosting the solution’s viscosity and reduc-
ing the ability of other components to dissolve.
[22]
The poor encapsulation efficiency reflects the lack of
particle formation for the cases of high tw and r of
1.2. The gap between the peak areas of b-sheet and
b-antiparallel of the powder obtained in these cases
and pure BSA reaches the value of 150, as shown in
Figure 7a. In addition, the mass peak area of the
dimer 1297 for r ¼1.2 and 1432 for r ¼0.6 for tw of
29.1 and 39.6 mg/ml, respectively. As seen in the SEM
images in Figure 3a, microparticles are not formed
and the BSA is not homogenously spread in the pow-
der and, thus, exposed to any damage. In fact, BSA
formed dimers; the mass peak area of the dimer 1297
for r ¼1.2 and 1432 for r ¼0.6 for tw of 29.1 and
39.6 mg/ml respectively. The dimer area disappears
when the tw goes below 19 mg/ml, showing also one
of the highest encapsulation efficiencies (g) (90.8),
Table 3. The encapsulation for these cases is con-
firmed by the difference between the area of the
monomer in mass spectrum of the spray freeze dried
microparticles and pure BSA (<1.05 %, Table 3). For
Table 3. High-Performance Liquid Chromatography (HPLC) analysis.
Type # Quantity [mg/ml] Monomer [%] Dimer area [mAUs] gEncapsulation
Total wt [mg/ml] r51.2 (r
a
¼0.2) 29.1 1 0.65 145 ± 5.55 1297 ± 145
25.6 2 0.74 107 ± 7.43 334 ± 98.5
22.1 3 0.73 137 ± 4.90 127 ± 32.4
18.6 4 0.75 0.34 ± 0.10 0 91.8 ± 1.42
14.6 5 0.78 1.03 ± 0.53 3.20 ± 1.30
Total wt [mg/ml] r50.6 (r
a
¼0.2) 39.6 6 0.74 94.2 ± 11.2 1432 ± 355 51.4 ± 2.56
28.1 7 0.75 83.1 ± 23.7 917 ± 125
23.1 8 0.53 192 ± 34.5 537 ± 158
rMannitol (wt % ¼17.1, r
a
¼0.2) 14 9 0.73 146 ± 21.4 11200 ± 899 27.9 ± 3.32
5 10 0.64 185 ± 36.7 6525± 673
2 11 0.74 125 ± 43.2 560 ± 135 59.2 ± 2.98
1.3 12 0.67 64 ± 16.6 432 ± 109
0.6 13 0.74 0.85 ± 0.13 0 90.8 ± 0.84
0.3 14 0.68 35.1 ± 2.35 0 79.9 ± 1.68
rLactose (wt % ¼17.1, r
a
¼0.2) 14 15 0.74 129 ± 44.4 1333 ± 326
5 16 0.75 85.6 ± 13.5 234 ± 98.4
2 17 0.72 162 ± 33.6 776 ± 183 63.4 ± 1.12
1.3 18 0.78 166 ± 14.3 897 ± 84.2
0.6 19 0.74 186 ± 22.7 787 ± 110
r
a
(wt % ¼17.1, r¼0.6) 0 20 0.69 176 ± 25.5 10.1 ± 1.85
0.05 21 0.75 31.6 ± 9.43 13.6 ± 7.31
0.09 22 0.61 20.1 ± 8.32 6.61 ± 1.53 84.4 ± 2.45
0.15 23 0.75 1.39 ± 0.92 0 90.5 ± 3.23
0.37 24 0.75 7.07 þ1.36 375 ± 78.3
0.69 25 0.75 31.1 ± 11.2 207 ± 81.1 74.9 ± 1.82
r
c
Mannitol/ Lactose (wt % ¼17.1, rof 0.6, r
a
¼0.2) 8 26 0.75 34.1 ± 9.36 64.4 ± 22.5 71.3 ± 3.53
2.7 27 0.75 64.4 ± 5.57 334 ± 84.2
1 28 0.74 114 ± 36.8 454 ± 83.1
0.3 29 0.75 127 ± 47.3 789 ± 112
0.1 30 0.74 134 ± 28.8 1133 ± 232 31.1 ± 4.52
Protein molecular weight [kDa] 18.3 32 –86.2 ± 11.4 –
36.8 33 –0.45 ± 0.27 –90.2 ± 1.19
64 34 –0.32 ± 0.16 –
355 35 –9.15 ± 1.36 –88.7 ± 2.08
450 36 –133 ± 23.6 –
PVP molecular weight [kDa] 55 37 0.75 17.7 ± 9.65 1222 ± 201
360 38 0.68 178 ± 46.7 136 ± 65.3 94.9 ± 1.42
Trileucine/ leucine 0.3 39 0.75 20.7 ± 8.42 860 ± 122
1 40 0.75 14.4 ± 3.69 375 ± 88.4
3 41 0.75 67.7 ± 14.3 139 ± 73.2
Alternative amino acid Glutamine 42 0.75 23.7 ± 10.5 5.50 ± 1.76 95.4 ± 2.77
Tyrosine 43 0.75 1.23 ± 0.84 0
Arginine 44 0.75 3.31 ± 0.34 0
Quantity of the protein in mg/ml, the difference in the peak area of the monomer with respect to the reference of free BSA, the area of the dimer in
mAUs, and the efficiency encapsulation are shown.
DRYING TECHNOLOGY 9
one of these cases, BSA is confirmed to be positioned
within the microparticle’s shell, Figure 6a.
4.2. Ratio between polymer and carrier
Due to the promising results achieved by employing a
tw below 19, a tw of 17.1 is selected to verify the
impact of r to the properties of SFD microparticles.
As described in the above sub-sections, the cases con-
taining PVP at weights above 10 mg/ml do not or par-
tially generate microparticles, Figure 3b. Such a
conclusion has been shown by Sou et al., who showed
that weight percentage ratios of 60 and above of PVP
enhance the protein stability of spray-dried pow-
der.
[23]
For r below than 5, a decrease in r contributes
to a decrease in da by maintaining the BSA located
inside the microparticle’shell, Figure 6b and c.A
reduction in r is achieved by reducing PVP and rais-
ing the amount of carrier, mannitol, or lactose. PVP
distributes first on the surface of the evaporating
droplet; the time to shell formation could be delayed
(even by a few milliseconds) when having a lesser
number of PVP molecules.
[5]
A delayed time to shell
formation would imply microparticles with a slightly
smaller diameter. An opposite trend is shown in
Figure 3b underlining the importance of the carrier.
As demonstrated by Eggerstedt et al., lower values of
r produced microparticles with a surface composed of
a small number of concentric patterns and an inner
core highly porous.
[9]
This might indicate that when
the droplet reaches the nitrogen, the shell is not fully
formed. The freeze-drying procedure removes the
remaining water. Due to the large molecular weight
and the large content in the initial formulation of
PVP 1300, the shell formation is expected to occur in
a few milliseconds at temperatures below 20 C.
[3,5,24]
The shell formation at 10 cm distance can be verified
by the fact that when increasing the speed of liquid
flow, no microparticles are formed, Figure S1 in the
SI. Therefore, it can be assumed that the first layer of
the shell is formed before reaching the liquid nitrogen
container. The porous structure is then derived by the
water leaving the formed shell during the freeze-dry-
ing. This assumption might be valid for most cases
shown under this category. A larger da for lower r
could be related to the effect of the remaining water
in SFD microparticles. Some coagulation is visible in
images of microparticles taken for r of 0.6 for both
mannitol and lactose as carriers, Figure 3b. The coa-
gulated appearance is much more visible for all cases
involving lactose as the sole carrier. Lactose has a
higher tendency to crystallize under a certain pressure,
at low levels of humidity, or exposed to high tempera-
tures.
[25]
This fragility of lactose could have damaged
the particles’structure during the freeze drying pro-
cedure where remaining water is removed at different
vacuum pressures and temperatures. For the cases
containing lactose solely, the difference between the
IR peaks b-sheet and b-turn is, on average, 50 %
higher than the corresponding mannitol-containing
cases, Figure 7b. The lowest g(63.4) is connected to
the case with a r of 2 and contains lactose, Table 3.
High dimer areas in the mass spectra of cases contain-
ing lactose and with high r are shown in Table 3. The
exact opposite result is obtained analyzing samples
containing mannitol and with a low r (0.6 and 0.3),
Table 3. This further remarks that mannitol is an
optimal carrier for the stability of drugs.
[26]
4.3. Ratio between free amino acid and carrier
Leucine has an impressive effect on the particle
formation generated by spray drying techniques.
[27]
L-leucine has a high Peclet number (Pe) and, thus,
during the drying procedure, it precipitates on the
surface of droplets producing a hydrophobic layer.
This layer restricts the dispersion of water and brings
the formation of indented particles.
[28]
Sou et al. iden-
tify the weight percentage of 20 as the optimal for
maintaining the protein stability in spray drying for-
mulations containing PVP and mannitol.
[23]
Therefore, values of L-leucine weights between 0 and
4.5 have been tested to understand the effect on BSA
stability. Without any leucine, at a ratio of 0 between
the amino acid and the carrier (ra), the particles look
highly porous and the surface highly cracked.
Therefore, the stability of BSA can be compromised.
All the b-peak areas of this case differ from the pure
BSA of an average of 100, Figure 3. The difference
between the monomer area with pure BSA is also rea-
sonably high, 176, Table 3. By increasing the weight
of leucine (up to reaching a ra of 0.15), the SFD
microparticles appear smoother and with a lower con-
tent of pores in their internal structure, Figure 3.
Moreover, by analyzing fluorescent images, the
BSA is ensured to be enclosed, Figure 6, by a layer of
L-leucine and PVP (first 10 nm of the microparticles
as listed in Table 2). Weights of L-leucine between 1.5
and 2.5 seem to be ideal for maintaining the BSA sta-
bility. From the IR and chromatography spectra ana-
lysis, none of the significant peaks show a change
compared to free BSA, as shown in Figure 7c and
Table 3. Beyond 2.5 mg/ml, L-leucine promotes a very
early shell formation, producing larger microparticles;
10 A. BALDELLI ET AL.
with an increase in ra from 0.2 to 1, the da raises
from 180 to 340 mm. However, the shell formed tends
to be weak, and, once frozen, a larger quantity of
water is entrapped within. The reasonably quick
freeze-drying procedure could provoke the rupture of
such a weak shell and the formation of a highly por-
ous internal structure. These two characteristics make
these microparticles very similar in morphology to the
case of 0 mg/ml of L-leucine, Figure 3c.
4.4. Ratio between two carriers
A co-spray drying of carriers, such as lactose and
mannitol, is often used.
[29]
For example, Ferdynand
and Nockhodchi show a ratio of 1:3 mannitol:lactose
ratio to offer the highest salbutamol sulfate stability in
spray-dried powders.
[29]
Here, we try to investigate
the conjunct action of two carriers to the particle for-
mation of SFD microparticles, including one or more
proteins. With an increase in the ratio between man-
nitol and lactose (rc), the particles show a higher da
and a smoother surface (Figure 4b). The larger diam-
eter is due to the presence of mannitol and with a
high Peclet number, it distributes quicker to the sur-
face of the evaporating droplet. The rougher surface
in cases of high lactose content is derived by, possibly,
the late time to shell formation. If a thin shell is
formed by the time evaporating droplets are collected
into the liquid nitrogen, the freeze-drying procedure
might break or crack it while the water is leaving.
Even though the BSA appears to be enclosed in all
cases tested under this category, Figure 6 shows that
BSA’s stability is spoiled by high lactose content. By
decreasing rc from 8 to 0.1, the dimer area increases
from 64 to 1133, the difference between the monomer
area of spray-dried microparticles and pure BSA
increases from 34 to 134, Table 3, and the IR peaks of
b-sheet and b-antiparallel deviate from the pure BSA
ones of more than 150 %, Figure 7. A high encapsula-
tion efficiency, 71, is reached for rc of 8. Again the
cause of this success can be linked to the early shell
formation supported by mannitol
[30]
and to the pos-
sible higher tendency of lactose to crystallize.
[31]
4.5. Interchange of one component
4.5.1. Encapsulated protein
Encapsulating proteins with a broad range of molecu-
lar weights indicates the formulation’s efficiency. We
select the weights of 6, 9, and 2 mg/ml for PVP,
Mannitol, and Leucine, respectively, due to the BSA
stability under these conditions (Figures 6 and 7, and
Table 3). As shown in Figure 4a, the morphology and
the da of SFD changes in protein do not alter micro-
particles. The micro and nano roughness appears
unmodified when different types of proteins, Figure 4.
However, a correlation between the microparticles
with the largest size and the degradation rate seems
proportional. Microparticles composed of whey, pea,
and soy protein show a da close to or larger than
200 mm, Figure 4a. The same cases show a difference
between the deconvoluted b-sheet of the freeze dried
microparticles and free BSA of about 100, 20, and 20
respectively, Figure 7h. Besides the molecular weight,
there are other differences between the proteins
selected. First of all, b-lactoglobulin and hemoglobulin
have a much shorter chemical composition than pea,
whey, and soy proteins. The last contains more than
20 components,
[32]
which could contribute to facile
degradation during SFD and freeze drying. Even so,
dimers were low or not present in the mass spectrum
of microparticles containing different types of pro-
teins, Table 3. A mild decrease in the stability can be
seen in the difference in the monomer area for micro-
particles containing whey and soy protein, 86.2 and
133 respectively, Table 3. Due to the high molecular
weights, whey and soy proteins appear more challeng-
ing to encapsulate by using SFD.
4.5.2. Polymer for shell formation
PVP can be available at different weight percentages.
High molecular weight polymers tend to distribute earlier
on the surface of an evaporating droplet.
[5]
Therefore,
PVP with a molecular weight of 1300 kDa generates
spherical and smooth microparticles, Figure 4b.Using
thesamepolymerwithalowermolecularweightshould
produce a late shell formation and, thus, possibly larger
microparticles in a common spray drying procedure.
However, in SFD, the use of PVP with a molecular
weight of both 360 and 55 kDa, the shell seems not to be
fully formed when reaching the liquid nitrogen. This
assumption is confirmed by the large and agglomerated
particles visualized in the SEM images, Figure 4b.Not
completing the shell formation would result in a sharp
decrease in BSA stability, as shown in the IR and mass
spectra peaks deconvolution in Figure 7f and Table 3.In
the case of polymers with low molecular weight, the shell
might have partially formed, and the pressure imposed
by the water evaporating during the freeze-drying would
disrupt such a weak shell.
[33]
4.5.3. Type of free amino acid
Leucine is commonly used in spray drying due to its
known quality in improving the particles’
DRYING TECHNOLOGY 11
aerodynamic properties for some types of drug deliv-
ery (i.e., pulmonary) and, like other hydrophobic
amino acids, in protecting spray dried formulations
against moisture.
[27]
Trileucine is considered a sur-
face-active molecule and improves the dispersibility of
particles without altering the morphology of the par-
ticle.
[3]
A high amount might generate a coating on
the surface and increase the cohesiveness of spray-
dried microparticles.
[3]
The rate of coagulated particles
and, thus, the number of particles with high cohesive-
ness appears to increase proportionally to a surge of
the ratio between trileucine and leucine, Figure 5a.As
a result, the average da increases from 178 to about
400 mm with an increase in the ratio of trileucine and
leucine from 0 to 3, as shown in Figure 5a. With the
same increase in the ratio, the dimer area of the mass
spectra increases from 0 to 890 mAUs, Table 3.
Amino acids can be divided into charged, polar,
amphipathic, and hydrophobic. Arginine falls in the
first category, where members having side chains can
form salt bridges. As a solution additive, arginine sta-
bilizes proteins against aggregation, especially in pro-
tein refolding.
[34]
Moreover, when distributed on the
surface of spray-dried microparticles, arginine enhan-
ces the dispersibility due to the positive charge, argin-
ine generates electrostatic repulsion between
particles.
[34]
As shown in Figure 5b, the separation
between microparticles containing arginine is sharper
than microparticles containing other amino acids.
This morphological property could be beneficial for
some types of drug delivery, i.e., aggregation affects
the location of deposition in nasal or lung delivery.
[8]
Arginine appears to maintain the stability of proteins
as shown with a maximum distance in the deconvo-
luted IR peaks concerning free BSA of only 2 %,
Figure 7g.
Glutamine is polar and tends to form hydrogen
bonds as a proton donor. However, glutamine is
shown to have a minimal effect on protein stabil-
ity.
[35]
Even though changes in the morphology com-
pared to microparticles containing other amino acids
is practically null Figure 5b, a slight degradation of
the BSA stability can be recognized. For example, the
difference between all IR peaks of spray-dried freeze
microparticles and free BSA is almost 10 %, and a
minor presence of a dimer area of 5.5 mAUs in the
IR spectra, Table 3.
Tyrosine belongs to the group of amphipathic since
it can show both a polar and non-polar behavior.
[36]
The oxygen bonds generated by -OH groups of tyro-
sine contribute highly to proteins’stability. This
amino acid is much less common in spray drying
since it does not contribute directly to the shell or
particle formation. Given enough time for the distri-
bution of the other components on the surface of
evaporating droplets, the use of tyrosine highly con-
tributes to the stability of proteins.
[36]
Microparticles
containing tyrosine show the lowest difference in
monomer area with respect to free BSA (1.23 in Table
3). All the IR peaks differ at most 4 % from the free
BSA peaks (Figure 7g).
5. Conclusions
Spray freeze drying is a valid technique for the encapsu-
lation of proteins. Using a few different formulations,
the protein of BSA is maintained stable. However, the
control over the properties of SFD microparticles can be
challenging. Several parameters and conditions impact
the appearance of SFD microparticles and the chemical
structure of encapsulated proteins. By experimenting
with a large matrix of conditions, we establish the fol-
lowing conclusions on the relation between formulation
and encapsulation efficiency of SFD:
An increase in total solids weight generates an
increase in the projected equivalent area of micro-
particles and decreases the stability of encapsulated
proteins, regardless of the ratio between polymer
and carrier;
High quantities of amino acid contained in the for-
mulation derive an increase in the projected
equivalent area of microparticles. Weights between
1.5 to 2.5 mg/ml show the lowest damage to the
encapsulated proteins;
Mannitol is more favorable than lactose in preserv-
ing the stability of encapsulated proteins;
The formulation composed of PVP, mannitol, and
leucine at the weights of 6, 9, and 2 mg/ml,
respectively, show the possibility to encapsulate
stable proteins of several types;
The polymer PVP would need to have a molecular
weight of 1300 kDa for ensuring its distribution on
the surface of microparticles;
While the morphology remains intact when chang-
ing the kind of amino acid, the stability can be
reduced in glutamine as an amino acid.
We believe that the above-listed conclusions could
establish a preliminary guideline for researchers plan-
ning to use SFD as a technique to encapsulate proteins
for drug delivery. However, this study involves some
limitations, such as the lack of measuring moisture con-
tent of SFD microparticles and the time to shell
12 A. BALDELLI ET AL.
formation. Moreover, the sugars employed in this pro-
ject are mannitol and lactose; future investigations could
use other sugars, such as trehalose or sucrose.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This research study has been funded by the Natural
Sciences and Engineering Research Council (NSERC)
CoVID-19 Alliance Grant (Grant number ALLRP
554607–20) to Anubhav Pratap-Singh. Equipment used in
the study were also purchased using Canadian Foundation
for Innovation John E. Evans Leaders Fund (CFI-JELF)
award (Award number 37498) to Anubhav Pratap-Singh.
ORCID
Alberto Baldelli http://orcid.org/0000-0002-6296-5460
Hui Xin Ong http://orcid.org/0000-0002-2882-1551
Daniela Traini http://orcid.org/0000-0002-7173-017X
Anubhav Pratap-Singh http://orcid.org/0000-0003-
1752-0101
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14 A. BALDELLI ET AL.
