Note: Descriptions are shown in the official language in which they were submitted.
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"Methods of Removing Residual Solvent from Nasal Drug Delivery Compositions"
s BACKGROUND
Field of Invention
The present invention relates to compositions for delivery of drugs intended
to
reside in the nose. The invention also pertains to:methods of making such
nasal
io drug delivery compositions and to improved methods of removing solvents
from
pharmaceutical preparations.
Back round
Is A number of intranasal preparations are known which are specifically
designed to deliver drugs across the nasal mucosal membranes to effect
systemic
drug administration. However, there remains a need for intranasal preparations
specifically designed to retain the drugs for extended periods of time in the
nose
without crossing the mucosal membranes.
Cortesi, R., Esposito, E., Menegatti, E., Gambri, R., and Nastruzzi, C.,
"Gelatin Microspheres as a New Approach for the Controlled Delivery of
Synthetic
Nucleotides and PCR-Generated DNA Fragments", Int. J. Pharm. 105:181-6 (1994),
disclose a gelatin solution emulsified in isopropyl palmitate as the oil phase
(without
2s surfactant). Gel beads are formed by cooling the emulsion, and these are
then
washed with acetone and collected on a sintered glass disk. The mean volume
particle diameter is 22 pm, with a range of 5-42 ~,m. As particles below 10
~.m tend
to deposit in the lungs, this range of particle size is not suitable for
administration of
pharmaceuticals intended for retention in the nose.
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Illum et al. , PCT/GB95/01735 describe a drug delivery composition for nasal
administration comprising ICAM-1 and bioadhesive compositions comprising
chitosan, a liquid polymeric material, or a variety of water-swellable
microspheres,
including gelatin. Illum discloses the concept of emulsifying a warm aqueous
s solution of gelatin and ICAM-1 in a vegetable oil containing a surfactant,
followed by
thermal gelation, hardening with acetone, harvesting the ICAM/gelatin
microspheres,
and drying.
However, the processes taught by Illum for the production of gelatin
to microspheres and the resulting product are not desirable for commercial
scale
pharmaceutical use. The process requires large amounts of oil and acetone. The
resulting product has been found to contain mainly aggregates of small primary
particles. These aggregates tend to deaggregate during handling, resulting in
mixtures of fragments and non-aggregated particles with about 3.5% by volume
of
~s particles with volume diameters below 10 p,m. Particles below 10 ~m volume
diameter are not suitable for administration of drugs intended for nasal
retention, as
they tend to enter the lower airway and lungs, which is not desirable from
either a
safety or commercial standpoint.
2o Accordingly, it is desirable to provide a pharmaceutical preparation for
delivery of drugs to the nose that maintains biological activity and physical
integrity,
exhibits extended intranasal residence time, and allows sustained release of
active
ingredient in the nasal cavity. The desired characteristics for an intranasal
formulation include:
2s (1) a pharmaceutically acceptable formulation with respect to process
consistency, scalability, and GRAS {generally regarded as safe)
components;
(2) sustained release of active drug over time;
{3) high quality and activity of released drug;
30 (4) restricted particle size range of volume diameter 10-100 p,m,
preferably 20-
80 pm, to minimize concerns about lung delivery;
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(5) lack of irritation to the mucociliary apparatus;
(6) convenient and reproducible administration into the nasal cavity;
(7) dry powder based;
(8) stability over time without refrigeration-;, and
s (9) residual solvent levels at or below pharmaceutically acceptable levels.
Criteria for a commercially suitable process for producing such a product
include minimal solvent consumption and demonstrated scalability to at least
300 g.
io One problem commonly found in pharmaceutical compositions in which the
production process involves solvents is unacceptably high levels of residua!
solvent
in the final product, so that the product does not meet safety and regulatory
requirements. Traditional methods of removing solvents are frequently
unsatisfactory. For example, evaporation under vacuum at room temperature may
is be inadequate and at elevated temperatures may cause degradation of the
active
ingredient. The alternative technique, extraction by supercritical fluid, is
not always
effective when applied to dry powders. Thus, there is a need for an improved
method of removing residual solvents from pharmaceutical formulations.
2o The present invention provides these and other advantages.
SUMMARY OF INVENTION
The present invention provides an improved process for production of
2s pharmaceutical gelatin-based microsphere compositions comprising one or
more
drugs to be delivered to and retained in the nose, and the drug-containing
microspheres produced by this process. The invention also concerns improved
methods of removing solvents from dry powder pharmaceutical compositions.
3o In general, the process for making the microsphere/drug composition
comprises the following steps:
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a) preparing a low-salt or salt-free aqueous solution of gelatin and the drug
to be
delivered at a temperature above the gelation temperature of the gelatin;
b) preparing a solution of a suitable oil and a suitable surfactant;
c) emulsifying a mixture of the solution of step (a} and the solution of step
(b) to
generate a water-in-oii emulsion wherein the volume ratio of (a) : (b) is
between 1:2
and 1:10;
to d) continuing emulsification until the target droplet size is achieved;
e) thereafter reducing the temperature of said emulsion to below the gelation
temperature of the gelatin at a controlled rapid rate to achieve gelation
before
droplet coalescence, thus allowing the formation of gelatin microspheres
associated
is with drug at the desired particle size;
the emulsion in steps (c, d) and (e) above being mixed at the maximum rate
consistent with a low-vortex or vortex-free circulation pattern;
2o f) separating the drug-containing microspheres from the oillsurfactant
phase; and
g) dehydrating the drug-containing microspheres to produce a dry powder.
The invention also comprises an improved method for removing residual
2s solvent from pharmaceutical matrices such as dry powders by contacting said
matrices with a humidified stream of a suitable gas under conditions which
permit
residual solvent to be entrained in the gas, and then drawing off the gas.
This can
be done in discreet batches or as part of a continuous flow process.
3o The resulting product is a free-flowing powder comprising gelatin
microspheres associated with drug, with a volume particle diameter of between
40 -
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60 p,m and wherein a preponderance of the microspheres exist as unaggregated
individual particles, rather than as aggregates of smaller particles.
The invention further comprises an intranasal drug delivery system
s comprising the above gelatin microspheres in association with a drug to be
delivered
to and retained in the nose.
DRAWINGS
~o Fig. 1 shows impellers suitable for use in the present invention: (A) the
A310
is a high-efficiency axial flow impeller; (B) the A200 is a four-blade axial
flow
impeller; and (C) the 8100 is a radial-flow impeller. All are manufactured by
Lightnin
Equipment.
is Fig. 2 shows results of the gelatin:oil ratio studies of Example 2.
Fig 3 shows the preferred position of the impellers in the emulsification step
in
(A) for volumes below 3 L and (B) for volumes of 3 L and above. (1) 3" radial
impeller (top left quadrant, 12 deg to vertical); {2) 4" radial impeller; (3)
baffles.
Fig. 4 shows a schematic representation of a batch emulsification process
suitable for preparation of microspheres. (1) gelatin solution {50 °C);
(2) oiI/Span
mixture (50 °C); (3) water in oil emulsion; (4) acetone; (5) solid
microspheres
suspended in oil; (6) acetone washings; (7) oil/acetone; (8) acetone; (9)
2s microspheres; (10) vacuum; (11 ) 75 pm mesh sieve; (12) dried microspheres.
Fig. 5 shows scanning electron micrographs showing structure of
tICAM(453)/gelatin microspheres made by the process of Example 8_(panels A and
B) and by the method of Illum et al., supra (panels C and D). The mean volume
so particle diameter of the preparation in panels A and B is 46.4 p,m, with
0.5% of
particles having a volume particle diameter >100~m and undetectable levels of
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WO 99/03452 PCT/US98/t4790
particles having a volume particle diameter <10pm. In contrast, the mean
volume
particle diameter for panels C and D is 123 pm, with 39% >100 p.m and 33% <10
p,m.
s Fig. 6 shows appearance of the emulsion droplets and microspheres at
various stages during the batch emulsification process of Example 15 using
baffles,
gradual cooling, and high mixing speed: (A) emulsion droplets at 50 °C;
(B)
emulsion droplets at 40 °C; (C} emulsion droplets at 35 °C; and
(D) emulsion
droplets at 30 °C.
~o
Fig. 7 shows appearance of the emulsion droplets and microspheres at
various stages during the batch emulsification process of Example 15 using
baffles,
moderate cooling, and high mixing speed: (A) emulsion droplets at 625 rpm (1
min);
(B) emulsion droplets at 625 rpm (20 min}; (C) cooled emulsion (< 15
°C); and (D)
is microspheres (washed and dried).
Fig. 8 shows appearance of the emulsion droplets and microspheres at
various stages during the batch emulsification process of Example 15 using
baffles,
rapid cooling, and a high mixing speed: (A) emulsion droplets immediately
following
2o gelatin addition at 375 rpm; (B) emulsion droplets following mixing at 625
rpm for 20
min; (C) gelatin microspheres suspended in oil following mixing for one hour
below
15 °C at 625 rpm; and (D) gelatin microspheres following washing,
filtration and
vacuum drying.
2s Fig. 9 shows appearance of the emulsion droplets and microspheres at
various stages during the low mixing speed process of Example 15 using baffles
and
rapid cooling: (A) emulsion at 425 rpm (30 min); (B) cooled emulsion at 15
°C; and
(C) microspheres (washed arid dried).
s
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WO 99/03452 PCTNS98/14790
Fig. 10 shows typical particle size distribution obtained by laser particle
sizing
of microspheres generated by the rapid cooling method of Example 15, using
baffles, higher impeller height, and lower mixing speed.
s DETAILED DESCRIPTION OF INVENTION
1. Method of making_oelatin microspheres (see fig. 4):
Preparation of aclueous solution of gelatin and drug:
io
An aqueous solution of gelatin and drug is prepared. Neither gelatin grade
nor gelatin concentration are critical to the size of the final microspheres.
Gelatin
should be food-grade, preferably at least NF grade. The gelatin should have a
bloom
strength of at least 80, and preferably of >_ 150. A bloom strength of 250 is
most
~ s preferred because its properties are fairly consistent from batch to batch
and it is
available year round. Its color is lighter and has a fainter odor than lower-
quality
gelatin grades. Higher bloom strengths provide superior color, odor, and nasal
residence time in the final product. Suitable gelatin is, e.g. P-8 grade, 250
bloom,
obtained from Hormel Foods (Austin, MN). The concentration of gelatin may
2o conveniently be in a range of from about 1 % to about 30% (w/w). Within
this range,
higher gelatin concentrations lead to reduced solvent and oil consumption, but
the
total solids content in the aqueous solution (including gelatin and drug to be
delivered) should not exceed 30% (w/w). Because gelatin concentration did not
appear to affect the ultimate volume particle diameter, 20%, which is the
highest
2s easily-handled concentration based on viscosity, is preferred.
The drug to be delivered may be any suitable chemical or biological
pharmaceutical agent that is capable of being dissolved or suspended in a
gelatin
solution and that is insoluble in the oil and insoluble in the dehydrating
solvent used
so in the process. Examples of suitable drugs include but are not limited to,
e.g., small
chemical entities; proteins, peptides, and polypeptides (e.g. ICAM-1 and other
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WO 99/03452 PCT/US98/14790
antiviral agents; vaccines, antigens, antibodies; lymphokines, and fragments
of any
of the foregoing); nucleotides (e.g. genes, DNA, RNA, and fragments of any of
the
foregoing) carbohydrates, etc. The concentration of the drug to be delivered
is
similarly determined by the total allowable concentration of the loaded
solids.
s
The aqueous solution of gelatin and drug may be prepared by any convenient
means. Solutions of gelatin and drug may be prepared individually and then
mixed,
or either the gelatin or the drug may be added to a solution of the other.
Persons
skilled in the art will understand that buffering or adjustment of the pH may
be
~o needed to protect the drug during processing. Also, for protein drugs it is
preferable
that the aqueous solution be low-salt or salt-free to avoid precipitating the
protein.
By "low salt" is meant < 200 mOs/kg.
When the drug is ICAM-1, a solution of ICAM-1 in histidine buffer, pH 7, is
is conveniently prepared and added to an aqueous solution of gelatin. L-
histidine is
obtained from, e.g., Calbiochem Corp. The term "ICAM" as used herein is
intended
to refer to ICAM-1 and any fragments, analog or derivative of ICAM-1 which
retains
the ability to bind to human rhinovirus of the major receptor group and
inhibit
infectivity. The ICAM may be prepared as set forth in Example 1 below.
zo
Gelatin-containing solutions should be held at a temperature above the
gelation temperature of the gelatin. For a gelatin with a bloom strength of
250, this
means at 40 °C or higher.
2s Preaaration of solution of oil and surfactant-
The oil is preferably a refined oil, e.g. mineral or vegetable oil, preferably
food-grade or NF grade vegetable oil. Examples of suitable vegetable oils are
corn,
soybean, and safflower. Hyperrefined vegetable oils from which color bodies,
3o hydrophilic substances, and natural surfactants have been removed do not
work as
well as less refined oils in this procedure. Corn oil and soybean oil are
preferred;
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WO 99/03452 PCT/US98/14790
corn oil is particularly preferred because it is more economical. Suitable
corn oil is
e.g. NF grade as obtained from, e.g., Ruger Chemical Co. (Welch, Holme and
Clark).
s Examples of suitable surfactants are Span 80 (sorbitan monooleate, obtained
from Ruger Chemical Co., ICI Americas Inc.), lecithin, and pluronic L 1011
(BASF).
Span 80 is preferred. In general, a hydrophilic-lipophiiic balance (HLB) of 4-
6 is
desired. Surfactants with HLB values of 3 or below (e.g. glyceryl monooleate,
sorbitan trioleate) are less effective. A surfactant concentration of 0.1 % is
too low,
l0 1 % is functional, and higher concentrations provide no further improvement
in the
result.
4) Emulsification:
Is The aqueous solution of gelatin and drug is emulsified with the solution of
oil
and surfactant is emulsified to generate a water-in-oil emulsion.
Methods of emulsification are well-known in the art. Generally, the aqueous
gelatin/drug solution is gradually added to the oil/surfactant solution with
stirring or
2o mixing. Proper agitation during emulsification is required to achieve the
desired
mean volume particle diameter (40-60 pm). It is possible to use a variety of
stirrers
or impellers, but it is preferred to minimize the formation of a vortex to
avoid air
entrainment. This can be achieved through the use of baffles or correct
positioning
of the impeller. initial specific power consumption is preferably at feast 1.4
watts/L.
2s High-shear mixing should be avoided. Mixing conditions affect the final
product and
so care must be taken to reproduce mixing conditions to insure reproducibility
of the
final product.
The volume ratio of gelatin/drug solution to oil/surfactant solution affects
the
3o quality of the resulting product. Preferably the volume ratio of
gelatinldrug solution
to oil/surfactant solution is between 1:2 and 1:10. Larger ratios are
desirable
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WO 99/03452 PCTNS98/14790
because the emulsion volume, and the oil and solvent requirements, are
reduced.
However, the mean volume particle diameter rises gradually with volume ratios
up to
1:2. At higher ratios, the mean volume particle diameter rises dramatically,
and at
even higher ratios, emulsion inversion takes place, making particle formation
s impossible. Volume ratios of 1:2 to 1:5 are preferred; 1:3 to 1:4 are
particularly
preferred.
Emulsification is continued until the target droplet size is obtained. Droplet
size is monitored by methods known to those in the art, e.g. by optical
microscopy.
io Droplets of 20-80 pm are preferred; 40-60 ~cm are particularly preferred.
For a
volume of 8 L, a mean volume particle size of approximately 50 ~m can be
achieved
in approximately 30 min (~ 10 min).
5) Cooling:
i5
The temperature of the emulsion is reduced to below the gelation temperature
of the gelatin at a controlled rapid rate to achieve gelation before droplet
coalescence, thus allowing the formation of gelatin microspheres with
associated
drug at the desired particle size. For example, for gelatin of bloom strength
of 250,
2o the emulsion is cooled to a temperature of 23 °C or lower.
Preferably the cooling rate is between 1-4 °C/min. A cooling rate of
between
2.0-2.5 °C/min is particularly preferred.
2s Seaaratingi the druglmicrospheres from the oil/surfactant phase'
In general, separation is most efficient if the microspheres are separated
from
as much of the oil as possible at the outset. This can be done by physical
means,
such as allowing the microspheres to settle under gravity or by centrifugation
3o followed by decantation. Separation can also be accomplished by washing
with
suitable solvents. It is important that the emulsification be agitated gently
during the
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washing process. If washing is used, there are several possible alternatives
to
remove the oil:
a) In the first method, the oil is removed by washing first with 'h volume
s hydrocarbon solvent such as heptane, then by washing with a water-miscible
solvent such as acetone.
The hydrocarbon solvent is chosen as follows:
i) it should be miscible with both the oil and the water-miscible wash chosen
i o below;
ii) it should not dissolve appreciable water; and
iii) its density should be less than mixtures of water and the water-miscible
solvent containing up to 20% water.
~s The hydrocarbon wash reduces the oil phase viscosity and density, allowing
the microspheres to settle easily with gravity. This permits a large portion
of the
hydrocarbon phase to be decanted immediately. A single wash with 0.5 emulsion
volumes is sufficient. The wash is carried out by brief stirring (5 min) at
room
temperature. Two such washes are sufficient to remove a majority of the oil.
The remaining washes are with a water-miscible solvent such as acetone
(HPLC grade, J.T. Baker) or low molecular weight alcohols. If the hydrocarbon
is
chosen according to the criteria set forth above, the initial water-miscible
wash will
lead to three phases (1) the gelatin microspheres, (2) a liquid phase (mostly
water-
2s miscible wash), and (3) another liquid phase floating on the water-miscible
wash and
containing mostly the hydrocarbon and the remaining oil. This splitting of the
two
liquid phases is a consequence of extracting the water from the gelatin
microspheres. The two organic phases separate easily from each other and from
the solids. An advantage of this approach is that the microspheres are
transferred
3o into the water-miscible solvent-rich phase, where they will remain
throughout the rest
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of the washes. Typically the hydrocarbon-rich phase is eliminated after the
initial
water-miscible solvent wash.
(b) The second method is to wash only with water-miscible solvent. This
process is
s carried out using sequential stirring, settling, and decantation steps as
above. This
has the virtue of requiring only one solvent, but is somewhat more complex
because
the initial water-miscible wash again results in a splitting of the liquid
into two
phases. In this case, however, the oil-rich phase is the lower one, and the
beads
are not separated immediately from the oil and surfactant. The acetone,
because it
~o also acts as a solvent, for water, does not appreciably reduce the oil-
phase viscosity,
and ordinarily at least two water-miscible washes of 1 emulsion volume each
are
necessary to remove the oil phase. Phase separation is slower than when
hydrocarbon is used as in (a) above.
is Dehvdratingi the drug/microspheres to produce a dry aowdev
The now-dehydrated gelatin/drug microspheres, suspended in water-miscible
solvent, are collected. Methods of separating the microspheres from the water-
miscible solvent are well-known to those in the art. Examples of suitable
means are
2o filtration using e.g. a Buechner funnel or centrifugation using ordinary or
basket
centrifuges.
Key factors in the above process are choice of oil, surfactant type,
surfactant
concentration, gelatin/oil ratio, the choice of mixing conditions, the thermal
history of
2s the emulsion and the cooling rate (temperature profile), and the washing
method
(including solvent choices).
The process of the present invention is suitable for scale-up more than 100 x
over that described by Illum et al.
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An advantage of the present process is that the microsphere product has no
particles having volume particle diameters less than 10 ~.m as measured by
laser
light scattering analysis. In the laser light scattering technique, the
angular variation
in intensity of light scattered from a plume of the particles in air is
measured, using
s as a light source a laser of defined wavelength. The scattering data are
deconvoluted to provide a volumetric particle size distribution. Instruments
suitable
for making these measurements are known to those skilled in the art and are
available from e.g. Malvern Instruments, Southboro, MA. The desired particle
size
range is achieved by use of high-bloom strength gelatin (preferably at least
150,
~o most preferably 250 or greater), use of a low-salt or salt-free buffer for
the aqueous
gelatin/drug solution, consistent low-vortex or vortex-free stirring during
emulsification to achieve consistent droplets of the target size, and rapid
cooling
upon attainment of desired droplet size to prevent aggregation. The improved
process eliminates the need for a centrifuge and facilitates large batch
processing.
is It also uses less solvent, is more efficient, costs less, and results in
better product
than the prior art processes.
The foregoing processes can be conducted either as batch processes or as
continuous or in-line processes according to techniques and using equipment
known
2o to those skilled in the art, and person skilled in the art will be able to
make
appropriate adjustments and modifications to implement each of these
techniques.
II. Reducing the residual solvent to~~harmaceutically acceptable level
2s The invention further comprises a novel method of removing volatile solvent
from a pharmaceutical matrix. The process involves contacting said
pharmaceutical
matrix with a suitable humidified gas under conditions which permit residual
solvent
to be entrained in the gas, and removing the solvent/gas mixture. Preferably,
the
humidified gas is passed through a fluidized bed of the pharmaceutical matrix.
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The pharmaceutical matrix may be any pharmaceutical formulation, including
but not limited to, e.g., beads, polymer or nonpolymer processed materials,
drug-
related raw materials, excipients and final products including
biopharmaceuticals,
including dry powder microspheres such as gelatin microspheres prepared
s according to the above process, and oil-based microspheres such as liposomes
known in the art. The invention is particularly suitable for use with matrices
which
tend to physically trap entrained solvent within the matrix, so that
procedures such
as heat or vacuum drying do not effectively remove the solvent.
Io The solvent may be any residual volatile solvent remaining after the basic
preparation process. Examples of solvents commonly used in the preparation of
pharmaceutical formulations include water, acetone, and aliphatic alcohols
(e.g.
methanol, ethanol, isopropyl alcohol), DMSO, chloroform, methylene chloride
etc.
is The gas may be any suitable gas which is nonreactive with the
pharmaceutical formulation and which is capable of removing and carrying the
solvent. Examples of suitable gases are air, nitrogen, argon, and carbon
dioxide.
The relative humidity in the gas should be from about 85 to about 96 %. The
2o purpose of the humidity is to retain sufficient moisture within the
pharmaceutical
matrix. For matrices which tend to trap solvent, low moisture content inhibits
escape
of the solvent. When the solvent is water, lower relative humidities should be
used.
Humidities of 0-50% are preferred, depending on the water sorption and
desorption
properties of the matrix.
2s
To increase the moisture content of the pharmaceutical matrix, the matrix
could simply be placed in a gas-tight chamber with an appropriate salt
solution to
obtain a specifrc high humidity. However, the evaporated solvent would not be
able
to leave the closed chamber and therefore, would equilibrate with the solvent
3o adsorbed in the matrix. In addition, stagnant dry matrix easily aggregates
under
high relative humidities. To continuously remove the evaporated solvent,
humidified
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WO 99/03452 PCT/US98/14790
gas could be passed over the matrix but three problems would still exist: (1)
a matrix
such as a powder would be difficult to contain; (2) dry matrix could be
stagnant and
could form aggregates; and (3) the evaporated solvent could be hazardous and
difficult to control.
To overcome these problems, a fluidized bed is prepared according to
methods known to those skilled in the art, see, e.g., Porter, H. F.,
McCormick, P. Y.,
Lucas, R. L., and Wells, D. F., "Gas-Solid Systems," in Chemical Engineers'
Handbook, 5'" ed., R. H. Perry and C. H. Chilton, eds. (McGraw-Hill, New
to York,1973). Conveniently, a column such as a chromatography column may be
used. The matrix containing the residual solvent is loaded into the column and
humidified gas is passed into the bottom of the column and through the
pharmaceutical matrix in order to fluidize the matrix bed. Advantages to using
such
a column include: (1) the humidified gas can be evenly distributed through the
is whole area of the bottom filter support, exposing the pharmaceutical matrix
homogeneously to the gas stream, and (2) the column can be easily dismantled
for
sample loading, unloading, and cleaning. Because of the gas flow, the
pharmaceutical matrix is completely fluidized inside the column. The
continuous
fluidization reduces aggregation of dry matrix.
Residual water is then removed by passing dry gas through the matrix and
removing the humidified gas. For this purpose "dry" means having less than 50%
relative humidity.
2s Apparatuses suitable for use in the present invention are available
commercially from, e.g., Fluid Air Inc., Aurora, IL; Niro inc., Columbia, MD.
Other
variations will be apparent to those skilled in the art, for example methods
of
modifying the apparatus and procedure as necessary for various matrices and
batch
sizes.
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When used in combination with the above method for making drug/gelatin
microspheres using acetone as a solvent, a humidity of 92% at room temperature
is
capable of reducing residual acetone level to 200 ppm or below within 8 hours.
It is
believed that acetone is adsorbed and bound strongly in the gelatin matrix by
s hydrogen bonding, which prevents its removal from the polymer under
relatively high
vacuum and/or even at temperatures higher than its boiling point. By
increasing the
moisture content of the gelatin, the hydrogen-bound acetone molecules are
replaced
quickly by water molecules. The decreased glass transition temperature of
gelatin at
higher moisture contents also increases the rate of acetone diffusion out of
the
io gelatin matrix.
The result is a dry powder having a pharmaceutically acceptable level of
residual solvent.
is III. Product
The resulting product is a free-flowing dry powder comprising gelatin
microspheres containg the desired drug. Specifically, the following parameters
are
preferred:
Mean volume particle diameter 20-80 ~cm
Span* < 1.0
of particles with >100 Nm volume <10%
particle diameter
of particles with <10 Nm volume <1 % (by laser light
particle diameter scattering)
Potency of released drug active
Process scale (dry-weight basis) >100 g
When the solvent is acetone,
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WO 99/03452 PCTNS98/14790
preferred residual level < 250 ppm
[* Span is a measure of the particle size distribution calculated from
percentiles.
s Span = w, 0.9) - D (v. 0.1 ) (1 )
D (v, 0.5)
where, D(v,0.9), D{v,0.1 ) and D(v,0.5) are the 90th, 10th and 50th percentile
volume
particle diameter of the microspheres, respectively.]
io
The drug microspheres of the present invention may be administered as
prepared above or may be compounded in a pharmaceutical preparation in which
such drug microspheres comprise the active ingredient or one of a plurality of
active
ingredients, or may be mixed with microspheres containing other drugs. Drug-
ts containing gelatin microspheres may be mixed with placebo gelatin
microspheres
containing no drug to achieve blends with lower concentration of drug per unit
volume or weight.
Suitable pharmaceutical preparations may, for example, take the form of
20 ointments, gels, pastes, creams, sprays (including aerosols), lotions,
powders,
suspensions, solutions and emulsions of the active ingredient in suitable
excipients.
Examples of suitable excipients include pharmaceutically acceptable fillers
and
extenders, binding agents, moisturizing agents, agents for retarding
dissolution,
disintegrating agents, resorption accelerators, surface active agents,
adsorptive
2s carriers, and lubricants. It is understood that the excipient(s) must be
chosen to
preserve the integrity and activity of the drug microspheres and to be
compatible
with the selected route of administration.
The pharmaceutical preparations which are powders and sprays can, for
3o example, contain appropriate diluents, e.g. lactose, talc, silicic acid,
aluminum
hydroxide, calcium silicate, and poiyamide powder or mixtures of these
substances.
17
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WO 99/03452 PCTNS98/14790
Aerosol sprays can, for example, contain the usual propellants, e.g.
chlorofluorohydrocarbons.
The pharmaceutical compositions which are ointments, pastes, creams and
s gels can, for example, contain appropriate diluents, e.g. animal and
vegetable fats,
waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene
glycols,
silicones, bentonites, silicic acid, talc and zinc oxide or mixtures thereof.
The pharmaceutical compositions which are solutions and emulsions can, for
io example, contain appropriate diluents and emulsifiers known to those in the
art, e.g.,
ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol,
benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide,
oils (e.g.
ground nut oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols
and fatty
acid esters of sorbital or mixtures thereof.
is
The pharmaceutical preparations which are suspensions can contain
appropriate diluents (e.g. ethyl alcohol, propylene glycol), surface-active
agents (e.g.
ethoxylated isostearyl alcohols, polyoxyethylene sorbite and sorbitane
esters),
microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and
2o tragacanth or mixtures thereof.
The pharmaceutical preparations of the present invention can also contain
coloring agents and preservatives as well as perfumes. The pharmaceutical
preparations of the present invention contain from 0.1 to 99.5% by weight,
preferably
2s from 0.5 to 95% by weight, of the drug-containing microspheres in the total
composition.
The production of the above pharmaceutical compositions is carried out by
suitable methods well-known to those in the art.
18
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WO 99/03452 PCT/US98l14790
There are a number of viruses, e.g. rhinovirus and adenovirus, which infect
warm-blooded animals via the mucosal surfaces. Antiviral agents suitable for
treatment of such infections are known but it is desired to provide
formulations for
enhanced delivery to such mucosal surfaces, particularly to the nasal passages
s which are one of the primary routes of infection for such viruses.
As the major routes of infection by rhinovirus are the mucosal surfaces of the
nose and eyes, it is desired to protect such mucosal surfaces with topical
application
of appropriate antirhinoviral agents.
io
Accordingly, it is desirable to have formulations of human major rhinovirus
receptor (ICAM-1 ) or fragments thereof which are capable of residing in the
nasal
passages for extended periods of time to effect protection against rhinoviral
infection
in a safe and cost-effective manner.
is
The gelatin microspheres of the present invention are particularly suitable
for
intranasal administration. By "intranasally" and "intranasal administration"
is meant
administration to the nasal cavity in a form intended to remain in the nose,
including
dry powders, nasal drops, nasal spray, and creams, gels, or other formulations
2o suitable for topical application to the nasal cavity. However, the
pharmaceutical
gelatinldrug microspheres of the present invention may also be formulated for
topical
administration, e.g. as creams, lotions, salves, ointments, etc., for topical
administration to e.g. to the eye, mouth, ear, skin. Oral formulations may
also
include troches and mucoadhesive buccai tablets, etc.
The invention will be described in further detail in connection with the
following examples, which are directed to administration of antirhinoviral
agents,
specifically ICAM-1 and fragments thereof, but the gelatin/drug microspheres
of the
present invention are also suitable for topical administration of other water-
soluble
3o pharmaceutical chemical and biological agents, e.g. small chemicals such as
antibiotics; proteins, peptides, and polypeptides (e.g., vaccines, antigens,
19
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WO 99/03452 PCTlUS98l14790
antibodies, lymphokines, and fragments of any of the foregoing); nucleotides
(e.g.
genes, DNA, RNA, and fragments of any of the foregoing); carbohydrates; and
antiviral agents such as Enviroxine, Pirodavir, interferon-alpha, sialidase
inhibitors,
acyclovir, adenosine, arabinoside, interferon and interferon -inducing agents.
The invention can be further understood upon consideration of the following
examples. As used hereinabove and below unless expressly stated to the
contrary,
all temperatures and temperature ranges refer to the centigrade system; the
term
percent or (%) refers to weight percent; and the term mole and moles refer to
gram
io moles.
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EXAMPLES
Example 1
Production of tICAM(453)
s
Rhinoviruses are members of the picornavirus family and are responsible for
50% of colds in humans over the course of one year. During peak season (mid-
September to mid-October) however, rhinoviruses account for up to 75% of all
colds.
Approximately 90% of all rhinoviruses bind to a major human rhinovirus
receptor
~o (HRR). An antiviral agent comprising the human major rhinovirus receptor or
fragments thereof is an effective inhibitor of rhinoviraf infection of
susceptible cells.
The human major rhinovirus receptor is the same as the protein known as
intercellular adhesion molecule-1 (ICAM-1 ), with the exception of a G>A at
nucleotide 1462, resulting in the amino acrd substitution Glu>Lys at amino
acid
is position 442. ICAM-1 is a glycoprotein with a molecular weight of 45-50 kD
(excluding carbohydrate) and 8 potential sites for N-linked carbohydrate
attachment;
the glycosylated protein has a molecular weight of 82-88 kD. ICAM-1 has 507
amino acids and consists of a cytoplasmic domain, a transmembrane domain, and
five extracellular immunoglobulin-like domains (amino acids 1-88, 89-185, 186-
284,
20 285-385, and 386-453). The presently preferred embodiment for antivirai
purposes is
a fragment consisting of the first 453 amino acids of the full HRR sequence,
which
retains rhinovirus binding activity.
Recombinant tICAM{453) was purified from fermentation fluid of continuous
2s cell culture of Chinese hamster ovary (CHO) cells containing cDNA coding
for
tICAM(453) maintained by continuous perfusion in a customized medium free of
all
plasma-derived components. By "tICAM(453)" is meant the first 453 amino acids
(domains 1-5) of ICAM-1. The sequence for ICAM-1 is known to those skilled in
the
art, as are methods of preparing the cDNA coding for tICAM(453). See, e.g"
3o Staunton, D.E., S.D. Marlin, C. Stratowa, M.L. Dustin, and T.A. Springer,
"Primary
Structure of ICAM-1 Demonstrates Interaction Between Members of the
21
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WO 99/03452 PCTNS98/14790
Immunoglobulin and Integrin Supergene Families", Cell 52:925-933 (1988);
European Patent Application Pulbication No. 362,531.tICAM(453) obtained from
clarified and concentrated fermentation fluid was purified by hydrophobic
chromatography, metal ion chromatography, precipitation at low pH, filtration,
anion
s exchange chromatography, hydroxyapatite adsorption, size exclusion
chromatography, and a second HAP adsorption step. Viral inactivation was
accomplished via low pH precipitation (demonstrated to reduce MuLV and
Reovirus
titers by 36 and 22 logs, respectively), and by pasteurization {shown to
reduce the
reference virus titers by an additional 5 logs). The tICAM(453)-containing
solution
~o was sterilized by filtration to yield the liquid drug substance, which was
stored at -35°
C until formulated into a gelatin-based microsphere powder. Some lots were
lyophilized in phosphate-buffered saline (PBS, or 0.15 M NaCI, 0.01 M Na2HP04,
adjusted to pH 7.0) and reconstituted before use. tICAM(453) was concentrated
to
>50 g/L and diafiltered into a low-ionic-strength 10 mM histidine buffer, pH
7Ø
is
Example 2
Process for Production of Gelatin Microspheres
A} Materials
tICAM(453): tICAM(453) was made as in Example 1.
Albumin: 25% albumin (human) USP (Bayer Corporation, Elkhart, IN)
2s Grades of Gelatin: Acid-type gelatin, extracted from porcine skin, was
obtained from
Hormel Foods (Austin, MN). Gelatin is normally graded by its Bloom strength, a
measure of gel strength under defined conditions (see, e.g., US Patent No.
1,540,979). These studies were conducted with gelatin with Bloom strengths of
225
(Hormel grade P-7), 250 (grade P-8) and 275 (grade P-9).
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WO 99/03452 PCT/US98/14790
Oils: A variety of oils was used. Food-grade corn oil was obtained from a
grocery
store (e.g. Mazola~, CPC International, Englewood Cliffs, NJ). Pharmaceutical
or
NF grade was supplied by Ruger Chemical (Irvington, NJ). Soybean oil was
either
food-grade or NF grade from Ruger Chemical.
s
Surfactants: Span 80~ (sorbitan monooleate), Span 85~ (trioieate), and Arlacel
186T"" (glyceryl monooleate) were supplied by ICI Americas (Wilmington, DE).
Pluronic L-1011T"" was from BASF (Mt. Olive, NJ). Lecithin was supplied by
Central
Soya (Ft. Wayne, IN).
~o
Solvents: Acetone and n-heptane were reagent grade, purchased from JT Baker
(Phillipsburg, NJ), EMScience (Gibbstown, NJ), and Mallinkrodt (Chesterfield,
MO).
B) Methods:
Is
As the supply of tICAM(453) was limited, this resource was conserved by
carrying out range-finding studies on a small scale first without tICAM(453)
present,
then evaluating the effects of adding tICAM(453) on the process, and finally
scaling
up the resulting process.
An emulsion volume of 200 mL was selected for these range-finding
experiments. This scale allowed the conduct of multiple experiments per day
while
providing enough material from each experiment for characterization. The
outcome
of each experiment was evaluated first by a visual examination of the flow
properties
2s of the resulting powder. Sticky or highly aggregated material was rejected
without
further analysis. Material acceptable by visual inspection was analyzed for
particle
size distribution by laser light scattering.
Once a process at the 200-mL scale (8-10 g dry-weight basis) had been
3o defined, tICAM(453) was included in order to assess its effect on the
process and to
evaluate the effect of the process on molecular integrity and biological
activity.
23
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WO 99/03452 PCT/US98/14790
Finally, the process established in the range-finding experiments was scaled
up to the 150-g level to establish commercial utility.
s C) General Scheme of Process:
Range-finding experiments were conducted by emulsifying a total volume of
200 mL in a 400-mL beaker (7.3 cm diameter). Either a 1.5" radial-flow
impeller
(item 8100, Lightnin Equipment, Milwaukee, WI) or a 2" high-efficiency axial-
flow
~o impeller (item A310) was used. The gelatin solution was added to the oil
phase
while stirring in initial experiments at 700 rpm. After 10 min, the stirring
speed was
then lowered to 400 rpm. The designs of the impellers is shown in Fig. 1. In
other
experiments, the stirring rate was held constant.
is The initial temperature of emulsification was at least 45 °C, and
cooling took
place by natural convection. Washing was carried out using two 100-mL washes
with heptane, followed by two 100-mL washes with acetone. For each wash, the
resulting suspension was either stirred by hand or by using a motor-driven
mixer.
The solids were allowed to settle after each wash by gravity, and the
supernatant
2o removed by decantation. The final powder was collected by vacuum filtration
on a
Buechner funnel, using #3 Whatman (Fairfield, NJ) filter paper. It was dried
overnight under vacuum at 34 - 40 °C.
D) Powder Characterization:
The suitability of the powder was evaluated in stages. Initial evaluation
required that the powder be free-flowing, without stickiness attributable to
residual
oil. Any lumps were removed by passing the powder through a 60- or 70-mesh
sieve. Because the goal was to have a powder falling into the size range 10 -
100
3o Nm, and preferably 20 - 80 Nm, most of the powder should pass easily
through
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WO 99/03452 PCT/US98/14790
either of these sieves, which have cut-off diameters of 250 and 212 pm,
respectively.
E) Scale Down and Scale Ua Studies:
s
The range-finding experiments in section C above served to determine a
suitable composition for the emulsion being prepared, i.e., oil and surfactant
choice,
concentrations, etc. Process reproducibility was examined at the 2.5-g scale
(50 mL
emulsion). This was considered the smallest scale at which the process could
be
io carried out without altering critical parameters.
F) Calculation of tICAM~453~ and Albumin Loadin_a_
Theoretical loading of tICAM(453) and albumin were calculated on the
is following basis:
i) The moisture content of the gelatin raw material and of the final product
were assumed to be similar. Thus, moisture content could be ignored.
ii) Only the polypeptide portion of the tICAM(453) molecule (and not the
carbohydrate portion) was taken into account.
These assumptions, when used consistently in calculating loading, pose no
difficulties. These assumptions do not apply and must be corrected for when
calculating the dry-weight powder yield.
2s The theoretical loading is given by:
Cr'~r
f_
~c% + c~m% + C'cmeP~
so This expression is based on a mass balance for the preparation of a gelatin
solution
containing tICAM(453) (or albumin). In the expression, f; is the mass fraction
(or
loading) of ICAM(453) (or albumin), c; is the concentration, in g/L, of
tICAM(453) (or
CA 02296620 2000-O1-12
WO 99/03452 PCT/US98/14790
albumin) in the original solution, p; is the density of the tICAM(453) (or
albumin}
solution, in g/L, and m; is the mass of the tICAM(453) (or albumin) solution
used.
The buffer salt concentration for the tICAM(453) (or albumin) solution is
given by cb
(in g/L); this can be significant if the tICAM(453) (or albumin) concentration
is low
s and the buffer is phosphate-buffered saline. For a low-salt buffer, the
correction
given by cb is small. The total mass of gelatin used is Ggm9 for the case
where a
gelatin solution is mixed with an tICAM(453) (or albumin) solution. m9
represents the
mass of gelatin solution, and G9 the mass fraction (g/g) of gelatin before
adding the
tICAM(453) (or albumin). The solution densities were measured and found to be
l0 1020 g/L for tICAM(453) at 50g/L and 1070 g/L for albumin at 250 g/L.
G) Results:
Surfactant choice:
is
Five oil-soluble surtactants were tested, having a variety of chemical
compositions. These were Span 80 and 85, Pluronic L-1011, Arlacel 186, and
soybean lecithin. Each of these was tested in corn oil at the 1 % level in the
200-mL
model system under the conditions shown in Table I:
Table I
Effect of Surfactant Choice on Particle Size Distribution
In common: Porcine 250-bloom type A gelatin 20% (w/w) in water, emulsified in
corn
2s OII
1:4 gelatin-oil ratio: A310 impeller, at 700/400 rpm (start/end)
SurtactantResult D[4,3], Nm
Span 80 89% through 60 mesh112
Arlacel coarse powder n.d.
186
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WO 99/03452 PCT/US98/14790
D[4,3] = mean volume particle diameter; the volume particle diameter is the
diameter
of a sphere having the same volume as the given particle
1:3 gelatin-oil ratio, 650 rpm
SurfactantResult D[4,3],
Nm
Span 80 99% through 70-mesh 57
Span 85 0% through 70-mesh n.d.
Lecithin oily powder; difficult n.d.
to wash
s These experiments showed that Span 80 and L-1011 were superior to the other
surfactants (data for L-1011 not shown). Given that L-1011 is not available in
a
compendial (USP or NF) grade, Span 80 was selected for use in further studies.
Span 80 concentration:
to
Surfactant concentration was investigated at 0.1, 1, and 2% (w/v), and at
gelatin:oil ratios of 1:2 to 1:4 (Table II) at 200 mL volume scale:
Table II
Effect of Span 80 Concentration
Conditions: Porcine 250 Bloom type A gelatin at 20% (w/w) in water; emulsified
in
corn oil
volume ratio; 2" A310 er at 0 rpm (startlend)
1:4 impell 700/40
through 60- D[4,3],
Concentrationmesh sieve Nm
0.1 % n.d.* 202
1.0% 89% 112
93% 113
2.0% n.d. 65
93% 116
*n.d. = not determined
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WO 99/03452 PCT/US98/14790
volume ratio (cont'd);1.5"
1:4 8100 impeller
at 700/400
rpm (start/end)
through 60- D[4,3],
Conc. mesh sieve Nm
0.1 % 82% 196
1.0% n.d. 87
90% 131
2.0% n.d. 117
1:3 volume ratio;1.5" 8100 impeller at 650 ram
through 70- D[4,3],
Conc. mesh sieve Nm
0.1% 0% n.d.
1.0% 98% 63
2.0% 92% 103
1:2 volume 00 rpm (startlend)
ratio;1.5"
8100 impeller
at 700/4
through D[4,3],
Conc. sieve Nm
0.1 % sticky n.d.
1.0% 93% (60-mesh)130
n.d. 120
2.0% 94% (70-mesh)133
1:2 volume (cont'd); 0 impellert 700/400 rpm (start/end)
ratio 2" A31 a
through 70- D[4,3],
Conc. mesh sieve Nm
0.1 % sticky
1.0% n.d. 129 Nm
99% 130
93% 180
2.0% 98% 116
The table shows that at the 1:4 ratio, particles decreased in size with
increasing
concentration of Span 80 up to and including 1 %, but that increasing the
surfactant
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WO 99/03452 PCT/US98/14790
concentration beyond 1 % had little effect. When the amount of gelatin was
increased relative to the oil, representing more difficult emulsification
conditions, this
effect was even more marked. Based on these experiments, 1 % Span 80 was used
in all further investigations.
Gelatin-Oil ratio:
This ratio was defined as the ratio, by volume, of a solution of 20% (w/w)
porcine 25 Bloom type A gelatin: corn oil containing 1 % Span 80. Ratios of
1:4 to
l0 1:1 were examined. Higher ratios are preferred because at a given scale,
oil and
solvent consumption and processing volume are reduced. At the same time, a
higher proportion of gelatin solution in the emulsion increases the likelihood
that
droplets or beads collide with each other, resulting in increased incidence of
coalescence or aggregation.
Indeed, multiple attempts to use a 1:1 ratio failed, for reasons including
inversion of the emulsion, difficulty in washing out the oil, and unacceptably
large
(>200 p,m) microspheres.
2o Microspheres of the desired size were obtained at a ratio of 1:3 in
multiple
runs at varying scales. The difference between ratios of 1:3 and 1:2 is
slight: at the
200-mL scale, 5 runs at a ratio of 1:3 led to sizes of 62.8 ~14.6 pm, while a
single
run at 1:2 led to 77.0-pm microspheres (Fig.2). Mixing was with a 1.5" 8100
(radial
flow) impeller, used at 6001700 RPM (start/end).
Oil Tvpe:
Corn and soybean oils were both evaluated over a period of time. The results
of these comparisons under two different conditions are shown in Table III:
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WO 99/03452 PCT/US98/14790
Table III
Soybean vs. Corn Oil in Paired Experiments
2 volume ratio;2" A310 impeller at 700/400 rpm (start/end)
through 70-
oil type mesh sieve D[4,3],
Nm
corn 99 129.9
soybean 96 148.8
1:3 volume ratio;1.5" 8100 impeller, 650-700 rpm
through 70-
oil type mesh sieve D[4,3],
Nm
com 99 53
soybean 100 53
corn 98 74
corn 8g g0
soybean 98 74
soybean 92 n.d.
soybean 94 n.d.
soybean 99 69
soybean g8 6g
soybean 93 81
soybean 93 90
corn gg g2
corn 99 64
corn 97 67
soybean 98 55
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The table suggests that there is no profound difference in the results between
normally-refined corn and soybean oils, as might be expected from their
similar
viscosities.
s Processing Conditions, Stirring Speed. Im~~eller Type, and Washing Method:
Selection of appropriate surfactant, oil type, etc. can be applied to larger
scales. Appropriate mixing conditions during emulsification are more scale-
dependent. Nevertheless, the above experiments illustrate the relative
importance
~o of these variables. As indicated in Tables II and III, the choice of
impeller had little
bearing on the final particle diameter at the 200-mL scale. This may be
because the
two impellers used at this scale were almost as large as the vessel itself:
the ratios
of impeller to vessel diameter were 0.52 and 0.70.
is The mixing and washing conditions at the 200 mL scale had more of a
bearing on the final size. Initially, washing was carried out by occasionally
stirring
the emulsion in solvent with a spatula. This was changed to continuous mixing
using a propeller mixer. At the same time, the procedure for emulsification
was
changed. Instead of stirring for 700 rpm for 10 min, and then at 400 rpm, the
stirring
2o speed was kept constant. In Table IV, the results of these changes are
summarized:
Table IV
The Effect of StirrincLS,peed (Emulsion) and Mixing Method Washing)
2s Conditions: 1:3 volume ratio of 20% (w/w) Porcine 250 Bloom type A gelatin
inwater:1 % Span 80 in corn oil, 200-240 mL scale
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WO 99/03452 PCT/US98/i4790
2" A310 impeller
Speed, rpm stirred wash?D(4,3],
(startlend) Nm
700/400 No 84
No 162
550/550 Yes 55
1.5" 8100 impeller
Speed, rpm stirred wash?D[4,3],
(startlend) Nm
700/400 13 runs No 109 34
2 runs Yes 63 4
all speeds 27 runs Yes 67 10
s The table shows that before implementing these changes, particles were large
and
varied in size from batch to batch. It also shows that mixing conditions
during
emulsion cooling do influence the final diameter.
Two runs were performed using two mixing speeds, but with continuous
io stirring during the washing phase. In these two runs, there was a reduction
in both
mean volume particle diameter and variability as measured by the between-run
standard deviation.
Numerous additional runs were carried out where both changes were
is implemented. In these runs (25) there was also a reduction in both mean
volume
particle diameter and standard deviation relative to the initial preparation
conditions,
using either the A310 or 8100 impellers.
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WO 99/03452 PCT/US98/14790
H) Summary:
The results of the experiments described above allowed definition of a
"standard process" before investigating the effects of incorporating drug. The
choice
s of surfactant was clear - Span 80 - and the concentration was set at 1 %
(w/v)
because higher concentrations offered little additional advantage. Corn and
soybean oils were equivalent in many respects: the particle size distribution
did not
differ significantly, both are available in compendia) grade, and both are
commonly
used as pharmaceutical excipients. Corn oil was chosen because its cost is
lower
i o than soybean oil.
The gelatin solution was set at 20% (wlw) solids. It is difficult to remove
entrained air or pour gelatin solutions at higher concentrations. Lower
concentrations appeared to offer little advantage, and would require extra oil
and
Is solvent per gram of final product.
The volume ratio of gelatin to oil was set to 1:3. It is desirable to use as
little
oil as possible. As described above, reducing the oil to 1:2 made it more
difficult to
obtain the desired particle-size distribution.
Rapid cooling or longer stirring reduced the mean volume particle diameter.
Gelation of gelatin solutions is a kinetic phenomenon, so that gel strength is
a
function of both time and temperature.
2s Example 3
Incorporation of tICAM~453~
tICAM(453) was incorporated into the gelatin microspheres made by the
process of Example 2 to evaluate the effect of the protein on particle size;
determine
3o protein loading; and evaluate the effect of processing conditions on
bioactivity.
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WO 99/03452 PCT/US98/14790
Emulsification conditions were 1:3 volume ratio of 20% porcine 250 Bloom type
A
gelatin in water (w/w):1 % Span 80 in corn oil.
Initial experiments were conducted as shown in Table V:
s
Table V
tICAM(453) Parameters at the 240-mL Scale
D[4,3] 72 Nm
total solids: 20%
~o tICAM(453) bioactivity:starting material: 0.43 Ng/mL
released from formulation: 0.43
(control) 0.29
Theoretical loading: 8.9%
Final loading: 7.8% (87% of theoretical)
is Control lots (2): D(4,3] =53, 60 Nm
In the top part of the table, the results of a run incorporating tICAM(453)
are
presented. In the lower part are the results of two control runs. The
tICAM(453)-
loaded material had a slightly higher mean volume particle diameter than the
2o controls. tICAM(453) released from the formulation had bioactivity
identical to the
starting material. The measured loading was 7.8%; this was 87% of the loading
expected based on the composition of the initial gelatin solution.
Example 4
2s Process Rearoducibility
Process reproducibility was investigated in several runs at the 50-60-mL
scale (2.5-3 g) under the same conditions as in Example 3, as shown in Table
VI:
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Table VI
Process Reproducibility at the 50-60-mL (2.5-3-g) Scale
Run #: 1 2 3 4 5
Scale 2.5 2.5 2.5 3 g 3 g
g g g
Target loading 11.5 10.9 11.7 14.0 13.8
% % % %
Actual loading 10.0 9.9 10.3 12.1 11.7
% % % %
of expected 87% 91 % 88% 86% 85%
140-635 mesh 88% 97% 95%
< 70 mesh 98% 100% 100% 97% 96%
tICAM(453) yield (%) 74% 81 % 83% 86% 78%
Solids yield (%) 97% 92% 94% 99% 96%
Light scattering:
D[4,3], Nm 61 53 49 95 61
of particles with <9.5 0 % 0.4 0 % 0.1 0.2
Nm % %
volume particle diameter
of particles with >101 7 % 2 % 1 % 34 % 7.6
Nm
volume particle diameter
Notes corn soybean
oil oil
Plaque assays were performed according to the method of Greve et al., J.
Virol.
s 65:6015-23 (1991) and results are shown below:
tICAM(453) tICAM(453)/Released
(starting gelatin tICAM(453) control
material) solution
0.26 0.39 0.23 0.30
0.48 0.68 0.34 0.39
0.41 0.42 0.46 0.25
0.32 0.32 0.34 0.34
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WO 99/03452 PCT/US98/14790
Potency results are expressed as a concentration in Ng/mL necessary to inhibit
viral
infection of susceptible cells. Thus, higher values reflect lower potency.
These experiments had several purposes: (1) to show that the formulation
s process, of Example 3, could be carried out reproducibly, (2) to demonstrate
that
tICAM(453) would remain active once released from the formulation, and (3) to
determine an appropriate initial loading required to obtain 10% tICAM{453) in
the
final formulation.
to Initially, two runs were performed to establish what the final loading of
tICAM(453), expressed as a percentage of the initial, theoretical loading.
These are
summarized in Table VI, runs 4 and 5. These two runs, plus the result shown in
Table V together showed that the tICAM(453) loading is between 85 and 87% of
that
expected from the gelatin solution. Therefore for a 10% loading the initial
is tICAM(453) content should be 11.8%.
Runs 1,2, and 3 in Table VI were carried out in order to show consistent
tICAM(453) loading, particle size distribution, and yield. In addition, the
bioactivity of
the tICAM(453) released from the powder was measured and found to be
Zo comparable to the starting material.
Interestingly, the tICAM(453) yield was <100%. The nephelometric assay for
tICAM(453) detects both active and inactive tICAM(453), so the loss of
material is
not assay-related. The most likely point at which tICAM(453) is lost is in the
initial
2s acetone wash. If all of the water present is extracted into the acetone
phase, it will
contain 17% water. This may be enough to dissolve tICAM(453) and perhaps
gelatin as well.
The biological activity of the tICAM(453) is not affected by the process
3o conditions.
36
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Example 5
Process Scale-Up
Parameters for the scale-up of the process were investigated. In initial
larger-
s scale experiments, it was assumed that only the mixing conditions need be
modified,
and that other process parameters could be kept constant.
A scale of 150 g was selected as the target. At this scale (3 L emulsion),
cooling under ambient conditions was too slow, therefore the mixing vessel was
io immersed in a refrigerated water bath at an initial temperature of 50 - 55
°C. The
gelatin solution was added to the warm oil, and the refrigeration unit was
immediately turned on. It took approximately 70 min before the emulsion
reached its
final temperature of 10°C.
~s Factors influencing particle size, where the emulsion composition was held
constant, were expected to be:
i) impeller size and type
ii) impeller position
iii) tank dimensions
2o iv) temperature profile with time
v) presence of tICAM(453) (keeping solids at 20%).
It is important to apply the necessary level of agitation without entrainment
of
air. The stirring speed is limited by the formation of a vortex in the
emulsion.
2s Because vortex formation was undesirable, particles of the desired diameter
could
not be obtained while stirring at constant speed, even when the impeller was
placed
in the optimal position. As the emulsion cooled, the emulsion viscosity
increased,
and it was possible to increase the stirring speed during the cooling process
without
increasing vortexing. When this was done, particles of the desired size were
3o achieved.
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Therefore it is critical that the stirring accomplish two things: first, to
provide
sufficient shear to break up the gelatin solution into sufficiently small
droplets, and
second, to provide sufficient fluid flow so that no stagnant zones form near
the walls.
It appeared that the tatter consideration was more important, given that as
the
s emulsion cooled, it seemed to become shear-thinning, particularly at a
gelatin-oil
ratio of 1:3. Under that circumstance, a small impeller could provide a high
degree of
shear and flow only in a zone surrounding the impeller.
Ordinarily, if there is a problem with vortexing, it can be solved by the
addition
io of baffles. Indeed, baffles did allow an increase in mixing speed over the
unbaffled
configuration. At the same time, the gap between the baffle and the tank wall
was
only 1/8" (3 mm), potentially allowing a build-up of cool, slowly moving
material to
accumulate. If baffling is used care must be taken to avoid this problem.
~s Two separate approaches to scale-up were used, each of which had its
advantages. In the first approach, the mixing shaft was positioned along the
axis of
the beaker. The impeller itself was as large as would feasibly fit into the
vessel, and
was positioned close to the bottom. In this configuration, a large, deep
vortex
formed, exposing the center portion of the impeller so that only the tips of
the blades
2o entered the solution. The emulsion was cooled by packing ice bags around
the
vessel. With this configuration, experiments with and without tICAM{453) were
conducted at the 3-L scale. The results are shown in Table VII:
Table VII
2s Batches at the 150-g Scale: Centered Impeller
1:3 volume ratio of 20% (w/w) porcine 250-Bloom type A gelatin in water :1 %
Span
80 in corn oil
3o emulsion volume 3 L; impeller: 3.5" (8.9-cm) marine propeller (10°
pitch)
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Mixing speedtICAM(453)
(rpm) -loaded? D[4,3],
Nm
760 No 67
850 No 56
850 No 55
850 Yes 49
The table shows that a 3'/2" {8.9 cm) marine propeller with shallow pitch
(~10°)
served, when driven at 850 rpm, to create microspheres with a mean diameter of
50
- 70 Nm. Incorporation of tICAM(453) at 10% did not have a significant effect
on the
s particle size.
This procedure did have some disadvantages. The propeller caused
splashing of the oil phase, particularly during the initial phase of the run,
and air
could potentially be entrained into the emulsion, causing tICAM{453)
denaturation or
~o foaming, which would interfere with the subsequent washing steps. Therefore
an
approach in which vortexing and entrainment of air were minimized was also
pursued.
At the larger scales {120 g and up), the beneficial rate of cooling adds to
the
is complexity of scale-up. Because cooling is carried out from the vessel
walls, there is
a boundary layer of cooler and therefore more viscous emulsion near the wall
of the
tank. This layer is kept thin by mixing action. At higher cooling rates, the
thermal
(and thus hydrodynamic) boundary layer becomes thicker, and more intensive
mixing is necessary to offset this effect.
With the lower cooling rate at larger scale, it was unclear whether any extra
stirring time to allow hardening is necessary. To avoid the possibility of
problems a
1-hr hold step was included in the process.
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Studies were carried out in order to determine suitable impellers for
evaluation. A 3-L emulsion containing 750 mL gelatin solution was held at 50
°C in
a beaker with a diameter of 20.3cm. The impellers listed below were tested in
as
many as three different positions: vertically mounted and centered, vertically-
s mounted and off center, and mounted at an angle of 10-15° from
vertical in the off
center position (see Fig. 3).
The maximum speed that each impeller could be turned was checked, and
the mixing behavior examined qualitatively at that speed. The results are
shown in
io Table VIII:
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Table VIII
Mixing Studies at the 3-L Scale
Max Tip
Size Speed Speed
ImpellerType (in) Mounting (rpm) {in/min)Notes
8100 radial 2 vertical, 900 5700
flow off center
angled, 940 5900 improved
off center pumping
vertical, 770 4800 poor flow
centered at walls
A200 axial 2 vertical, 1000 6300 eddies,
flow
(4 off-center one deep
blades, vortex
45 pitch)
angled, 1000 6300 same
off-center
A310 high 2.5 angled, 1160 9100 narrow
efficiency off center vortex,
axial leading
flow to
air
entrainme
nt and
loss of
mixing.
Dead
spots.
Marine 45 pitch 3 angled, 490 4600
off center
Marine 10 pitch 3.5 vertical, 630 6900 poor
off center vertical
mixing,
stagnation
at walls
angled, 660 7300 same
off center
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Direct comparisons of the different impeller designs was difficult because the
diameters of the impellers available were not all the same. Nevertheless. it
appeared that the most attractive design was the radial-flow impeller, and
that the
impeller should be positioned off-center.
It was decided to position the impeller at an angle in the "upper-left"
quadrant
of the mixing vessel, as shown in Fig. 3. This position minimizes the
formation of a
vortex. A number of experiments are summarized in Table IX, arranged in order
of
increasing scale, but incorporating a variety of impellers, vessel diameters,
and
io mixing speeds:
Table IX
Scale-Up Studies
Scale Volume Vessel mixing D[4,3),
(dry (L) dia. impellerspeed, drying Nm notes
basis), (cm) rpm method
9
150 3.0 20.3 8100 1000 100* impeller at
(2") constant speed
150 3.0 20.3 8100 126* gradually
(2") increased
speed to 1600
rpm
150 3.0 17.1 8100 400- 50* final emulsion
(3") 640 kept cool
180 3.6 20.5 A310 440 o/n n.d. 52% <140
(3.8") vacuum mesh when
sieved.
Propeller
incorrectly
positioned
300 6.0 20.3 A310 500- ambien 91 poor washing
(3.8") 550 t
300 6.0 20.3 A310 520 fluidize74 stirrer speed
(3.8") d bed increased
to
640 rpm when
T=42C. Result
close to target.
n1n arninh+I -_ e+~,..".,;....,.~.*t~,..r.._.._.:__
= nv n r .,.,+..I ~
__J___, ...~. ..~~ ~wvwynvr, vVIVl1"r Vlr,~'
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Table IX shows that the particles obtained at the 300-g scale were
unacceptably
large. Exhaustive experimentation was not performed because one specific
configuration was successful at the 150-g scale: a 3" (7.6 cm) radial-flow
(R100)
s impeller mounted in a 4.5-L (17.1 cm diameter) beaker.
Minor adjustment of conditions may be needed to achieve the desired particle
size distribution; these are within the purview of those skilled in the art.
For
example, as increasingly larger vessels of 7.3 cm to 20 cm diameter were used,
the
impeller-to-tank diameter ratio actually increased. This may be related to
thixotropy
io of the cooled emulsion, which tends to limit the zone of active fluid
motion to a
region around the impeller. At substantially higher scales (e.g., >1000 g,
where the
emulsion volume will be >25 L), the above approach may become impractical and
other methods must be substituted to intensify the level of mixing. One
alternative is
a semi-batch form of operation, where mixing is done in-fine. Modest changes
in
is scale would be accomplished via changes in the total processing time.
Example 6
Adjustments to Avoid Precipitation of Druq
2o If the drug to be delivered is a protein having an isoelectric point such
that it
tends to precipitate during the process of Examples 3 and 5, adjustments can
readily
be made to avoid such precipitation. For example, albumin precipitates when
added
to warm gelatin solution, because gelatin solutions at 20 wt% have pH values
of
approximately 5, close to the isoelectric point of albumin. Precipitation was
avoided
2s by adjusting the pH of the gelatin solution to 6.0-6.1 or by dissolving the
gelatin
initially in 0.019 M NaOH.
Similar adjustments to avoid such problems with other drugs are expected to
occur to those skilled in the art.
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Example 7
General Process for Preparation of Pharmaceutical Grade tICAM(453n/gelatin
microspheres
s The gelatin/tICAM(453) microspheres are prepared by a process that begins
by mixing a solution of tICAM(453) and gelatin in corn oil to form a water-in-
oil
emulsion. This emulsification process uses corn oil, gelatin, Span 80, water
for
injection, and tICAM(453) in L-histidine buffer as the raw materials. The wet
gelatin
beads are subsequently washed with acetone to remove the oil and surfactant,
and
Io to dehydrate the gelatin beads into a dry microsphere powder. Residual
acetone is
removed and the final moisture content is fixed by fluidizing a bed of the
microspheres with an air stream of controlled humidity.
Table X lists the quantitative composition of tICAM{453)/gelatin microspheres
is and placebo (gelatin microspheres only) bulk powder preparations prepared
by this
process.
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Table X:
Unit formula listin4 components for placebo and tICAM(453)/_gelatin
microsphere
bulk powder preparation.
Placebo tICAM(453)/gelatin
microsphere
(10% load)
Gelatin 150 g 104.7 g
L-Histidine 0.4 g
tICAM(453) 14.9 g
Batch size (total150 g 120 g
of
above)
Water for injection*600 g 480 g
Total aqueous 750 mL 600 mL
volume
Corn oil**
Weight 2070 g 2208 g
Volume 2250 mL 2400 mL
Span 80** 22.5 g 24.0 g
Acetone** approx. approx. 15 L
15 L
s
* including that added along with ICAM. Partially removed
during processing.
** Removed during processing. Span 80 and corn oil are
reduced to respective levels of x0.14 and <_0.3 mg/g of gelatin
~o microsphere powder. Acetone at the end of the process
complies to a limit of s250 ppm.
Example 8
is General Process for Preparation of Pharmaceutical Grade ttCAM(453)/gelatin
Microspheres
The following is a standard general procedure used repeatedly for the
production of tICAM(453)/gelatin microspheres:
1. tICAM(453) is concentrated to > 50 g/L and diafiltered into a low-ionic
strength 10 mM histidine buffer, pH 7Ø The ultrafiltrationldiafiltration
(UF/DF)
is carried out using a model S10Y30 (30,000 MWCO) tangential-flow
CA 02296620 2000-O1-12
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ultrafiltration cartridge (Amicon, Beverly, MA) with at least 7 volumes of the
histidine diafiltration buffer.
2. The final UFIDF formulated tICAM(453) bulk is sterile-filtered through a
0.2-
s micron filter into sterile polyethylene terephthalate copolymer (PETG)
bottles
and stored at not more than -30°C. Aliquots of the sterile formulated
bulk are
used to quantitate tICAM{453) content by immunonephelometry, tICAM(453}
integrity by SDS-PAGE, bioactivity by the cell-based plaque assay, and
microbial load. Immunonepheiometry is a method for assaying the
to concentration of a specific protein in solution based on measurement of the
intensity of light scattered from precipitation formed by mixing the protein
of
interest with antibodies reactive against it. Automated instruments for
carrying out this assay are manufactured, for exampie, by Behring
Diagnostics, Inc. (Somerville, NJ).
Is
3. At the time of formulation, the tICAM(453) is thawed by placing the bottle
in a
water bath set at not more than 40°C.
4. A solution composition of 104.7 g of gelatin, 14.9 g of tICAM(453) in
histidine
2o buffer from step 1, and sufficient water for injection for a total weight
of 600 g
is prepared in a 1 L glass beaker. This tICAM(453) gelatin solution has a
total
solids content of 20% (w/w) with tICAM(453) comprising 12.4% of the total
solids. The mixture is stirred while covered for not more than 30 minutes in a
50°C water bath. In a separate 4.5 L stainless steel beaker, 2208 g
(2400 mL)
2s of corn oil and 24 g of Span 80 (=1 %) are mixed at 200 rpm in a
50°C water
bath for not less than 30 minutes using a 3-inch radial flow impeller attached
to a Lightnin~ mixer (Lightnin Equipment Co., Milwaukee, WI).
5. To generate the water-in-oil emulsion, the speed of the mixer in the oil
so solution is increased to 300 rpm, and the warm tICAM(453) gelatin solution
is
gently poured into the oiUsurfactant solution over a period of approximately 1
46
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WO 99/03452 PCT/US98/14790
minute. The mixer speed is then increased to 425 rpm to form gelatin-water
droplets in the oil/surfactant phase. The water bath set-point is reduced to
10°C and the emulsion mixed until its temperature reaches 15°C.
During this
period, the mixer speed is increased at 10-minute intervals to the maximum
s speed attainable without causing air entrainment. This process cools and
solidifies the gelatin-tICAM(453) aqueous droplets into gel beads. Once the
emulsion temperature reaches 15°C, the emulsion is mixed for not less
than
60 minutes at a mixing speed of not more than 640 rpm. This step hardens
and stabilizes the gelatinItICAM(453) beads.
~o
6. The cooled emulsion is transferred into a 7.6 L stainless steel beaker and
the
gel beads are subsequently separated from the oil and surfactant by washing
the emulsion with acetone. Six washes with acetone remove the oil/surfactant
phase of the emulsion and dehydrate the tICAM(453)/gelatin into
is microspheres of the target particle size.
7. The microspheres are collected on a 3.0-micron Teflon-type FS filter
{Millipore, Bedford, MA) in a Buechner funnel set on a filtration flask
connected to a vacuum line. The microspheres are dried into a free flowing
2o powder by drawing a vacuum until no further filtrate emerges from the
funnel.
All the microspheres from the Buechner funnel are transferred to a 60-mesh
stainless steel sieve and sieved into a collecting pan. This powder
corresponds to one of two sub-batches of the tICAM(453)/gelatin microsphere
bulk powder batches that are combined prior to the next manufacturing step.
2s The powder is transferred into a type-III flint glass bottle capped with a
Teflon-lined lid and stored at 2-8°C until removal of the acetone as
set forth
in Example 15 below.
tICAM(453)/gelatin microspheres made by this procedure had
3o approximately a 10% tICAM(453) content. Particles made by this procedure
are
shown in Figure 5.
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Example 9
General Procedure for Preparation of Placebo
s The following is a standard general procedure used repeatedly for the
production of tICAM(453)/gelatin microspheres:
Placebo gelatin microspheres (no drug loading) are prepared by a procedure
which follows steps 4 to 7 of the procedure of Example 8 above, omitting the
~o addition of tICAM(453). The process steps used for manufacturing both
tICAM(453)-
loaded microspheres and placebo microspheres are identical except that
slightly
different gelatin:oil ratios are employed in the emulsification processes for
tICAM(453)-loaded microspheres compared with the placebo gelatin microspheres
in order to obtain similar particle sizes. This is achieved by changing the
gelatin:oil
is ratio from 1:4 (for tICAM(453)-loaded microspheres) to 1:3 (for placebo
gelatin
microspheres) while keeping the emulsion volume constant for both.
Example 10
2o General Procedure for Preparation of albumin/gelatin Microspheres
The following is a standard general procedure used repeatedly for the
production of albumin/gelatin microspheres:
2s Albumin/geiatin microspheres are prepared according to the process of
Example 8, with the following changes to the preparation of the gelatin
solution: A
solution of gelatin and albumin is prepared by dissolving 104.8 g of 250-Bloom
gelatin in 43fi.3 g of 0.019 M NaOH at 50 °C. 58.7 g of 25% albumin
(human) USP
(Bayer Corp, Elkhardt, IN) is added. This corresponds to a solids
concentration of
30 20% and an albumin loading of 12.3% and leads to an albumin loading of 10%
in the
final product. The presence of the NaOH in the albuminlgelatin solution is
needed to
48
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prevent isoelectric precipitation of albumin. The gelatin solution at this
concentration
would otherwise have a pH of 4-5. This albumin/gelatin solution is emulsified
in corn
oil and processed as in steps 4-7 of Example 8.
s When the powder recovered from two runs prepared according to the above
process was combined after the vacuum filtration, and treated by the acetone
removal process described in Example 13 and the moisture removal process of
Example 14, gelatin microspheres having a mean volume particle diameter of 43
Vim, with no detectable particles of volume diameter <10 pm or > 100 pm were
io obtained. The final albumin loading was 10.6% by weight.
Example 11
Adjustments for Particle Size
is Comparisons were made between placebo (gelatin only) microspheres
prepared by the method of Example 9 and albumin-loaded microspheres prepared
by the method of Example 10. Results are shown in Table XI:
Table XI
2o Effect of Albumin on Particle Size
Volume: 3L; 17.3 cm beaker;1:3 volume ratio gelatin:oi1;20% solids; 3" 8100
impeller
Loading D[4.3], p.m % > 101 ~m
0% 50 1.1
0% 68 9.6
0% 64 4.8
10% albumin 96 35
10% albumin 118 68
10% albumin 98 40
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This shows clearly that the presence of albumin increased the mean volume
particle
diameter.
tICAM(453) also increased the mean volume particle diameter. To study this,
s experiments were carried out at an intermediate scale, i.e., with an
emulsion volume
of 800 mL in a 12.5-cm vessel and a 2" 8100 impeller (40 g dry weight). At the
800-
mL scale (Table XII), and with gelatin:oil ratio of 1:3, albumin loaded
microspheres
were only slightly larger than placebo microspheres (73 versus 62 ~.m}. At a
gelatin:oil ratio of 1:4, albumin-loaded microspheres had a mean volume
particle
to diameter of 57 ~m compared to 47 pm for placebo.
Table XII
Comparison of Placebo Gelatin Microspheres Albumin-Loaded Gelatin
Microsaheres, and tICAM(453)-Loaded Gelatin Microspheres
loading volume Gelatin-Oil D[4,3], % % solids
(mL) Ratio pm >101 ~.m
placebo 800 1:3 62 4.6 20
albumin 800 1:3 73 9.0 20
tICAM(453) 800 1:3 92 31 20
tICAM(453) 800 1:3 165 92 20
placebo 800 1:4 47 0.9 20
albumin 800 1:4 57 2.0 20
tiCAM(453) 800 1:4 63 4.1 20
tICAM(453) 3000 1:4 63 5.0 20
However, the mean volume particle diameters of tICAM(453)-loaded
microspheres were much larger than the placebo (92 and 165 Nm vs. 62 ~.m,
respectively). These experiments showed that tICAM(453) causes an increase in
2o the mean volume particle.
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The effect of the loaded protein on particle size was readily minimized by
changing the gelatin:oil ratio from 1:3 to 1:4 while keeping the emulsion
volume
constant. This was first shown at the 800-mL scale (Table XII}. At the
oif:gelatin
ratio 1:4, both albumin and tICAM(453)-loaded microspheres were larger than
the
s placebo microspheres, but more importantly, both the albumin-loaded and
tICAM(453)-loaded microspheres exhibited a pharmaceutically acceptable size
distribution.
Example 12
Process for Producinct tICAM(453)-Loaded Gelatin Microspheres in Largier
i o Batches
The process used above in Table XII (3000 mL) was used to prepare larger
batches and is further shown below:
is Materials
250-Bloom gelatin: 105 g
tICAM(453) in histidine buffer 15 g (12.4% theoretical loading)
corn oil (p = 0.92) 2200 g
Span 80 24 g
Emulsifcation
Gelatin-tICAM(453) solution weight: 600 g
Beaker diameter 17.3 cm
Impeller: 76 mm 8100 in off-center position, shaft 10-15° from
vertical
2s Speed: 400, increased to 640 rpm during
cooling
Temperatures
Initial: 45 - 50 °C
Cooling to 15 °C over ~70 min
3o Hardening for 1 hr at <15 °C
Washing
Seven washes with acetone, each with stirring for 10 min and 10-min settling
time.
3s Wash volumes: 1 3 L
2-5 2 L
6-7 1 L (5 min stirring and settling)
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Filtration
in Buechner funnel, using 3.0-Nm Millipore Fluoropore filter
s Results are shown in Table XIII below:
Table XIII
to Characterization and Stability of Preclinical Microsahere Formulations
ICAM-loaded ICAM-loaded
#1 #2
Assay Results Initial 3 month Initial 3 month
5C 5C
Mean Volume Particle Diameter,47 46 46 47
~,
<10~. -- 0 0 0 0
>100 ~,t 0 1 0 1
Moisture, % 14.9 15.4 14.5 15.7
Residual Acetone, ppm <150 g0
Loading, % (by 10.5 12.1 11.1 12.4
immunonephelometry)
Potency, ~g/ml (by plaque 0.28 0.45 0.28 0.40
assay)
Identity/Integrity (Western Intact Intact Intact Intact
blot)
Example 13
is General Procedure for Removal of Residual Acetone
The following is a standard general procedure used repeatedly for the
removal of residual acetone from pharmaceutical compositions comprising
gelatin
microspheres:
The product of Example 8 (240 g theoretical weight) is charged into a 13-cm
Amicon Vantage~ S2 column (Amicon, Beverly, MA) connected to a sterile air
supply and two gas washing bottles. One bottle is immersed in a water bath set
at
24°C, and the second bottle is set as a moisture trap. The air is
humidified during its
2s passage through the first bottle at a flow rate of 15 Umin. The relative
humidity is set
to 90-95% by adjusting the water bath temperature. At 15 Umin, the powder
evenly
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fluidizes the powder bed. Treatment is carried out for approximately 11 hours,
by
which time the residual acetone level in the powder is reduced to below 250
ppm.
Example 14
s General Procedure for Moisture Reduction
The following is a standard general procedure used repeatedly for moisture
reduction in pharmaceutical compositions comprising gelatin microspheres:
~o A similar setup as in Example 12 is employed to reduce the moisture content
of the microspheres. In this case, the relative humidity is 35-40%, controlled
by
setting the water-bath temperature to 2-4 °C, and the target range for
the final
moisture content in the microspheres is 12-18%. The bulk tICAM(453)
microspheres
are removed from the column and stored in type-III flint glass jars with
Teflon-lined
is lids at 2-8°C.
Example 15
Process for Making 400a Gelatin/tICAM(453) Microspheres
2o Porcine gelatin (type A) was purchased from Hormel Foods (Austin, MN).
Corn oil and Span 80 (sorbitan monooleate, NF grade) were supplied by Ruger
Chemical Co. Inc. (Irvington, NJ). Acetone (reagent grade) was purchased from
EM
Science (Gibbstown, NJ). Purified ICAM was prepared according to Example 1 in
a
10mM histidine buffer pH 7.0 ( ~50mg/ml). Human serum albumin (HAS Lot #
2s 684P067A , commercial stock) was obtained from Bayer Corporation,
Biological
Products, Clayton NC. The vessels, impellers, shafts and baffles used in these
experiments were made of Stainless steel (SS 316).
Strategies for a geometric scale up of the manufacturing process from the
30 1508 scale of Example 8, 9, and 10 to larger scales were based on
principles of
constant mixing power per unit volume. A geometric scaled-up process from the
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150g to 400g batch size would involve a proportional increase in vessel size
keeping
the height to diameter ratio (HID) of the emulsion bed constant.
The following derivation was used to determine the formula required for
s calculating geometrically scaled-up process parameters for larger batch
sizes based
on known process parameters at the smaller 150g scale.
Geometric scale-up:
Volume of the emulsion bed = ~ R2 H
where R is the radius of the bucket and H is the emulsion height.
Dividing the volume of the emulsion bed at a larger geometrically scaled up
batch
is size (~ RL 2 HL ) by the emulsion volume at the smaller 150g scale (~ Rs2
Hs),
produces
VL- ~ RL? HL
Vs ~ Rs2 Hs
Substituting the radius by the bucket diameter (R=D/2)
VL- ~L?I~L
Vs ~t (Ds2/4) Hs
X and = the numerator and denominator by D~ and DS, respectively, produces
VL - ~L 3L4) (HLID )
Vs T~ (Ds314) (HsIDs)
For geometric similarity HID is constant at all scales, hence the above
equation
reduces to,
VLNs = (DL3IDS3)
3s DLIDs= NLNs)'rs
DL=NLNS)~/3DS
where,
Ds - diameter of the vessel used at the smaller 150g scale ( 6.7")
4o Vs - volume of the emulsion at the smaller 150g scale (3.0 L)
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D~ - diameter of the vessel at any scale larger than 150g
V~ - volume of the emulsion at any scale larger than 150g
Thus, knowing the emulsion volume and vessel diameter at the smaller 150g
s scale and the emulsion volume for a larger scale (fixed for a given scale),
the vessel
diameter to be used for that larger scale (D~) can be determined.
Power per unit volume (P/v):
io Power P x N3d2 (2)
and (Plv)t _
(P l v)S - I
where,
N - mixing speed (rpm)
d - impeller diameter
is (P/v)s - Mixing power per unit volume generated at the smaller 150g scale
(P/v)~ - Mixing power per unit volume to be generated at any scale larger than
150g.
The power per volume equations describe dimensional and dynamic similarity
and are applicable in the turbulent flow regime where the power number is
constant
zo for a given impeller design for a wide range of Reynolds numbers [Rushton,
J.H., E.
W. Costich, H.J. Everett, Chem. Eng. Progress 46(8):395-404 (1950)].
Equations 1 and 2 were used to calculate the geometric scaled up
parameters for various larger scales ranging from 300 to 10008. These values
are
2s listed in Table XIV and account for a proportionate increase in emulsion
volume and
impeller diameter to generate a similar mixing power per unit volume within
the
emulsion as that generated at the 1508 scale:
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Table XIV
Process parameters for various batch sizes calculated using equations for
geometric
scale-up and constant mixing power per unit volume
s
Scale (g) 150 300 400 500 750 1000
Emulsion volume (L) 3.0 6.0 8.0 10.0 15.0 20.0
Bucket diameter (inch)6.7 8.4 9.3 10.0 11.4 12.5
Impeller diameter 3.0 3.8 4.1 4.5 5.1 5.6
(inch)
Emulsion height (inch)5.2 6.5 7.2 7.7 8.8 9.7
RPM 425 365 343 327 299 281
In addition, an emulsification system is more complex than a simple mixing
process, and increased shear forces are important in addition to mixing
forces.
~o Mixing times were not kept constant since they were are dependent on the
batch
size and rate of cooling.
Emulsification set-up for the scaled up process at the 400:
is The relative amounts of gelatin, oil and Span 80 used in the scaled up
process at the 400g scale are outlined in Table XV. As opposed to the 150g
scale
method, this process utilizes the same gelatin to oil ratio for the production
of both
gelatin microspheres and ICAM/gelatin microspheres.
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Table XV
Retative amounts of gelatin, oil and Span 80 used for production at the 150 g
scale
and at the 4008 scale
s
Amount of Gelatin Gelatin: Volume of Amount of Span
(g) Oil Oil 80
Ratio (ml) (g)
1. 150 g scale
Gelatin Microspheres
150 g 1:3 2250 ml 22.5 g
(20 % solution = 750 (1% w/v)
ml)
ICAMIGelatin Microspheres
(11.5% theoretical
loading)
105 g gelatin 1:4 2400 ml 24.0
+ 15 g ICAM (1 % w/v)
(20 % solution = 600
ml)
2. 400g scale
Gelatin Microspheres
400 g 1:3 6000 ml 60 g
(20 % solution = 2000 (1 % w/v)
ml)
ICAM/Gelatin Microspheres
(11.5% theoretical
loading)
354 g gelatin 1:3 6000 ml fi0.0
+ 46 g ICAM (1 % w/v)
(20 % solution = 2000
ml)
The emulsification process at the 400g scale (8L emulsion volume) required a
~0 9.3" diameter vessel with a 4.0" radial impeller at an initial mixing speed
of 343rpm,
as seen in Table XIV. A gelatin solution was prepared at a mixing speed of 200
rpm
using a 3" radial impeller in a 3 L stainless steel vessel placed in a water
bath
maintained at 50°c. A mixture of corn oil and Span 80 was mixed at 250
rpm using a
4" radial impeller in a 12 I_ stainless steel vessel placed in a water bath
maintained at
is 50°C. The dissolved gelatin solution was added to the oiI/Span
mixture and the
emulsification was performed under various experimental conditions (with and
without
baffles) and cooling rates (gradual, moderate and rapid) to generate
microspheres in
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the desired 50~,m size range. Photographs of the emulsion at various points
during
the emulsification process were captured by an optical microscope (Zeiss,
Germany)
fitted with a camera attachment (Polaroid Microcam, UK) to evaluate the effect
of
various process parameters on the size of the emulsion droplets and
microspheres
s generated. The microspheres were acetone washed to remove traces of oil and
moisture from the microsphere preparation. The acetone washed microspheres
were
then vacuum filtered using a Buechner funnel and sieved using a 75pm mesh
sieve to
eliminate large particles and aggregates from the microsphere preparation.
This was
followed by an acetone removal step performed by fluidizing the powder bed
with
io humidified air (RH~90%) for ~16 hours in a chromatography column. Following
acetone removal the moisture level of the microspheres was adjusted to 13 -15
% by
circulating clean air (RH~35%) through the column. The dry microspheres were
stored in glass containers.
Is Emulsification Without Baffles (similar to the 150g scale process):
The manufacturing process used was similar to that used at the smaller 150g
scale. A 4.0" radial impeller was placed in the top left quadrant and
positioned off
center at a 12 degree angle to the vertical in a 9.25" diameter vessel.
Emulsification With Baffles:
The emulsion volume (8L), bucket diameter (9.25") and impeller size (4.0")
were the same as that used in the experimental set-up without baffles. In
addition,
2s four stainless steel baffles (10 inches high and 3/4" wide) were positioned
vertically
and equidistant (90° apart) from each other and 3/8" inches from the
sides of the
vessel. The impeller was positioned at the center (straight, no angle) of the
vessel
as seen in Figure 3b.
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Cooling Methods:
Three cooling methods were evaluated as described below:
Gradual Cooling:
s
The cooling process used was similar to that used at the smaller 150g scale.
The gelatin solution (50°C) was added to the vessel containing the
oil/Span mixture
(placed in a 50°C water bath) and mixed at the preset initial mixing
speed.
Immediately following addition, the water bath set-point temperature was
changed
to from 50 to 10°C and the mixing speed was increased as the emulsion
gradually
cooled to 10°C.
A gradual cooling rate was evaluated for emulsification runs with and without
baffles. For emulsification runs performed without baffles, three initial
mixing speeds
rs of 343, 375 and 415 rpm were utilized. Experiments with baffles were
evaluate at
two initial mixing speeds of 425 and 475 rpm.
Moderate Cooling:
2o Experiments were performed in an attempt to achieve a higher cooling rate
{~1.0 deg./min) than that achieved at the 1508 scale (~0.47 deg./min). The
gelatin
solution (50°C) was added to the oiI/Span mixture (50°C) and
mixed at the preset
initial mixing speed. immediately following addition, the bath was turned off,
and ice
was gradually added to the warm bath water at intervals to maintain a cooling
rate of
2s 1.0 deg./min. Moderate cooling was performed with baffles, and evaluated
for two
initial mixing speeds of 625 and 640 rpm.
Rapid Cooling:
3o A rapid cooling rate (~2.3 deg./min) was achieved as follows. Immediately
following the addition of the gelatin solution to the oil/Span mixture at
50°C, the initial
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mixing speed was increased to a predetermined rpm. The emulsion was mixed at
this
speed for a fixed time (20-40 min) at 50° C to generate droplets in the
50pm size
range. The emulsion was then rapidly cooled to 15°C by draining the
warm water from
the water bath and replacing it with a slurry of ice and water. The bath
temperature
s was set to 0.1 °C. The mixing speed was increased as the emulsion
rapidly cooled
from 50 to 15°C. When the emulsion reached a temperature of
15°C, the bath
temperature was increased to 10°C and the microspheres were held at
this
temperature and stirred for 1 hour at 525 rpm.
to The rapid cooling process with baffles was utilized to produce gelatin
microspheres, albumin-loaded gelatin microspheres, and ICAM-loaded gelatin
microspheres under the following two experimental conditions:
High Mixing Speed:
IS
The impeller was positioned at a height of 1.5 " from the bottom of the
vessel,
and three initial mixing speeds of 600, 625 and 650 rpm were evaluated, with a
final
mixing speed of 780 rpm. In addition, initial mixing times of 20, 30 and 40
minutes at
650 rpm were tested.
Low Mixing Saeed:
In order to decrease the mixing speed and still generate emulsion droplets in
the desired size range (~50pm) the impeller was raised to 2.5" from the
bottom, and
2s an initial mixing speed of 425 rpm with a final rpm of 605 during cooling
was
evaluated.
Studies on release of ICAM from gelatin microspheres~
3o Release studies on ICAM/gelatin microspheres were performed as follows:
20mg of ICAM/gelatin microspheres were weighed into a 12X75mm polypropylene
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test tube and 2.0 ml of phosphate buffered saline (pH 7.2) was added to the
tube.
The tube was vortexed briefly to suspend the microspheres and was placed in a
40°C water bath. The tube was vortexed at intervals of 5 minutes for a
total of 1.0
hr. The resulting solution was filtered through a syringe filter (0.2p,m, 25mm
Gelman
s Acrodisk (Fisher Scientific, Norcross, GA), low protein binding membrane)
and
submitted in duplicate for ICAM determination by immunonephelometry
Emulsification set-ua for the scaled up process at the 400g scale:
io Gelatin microspheres and ICAM/gelatin microspheres were prepared by an
emulsification process carried out in a 9.25" diameter vessel with a 4" radial
impeller
at a predetermined initial mixing speed, under various experimental conditions
(with
and without baffles) utilizing several cooling methods (gradual, moderate and
rapid
cooling). The results obtained under these varying conditions are summarized
~ s below.
Emulsification without baffles Gradual Cooling):
Three emulsification runs with initial mixing speeds of 343, 375 and 415 rpm
2o were evaluated. The maximum initial mixing speed that was possible without
causing excessive splashing and air entrapment was 415 rpm. The lowest mixing
speed of 343 rpms with a 4" radial impeller was determined by geometric scale-
up
calculations (values listed in Table XIV) and provided similar mixing
conditions as
that obtained at the 150g batch (6.7" diameter vessel, 3.0" impeller and an
initial
2s mixing speed of 425 rpm).
The results for the above three runs are outlined in Table XVI:
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Table XVI
Experimental conditions and particle size data for microspheres prepared by
the
batch emulsification process utilizina Gradual cooling (a) without baffles and
(b) with
s baffles
(a) Gradual cooling without baffles
Impeller Position12 deg 12 deg. 12 deg.
Baffles NO NO NO
RPM 343 375 415
Cooling Rate Gradual Gradual Gradual
Particle Size, 83 72 71
pm
Span 0.86 0.82 0.81
<10~m 0 0 0
> 100 p.m 21 11 9
io {b) Gradual cooling with baffles
Impeller PositionCenter Center
Baffles YES YES
RPM 425 475
Cooling Rate Gradual Gradual
Particle Size, 65 70
p.m
Span 0.78 0.67
<10~tm 0 0
> 100 p,m 6 9
Gradual Cooling Rate ~ 0.25 deg.lmin
~s The average particle size of the microspheres generated by all three runs
was large
(> 70~m), which were much higher than the acceptable product specification of
50pm. The larger emulsion volume (8L) at the 400g scale required ~2.5 hr to
gradually cool from 50 to 15°C. This corresponded to a cooling rate of
0.25°Clmin
which was much slower than 0.47°Clmin observed at the 150g scale (3L
emulsion
2o volume). During this slow cooling period, the three mixing speeds evaluated
were
insufficient to provide the shear necessary to produce and maintain small
emulsion
droplets. These droplets coalesced to large droplets, which on cooling
resulted in
large particles. In addition a high percentage (10-20%) of the microspheres
was
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above 100~m, which was outside the acceptable limit of <10%. No microspheres
below 10~m were generated by this process.
The principles of geometric scale-up and constant mixing power per unit
s volume assume that the mixing process dictates the particle size of the
microspheres. However, the experiments performed thus far demonstrate that
droplet coalescence during cooling (due to low shear rates) and the low
cooling
rates are important factors affecting particle size. These two issues were
addressed
by utilizing baffles and a rapid cooling rate, as described below.
io
Emulsification with baffles:
A high shear rate associated with a high mixing speed could not be achieved
without a secondary source to enhance turbulence and shear in the system.
Baffles
is were used to increase the mixing speed and shear in the system during the
emulsification step to produce smaller droplets and subsequently smaller
microspheres. A schematic representation of the batch emulsification process
with
baffles can be seen in Figure 4. Higher initial mixing speeds up to 650 rpms
with
minimal splashing and air entrapment were achieved using baffles.
The emulsification process with baffles was evaluated using three different
cooling methods.
Gradual cooling (baffles):
2s
The microspheres prepared possessed average diameters > 65~m (Table
3XVIb). Photographs of the emulsion droplets and resulting microspheres at
various
stages during the process can be seen in Figure 6. Emulsion droplets in the
desired
size range (~50pm) were achieved during the emulsification process due to the
3o higher shear rates achieved with baffles. However, during the long cooling
period
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the emulsion droplets coalesced to form larger emulsion droplets which, on
cooling,
resulted in large microspheres (>65~m).
The above experiments demonstrated that a slow cooling rate of 0.25
s deg./min was not successful in producing 50pm microspheres. Subsequent
experiments (moderate and rapid cooling) investigated higher cooling rates.
Moderate cooling:
~o A moderate cooling rate of 1.0 deg./min also resulted in droplet
coalescence
and large particles (> 135pm) and ~80% of the particles above 100~m. These
results are summarized in Table XVII:
Table XVII
~s
Experimental conditions and particle size data for microspheres prepared by
the
batch emulsification process utilizin4 manually controlled cooling with
baffles
Impeller PositionCenter Center
Baffles YES YES
RPM 625 640
Cooling Rate Moderate Moderate
Particle Size, 138 144
~m
Span 0.85 0.87
<10N,m 0 0
> 100 ~tm 81 83
Moderate Cooling Rate ~ 1.0 deg./min
Figure 7 shows the appearance of the emulsion droplets and solid
microspheres at various stages during the moderate cooling process. Emulsion
2s droplets in the desired size range (~50p.m) were achieved during the
emulsification
step, with a higher mixing speed of 625 rpm. However, during the cooling from
50 to
15°C (35 min) the small emulsion droplets coalesced to yield large
microspheres. A
higher initial mixing speed could have resulted in smaller droplets which were
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thermodynamically unstable resulting in a greater degree of coalescence. This
could explain the higher particle sizes and greater percentages of particles
above
100~,m seen in the moderate cooling process (Table XVII) over that observed
using
the gradual cooling process (Table XVI).
s
In an attempt to gel the ~50pm emulsion droplets into 50wm microspheres
rapidly, thus preventing them from coalescing, a rapid cooling method was
evaluated.
to Ranid cooling_
A rapid cooling rate of ~2.3 deg./min was achieved under two different
experimental conditions (high mixing and low mixing), as described earlier.
The
emulsion cooled from 50 to 15°C in ~15 min.
High Mixing Seed:
Three initial emulsification speeds of 600, fi25 and 650 rpms were evaluated.
An emulsification time of 20 min at the above mixing speeds produced droplets
in
2o the 50~m size range, which were rapidly cooled to generate microspheres
that
possessed average particle volume diameters of 57pm, 60, 69 and 62pm, no
fines,
and less than 10% of the particles above 100pm. These results are summarized
in
Table XVIII:
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Table XVIII
Experimental conditions and particle size data for gelatin microspheres
prepared by
the batch emulsification process utilizing the rapid cooling method with
baffles and
s (a) an initial emulsification time of 20 and 40 minutes and (b) an initial
emulsification
time of 30 min.
(a) Initial emulsification times of 20 and 40 min
Gela tin Microspheres
Emulsification 20 min 20 min 20 min 20 min 40 min
time
Impeller Position Center Center Center Center Center
Baffles YES YES YES YES YES
RPM 600 625 625 650 650
Cooling Rate Rapid Rapid Rapid Rapid Rapid
Particle Size, 57 60 69 62 63
p.m
Span 0.92 0.85 0.87 0.79 0.86
<10~m 0 0 0 0 0
> 100 p,m 5 4 9 5 6
io
(b) initial emulsification time of 30 min
Gelatin Microspheres
Emulsification time 30 min 30 min 30 min
Impeller Position Center Center Center
Baffles YES YES YES
RPM 650 650 650
Cooling Rate Rapid Rapid Rapid
Particle Size, wm 56.71 52.75 55.43
Span 0.81 0.79 0.78
<10~m 0 0 0
> 100 p.m 3 2 2
rcap~a ~ooung rcaie ~ ~.~ aegimin
The process to be transferred to the pilot plant required a range of
Is initial emulsification times to be specified, rather than a rigid 20 min
mixing time.
The minimum emulsification time required to generate ~50~m emulsion droplets
prior to rapid cooling, was 20 min. This process was repeated for an
emulsification
time of 40 min, followed by rapid cooling which generated particles with a
size of
63p,m (Table XVllla).
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Since the process worked at 20 and 40min., an initial emulsification time of
30
~ 10 min. at 650rpm could be specified. Three replicates performed with a 30
min.
emulsification time followed by rapid cooling resulted in average particles
sizes of
55~.m. These results are summarized in Table XVlllb.
s
This process can be summarized as follows:
Time Emulsion Temp. (C) RPM
50 375 Gelatin added to oil
io 0 50 650
50 650
50 650
50 650
is The emulsion generated was rapidly cooled as describe earlier. During the
cooling process, the rpms were increased at set temperatures as indicated
below.
Emulsion Temp. (C) RPM
700
20 35 715
30 730
28 750
23 765
18 780
2s 15 780
The microspheres were then acetone washed, filtered and dried.
Albumin/gelatin microspheres generated by the above process possessed
3o average sizes of 61, 58 and 70p,m, and ICAM/gelatin microspheres were 66~m
in
volume diameter. The results are summarized in Table XIX and shown in Fig. 8:
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Table XIX
Experimental conditions and particle size data for Albumin/Gelatin
microspheres and
ICAMlgelatin microspheres prepared by the batch emulsification process
utilizingi the
s rapid cooling method with baffles
Albumin/Gelatin ICAM/Gelatin
Microspheres Microspheres
Impeller Position Center Center Center Center
1.5" from1.5" from 1.5" from 1.5" from
base base base base
Emulsifrcation 30 min 30 min 30 min 30 min
time
Baffles YES YES YES YES
RPM 650 650 650 650
Cooling Rate Rapid Rapid - Rapid Rapid
Particle Size, 61 58 70 66
pm
Span 0.70 0.70 0.75 0.84
<10pm 0 0 0 0
>100pm 3 2 8 8
Rapid Cooling Rate ~ 2.3 deg/min
to Low mixing speed:
The high mixing speed process resulted in splashing during the emulsification
process, and air entrapment during the holding step (780 rpm for 1 hr at
15°C). To
minimize splashing, the impeller was raised to 2.5" from the bottom of the
vessel and
Is was used at a lower mixing speed. The initial mixing speed evaluated was
425 rpm
with a final rpm of 605 during the cooling process.
The process evaluated was as follows:
Zo Time (min) Emulsion Temp. (°C) RPM
50 375 Gelatin added to oil
0 50 425
50 425
2s 20 50 425
30 50 425
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The emulsion generated was rapidly cooled as describe earlier. As the
emulsion cooled, the rpms were increased at set temperatures as indicated
below.
Emulsion Temp. (°C) RPM
s 45 475
40 525
35 550
30 575
25 590
20 605
605
The microspheres were then acetone washed, filtered and dried.
Gelatin microspheres, albuminlgelatin microspheres, and ICAM/gelatin
microspheres (varying loads) prepared by the low mixing speed process were in
50m size range, with spans of around 0.8, no fines below 10~m, and less than
3.5%
above 100wm. These results are summarized in Tables XX and XXI and shown in
2o Fig. 9:
Table XX
Experimental conditions and particle size data for gelatin microspheres and
albumin/aelatin microspheres prepared by the batch emulsification process
2s utilizing the modified rapid coolinqprocess with baffles
Gelatin Albumin/Gelatin
Microspheres Microspheres
Impeller PositionCenter Center Center Center
2.5" from 2.5" from 2.5" from 2.5" from
base base base base
Baffles YES YES YES YES
RPM 425 425 425 425
Coolin Rate Rapid Rapid Rapid Rapid
Particle Size, 53 50 55 54
wm
Span 0.89 0.81 0.80 0.75
<10~,m 0 0 0 0
>100~m 2 1 2 2
ss
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Table XXI
Exae~imental conditions and particle size data for ICAM/gelatin microspheres
prepared by the batch emulsification process utilizing the modified rapid
cooling
process with baffles
ICAMIGelatin
Microspheres
loading of ICAM 10% 10% 10% 5% 1
Impeller PositionCenter Center Center Center Center
2.5" 2.5" 2.5" 2.5" 2.5" from
from from from from base
base base base base
Baffles YES YES YES YES YES
RPM 425 425 425 425 425
Cooling Rate Rapid Rapid Rapid Rapid Rapid
Particle Size, 54 57 58 54 52
pm
Span 0.76 0.75 0.84 0.82 0.86
<10pm 0 0 0 0 0
>100N.m 1 2 3 2 2
io Typical particle sizes of microspheres obtained by this process are shown
in
Table XXII and Fig. 10:
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Table XXII
Typical particle sizes obtained by the above process
Size (Low)Result in Size (Hi) Result
~m % wm Below
0.50 0.00 1.32 0.00
1.32 0.00 1.60 0.00
1.60 0.00 1.95 0.00
1.95 0.00 2.38 0.00
2.38 0.00 2.90 0.00
2.90 0.00 3.53 0.00
3.53 0.00 4.30 0.00
4.30 0.00 5.24 0.00
5.24 0.00 6.39 0.00
6.39 0.00 7.78 0.00
7.78 0.00 9.48 0.00
9.48 0.00 11.55 0.00
11.55 0.00 14.08 0.00
14.08 0.00 17.15 0.00
17.15 0.26 20.90 0.25
20.90 1.32 25.46 1.57
25.46 3.04 31.01 4.61
31.01 7.27 37.79 11.88
37.79 16.58 46.03 20.45
46.03 27.01 56.09 55.46
56.09 24.35 68.33 79.82
68.33 79.82 83.26 92.64
83.26 5.21 101.44 97.85
101.44 1.77 123.59 99.62
123.59 0.37 150.57 100.00
150.57 0.00 183.44 100.00
183.44 0.00 223.51 100.00
223.51 0.00 272.31 100.00
272.31 0.00 331.77 100.00
331.77 0.00 404.21 100.00
404.21 0.00 492.47 100.00
492.4 0.00 600.00 100.00
s
As can be seen from the particle size table the average particle size [D(4,3)]
is 56.36 Vim, with a span of 0.78, no particles with a volume particle
diameter <
10p,m, and ~ 3% particles with a volume particle diameter above 100pm.
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Studies on release of ICAM from Gelatin micros~heres:
ICAM/gelatin microspheres prepared with varying theoretical loads of 12.5,
10.5, 10.0, 5.0, 2.5 and 1.5 % were dissolved as described above in this
example,
s and assayed for ICAM by immunonephelometry. All batches exhibited 100%
actual loading, within assay variability. A ICAM/gelatin microsphere
preparation was
dissolved, and the sample bioassayed. The ICAM/gelatin microsphere formulation
was comparable to ICAM standard (103% of standard). The ED5° was 0.298
compared to 0.303 for the standard. These results indicated that ICAM was
still
~o potent following the formulation and washing procedures.
Conclusions:
The strategy for scaling up the microsphere manufacturing process to the
is 4008 scale was a geometric scale-up with constant mixing power per unit
volume,
based on a water-in-oil emulsification process developed at the smaller 150g
scale.
Several experimental conditions (with and without baffles) and various cooling
methods (gradual, moderate, and rapid) were evaluated. The gradual cooling
process without baffles required ~2.5 hr to cool from 50 to 15°C. The
mixing speeds
2o evaluated (343, 375 and 415 rpm) did not provide the shear necessary to
produce
and maintain small emulsion droplets, which on cooling resulted in large
microspheres. The mixing speed could not be increased above 415 rpm without
causing significant splashing and air entrapment. Baffles were introduced into
the
system to increase the mixing speed and shear forces in the system. The
2s emulsification speeds were increased to 650 rpm resulting in emulsion
droplets in
the 50m size range. However, during the gradual (2.5 hr cooling time) and
moderate
cooling (35 min cooling time) steps these 50~,m emulsion droplets coalesced,
which
on cooling, resulted in very large particles. A rapid cooling process was
developed
to gel the 50~,m emulsion droplets rapidly before they coalesced. This process
was
3o evaluated under two conditions; a high mixing speed with a low impeller
height and a
iow mixing speed at a higher impeller height. Both methods consistently
produced
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microspheres in the 50p,m size range. Three factors were identified as
critical for
achieving microspheres in the desired 50wm size range. The include; initial
emulsification time (~30min), appropriate vessel geometry and mixing speeds,
and a
rapid Goofing rate (~2.3 deg./min).
s
~o
Numerous modifications and variations in the invention as described in the
above illustrative examples are expected to occur to those skilled in the art
and
consequently only those limitations as appear in the appended claims should be
placed thereon.
Accordingly it is intended in the appended claims to cover all such equivalent
variations which come within the scope of the invention as claimed.
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