U.S. patent application number 16/716886 was filed with the patent office on 2020-04-30 for coiled tube emulsification methods.
This patent application is currently assigned to Rezolute, Inc.. The applicant listed for this patent is Rezolute, Inc.. Invention is credited to Kathleen M. Campbell, Isaac J. Dillon, Sankaram Mantripragada.
Application Number | 20200129443 16/716886 |
Document ID | / |
Family ID | 65719702 |
Filed Date | 2020-04-30 |
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United States Patent
Application |
20200129443 |
Kind Code |
A1 |
Dillon; Isaac J. ; et
al. |
April 30, 2020 |
COILED TUBE EMULSIFICATION METHODS
Abstract
Embodiments of the present technology may include a method of
forming an emulsion. The method may include flowing an oil stream
and an aqueous stream into a coiled tube to form a mixture of an
oil phase and an aqueous phase in the coiled tube. The method may
also include flowing the mixture in the coiled tube against gravity
and under laminar conditions. A plurality of beads may be disposed
within the coiled tube. The method may further include mixing the
oil phase and the aqueous phase in the coiled tube until the
emulsion is formed.
Inventors: |
Dillon; Isaac J.; (Aurora,
CO) ; Campbell; Kathleen M.; (Firestone, CO) ;
Mantripragada; Sankaram; (Windsor, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rezolute, Inc. |
Redwood City |
CA |
US |
|
|
Assignee: |
Rezolute, Inc.
Redwood City
CA
|
Family ID: |
65719702 |
Appl. No.: |
16/716886 |
Filed: |
December 17, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
15705818 |
Sep 15, 2017 |
10561620 |
|
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16716886 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/5052 20130101;
B01F 13/103 20130101; B01F 3/0861 20130101; B01F 2215/0418
20130101; B01F 3/2261 20130101; B01F 5/0647 20130101; B01F 15/0243
20130101; B01F 5/0696 20130101; A61K 9/1694 20130101; A61K 9/1647
20130101; B01J 13/046 20130101; B01F 13/0052 20130101; B01F 3/0811
20130101; B01F 2003/0834 20130101; B01F 2003/0842 20130101; B01F
2015/0221 20130101; B01F 3/088 20130101; B01F 3/0807 20130101 |
International
Class: |
A61K 9/50 20060101
A61K009/50; B01F 3/08 20060101 B01F003/08; B01F 13/00 20060101
B01F013/00; B01J 13/04 20060101 B01J013/04; B01F 5/06 20060101
B01F005/06; B01F 15/02 20060101 B01F015/02; A61K 9/16 20060101
A61K009/16; B01F 13/10 20060101 B01F013/10; B01F 3/22 20060101
B01F003/22 |
Claims
1. A method of forming an emulsion, the method comprising: flowing
an oil stream and an aqueous stream into a coiled tube to form a
mixture of an oil phase and an aqueous phase in the coiled tube;
flowing the mixture in the coiled tube against gravity and under
laminar conditions, wherein a plurality of beads are disposed
within the coiled tube; and mixing the oil phase and the aqueous
phase in the coiled tube until the emulsion is formed.
2. The method of claim 1, wherein: the oil stream comprises a
biodegradable polymer, and the method further comprising: diluting
the emulsion with additional water, and forming microparticles from
the emulsion.
3. The method of claim 2, wherein: the oil stream comprises a
physiologically active substance, and the microparticles comprise
the physiologically active substance.
4. The method of claim 2, wherein: the oil stream comprises a
protein or peptide compound, and the microparticles comprise the
protein or peptide compound.
5. The method of claim 4, wherein the protein or peptide compound
comprises insulin, human growth hormone, glucagon-like peptide-1,
parathyroid hormone, a fragment of parathyroid hormone,
enfuvirtide, or octreotide.
6. The method of claim 4, wherein the protein or peptide compound
comprises a protein-PEG conjugate.
7. The method of claim 2, wherein: the aqueous stream comprises a
physiologically active substance, and the microparticles comprise
the physiologically active substance.
8. The method of claim 2, wherein: forming microparticles comprises
removing water and solvent from the emulsion.
9. The method of claim 1, further comprising flowing a plurality of
oil streams and a plurality of aqueous streams into a plurality of
coiled tubes.
10. The method of claim 2, wherein: the microparticles comprise a
median diameter in a range from 30 to 50 .mu.m.
11. The method of claim 1, wherein: flowing the oil stream is at a
flow rate in a range from 20 to 100 ml/min, and flowing the aqueous
stream is a flow rate in a range from 100 to 200 ml/min.
12. The method of claim 1, wherein the flowing the mixture in the
coiled tube is at a Reynolds number ranging from 0.1 to 10,000.
13. The method of claim 1, wherein: the oil stream is a first oil
stream, the aqueous stream is a first aqueous stream, the coiled
tube is a first coiled tube, the mixture is a first mixture, the
oil phase is a first oil phase, the aqueous phase is a first
aqueous phase, the plurality of beads is a first plurality of
beads, and the emulsion is a first emulsion, further comprising:
flowing a second oil stream and a second aqueous stream into a
second coiled tube to form a second mixture of a second oil phase
and a second aqueous phase in the second coiled tube, flowing the
second mixture in the second coiled tube against gravity and under
laminar conditions, wherein a second plurality of beads are
disposed within the second coiled tube, mixing the second oil phase
and the second aqueous phase in the second coiled tube until a
second emulsion is formed, and mixing the first emulsion and the
second emulsion to form a third emulsion.
14. The method of claim 13, wherein: the first emulsion comprises a
first physiologically active substance, the second emulsion
comprises a second physiologically active substance, and the first
physiologically active substance is different from the second
physiologically active substance.
15. The method of claim 13, wherein: the first emulsion comprises a
physiologically active substance at a first concentration, the
second emulsion comprises the physiologically active substance at a
second concentration, and the first concentration is different from
the second concentration.
16. The method of claim 13, further comprising: flowing a third oil
stream and a third aqueous stream into a third coiled tube to form
a third mixture of a third oil phase and a third aqueous phase in
the third coiled tube, flowing the third mixture in the third
coiled tube against gravity and under laminar conditions, wherein a
third plurality of beads are disposed within the third coiled tube,
mixing the third oil phase and the third aqueous phase in the third
coiled tube until a fourth emulsion is formed, and mixing the
fourth emulsion with the first emulsion and the second emulsion to
form the third emulsion.
17. The method of claim 1, wherein: the mixture is a first mixture,
further comprising: mixing the emulsion with an aqueous solution
comprising an emulsifier to form a second mixture, and evaporating
water from the second mixture to form microparticles.
18. The method of claim 16, further comprising: removing
wastewater, fines, or aggregates from the second mixture using a
plurality of screens.
19. The method of claim 1, wherein: the coiled tube is coiled
around a longitudinal axis, and the longitudinal axis is
vertical.
20. The method of claim 1, wherein the emulsion is characterized by
particles having a unimodal particle size distribution profile.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a divisional of and claims the benefit
of priority to U.S. patent application Ser. No. 15/705,818, filed
Sep. 15, 2017, entitled "COILED EMULSIFICATION SYSTEMS AND
METHODS," the entire contents of which are herein incorporated by
reference.
BACKGROUND
[0002] Biodegradable microparticles may be used to deliver
physiologically active substances such as, small molecule drugs,
hormones, proteins, diagnostics, and other medically active agents
to a patient. Microparticles are suspended in an aqueous diluent to
make a suspension, which can be injected parenterally through a
needle. They may also be implanted as a solid. After injection, the
microparticles degrade and gradually release agents to the body.
Biodegradable microparticles may reduce the frequency of
injections, as the physiologically active substance is released
gradually into the body. The microparticle size distribution
affects the required gauge and other characteristics of the needle.
More flowable microparticles may be easier to fill into vials and
may be more easily injected with a large gauge (smaller diameter)
needle. Once in the body, the rate of release and the concentration
of the physiologically active substance may be related to the
microparticle size, the microparticle size distribution, the
initial concentration of the physiologically active substance, and
other characteristics of the microparticles. Such biodegradable
microparticles also need to meet health and safety regulations for
contaminant concentrations including the solvents used to prepare
the microparticles. Thus, a need for microparticles with superior
syringability, injectability, flowability, uniformity, and purity
characteristics exists. Forming microparticles involves forming an
emulsion from an oil component and an aqueous component. The
process for forming an emulsion can affect the characteristics of
the microparticles, and the efficiency of the emulsion forming
process may impact the availability and acceptability of
microparticles with physiologically active substances. Further,
there is a need for a process for the production of microparticles
that requires less space than conventional processes. The methods
and systems described herein provide solutions to these and other
needs.
BRIEF SUMMARY
[0003] Embodiments of the present technology may allow for forming
an emulsion efficiently and with high homogeneity. Embodiments may
use a configuration for mixing an oil phase and an aqueous phase
that reduces unwanted chaotic mixing, using laminar flow, which
allows for a gentle mixing of components. The configuration used is
a coiled or helical tube packed with beads, with the flow directed
against the direction of gravity. In addition, the helical or
coiled configuration may reduce the footprint of emulsifiers.
Several coiled tubes may be nested together in the same or similar
space as one coiled tube. As a result, embodiments may include a
more efficient and economical process of forming an emulsion. In
addition, embodiments of the present technology may produce a
targeted distribution of microparticles from the emulsion.
Microparticles may be classified by a plurality of screens with
recirculating flow from a stirred tank, which may better control
the microparticles produced.
[0004] Embodiments of the present technology may include a system
for forming an emulsion. The system may include a coiled tube. The
coiled tube may have a first end and a second end. The second end
may be located at a position higher than the position of the first
end. The system may also include a plurality of beads disposed
within the coiled tube. The system may further include a first
inlet fluidly connected to the coiled tube. The first inlet may be
configured to deliver a first fluid to the first end before the
second end. In addition, the system may include a second inlet
fluidly connected to the coiled tube. The second inlet may be
configured to deliver a second fluid to the first end before the
second end.
[0005] Embodiments of the present technology may include a system
for forming microparticles from the emulsion by removing the
solvent and the water. Microparticles may be prepared by a single
emulsification process or a double emulsification process. In the
single emulsification process, an organic solvent phase containing
a biodegradable polymer, an aqueous solution containing an
emulsifier, such as polyvinyl alcohol, and a physiologically active
substance may be homogenized to produce an emulsion. The solvent
may be evaporated, and water from the resulting hardened
microspheres may be removed by air-drying or freeze-drying. In the
double emulsification process, an aqueous solution that may contain
a physiologically active substance and an organic solvent phase
containing a biodegradable polymer may be homogenized to form an
emulsion. The emulsion may be mixed with another aqueous solution,
which contains an emulsifier such as polyvinyl alcohol. Evaporation
of the solvent and water may produce microspheres. When a
physiologically active substance is soluble in the organic solvent
phase, the method may be single emulsification because it may
produce uniform mixing of the biodegradable molecules and the
physiologically active substance molecules. When the
physiologically active substance is not soluble in the organic
solvent phase and is soluble in the aqueous solution, the method
may be double emulsification.
[0006] Embodiments of the present technology may also include a
system for forming an emulsion. The system may include coiled tubes
nested together. The system may include a plurality of coiled
tubes. For each tube of the plurality of coiled tubes, the coiled
tube may include a first end and a second end. The second end may
be disposed at a position higher than the position of the first
end. For each coiled tube, a first inlet may be fluidly connected
to the coiled tube, where the first inlet is configured to deliver
a first fluid to the first end before the second end. The system
may further include a second inlet. Also for each coiled tube, a
second inlet may be fluidly connected to the coiled tube, where the
second inlet is configured to deliver a second fluid to the first
end before the second end. A plurality of beads may be disposed
within the coiled tubes. Each coiled tube may be coiled around a
longitudinal axis. Each coiled tube may be characterized by a first
width in a direction perpendicular to the longitudinal axis. The
plurality of coiled tubes may be coaxial with the longitudinal
axis. In addition, the plurality of coiled tubes may be
characterized by a second width in a direction perpendicular to the
longitudinal axis. The first width may equal the second width.
[0007] Embodiments of the present technology may include a method
of forming an emulsion. The method may include flowing an oil
stream and an aqueous stream into a coiled tube to form a mixture
of an oil phase and an aqueous phase in the coiled tube. The method
may also include flowing the mixture in the coiled tube against
gravity and under laminar conditions. A plurality of beads may be
disposed within the coiled tube. The method may further include
mixing the oil phase and the aqueous phase in the coiled tube until
the emulsion is formed.
[0008] A better understanding of the nature and advantages of
embodiments of the present invention may be gained with reference
to the following detailed description and the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1A and FIG. 1B show a helical mixer according to
embodiments of the present technology.
[0010] FIG. 2 shows a system for forming an emulsion and
microparticles according to embodiments of the present
technology.
[0011] FIG. 3A and FIG. 3B show a set of three helical tubes
according to embodiments of the present technology.
[0012] FIG. 4 shows a method of forming an emulsion according to
embodiments of the present technology.
[0013] FIG. 5 shows a coiled tube mixer according to embodiments of
the present technology.
[0014] FIG. 6 shows a helical mixer according to embodiments of the
present technology.
[0015] FIG. 7 shows a triple helical mixer according to embodiments
of the present technology.
DETAILED DESCRIPTION
[0016] Conventional emulsification methods may not result in a
homogeneous emulsion. Conventional methods may result in convection
currents that may create local eddies that disrupt flow and
uniformity. In addition, conventional methods may include
emulsifying mixers that occupy a large volume or footprint for the
amount of emulsion produced. This large volume or footprint
increases demand for manufacturing space and therefore increases
the price of the process and final product.
[0017] Embodiments of the present technology may provide for a
homogenous emulsion more efficiently than conventional methods. An
aqueous phase and an oil phase may be flowed through a coiled tube
to form an emulsion. Flow through a coiled tube may result in the
formation of a secondary flow due to centrifugal forces. This
secondary flow may create two symmetrical vortices perpendicular to
the axial flow through the tube, stabilizing the fluid and
preventing local eddies and random turbulence, which may allow
mixing in a predictable manner. In order to further reduce
turbulent flow and unpredictable mixing, the coiled tube may be
packed with beads to reduce the available flow path and therefore
reduce the Reynolds number. The beads may also serve to break up
the fluids and aid in mixing.
[0018] In the bulk fluid, chaotic convection currents may develop,
which may create non-uniformities in the emulsion. Convection
currents may result within a mixture of a heavier component and a
lighter component. The heavier component may move in the direction
of gravity relative to the lighter component, while the lighter
component may move against the direction of gravity relative to the
heavier component, resulting in convection. Emulsions may include
immiscible fluids of varying densities, which may result in
temperature-independent convection. For example, emulsions may have
a heavier oil phase and a lighter aqueous phase. To avoid this
phenomenon, the mixture of the oil phase and the aqueous phase are
flowed against the direction of gravity. As a result, because of
the direction of flow, both the oil phase and the water phase may
move in the same direction, reducing gravitational effects and
therefore negating chaotic convection currents. In addition, when
the flow through a mixer is in the same direction as gravity, the
heavier component may move faster in the direction of gravity
relative to the lighter component and therefore flow uncontrolled
at a faster volumetric flow rate than the lighter component, again
leading to non-uniformities in the emulsion or concentration
gradients through the mixer. To avoid this and have better control
of flow rates and a more controlled process, the mixture of the oil
phase and the aqueous phase may be flowed against the direction of
gravity. Furthermore, if the emulsion experiences a pressure drop a
small amount of the organic solvent in the oil phase can possibly
flash or can possibly be converted to a gaseous form. If this
occurs, the gas bubbles may travel in the direction against
gravity. The flow path of the emulsion may be in the same direction
as any bubbles to avoid inadvertent turbulence generated by the
emulsion and gas going in two different directions.
[0019] A coiled tube may also reduce the volume and footprint
needed for emulsification. The vertical orientation of the coiled
tube may reduce the footprint over a conventional mixer that may
not be vertical. In addition, several coiled tubes can be nested
together without increasing the footprint. Two coiled tubes nested
together may resemble the structure of a double helix seen with
DNA. Three coiled tubes nested together may resemble the structure
of a triple helix similar to a collagen helix.
[0020] A mixed emulsion may be formed by forming one emulsion with
an oil phase and an aqueous phase through a tube, another emulsion
with an oil phase and an aqueous phase through another tube, and
combining both emulsions. An advantage of using a mixed emulsion
may be physically separating compounds that may interact with each
other. The compounds that are physically separated may be two
different physiologically active substances. Another advantage of
using a mixed emulsion may be controlling the way the
physiologically active substance is released. In this instance, one
emulsion may include a different biodegradable polymer than the
other. The two emulsions may differ in the concentration of the
physiologically active substance.
[0021] A mixed emulsion may also be formed by mixing emulsions
produced by the same oil phase and oil phase flowing through
several different tubes. By nesting multiple tubes together, such
as the triple helical assembly, mixed emulsions may be used to
scale up production, without increasing the laboratory bench space
or manufacturing space. Examples of a triple helical assembly are
shown in FIG. 3A and FIG. 7 and described below.
I. PHYSIOLOGICALLY ACTIVE SUBSTANCES
[0022] Physiologically active substance means a natural, synthetic,
or genetically engineered chemical or biological compound that
modulates physiological processes in order to afford diagnosis of,
prophylaxis against, or treatment of an undesired existing
condition in a living being. Physiologically active substances
include drugs such as antianginas, antiarrhythmics, antiasthmatic
agents, antibiotics, antidiabetics, antifungals, antihistamines,
antihypertensives, antiparasitics, antineoplastics, antitumor
drugs, antivirals, cardiac glycosides, herbicides, hormones,
immunomodulators, monoclonal antibodies, neurotransmitters, nucleic
acids, proteins, radio contrast agents, radionuclides, sedatives,
analgesics, steroids, tranquilizers, vaccines, vasopressors,
anesthetics, peptides, and the like. The physiologically active
substance may include a small molecule. The small molecule may
include budesonide or albuterol sulfate.
[0023] Prodrugs, which undergo conversion to the indicated
physiologically active substances upon local interactions with the
intracellular medium, cells, or tissues, can also be employed in
embodiments. Any acceptable salt of a particular physiologically
active substance, which is capable of forming such a salt, is also
envisioned as useful in the present invention, including halide
salts, phosphate salts, acetate salts, and other salts.
[0024] The physiologically active substances may be used alone or
in combination. The amount of the substance in the pharmaceutical
composition may be sufficient to enable the diagnosis of,
prophylaxis against, or the treatment of an undesired existing
condition in a living being. Generally, the dosage will vary with
the age, condition, sex, and extent of the undesired condition in
the patient, and can be determined by one skilled in the art. The
dosage range appropriate for human use includes a range of 0.1 to
6,000 mg of the physiologically active substance per square meter
of body surface area.
[0025] The pharmaceutical compositions of the invention can be
administered parenterally by injection or by implantation. The
compositions can be administered intravenously, intraperitoneally,
intramuscularly, subcutaneously, intracavity, or transdermally.
Other methods of administration will be known to those skilled in
the art. For some applications, such as subcutaneous
administration, the dose required may be quite small, but for other
applications, such as intraperitoneal administration, the required
dose may be very large. While doses outside the foregoing dosage
range may be given, this range encompasses the breadth of use for
practically all physiologically active substances. The
pharmaceutical compositions of the invention can also be
administered enterally.
[0026] Of particular interest are physiologically active substance
that are proteins or peptides. The microparticles may include a
protein or peptide compound. Proteins or peptides include insulin,
human growth hormone, glucagon-like peptide-1, parathyroid hormone,
a fragment of parathyroid hormone, enfuvirtide, or octreotide.
[0027] Insulin is normally produced by the pancreas. Insulin
regulates the metabolism of glucose in the blood. A high level of
glucose or other high blood sugar may be an indication of a
disorder in the production of insulin and may be an indication of
diabetes. Insulin is often administered by injection as a treatment
for diabetes.
[0028] Another protein that may be used as a physiologically active
substance is glucagon-like peptide-1 (GLP-1). GLP-1, a 31 amino
acid peptide, is an incretin, a hormone that can decrease blood
glucose levels. GLP-1 may affect blood glucose by stimulating
insulin release and inhibiting glucagon release. GLP-1 also may
slow the rate of absorption of nutrients into the bloodstream by
reducing gastric emptying and may directly reduce food intake. The
ability of GLP-1 to affect glucose levels has made GLP-1 a
potential treatment for type 2 diabetes and other afflictions. In
its unaltered state, GLP-1 has an in vivo half-life of less than
two minutes as a result of proteolysis.
[0029] Proteins or peptides include human growth hormone. Human
growth hormone (hGH), a 191 amino acid peptide, is a hormone that
increases cell growth and regeneration. hGH may be used to treat
growth disorders and deficiencies. For instance, hGH may be used to
treat short stature in children or growth hormone deficiencies in
adults. Conventional methods of administering hGH include daily
subcutaneous injection.
[0030] Similar to hGH and GLP-1, enfuvirtide (Fuzeon.RTM.) is a
physiologically active substance that may face challenges when
administered to patients. Enfuvirtide may help treat HIV and AIDS.
However, enfuvirtide may have to be injected subcutaneously twice a
day. Injections may result in skin sensitivity reaction side
effects, which may discourage patients from continuing use of
enfuvirtide. A enfuvirtide treatment with less frequent
administrations or extended duration may be needed to increase
patient compliance, lower cost, and enhance the quality of life for
patients with HIV and AIDS.
[0031] Another physiologically active substance is parathyroid
hormone (PTH) or a fragment of PTH. PTH is an anabolic (bone
forming) agent. PTH may be secreted by the parathyroid glands as a
polypeptide containing 84 amino acids with a molecular weight of
9,425 Da. The first 34 amino acids may be the biologically active
moiety of mineral homeostasis. A synthetic, truncated version of
PTH is marketed by Eli Lilly and Company as Forteo.RTM.
Teriparatide. PTH or a fragment of PTH may be used to treat
osteoporosis and hypoparathyroidism. Teriparatide may often be used
after other treatments as a result of its high cost and required
daily injections.
[0032] As with other physiologically active substances, a PTH
treatment with less frequent administrations or extended duration
may be desired.
[0033] Additional information on the proteins and conjugates of the
proteins can be found in U.S. patent application Ser. No.
10/553,570, filed Apr. 8, 2004 (issued as U.S. Pat. No. 9,040,664
on May 26, 2015). Information regarding the concentration release
profiles of proteins and conjugates can be found in U.S. patent
application Ser. No. 14/954,701, filed Nov. 30, 2015. The contents
of patent applications, publications, and all other references in
this disclosure are incorporated herein by reference for all
purposes.
II. SYSTEM
[0034] Embodiments of the present technology may include a system
for forming an emulsion. The system may include a coiled tube or a
helix. The coiled tube or helix may include chemically resistant
materials such as stainless steel, ceramic, glass, various plastics
(e.g., polytetrafluoroethylene [PTFE]), or other materials with a
chemically resistant lining. As shown in FIG. 1A, coiled tube 102
may be coiled around a longitudinal axis 104. Longitudinal axis 104
may be vertical or substantially vertical. For example, the
longitudinal axis may be within 0 degrees, 5 degrees, 10 degrees,
30 degrees, or 45 degrees off of vertical. Vertical may be in the
direction of gravity. Coiled tube 102 may have a first end 106 and
a second end 108. Second end 108 may be located at a position
higher than the position of first end 106. Higher may mean away
from the Earth.
[0035] Coiled tube 102 may be characterized by a helix. Coiled tube
102 may be characterized by a helix angle ranging from 2 to 85
degrees. A helix angle of 0 degrees may be horizontal, and a helix
angle of 90 degrees may be vertical. Coiled tube 102 may be
characterized by a pitch, p, which describes the linear distance
between a point on a turn of the coil and the corresponding point
on an adjacent turn of the coil. A turn of the coil may be defined
as a full revolution around the longitudinal axis. Coiled tube 102
may have an overall length, L.sub.o, along longitudinal axis 104.
Coiled tube 102 may be a tube with an inner diameter of 1/8 inch to
10 inches.
[0036] The terms coil and helix may be distinguished based on the
pitch, p, and helix angle, .alpha.. A helix is a type of coil. A
coil can have little gap or no gaps between the coil. As a result,
for a coil, the pitch can be zero or slightly greater than zero. A
coil, however, is not limited to small pitches. For a helix, the
pitch is greater than zero and is not zero. The helix angle,
.alpha., can be a small number for a coil because there may be no
gaps between within the coil. For a helix, the angle is greater
than zero and less than 90. An angle of 90 represents a linear tube
that is neither a coil nor a helix. The coil or the helix may be
right handed or left handed. FIG. 5 shows a coil, and FIG. 7 shows
a helix. Coiled tubes described herein may include both helical
tubes and non-helical, coiled tubes unless the context dictates
otherwise. Embodiments may also exclude helical tubes or
non-helical, coiled tubes.
[0037] FIG. 1B shows an axial view of coiled tube 102. Coiled tube
102, when viewed axially, may appear to be a circle or an ellipse.
The circle may be characterized by an outside diameter (O.D.), the
distance from one outside edge of the circle to the farthest
outside edge of the circle in a direction perpendicular to the
longitudinal axis. The circle may be characterized by an inside
diameter (ID.), the distance from an inside edge of the circle to
the farthest inner edge of the circle in a direction perpendicular
to the longitudinal axis. The difference between the inside
diameter and the outside diameter may be the outer diameter, d, of
the tube. The circle may have a mean diameter that is the mean
average of the inside and outside diameters. If a coiled tube
viewed axially is an ellipse, then the ellipse may be characterized
by a major axis and a minor axis. The helix angle, .alpha., may be
related to the pitch, p, and the mean diameter, D.sub.m, by the
following equation:
.alpha. = tan - 1 ( p .pi. D m ) . ##EQU00001##
[0038] The coiled tube may have a number of turns around the
longitudinal axis ranging between 0.3 and 100, including from 0.3
to 1, from 1 to 10, from 10 to 20, from 20 to 30, from 30 to 50,
from 50 to 75, or from 75 to 100. The coiled tube, if straightened
out, may have an unwound length sufficient to create an average
particle residence time of 0.5 seconds to 20 minutes.
[0039] A plurality of beads may be disposed within the coiled tube.
The plurality of beads may be characterized by a median diameter of
2 mm, 1 mm, or 0.327 mm or any median diameter from 1 .mu.m and 4
mm. A segregated combination of bead median diameters may also be
used. The beads may include glass, borosilicate, ceramics, various
plastics, or polymer materials. Preferably, the beads may include
materials that are chemically resistant to interactions with the
fluids flowing through the tube.
[0040] The tube may be filled with beads of different median
diameters. For example, the bottom of the tube may be filled with a
first plurality of beads of a certain median diameter, and the
remainder of the tube may be filled with beads of monotonically
decreasing or monotonically increasing median diameter. In other
words, the tube may include a gradient of different median
diameters. For example, a first plurality of beads having a median
diameter of 1 mm may be used in combination with a second plurality
of beads having a median diameter of 2 mm. The number of different
pluralities of beads that differ in the median diameter may range
from 2 and 10. Each median diameter for the different pluralities
of beads may be statistically different from the others.
[0041] FIG. 2 shows a system 200 for forming an emulsion and
microparticles. System 200 may include a coiled tube 202. Coiled
tube 202 may be any tube described herein. System 200 may further
include a first inlet 204 fluidly connected to coiled tube 202.
First inlet 204 may be configured to deliver a first fluid to first
end 206 before second end 208. Coiled tube 202 may have its
longitudinal axis aligned with the direction of gravity, as
described herein. Hence, second end 208 may be above first end 206.
The first fluid may be driven by a pump 210. The first fluid may be
any oil stream or any aqueous stream described herein. In some
embodiments, a screen may be located at either or both of first end
206 and second end 208.
[0042] In addition, system 200 may include a second inlet 212
fluidly connected to coiled tube 202. Second inlet 212 may be
configured to deliver a second fluid to first end 206 before second
end 208. The second fluid may be driven by pump 214. The second
fluid may be any oil stream or any aqueous stream described herein.
The second fluid may be a different stream than the first fluid.
The first fluid and second fluid may both enter coiled tube
202.
[0043] A pump, such as pump 210 or pump 214, may be fluidly
connected to the coiled tube. The pump may be configured to drive a
flow of fluid from first end 206 to second end 208. The pump
flowrates may be set to correspond with a Reynolds number from
significantly less than 1 to 10,000, including from 0.1 to 0.5,
from 0.5 to 1, 1 to 100, from 100 to 500, from 500 to 1,000, from
1,000 to 2,000, from 2,000 to 5,000, or from 5,000 to 10,000. The
flow may also be driven without using pumps. For example, flow may
be driven by applying pressure. The pressure may be a positive
pressure, which is applied by forcing compressed air or a
compressed gas to move a fluid from one location to another. The
pressure may be a negative pressure, which is applied by using a
vacuum to move a fluid from one location to another. System 200 may
include a device for applying pressure to the fluid.
[0044] System 200 may include a third inlet 216 fluidly connected
to coiled tube 202. Third inlet 216 may be in closer fluid
communication with second end 208 than first end 206. A fluid
entering through the third inlet may not enter coiled tube 202.
Instead, the fluid entering through third inlet 216 may mix with
the output of coiled tube 202. For example, third inlet 216 may
deliver dilution water to mix with the emulsion formed after mixing
an oil stream and an aqueous stream in the coiled tube. The fluid
from third inlet 216 may be delivered using pump 218. The emulsion
may form microparticles after being diluted with water or other
diluents.
[0045] Third inlet 216 may lead to unit operations for
concentrating microparticles and filtering microparticles. After
being diluted, the microparticles may enter a first stirred tank
reactor 220.
[0046] The outlet of first stirred tank reactor 220 may be pumped
by pump 222. System 200 may include a fourth inlet 224. Fourth
inlet 224 may be in closer fluid communication with second end 208
than first end 206. A fluid entering through the fourth inlet may
not enter coiled tube 202. Instead, the fluid entering through
fourth inlet 224 may mix with the output of first stirred tank
reactor 220. For example, fourth inlet 224 may deliver dilution
water to mix with the output of first stirred tank reactor 220. The
fluid from fourth inlet 224 may be delivered using pump 226. The
emulsion may form microparticles after being diluted with water or
other diluents.
[0047] The mixture of fluid from fourth inlet 224 and the output of
first stirred tank reactor 220 may flow to second stirred tank
reactor 228. Second stirred tank reactor 228 may be fluidly
connected to a plurality of screens. Screen 230 may remove
wastewater and fines. Screen 230 may have a size ranging from 5
.mu.m to 40 .mu.m, including about 25 .mu.m. Fines and wastewater
may pass through screen 230 and be sent to waste outlet 232.
[0048] Second stirred tank reactor 228 may be fluidly connected to
screen 234. Screen 234 may have a size of 50 .mu.m to 250 .mu.m,
including about 100 .mu.m. Process fluid including spheres of a
desired size flow through screen 234 and proceed to a drying step
through outlet 236. Larger size particles are rejected by screen
234. Coiled tube 202 may be fluidly connected to screens 230 and
234 through second stirred tank reactor 228. The plurality of
screens may be in closer fluid communication with second end 208
than first end 206. Screens 230 and 234 may be simultaneously
processing fluid from second stirred tank reactor 228. Flow may
recirculate between screen 230, screen 234, and second stirred tank
reactor 228.
[0049] In some embodiments, coiled tube 202 may be a first coiled
tube out of a plurality of coiled tubes. The first coiled tube may
be coiled around a longitudinal axis. The first coiled tube may be
characterized by a first width in a direction perpendicular to the
longitudinal axis. For example, the first width may be the outside
diameter, inner diameter, or mean diameter in FIG. 1B. The system
may include a second coiled tube. The second coiled tube may
include a second plurality of beads disposed therein. The second
coiled tube may be coaxial with the longitudinal axis. The second
coiled tube may be characterized by a second width in a direction
perpendicular to the longitudinal axis. For example, the second
width may be the corresponding diameter as for the first coiled
tube. The first coiled tube and the second coiled tube are arranged
such that a pair of the first coiled tube and the second coiled
tube may be characterized by a third width perpendicular to the
longitudinal axis. The third width may equal to the first width and
to the second width. The third width may be the corresponding
diameter for the two coiled tubes together. In some embodiments,
the system may include a third coiled tube nested with the two
coiled tubes.
[0050] FIG. 3A and FIG. 3B show a set 300 of three coiled tubes
(coiled tube 302, coiled tube 304, coiled tube 306) nested
together. Although three coiled tubes are shown, any other
plurality of coiled tubes may be nested together. Each coiled tube
may be any tube described herein. For each tube of the plurality of
coiled tubes, the coiled tube may include a first end in region 308
and a second end in region 310. The second end may be disposed at a
position higher than the position of the first end. Set 300 may be
substituted for coiled tube 202 in FIG. 2. For each coiled tube, a
first inlet may be fluidly connected to the coiled tube, where the
first inlet is configured to deliver a first fluid to the first end
before the second end. Also for each coiled tube, a second inlet
may be fluidly connected to the coiled tube, where the second inlet
is configured to deliver a second fluid to the first end before the
second end. A plurality of beads may be disposed within the coiled
tubes. The first inlet, the second inlet, and the plurality of
beads may be any described herein.
[0051] Each coiled tube may be coiled around a longitudinal axis.
Each coiled tube may be characterized by a first width in a
direction perpendicular to the longitudinal axis. The first width
may be the outer diameter, inner diameter, or mean diameter. The
plurality of coiled tubes may be coaxial with the longitudinal
axis. In addition, the plurality of coiled tubes may be
characterized by a second width in a direction perpendicular to the
longitudinal axis. The second width may be the outer diameter,
inner diameter, or mean diameter for the plurality of tubes. The
first width may equal the second width. For example, the second
width may be outer diameter 312.
[0052] Each coiled tube of the plurality of coiled tubes may be
characterized by a first height in the direction of the
longitudinal axis. The plurality of coiled tubes may be
characterized by a second height in the direction of the
longitudinal axis. The first height may be equal to the second
height. Each coiled tube may have the same helix angle, pitch,
length, tube outer diameter, and/or tube inner diameter as the
other coiled tubes. In other words, each coiled tube may be
substantially identical to the other coiled tubes.
III. METHODS
[0053] FIG. 4 shows a method 400 of forming an emulsion. Method 400
may include flowing an oil stream and an aqueous stream into a
coiled tube to form a mixture of an oil phase and an aqueous phase
in the coiled tube (block 402). The coiled tube may be any coiled
tube described herein. Method 400 may be performed using system 200
and/or set 300.
[0054] The oil stream may include a biodegradable polymer. The
biodegradable polymer may include a polylactide, a polyglycolide, a
poly(d,l-lactide-co-glycolide), a polycaprolactone, a
polyorthoester, a copolymer of a polyester and a polyether, or a
copolymer of polylactide and polyethylene glycol. The biodegradable
polymer may exclude any of these polymers or groups of these
polymers. The molecular weight of the biodegradable polymer may be
adjusted to produce a desired pharmacokinetic profile.
[0055] Poly(d,l-lactide-co-glycolide) (PLGA) may have a molecular
weight from 5,000 Da to 7,000 Da, 7,000 Da to 17,000 Da, 17,000 Da
to 20,000 Da, 20,000 Da to 24,000 Da, 24,000 Da to 38,000 Da,
38,000 Da to 40,000 Da, or 40,000 Da to 50,000 Da, in examples.
PLGA may have a molar ratio of lactide to glycolide of 50:50 or
75:25. In some examples, PLGA may have a ratio of lactide to
glycolide ranging from 40:60 to 50:50, from 50:50 to 60:40, from
60:40 to 70:30, from 70:30 to 75:25, or from 75:25 to 90:10. The
ratio of lactide to glycolide may be less than or equal to 50:50,
less than or equal to 60:40, or less than or equal to 75:25, where
less than refers to a smaller proportion of lactide compared to
glycolide. The hydrophobic anion of the organic acid may improve
the release characteristics of some PLGAs but not others.
[0056] Possible PLGAs may include PLGA 502, PLGA 503, PLGA 752, and
PLGA 753. PLGA 502 may be a polymer with a lactide to glycolide
ratio of 50:50, an inherent viscosity from 0.16 to 0.24 dL/g, and a
molecular weight from 7,000 to 17,000 Da. PLGA 503 may be a polymer
with a lactide to glycolide ratio of 50:50, an inherent viscosity
from 0.32 to 0.44 dL/g, and a molecular weight from 24,000 to
38,000 Da. PLGA 752 may be a polymer with a lactide to glycolide
ratio of 75:25, an inherent viscosity from 0.14 to 0.22 dL/g, and a
molecular weight from 4,000 to 15,000 Da. PLGA 753 may be a polymer
with a lactide to glycolide ratio of 75:25, an inherent viscosity
from 0.32 to 0.44 dL/g, and a molecular weight from 24,000 to
38,000 Da. The PLGA polymer may also be acid end-capped or ester
end-capped.
[0057] The oil stream may include a physiologically active
substance The physiologically active substance may be a protein,
peptide compound, or a small molecule. The protein or peptide
compound may include a protein-PEG conjugate or a peptide-PEG
conjugate. The protein or peptide compound may be any protein or
peptide compound described herein. Physiologically active
substances may include those that dissolve in the organic solvent
in the presence of the biodegradable polymer.
[0058] The oil stream may include an organic solvent. The organic
solvent may include methylene chloride, benzyl benzoate,
dichloromethane, chloroform, ethyl ether, ethyl acetate, acetic
acid isopropyl ester (isopropyl acetate), acetic acid sec-butyl
ester, acetophenone, n-amyl acetate, aniline, benzaldehyde,
benzene, benzophenone, benzyl alcohol, benzyl amine, bromobenzene,
bromoform, n-butyl acetate, butyric acid methyl ester, caproic
acid, carbon disulfide, carbon tetrachloride, o-chloroaniline,
chlorobenzene, 1-chlorobutane, chloromethane, m-chlorophenol,
m-cresol, o-cresol, cyanoethane, cyanopropane, cyclohexanol,
cyclohexanone, 1,2-dibromoethane, dibromomethane, dibutyl amine,
m-dichlorobenzene, o-dichlorobenzene, 1,1-dichloroethane,
1,2-dichloroethane, dichlorofluoromethane, diethyl carbonate,
diethyl malonate, diethyl sulfide, diethylene glycol dibutyl ether,
diisobutyl ketone, diisopropyl sulfide, dimethyl phthalate,
dimethyl sulfate, dimethyl sulfide, N,N-dimethylaniline, enanthic
acid, ethyl acetoacetate, ethyl benzoate, ethyl propionate,
ethylbenzene, ethylene glycol monobutyl ether acetate, exxate 600,
exxate 800, exxate 900, fluorobenzene, furan,
hexamethylphosphoramide, 1-hexanol, n-hexyl acetate, isoamyl
alcohol (3-methyl-1-butanol), isobutyl acetate, methoxybenzene,
methyl amyl ketone, methyl benzoate, methyl formate, methyl isoamyl
ketone, methyl isobutenyl ketone, methyl isobutyl ketone, methyl
n-butyl ketone, methyl propyl ketone, 4-methyl-2-pentanol,
N-methylaniline, nitrobenzene, nitroethane, 1-nitropropane,
2-nitropropane, 1-octanol, 2-octanol, 1-pentanol, 3-pentanone,
2-phenylethanol, n-propyl acetate, quinoline, styrene,
1,1,2,2-tetrachloroethane, 1,1,2,2-tetrachloroethylene, toluene,
1,1,1-trichloroethane, 1,1,2-trichloroethane,
1,1,2-trichloroethylene, trifluoromethane, valeric acid, m-xylene,
o-xylene, p-xylene, 2,4-xylenol, or mixtures thereof. The organic
solvent may exclude any solvent or any groups of solvents.
[0059] Methods may include a mixture of solvents. The mixture of
solvents may include a solvent that is miscible in water, but the
mixture of solvents may be immiscible in water. For examples, a
water-miscible solvent such as dimethyl sulfoxide (DMSO), methanol,
dimethylformamide (DMF), acetonitrile, tetrahydrofuran, or mixtures
thereof may be added to the water immiscible solvent.
[0060] The oil stream may include a hydrophobic anion. The
hydrophobic anion may include anions associated with the
hydrophobic organic acids. For example, the hydrophobic anion may
include a pamoate anion, a docusate anion, or a furoate anion. In
these or other examples, the hydrophobic anion may be a fatty acid
anion, a phospholipid anion, a polystyrene sulfonate anion, or
mixtures thereof. The phospholipid of the phospholipid anion may
include phosphatidylcholine, phosphatidylglycerol,
phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine,
phosphocholine, or mixtures thereof. The hydrophobic anion may also
exclude any anion described or any group of anions described. The
hydrophobic anion may attach to a specific side chain on the
protein or it may attach to multiple side chains on the protein.
The hydrophobic anion may have a logP greater than 1. The logP is
the water-octanol partition coefficient and may be defined as the
logarithm of the concentration of the protein salt in octanol to
the concentration of the protein salt in water. A logP greater than
1 may result in a concentration in octanol that is 10 times greater
than that in water. The water-octanol partition coefficient may be
useful in comparing different molecules for their ability to
partition into a hydrophobic phase, when the molecules themselves
may be amphipathic. Methods may also include adding cationic
detergents, such as dodecylamine hydrochloride or
cetyltrimethylammonium bromide (CTAB), which may counter the charge
of negatively charged peptides and may increase the
hydrophobicity.
[0061] The aqueous stream may include water and an emulsion
stabilizer such as polyvinyl alcohol (PVA), may contain some
organic solvent, buffers, salts, and/or hydrophobic ions. The
aqueous stream may contain a physiologically active substance.
Physiologically active substances may include water soluble
proteins, peptides, or small molecules. The physiologically active
substance may also include PEG-conjugates or any physiologically
active substance described herein.
[0062] At block 404, method 400 may also include flowing the
mixture in the coiled tube against gravity and under laminar
conditions. The flow of the mixture in the coiled tube may have a
Reynolds number ranging from significantly less than 1 to 10,000. A
plurality of beads may be disposed within the coiled tube. The
beads may be any beads described herein. The flow in the coiled
tube may reduce, minimize, or eliminate chaotic convection
mixing.
[0063] At block 406, method 400 may further include mixing the oil
phase and the aqueous phase in the coiled tube until the emulsion
is formed. The emulsion formed may be homogenous. Homogeneity of
the emulsion may be determined by the particle size distribution.
Particle size distribution profiles may be predominantly unimodal.
Particles that are not part of the unimodal particle size
distribution profile may be no more than 25 vol % of the particles.
For example, microparticles with diameters smaller than the lower
end of the unimodal particle size distribution may total less than
25 vol %, less than 10 vol %, less than 5 vol, %, less than 2 vol.
%, or less than 1 vol. % of the total.
[0064] Method 400 may further include diluting the emulsion with
water. Method 400 may also include forming microparticles from the
emulsion. Forming microparticles may include removing water and
solvent from the emulsion. The microparticles may include a protein
or peptide compound, a PEG conjugate or a small molecule. The
microparticles may have a median diameter in a range from 1 to 99
.mu.m. The microparticles may be microspheres. The diameter of the
microparticles may be chosen based on the route of administration.
When the microparticles are intended to be implanted in the body as
a solid, the diameter may be in the range of less than 1 .mu.m and
several centimeters. The upper range may be an inch. When the
microparticles are intended to be injected as a suspension under
the skin or into the muscle, the microparticles may have a smaller
diameter and may be based on the dimensions of a needle. The inner
diameter of needles used to inject suspensions under the skin or
into the muscle may be in the range of several hundred to several
thousands of micrometers. For example, a needle of gauge 7 has an
inner diameter of approximately 3.81 mm. A needle of gauge 34 has
an inner diameter of approximately 0.0826 mm. Microparticles
injected using needles in the gauge range of 7 and 34 may have
diameters in the range of less than 1 .mu.m and 3,000 .mu.m.
Diameters of microparticles for narrower gauge needles may range
from 10 .mu.m to 90 .mu.m, 20 .mu.m to 70 .mu.m, or 25 .mu.m to 63
.mu.m.
IV. EXAMPLES
[0065] For the examples, the Reynolds number was calculated in two
different ways. For the helical emulsifiers without packing, the
Reynolds number, Re, may be related to the fluid velocity, V, the
diameter of the tube, D.sub.tube, and the kinematic viscosity of
the fluid, v, by the following equation:
Re = VD tube v . ##EQU00002##
[0066] For the packed helical emulsifiers, the Reynolds number may
be related to the superficial fluid velocity, V, the average
particle diameter of the packing, D.sub.p, and the kinematic
viscosity of the fluid, v, by the following equation:
Re = VD p v . ##EQU00003##
[0067] The critical Reynolds number, the Reynolds number that
corresponds with a maximum in the laminar flow regime, for straight
tubes is 2100. However, for coiled tubes, when a fluid is forced to
follow a curved path, centrifugal forces may create Dean vortices,
or a secondary flow perpendicular to the axial, primary flow. This
secondary flow may have a stabilizing effect. Flow through a coil,
therefore, may suppress turbulent fluctuations and smooths the
emergence of turbulence, increasing the value of the critical
Reynolds number, as compared to that of as straight pipe. The
critical Reynolds number through a coiled tube, Re.sub.cr, may be
related to the diameter of the tube, D.sub.tube, and the diameter
of the coil, D.sub.c, by the following equation:
Re cr = 2100 ( 1 + 12 D tube D c ) . ##EQU00004##
This stabilizing effect allows for larger diameters of process
equipment, or higher flow rates, and therefore higher throughput
and shorter processing time while still allowing for gentle mixing
of the emulsion.
[0068] In order to report a practical range of possible Reynolds
numbers through the helical emulsifiers in these examples, two
kinematic viscosities were used. An upper bound was determined by
assuming the kinematic viscosity to be that of pure water at
20.degree. C., 1.002 centistokes. The kinematic viscosity of the
emulsion was also experimentally determined using a Cannon-Fenske
viscometer. The experimental viscosity of the emulsion, 17.6
centistokes, was significantly greater than that of water yielding
a lower bound on the calculated Reynolds number range for each
example.
A. Examples 1-3
[0069] Examples 1-3 show the viability of the helical emulsifier
for making an emulsion that can be used to make microspheres. The
particle size can be tuned by adjusting the number of coils or the
diameter of the helix. The particle size increases as the number of
coils increases.
Example 1
[0070] A coiled tube mixer, shown in FIG. 5, for the preparation of
polymer microspheres was created by wrapping 1/8 inch PTFE tubing (
1/16'' inner diameter) around a 1.1-inch diameter cylinder for a
total of 35 complete coils. The resulting coil has a mean diameter
of 1.2 inches and a helix angle of 2 degrees.
[0071] These dimensions increase the critical Reynolds number to a
value of 7,851. A tee was connected at the inlet for the
introduction of two unmixed liquid phases. A second tee was
connected to the outlet of the helix for the introduction of an
emulsion dilution phase.
[0072] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
solution (Water Phase) was made by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 milliliters of deionized water
overnight. A dilution phase was prepared by tempering deionized
water to a temperature of 19.degree. C. The Oil Phase was pumped
through the assembly at a rate of 61 ml/min while the Water Phase
was concurrently pumped through the same assembly at a rate of 160
ml/min. The resulting Reynolds number through the apparatus was
laminar, falling between 168 and 2,948, which is well below the
critical Reynolds number of 7,851 for this mixer. Upon leaving the
helical apparatus, the emulsion was diluted using deionized water
pumped at a rate of 1,280 ml/min. The particle size distribution of
the emulsion was then analyzed using laser diffraction (Beckman
Coulter LS 13 320). The median particle size (d50) of the emulsion
was found to be 65 microns with a d10 of 31 .mu.m and a d90 of 130
.mu.m. The percentage of particles between 25 and 63 microns was
45% by volume.
Example 2
[0073] A helical mixer for the preparation of polymer microspheres
was created by wrapping 1/8 inch PTFE tubing ( 1/16'' inner
diameter) around a 1.1-inch diameter cylinder for a total of 70
complete coils. The resulting helix has a mean diameter of 1.3
inches and a helix angle of 2 degrees. In the current example,
these dimensions increase the critical Reynolds number to a value
of 7,625. A tee was connected at the inlet for the introduction of
two unmixed liquid phases. A second tee was connected to the outlet
of the helix for the introduction of an emulsion dilution
phase.
[0074] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
solution (Water Phase) was made by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 milliliters of deionized water
overnight. A dilution phase was prepared by tempering deionized
water to a temperature of 19.degree. C. The Oil Phase was pumped
through the assembly at a rate of 30 ml/min while the Water Phase
was concurrently pumped through the same assembly at a rate of 160
ml/min. The resulting Reynolds number through the apparatus was
laminar, falling between 144 and 2,535, which is well below the
critical Reynolds number of 7,625 for this mixer. Upon leaving the
helical apparatus, the emulsion was diluted using deionized water
pumped at a rate of 1,280 ml/min. The particle size distribution of
the emulsion was then analyzed using laser diffraction (Beckman
Coulter LS 13 320). The median particle size (d50) of the emulsion
was found to be 96 microns with a d10 of 51 .mu.m and a d90 of 133
.mu.m. The percentage of particles between 25 and 63 microns was
14% by volume.
Example 3
[0075] A helical mixer for the preparation of polymer microspheres
was created by wrapping 1/8 inch PTFE tubing ( 1/16'' inner
diameter) around a 0.63-inch diameter cylinder for a total of 55
complete coils. The resulting helix has a mean diameter of 0.75
inches and a helix angle of 3 degrees. In the current example,
these dimensions increase the critical Reynolds number to a value
of 9,375. A tee was connected at the inlet for the introduction of
two unmixed liquid phases. A second tee was connected to the outlet
of the helix for the introduction of an emulsion dilution
phase.
[0076] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
solution (Water Phase) was made by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 milliliters of deionized water
overnight. A dilution phase was prepared by tempering deionized
water to a temperature of 19.degree. C. The Oil Phase was pumped
through the assembly at a rate of 61 ml/min while the Water Phase
was concurrently pumped through the same assembly at a rate of 160
ml/min. The resulting Reynolds number through the apparatus was
laminar, falling between 168 and 2,948, which is well below the
critical Reynolds number of 9,375 for this mixer. Upon leaving the
helical apparatus, the emulsion was diluted using deionized water
pumped at a rate of 1,230 ml/min. The particle size distribution of
the emulsion was then analyzed using laser diffraction (Beckman
Coulter LS 13 320). The median particle size (d50) of the emulsion
was found to be 88 microns with a d10 of 40 .mu.m and a d90 of 320
.mu.m. The percentage of particles between 25 and 63 .mu.m was 21%
by volume.
Examples 1-3 Summary:
TABLE-US-00001 [0077] TABLE 1 Resulting Particle Size Distribution
of Process Parameters Microspheres Helical Emulsifier Total Flow
Particles Mean Tubing Through Measured between 25 um Number
Diameter ID Emulsifier Reynolds Median d10 d90 and 63 um Example of
Coils (in) (in) (ml/min) Number (.mu.m) (.mu.m) (.mu.m) (vol %) 1
35 1.18 0.063 221 168 65 31 130 45 2 70 1.26 0.063 190 144 96 51
133 14 3 55 0.75 0.063 221 168 88 40 320 21
[0078] These examples show that the helical mixer can be used to
make an emulsion that is appropriate for forming microspheres. The
resulting particle size distribution is larger and more variable
than desired for injection through large gauge (small diameter)
needles. For injection through small diameter needles, a particle
size range from 25 to about 63 .mu.m is desired. The percent of
material in the desired particle size range is less than 45% for
these examples. These data also show that the particle size
distribution can be adjusted by changing both the number of coils
and the mean diameter of the coils.
B. Examples 4-6
[0079] Examples 4-6 show continued functionality of the helical
mixer (without packing with beads) for making an emulsion that can
be used to make microspheres. In these examples, the particle size
is adjusted by using different flow rates. Faster flow rates result
in smaller particle size.
[0080] A helical mixer for the preparation of polymer microspheres
was created by wrapping 1/8 inch PTFE tubing ( 1/16'' inner
diameter) around a 0.62-inch diameter cylinder for a total of 22
complete coils. The resulting helix has a mean diameter of 0.75
inches and a helix angle of 3 degrees. For this apparatus, these
dimensions increase the critical Reynolds number to a value of
9,375. A tee was connected at the inlet for the introduction of two
unmixed liquid phases. A second tee was connected to the outlet of
the helix for the introduction of an emulsion dilution phase. This
assembly was used during the following three examples.
Example 4
[0081] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
solution (Water Phase) was made by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 milliliters of deionized water
overnight. A dilution phase was prepared by tempering deionized
water to a temperature of 19.degree. C. The Oil Phase was pumped
through the helical apparatus at a rate of 61 ml/min while the
[0082] Water Phase was concurrently pumped through the helical
apparatus at a rate of 160 ml/min. The resulting Reynolds number
through the apparatus was laminar, falling between 168 and 2,948,
which is well below the critical Reynolds number of 9,375 for this
mixer. Upon leaving the helical apparatus the emulsion was diluted
using deionized water pumped at a rate of 1230 ml/min. The particle
size distribution of the emulsion was then analyzed using laser
diffraction (Beckman Coulter LS 13 320). The volumetric median
particle size of the emulsion was found to be 62 .mu.m with a d10
of 25 .mu.m and a d90 of 119 .mu.m. The percentage of particles
between 25 and 63 microns was 44% by volume.
Example 5
[0083] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
solution (Water Phase) was made by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 milliliters of deionized water
overnight. A dilution phase was prepared by tempering deionized
water to a temperature of 19.degree. C. The Oil Phase was pumped
through the helical apparatus at a rate of 61 ml/min while the
Water Phase was concurrently pumped through the helical apparatus
at a rate of 250 ml/min. The resulting Reynolds number through the
apparatus was laminar, falling between 236 and 4,149, which is well
below the critical Reynolds number of 9,375 for this mixer. Upon
leaving the helical apparatus the emulsion was diluted using
deionized water pumped at a rate of 1,230 ml/min. The particle size
distribution of the emulsion was then analyzed using laser
diffraction (Beckman Coulter LS 13 320). The volumetric median
particle size of the emulsion was found to be 41 .mu.m with a d10
of 13 .mu.m and a d90 of 90 .mu.m. The percentage of particles
between 25 and 63 microns was 59% by volume.
Example 6
[0084] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (-19C). A second solution (Water
Phase) was made by dissolving 2.67 grams of poly(vinyl alcohol)
(PVA) in 267 milliliters of deionized water overnight. A dilution
phase was prepared by tempering deionized water to a temperature of
19.degree. C. The Oil Phase was pumped through the helical
apparatus at a rate of 9 ml/min, while the Water Phase and the
dilution water were concurrently pumped through the helical
apparatus at a rate of 21 ml/min and 120 ml/min, respectively. The
resulting Reynolds number through the apparatus was laminar,
falling between 114 and 2,001, which is well below the critical
Reynolds number of 9,375 for this mixer. The particle size
distribution of the emulsion was then analyzed using laser
diffraction (Beckman Coulter LS 13 320). The median particle size
of the emulsion was found to be 164 microns with a d10 of 83 .mu.m
and a d90 of 221 .mu.m. The percentage of particles between 25 and
63 microns was 3.5% by volume.
Examples 4-6 Summary:
TABLE-US-00002 [0085] TABLE 2 Resulting Particle Size Distribution
of Process Parameters Microspheres Helical Emulsifier Total Flow
Particles Mean Tubing Through Measured between 25 um Number
Diameter ID Emulsifier Reynolds Median d10 d90 and 63 um Example of
Coils (in) (in) (ml/min) Number (.mu.m) (.mu.m) (.mu.m) (vol %) 4
22 0.75 0.063 221 168 62 25 119 44 5 22 0.75 0.063 311 236 41 13 90
59 6 22 0.75 0.063 150 114 164 83 221 3.5
[0086] These examples show that the helical mixer can be used to
make an emulsion that is appropriate for forming microspheres. The
flow rate through the emulsifier directly affects the particle size
distribution, with faster flow resulting in smaller particles. The
particle size is larger and more variable than desired for
injection through large gauge (small diameter) needles. In these
examples, the percent of material in the desired particle size
range is less than or equal to 59% by volume.
C. Examples 7-17
[0087] Examples 7-17 show continued functionality of the helical
mixer (without packing with beads) for making an emulsion that can
be used to make microspheres. In these examples, the particle size
is adjusted by using screens on the entrance and/or exit of the
mixer to adjust the particle size distribution.
[0088] A helical mixer for the preparation of polymer microspheres
was created by wrapping 1/8 inch PTFE tubing ( 1/16'' inner
diameter) around a 0.62-inch diameter cylinder for a total of 22
complete coils. The resulting helix has a mean diameter of 0.75
inches and a helix angle of 3 degrees. For this apparatus, these
dimensions increase the critical Reynolds number to a value of
9,375. A tee was connected at the inlet for the introduction of two
unmixed liquid phases. A second tee was connected to the outlet of
the helix for the introduction of an emulsion dilution phase. This
assembly was used during the following eleven examples.
Example 7
[0089] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
solution (Water Phase) was made by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 milliliters of deionized water
overnight. A dilution phase was prepared by tempering deionized
water to a temperature of 19.degree. C. A screen was placed between
the inlet tee and the helical apparatus with a mesh size of 120 by
500 (35 .mu.m approximate retention). The Oil Phase was pumped
through the assembly at a rate of 61 ml/min while the Water Phase
was concurrently pumped through the same assembly at a rate of 190
ml/min. The resulting Reynolds number through the apparatus was
laminar, falling between 191 and 3,349, which is well below the
critical Reynolds number of 9,375 for this mixer. Upon leaving the
helical apparatus, the emulsion was diluted using deionized water
pumped at a rate of 1,200 ml/min. The particle size distribution of
the emulsion was then analyzed using laser diffraction (Beckman
Coulter LS 13 320). The median particle size (d50) of the emulsion
was found to be 45 microns with a d10 of 20 .mu.m and a d90 of 80
.mu.m. The percentage of particles between 25 and 63 microns was
65% by volume.
Example 8
[0090] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
solution (Water Phase) was made by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 milliliters of deionized water
overnight. A dilution phase was prepared by tempering deionized
water to a temperature of 19.degree. C. A screen was placed between
the inlet tee and the helical apparatus with a mesh size of 120 by
500 (35 .mu.m approximate retention). The Oil Phase was pumped
through the assembly at a rate of 61 ml/min while the Water Phase
was concurrently pumped through the same assembly at a rate of 160
ml/min. The resulting Reynolds number through the apparatus was
laminar, falling between 168 and 2,948, which is well below the
critical Reynolds number of 9,375 for this mixer. Upon leaving the
helical apparatus, the emulsion was diluted using deionized water
pumped at a rate of 1,200 ml/min. The particle size distribution of
the emulsion was then analyzed using laser diffraction (Beckman
Coulter LS 13 320). The median particle size (d50) of the emulsion
was found to be 48 microns with a d10 of 21 .mu.m and a d90 of 75
.mu.m. The percentage of particles between 25 and 63 microns was
67% by volume.
Example 9
[0091] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
solution (Water Phase) was made by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 milliliters of deionized water
overnight. A dilution phase was prepared by tempering deionized
water to a temperature of 19.degree. C. A screen was placed between
the outlet of the helical apparatus and the dilution tee with a
mesh size of 100 (140 .mu.m approximate retention). The Oil Phase
was pumped through the assembly at a rate of 61 ml/min while the
Water Phase was concurrently pumped through the same assembly at a
rate of 160 ml/min. The resulting Reynolds number through the
apparatus was laminar, falling between 168 and 2,948, which is well
below the critical Reynolds number of 9,375 for this mixer. Upon
leaving the helical apparatus, the emulsion was diluted using
deionized water pumped at a rate of 1,200 ml/min. The particle size
distribution of the emulsion was then analyzed using laser
diffraction (Beckman Coulter LS 13 320). The median particle size
(d50) of the emulsion was found to be 62 .mu.m with a d10 of 25
.mu.m and a d90 of 122 .mu.m. The percentage of particles between
25 and 63 microns was 44% by volume.
Example 10
[0092] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
solution (Water Phase) was made by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 milliliters of deionized water
overnight. A dilution phase was prepared by tempering deionized
water to a temperature of 19.degree. C. A screen was placed between
the inlet tee and the helical apparatus, as well as between the
outlet of the helical apparatus and the dilution tee. Both screens
had a mesh size of 100 (140 .mu.m approximate retention). The Oil
Phase was pumped through the assembly at a rate of 61 ml/min while
the Water Phase was concurrently pumped through the same assembly
at a rate of 200 ml/min. The resulting Reynolds number through the
apparatus was laminar, falling between 198 and 3,482, which is well
below the critical Reynolds number of 9,375 for this mixer. Upon
leaving the helical apparatus, the emulsion was diluted using
deionized water pumped at a rate of 1200 ml/min. The particle size
distribution of the emulsion was then analyzed using laser
diffraction (Beckman Coulter LS 13 320). The median particle size
(d50) of the emulsion was found to be 44 microns with a d10 of 15
.mu.m and a d90 of 69 .mu.m. The percentage of particles between 25
and 63 microns was 68% by volume.
Example 11
[0093] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
solution (Water Phase) was made by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 milliliters of deionized water
overnight. A dilution phase was prepared by tempering deionized
water to a temperature of 19.degree. C. A screen was placed between
the inlet tee and the helical apparatus with a mesh size of 100
(140 .mu.m approximate retention). The Oil Phase was pumped through
the assembly at a rate of 61 ml/min while the Water Phase was
concurrently pumped through the same assembly at a rate of 160
ml/min. The resulting Reynolds number through the apparatus was
laminar, falling between 168 and 2,948, which is well below the
critical Reynolds number of 9,375 for this mixer. Upon leaving the
helical apparatus, the emulsion was diluted using deionized water
pumped at a rate of 1,200 ml/min. The particle size distribution of
the emulsion was then analyzed using laser diffraction (Beckman
Coulter LS 13 320). The median particle size (d50) of the emulsion
was found to be 51 microns with a d10 of 21 .mu.m and a d90 of 73
.mu.m. The percentage of particles between 25 and 63 microns was
64% by volume.
Example 12
[0094] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
solution (Water Phase) was made by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 milliliters of deionized water
overnight. A dilution phase was prepared by tempering deionized
water to a temperature of 19.degree. C. A screen was placed between
the inlet tee and the helical apparatus, as well as between the
outlet of the helical apparatus and the dilution tee. Both screens
had a mesh size of 100 (140 .mu.m approximate retention). The Oil
Phase was pumped through the assembly at a rate of 59 ml/min while
the Water Phase was concurrently pumped through the same assembly
at a rate of 150 ml/min. The resulting Reynolds number through the
apparatus was laminar, falling between 159 and 2,788, which is well
below the critical Reynolds number of 9,375 for this mixer. Upon
leaving the helical apparatus, the emulsion was diluted using
deionized water pumped at a rate of 1,100 ml/min. The particle size
distribution of the emulsion was then analyzed using laser
diffraction (Beckman Coulter LS 13 320). The median particle size
(d50) of the emulsion was found to be 45 microns with a d10 of 15
.mu.m and a d90 of 70 .mu.m. The percentage of particles between 25
and 63 microns was 67% by volume.
Example 13
[0095] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
solution (Water Phase) was made by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 milliliters of deionized water
overnight. A dilution phase was prepared by tempering deionized
water to a temperature of 19.degree. C. A screen was placed between
the inlet tee and the helical apparatus, as well as between the
outlet of the helical apparatus and the dilution tee. Both screens
had a mesh size of 100 (140 .mu.m approximate retention). The Oil
Phase was pumped through the assembly at a rate of 51 ml/min while
the Water Phase was concurrently pumped through the same assembly
at a rate of 140 ml/min. The resulting Reynolds number through the
apparatus was laminar, falling between 148 and 2,548, which is well
below the critical Reynolds number of 9,375 for this mixer. Upon
leaving the helical apparatus, the emulsion was diluted using
deionized water pumped at a rate of 1,000 ml/min. The particle size
distribution of the emulsion was then analyzed using laser
diffraction (Beckman Coulter LS 13 320). The median particle size
(d50) of the emulsion was found to be 55 microns with a d10 of 21
.mu.m and a d90 of 84 .mu.m. The percentage of particles between 25
and 63 microns was 55% by volume.
Example 14
[0096] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
solution (Water Phase) was made by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 milliliters of deionized water
overnight. A dilution phase was prepared by tempering deionized
water to a temperature of 19.degree. C. A screen was placed between
the inlet tee and the helical apparatus, as well as between the
outlet of the helical apparatus and the dilution tee. Both screens
had a mesh size of 100 (140 .mu.m approximate retention). The Oil
Phase was pumped through the assembly at a rate of 50 ml/min while
the Water Phase was concurrently pumped through the same assembly
at a rate of 120 ml/min. The resulting Reynolds number through the
apparatus was laminar, falling between 129 and 2,268, which is well
below the critical Reynolds number of 9,375 for this mixer. Upon
leaving the helical apparatus, the emulsion was diluted using
deionized water pumped at a rate of 900 ml/min. The particle size
distribution of the emulsion was then analyzed using laser
diffraction (Beckman Coulter LS 13 320). The median particle size
(d50) of the emulsion was found to be 59 microns with a d10 of 24
.mu.m m and a d90 of 92 .mu.m. The percentage of particles between
25 and 63 microns was 50% by volume.
Example 15
[0097] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
solution (Water Phase) was made by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 milliliters of deionized water
overnight. A dilution phase was prepared by tempering deionized
water to a temperature of 19.degree. C. A screen was placed between
the outlet of the helical apparatus and the dilution tee with a
mesh size of 100 (140 .mu.m approximate retention). The Oil Phase
was pumped through the assembly at a rate of 61 ml/min while the
Water Phase was concurrently pumped through the same assembly at a
rate of 160 ml/min. The resulting Reynolds number through the
apparatus was laminar, falling between 168 and 2,948, which is well
below the critical Reynolds number of 9,375 for this mixer. Upon
leaving the helical apparatus, the emulsion was diluted using
deionized water pumped at a rate of 1,230 ml/min. The particle size
distribution of the emulsion was then analyzed using laser
diffraction (Beckman Coulter LS 13 320). The median particle size
(d50) of the emulsion was found to be 50 microns with a d10 of 18
.mu.m and a d90 of 81 .mu.m The percentage of particles between 25
and 63 microns was 59% by volume.
Example 16
[0098] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
solution (Water Phase) was made by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 milliliters of deionized water
overnight. A dilution phase was prepared by tempering deionized
water to a temperature of 19.degree. C. A screen was placed between
the inlet tee and the helical apparatus with a mesh size of 100
(140 .mu.m approximate retention). The Oil Phase was pumped through
the assembly at a rate of 61 ml/min while the Water Phase was
concurrently pumped through the same assembly at a rate of 160
ml/min. The resulting Reynolds number through the apparatus was
laminar, falling between 168 and 2,948, which is well below the
critical Reynolds number of 9,375 for this mixer. Upon leaving the
helical apparatus, the emulsion was diluted using deionized water
pumped at a rate of 1230 ml/min. The particle size distribution of
the emulsion was then analyzed using laser diffraction (Beckman
Coulter LS 13 320). The median particle size (d50) of the emulsion
was found to be 55 microns with a d10 of 24 .mu.m and a d90 of 92
.mu.m. The percentage of particles between 25 and 63 microns was
59% by volume.
Example 17
[0099] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
solution (Water Phase) was made by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 milliliters of deionized water
overnight. A dilution phase was prepared by tempering deionized
water to a temperature of 19.degree. C. A screen was placed between
the inlet tee and the helical apparatus, as well as between the
outlet of the helical apparatus and the dilution tee. Both screens
had a mesh size of 100 (140 .mu.m approximate retention). The Oil
Phase was pumped through the assembly at a rate of 61 ml/min while
the Water Phase was concurrently pumped through the same assembly
at a rate of 160 ml/min. The resulting Reynolds number through the
apparatus was laminar, falling between 168 and 2,948, which is well
below the critical Reynolds number of 9,375 for this mixer. Upon
leaving the helical apparatus, the emulsion was diluted using
deionized water pumped at a rate of 1,230 ml/min. The particle size
distribution of the emulsion was then analyzed using laser
diffraction (Beckman Coulter LS 13 320). The median particle size
(d50) of the emulsion was found to be 39 microns with a d10 of 10
.mu.m and a d90 of 62 .mu.m. The percentage of particles between 25
and 63 microns was 68% by volume.
Examples 7-17 Summary:
TABLE-US-00003 [0100] TABLE 3 Particles Total Flow between Through
Measured 25 um and Screen Mesh Emulsifier Reynolds Median d10 d90
63 um Example (inlet/outlet) (ml/min) Number (.mu.m) (.mu.m)
(.mu.m) (vol %) 7 120 .times. 500/None 251 191 45 20 80 65 8 120
.times. 500/None 221 168 48 21 75 67 9 None/100 .sup. 221 168 62 25
122 44 10 100/100 261 198 44 15 69 68 11 .sup. 100/None 221 168 51
21 73 64 12 100/100 209 159 45 15 70 67 13 100/100 191 145 55 21 84
55 14 100/100 170 129 59 24 92 50 15 None/100 .sup. 221 168 50 18
81 59 16 .sup. 100/None 221 168 55 24 92 59 17 100/100 221 168 39
10 62 68
[0101] These examples show that the helical mixer can be used to
make an emulsion that is appropriate for forming microspheres.
Using screens on the entrance and/or exit of the helical mixer
affects the resulting particle size distribution, by reducing the
mean particle size distribution compared to an emulsion made
without screens. Using screens was observed to result in a tighter
particle size distribution and more material in the desired
particle size range (25-63 .mu.m) than without screens. In these
examples the volume percent of microspheres with the desired
particle size distribution is less than or equal to 68%.
D. Examples 18-30
[0102] Examples 18-30 use a triple helical mixer. Three identical,
intertwined, right-handed helical mixers, shown in FIG. 7, for the
preparation of polymer microspheres were built out of three pieces
of 0.75-inch outer diameter, 0.065-inch wall 316L stainless steel
tubing, each piece of tubing was wound around one another in a
characteristic right-handed triple helix. The length of the tubing
was sufficient so that each helix was twisted into one complete
coil (i.e. a projection onto a plane perpendicular to the axis
would yield a complete circle). Each individual helix has a helical
length of 12.16 inches and a mean diameter of 0.885 inches, with 45
degree 0.75-inch sanitary elbows welded to each end. The three
mixers were attached together by a 2.5-inch diameter mounting plate
near each end of the assembly. The complete apparatus has an
overall length of 15.5 inches and a diameter of 2.7 inches at the
widest point. For the following examples, one, two, or three of the
helices were connected to two peristaltic pumps using 1/4'' tubing
and compression fittings, for the introduction of two unmixed
liquid phases. When a single helix was used, a tee was placed after
the outlet for the introduction of a liquid dilution phase. When
multiple helices were used, the mixer outlets were first recombined
using 1/4'' tubing and compression fittings, then fed into a tee
for the introduction of a liquid dilution phase.
Example 18 (No Beads)
[0103] An emulsion was made using the emulsifier described above
without packing with beads. An 8.8% w/w polymer-in-oil phase (Oil
Phase) was prepared by dissolving 25.5 grams of 50:50
poly(lactic-co-glycolic acid) (PLGA) (Resomer Select 5050 DLG 2A,
Lot number LP1321, Evonik Corp.) in 265.5 grams of dichloromethane
(DCM).The solution was allowed to stir overnight at room
temperature (.about.19.degree. C.). A second phase, the Water
Phase, was prepared by dissolving 8 grams of poly(vinyl alcohol)
(PVA) in 800 grams of deionized water. The solution was stirred for
one hour at 60.degree. C. then stirred overnight at room
temperature. A dilution water phase was made by setting the
temperature of a vessel containing deionized water to 19.degree. C.
One of the three helical mixers, having 100 mesh screens (140 .mu.m
approximate retention) on both the inlet and outlet, was used.
Approximately 140 ml of the Oil Phase and 330 ml of the Water Phase
were pumped simultaneously upwards, against gravity, through the
apparatus at rates of 9 ml/min and 21 ml/min, respectively. The
resulting Reynolds number through the apparatus was laminar,
falling between 2 and 40. The outgoing emulsion was met with the
dilution water phase at a flowrate of 168 ml/min. Laser diffraction
(Beckman Coulter LS 13 320) was used to analyze the particle size
distribution of the resulting emulsion. The volumetric median
particle size was found to be 169 .mu.m with a d10 of 79 .mu.m and
d90 of 300 .mu.m.
[0104] This example shows that the helical mixer can be used to
make an emulsion that is appropriate for forming microspheres, at a
larger microsphere scale than with the previous examples. The
particle size distribution had only 4.3% of the particles in the
desired 25 to 63 .mu.m range.
Example 19 (No Beads, Faster Flow)
[0105] This example is similar to Example 18, except that the flow
through the helical mixer is faster. An 8.8% w/w polymer-in-oil oil
phase (Oil Phase) was prepared by dissolving 51 grams of 50:50
poly(lactic-co-glycolic acid) (PLGA) (Resomer Select 5050 DLG 2A,
Lot number LP1321, Evonik Corp.) in 528 grams of dichloromethane
(DCM).The solution was allowed to stir overnight at room
temperature (.about.19.degree. C.). A second phase, the Water
Phase, was prepared by dissolving 8 grams of poly(vinyl alcohol)
(PVA) in 800 grams of deionized water. The solution was stirred for
one hour at 60.degree. C. then stirred overnight at room
temperature. A dilution water phase was made by setting the
temperature of a vessel containing deionized water to 19.degree. C.
One of the three helical mixers, having 100 mesh screens (140 .mu.m
approximate retention) on both the inlet and outlet, was used.
Approximately 120 ml of the Oil Phase and 400 ml of the Water Phase
were pumped simultaneously upwards, against gravity, through the
helical mixer at rates of 61 ml/min and 186 ml/min, respectively.
The resulting Reynolds number through the apparatus was laminar,
falling between 19 and 332. The outgoing emulsion was met with the
dilution water phase at a flowrate of 542 ml/min. Laser diffraction
(Beckman Coulter LS 13 320) was used to analyze the particle size
distribution of the resulting emulsion. The volumetric median
particle size was found to be 67.52 .mu.m with a d10 of 29.6 .mu.m
and d90 of 74.5 .mu.m.
[0106] This example shows that the helical mixer can be used to
make an emulsion that is appropriate for forming microspheres, at a
larger microsphere scale. The particle size distribution was better
than the previous example, with 36% of the particles in the desired
range.
Example 20 (Large Beads)
[0107] For this example, a helical mixer packed with 2 mm glass
beads was used to create an emulsion that can be used to make
microspheres. Using packing allows reduced flow rates, which reduce
convection currents, resulting in a less turbulent environment for
the emulsion droplets and the fragile physiologically active
substances
[0108] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 51 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1321, Evonik
Corp.) in 529 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
phase, the Water Phase, was prepared by dissolving 32 grams of
poly(vinyl alcohol) (PVA) in 3,200 grams of deionized water. The
solution was stirred for one hour at 60.degree. C. then stirred
overnight at room temperature. A dilution water phase was made by
setting the temperature of a vessel containing deionized water to
19.degree. C. One of the three helical mixers was packed with
2-millimeter diameter glass beads and 100 mesh screens (140 .mu.m
approximate retention) were placed on both the inlet and outlet.
Approximately 50 ml of the Oil Phase and 200 ml of the Water Phase
were pumped simultaneously upwards, against gravity, through the
helical mixer apparatus at rates of 37 ml/min and 180 ml/min,
respectively. The resulting Reynolds number through the apparatus
was laminar, falling between 2 and 37. Laser diffraction (Beckman
Coulter LS 13 320) was used to analyze the particle size
distribution of the resulting emulsion. The volumetric median
particle size was found to be 41.83 .mu.m with a d10 of 14.17 .mu.m
and d90 of 56.38 .mu.m.
[0109] The particle size distribution was better than the previous
example, with 81.6% of the particles in the desired range,
suggesting that packing the emulsifier with beads has a positive
effect on emulsion quality.
Example 21 (Large Beads)
[0110] This example is similar to Example 20, except that the flow
through the mixer was adjusted. An 8.8% w/w polymer-in-oil oil
phase (Oil Phase) was prepared by dissolving 51 grams of 50:50
poly(lactic-co-glycolic acid) (PLGA) (Resomer Select 5050 DLG 2A,
Lot number LP1321, Evonik Corp.) in 529 grams of dichloromethane
(DCM) and allowed to stir overnight at room temperature
(.about.19.degree. C.), resulting in approximately 400 mL of Oil
Phase. A second phase, the Water Phase, was prepared by dissolving
32 grams of poly(vinyl alcohol) (PVA) in 3200 grams of deionized
water. The solution was stirred for one hour at 60.degree. C. then
stirred overnight at room temperature. A dilution water phase was
made by setting the temperature of a vessel containing deionized
water to 19.degree. C. One of the three helical mixers was packed
with 2-millimeter diameter glass beads and 100 mesh screens (140
.mu.m approximate retention) were placed on both the inlet and
outlet. Approximately 50 ml of the Oil Phase and 440 ml of the
Water Phase were pumped simultaneously upwards, against gravity,
through the helical mixer apparatus at rates of 37 ml/min and 220
ml/min, respectively. The resulting Reynolds number through the
apparatus was laminar, falling between 2.5 and 44. Laser
diffraction (Beckman Coulter LS 13 320) was used to analyze the
particle size distribution of the resulting emulsion. The
volumetric median particle size was found to be 40.89 .mu.m with a
d10 of 13.35 .mu.m and d90 of 56.58 .mu.m.
[0111] The particle size distribution was similar to Example 20,
with 79.2% of the particles in the desired range.
Example 22 (Small Beads)
[0112] For examples 22-26, the emulsifier was filled with smaller
beads (.about.327 .mu.m median diameter), to determine if further
improvements could be made to the particle size distribution.
[0113] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 51 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1321, Evonik
Corp.) in 529 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
phase, the Water Phase, was prepared by dissolving 32 grams of
poly(vinyl alcohol) (PVA) in 3,200 grams of deionized water. The
solution was stirred for one hour at 60.degree. C. then stirred
overnight at room temperature. A dilution water phase was made by
setting the temperature of a vessel containing deionized water to
19.degree. C. One of the three helical mixers was packed with 327
.mu.m borosilicate glass beads (MO-SCI Health Care,
GL0179B5/300-355) and 100 mesh screens (140 .mu.m approximate
retention) were placed on both the inlet and outlet. Approximately
50 ml of the Oil Phase and 100 ml the Water Phase were pumped
simultaneously upwards, against gravity, through the packed bed
apparatus at rates of 9 ml/min and 21 ml/min, respectively. The
resulting Reynolds number through the apparatus was laminar,
falling between 0.05 and 0.83. The outgoing emulsion was met with
the dilution water phase at a flowrate of 168 ml/min. Laser
diffraction (Beckman Coulter LS 13 320) was used to analyze the
particle size distribution of the resulting emulsion.
[0114] The volumetric median particle size was found to be 40.49
.mu.m with a d10 of 24.7 .mu.m and d90 of 50.57 .mu.m. The
distribution was better than Example 21, with 90% of the particles
in the desired range, suggesting that using smaller beads and
slower flow rates in the emulsifier, increases the quality of the
resulting emulsion.
Example 23 (Small Beads PEG-Insulin Microspheres)
[0115] For this example, a small batch of drug-loaded microspheres
was made with PEG-insulin using the same emulsion step as Example
22. A 10% w/w polymer-in-oil oil phase (Oil Phase) was prepared by
dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)
(Resomer Select 5050 DLG 2A, Lot number LP1321, Evonik Corp.) in 88
grams of dichloromethane (DCM) along with 1.5 grams of PEGylated
Insulin Drug Substance (Lot 102516). The solution was stirred
overnight at room temperature (.about.19.degree. C.). A second
phase, the Water Phase, was prepared by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 grams of deionized water. The
solution was stirred for one hour at 60.degree. C. then stirred
overnight at room temperature. A dilution water phase was made by
setting the temperature of a vessel containing deionized water to
19.degree. C. One of the three helical mixers was packed with 327
.mu.m borosilicate glass beads (MO-SCI Health Care,
GL0179B5/300-355) and 100 mesh screens (140 .mu.m approximate
retention) were placed on both the inlet and outlet.
[0116] The Oil Phase and Water Phase were pumped simultaneously
upwards, against gravity, through the packed helical apparatus at
rates of 9 ml/min and 21 ml/min, respectively. The resulting
Reynolds number through the apparatus was laminar, falling between
0.05 and 0.83. The emulsion leaving the apparatus was met with the
dilution water phase at a flowrate of 168 ml/min. Flow through the
helical mixer slowed after approximately half of the Oil and Water
Phases had been passed through the mixer. The flow eventually
stopped, due to apparent clogging. The remaining Oil Phase and
Water Phase were pumped through a different but identical helical
mixer. Once all of the remaining Oil Phase had been passed through
the mixer, the emulsion was held in the primary tank while stirring
for 30 minutes. After the 30-minute hold, the contents of the
primary tank were transferred to a secondary tank at a flowrate of
24 ml/min. Dilution water, in line with the emulsion transfer, was
pumped at a rate of 115 ml/min.
[0117] Once the volume in the secondary tank reached 0.5 liters,
cross-flow filtration (CFF) was started using a 5 .mu.m ceramic
membrane. The microspheres were recirculated through the CFF
membrane at a rate of 1.9 L/min with the permeate waste exiting the
CFF membrane at 139 ml/min, in order to maintain the volume in the
secondary tank at 0.5 L. Once the entirety of the primary tank had
been transferred to the secondary tank, the temperature of the tank
jackets was increased to 25.degree. C. and the dilution water
flowrate was reduced to 25 ml/min. Additionally, the permeate
flowrate was reduced to 25 ml/min for the diafiltration step. This
process was continued until a total of six diavolumes, or 3 liters,
had been exchanged and then the temperature of the secondary tank
was increased and held at 35.degree. C. for two hours. At the end
of the temperature hold, the microspheres were cooled to 4.degree.
C. before being loaded onto the 25 .mu.m screen of the filter
dryer. The secondary tank was rinsed with 600 ml of chilled Milli-Q
water and this rinse water was also added to the filter dryer. The
filter dryer was vibrated in the forward direction while loading,
and the liquid permeate was drained off, leaving the microsphere
product on the screen. An air sweep of 5 sLpm was applied to the
filter dryer overnight to facilitate in drying of the microspheres.
After approximately 24 hours, the microspheres were harvested from
the screen.
[0118] The total harvested mass was 1.56 grams of product. This low
yield was likely due to losses caused by the clogging of the
helical mixer. The clogging might be because the small glass bead
size is increasing pressure through the helical mixer, which the
pumps cannot overcome. Laser diffraction (Beckman Coulter LS 13
320) was used to analyze the particle size distribution of the
final microspheres. The volumetric median particle size was found
to be 43.3 .mu.m with a d10 of 31.0 .mu.m and d90 of 53.7
.mu.m.
Example 24(PTFE Pump Heads)
[0119] Pumps were used which were rated to provide up to 100 psi to
overcome the back pressure experienced during the previous
examples. An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1321, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
phase, the Water Phase, was prepared by dissolving 8 grams of
poly(vinyl alcohol) (PVA) in 800 grams of deionized water. The
solution was stirred for one hour at 60.degree. C., then stirred
overnight at room temperature. A dilution water phase was made by
setting the temperature of a vessel containing deionized water to
19.degree. C. One of the three helical mixers was packed with 327
.mu.m borosilicate glass beads (MO-SCI Health Care,
GL0179B5/300-355) and 100 mesh screens (140 .mu.m approximate
retention) were placed on both the inlet and outlet. The Oil Phase
and the Water Phase were pumped simultaneously upwards, against
gravity, through the packed bed apparatus at rates of 9 ml/min and
21 ml/min, respectively. The resulting Reynolds number through the
apparatus was laminar, falling between 0.05 and 0.83. The pumps
used for this example were able to create up to 100 psi of pressure
to overcome the back pressure experienced during the previous
examples. The outgoing emulsion was met with the dilution water
phase at a flowrate of 168 ml/min. The pressure was measured near
the junction of the Oil and Water Phases at the inlet of the
helical mixer and was found to be 35 psi. Laser diffraction
(Beckman Coulter LS 13 320) was used to analyze the particle size
distribution of the resulting emulsion. The volumetric median
particle size was found to be 41.7 .mu.m with a d10 of 13.6 .mu.m
and d90 of 48.7 .mu.m.
[0120] The particle size distribution had a high percentage in the
targeted range, with 88.1% of the particles in the targeted range.
The pumps were able to provide enough pressure to overcome the 35
psi of backpressure created by the smaller packing.
Example 25 (PTFE Pump Heads, Slower Flow Rates)
[0121] Pumps were used which were able to provide up to 100 psi in
order to overcome the back pressure experienced during the previous
examples. In addition, lower flow rates (half of the flowrates used
in Example 24) were used to reduce the pressure drop through the
packed helical mixer.
[0122] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1321, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
phase, the Water Phase, was prepared by dissolving 8 grams of
poly(vinyl alcohol) (PVA) in 800 grams of deionized water. The
solution was stirred for one hour at 60.degree. C. then stirred
overnight at room temperature. A dilution water phase was made by
setting the temperature of a vessel containing deionized water to
19.degree. C. One of the three helical mixers was packed with 327
.mu.m borosilicate glass beads (MO-SCI Health Care,
GL0179B5/300-355) and 100 mesh screens (140 .mu.m approximate
retention) were placed on both the inlet and outlet. The Oil Phase
and the Water Phase were pumped simultaneously upwards, against
gravity, through the packed bed apparatus at rates of 4.5 ml/min
and 10.5 ml/min, respectively. The resulting Reynolds number
through the apparatus was laminar, falling between 0.02 and 0.42.
The pumps used for this example were able to create up to 100 psi
of pressure to overcome the back pressure experienced during the
previous examples. The outgoing emulsion was met with the dilution
water phase at a flowrate of 84 ml/min. The pressure was measured
near the junction of the Oil and Water Phases at the inlet of the
helical mixer and was found to be 25 psi. Laser diffraction
(Beckman Coulter LS 13 320) was used to analyze the particle size
distribution of the resulting emulsion. The volumetric median
particle size was found to be 41.7 .mu.m with a d10 of 26.2 .mu.m
and d90 of 50.3 .mu.m.
[0123] The particle size distribution had a high percentage (90.6%)
of the particles in the desired range. This batch had slightly
better particle size distribution than the previous example,
possibly due to the reduction in flowrate and backpressure.
Example 26 (PTFE Pump Heads, Slower Flow Rates)
[0124] Pumps were used which were able to provide up to 100 psi in
order to overcome the back pressure experienced during the previous
examples. In addition, lower flow rates (one quarter of the flow
rates used in Example 24) were used to reduce pressure.
[0125] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1321, Evonik
Corp.) in 88 grams of dichloromethane (DCM) and allowed to stir
overnight at room temperature (.about.19.degree. C.). A second
phase, the Water Phase, was prepared by dissolving 8 grams of
poly(vinyl alcohol) (PVA) in 800 grams of deionized water. The
solution was stirred for one hour at 60.degree. C. then stirred
overnight at room temperature. A dilution water phase was made by
setting the temperature of a vessel containing deionized water to
19.degree. C. One of the three helical mixers was packed with
borosilicate glass beads (MO-SCI Health Care, GL0179B5/300-355) and
100 mesh screens (140 .mu.approximate retention) were placed on
both the inlet and outlet. The Oil Phase and the Water Phase were
pumped simultaneously upwards, against gravity, through the packed
bed apparatus at rates of 2.25 ml/min and 5.25 ml/min,
respectively. The resulting Reynolds number through the apparatus
was laminar, falling between 0.01 and 0.21. The pumps used for this
example were able to create up to 100 psi of pressure to overcome
the back pressure experienced during the previous examples. The
outgoing emulsion was met with the dilution water phase at a
flowrate of 42 ml/min. The pressure was measured near the junction
of the Oil and Water Phases at the inlet of the helical mixer and
was 25 psi. Laser diffraction (Beckman Coulter LS 13 320) was used
to analyze the particle size distribution of the resulting
emulsion. The volumetric median particle size was found to be 45.8
.mu.m with a d10 of 29.44 .mu.m and d90 of 54.9 .mu.m.
[0126] The particle size distribution had a high percentage (92.6%)
in the targeted range. This batch had slightly better particle size
distribution than the previous example, possibly due to the
reduction in flowrate and backpressure. Although the slower
flowrates seem to reduce pressure and improve particle size
distribution, slower flow rates also increase process time.
Example 27 (Nixed Beads)
[0127] This example used half 1 mm beads and half 327 .mu.m beads
to reduce the pressure drop through the mixer. An 8.8% w/w
polymer-in-oil oil phase (Oil Phase) was prepared by dissolving 8.5
grams of 50:50 poly(lactic-co-glycolic acid) (PLGA) (Resomer Select
5050 DLG 2A, Lot number LP1321, Evonik Corp.) in 88 grams of
dichloromethane (DCM) and allowed to stir overnight at room
temperature (.about.19.degree. C.). A second phase, the Water
Phase, was prepared by dissolving 8 grams of poly(vinyl alcohol)
(PVA) in 800 grams of deionized water. The solution was stirred for
one hour at 60.degree. C. then stirred overnight at room
temperature. A dilution water phase was made by setting the
temperature of a vessel containing deionized water to 19.degree. C.
One of the three helical mixers was packed with 58.89 g of 1 mm
borosilicate glass beads (MO-SCI Health Care, GL01915B/1000) then
the remaining mixer volume was filled with 327 .mu.m borosilicate
glass beads (MO-SCI Health Care, GL0179B5/300-355) and 100 mesh
screens (140 .mu.m approximate retention) were placed on both the
inlet and outlet. The Oil Phase and the Water Phase were pumped
simultaneously upwards, against gravity, through the packed bed
apparatus at rates of 9 ml/min and 21 ml/min, respectively. The
resulting Reynolds number through the apparatus was laminar,
falling between 0.05 and 0.83. The outgoing emulsion was met with
the dilution water phase at a flowrate of 168 ml/min. The pressure
was measured near the junction of the Oil and Water Phases at the
inlet of the helical mixer and was found to be 15 psi. Laser
diffraction (Beckman Coulter LS 13 320) was used to analyze the
particle size distribution of the resulting emulsion. The
volumetric median particle size was found to be 47.7 .mu.m with a
d10 of 33.5 .mu.m and d90 of 59.0 .mu.m.
[0128] The particle size distribution had a high percentage (94.9%)
of the particles in the desired range. This batch had slightly
better particle size distribution than the previous example,
possibly due to the reduction in backpressure. This example also
used higher flow rates which is advantageous because it would
result in shorter process times and still be able to achieve better
particle size distribution than Example 26.
Example 28 (PEG-Insulin Microspheres)
[0129] A batch of microspheres was made with PEG-insulin using the
same emulsion step as Example 27. A 10% w/w polymer-in-oil oil
phase (Oil Phase) was prepared by dissolving 8.5 grams of 50:50
poly(lactic-co-glycolic acid) (PLGA) (Resomer Select 502H, Lot
number 577, Evonik Corp.) in 88 grams of dichloromethane (DCM)
along with 1.5 grams of PEGylated Insulin Drug Substance (Lot
102516). The solution was allowed to stir overnight at room
temperature (.about.19.degree. C.). A second phase, the Water
Phase, was prepared by dissolving 2.67 grams of poly(vinyl alcohol)
(PVA) in 267 grams of deionized water. The solution was stirred for
one hour at 60.degree. C. then stirred overnight at room
temperature. A dilution water phase was made by setting the
temperature of a vessel containing deionized water to 19.degree. C.
One of the three helical mixers was packed with 39.45 g of 1 mm
borosilicate glass beads (MO-SCI Health Care, GL01915B/1000) then
the remaining mixer volume was filled with 327 .mu.m borosilicate
glass beads (MO-SCI Health Care, GL0179B5/300-355) and 100 mesh
screens (140 .mu.m approximate retention) were placed on both the
inlet and outlet. The Oil Phase and Water Phase were pumped
simultaneously upwards, against gravity, through the packed bed
apparatus at rates of 9 ml/min and 21 ml/min, respectively. The
resulting Reynolds number through the apparatus was laminar,
falling between 0.05 and 0.83. The outgoing emulsion was met with
the dilution water phase at a flowrate of 168 ml/min. The maximum
pressure through the mixer was measured at the inlet of the mixer
and found to be 20 psi.
[0130] Once all of the remaining Oil Phase had been passed through
the mixer, the emulsion was held in the primary tank while stirring
for 30 minutes. After the 30-minute hold, the contents of the
primary tank were transferred to a secondary tank at a flowrate of
24 ml/min. Dilution water, in line with the emulsion transfer, was
pumped at a rate of 115 ml/min. Once the volume in the secondary
tank reached 0.5 liters, cross-flow filtration (CFF) was started
using a 5 .mu.m ceramic membrane. The microspheres were
recirculated through the CFF membrane at a rate of 1.9 L/min with
the permeate waste exiting the CFF membrane at 139 ml/min, in order
to maintain the volume in the secondary tank at 0.5 L. Once the
entirety of the primary tank had been transferred to the secondary
tank, the temperature of the tank jackets was increased to
25.degree. C. and the dilution water flowrate was reduced to 25
ml/min. Additionally, the permeate flowrate was reduced to 25
ml/min for the diafiltration step. This process was continued until
a total of six diavolumes, or 3 liters, had been exchanged and then
the temperature of the secondary tank was increased and held at
35.degree. C. for two hours.
[0131] At the end of the temperature hold, the microspheres were
cooled to 4.degree. C. before being loaded onto the 25 .mu.m screen
of the filter dryer. The secondary tank was rinsed with 600 ml of
chilled 0.5% sodium bicarbonate and this rinse was added to the
filter dryer. The filter dryer was vibrated in the forward
direction while loading and the liquid was drained off leaving the
microspheres on the screen. An air sweep of 5 sLpm was applied to
the filter dryer overnight to facilitate in drying of the
microspheres. After approximately 24 hours, the microspheres were
harvested from the screen.
[0132] The total harvested mass was 6.06 grams of product. This
yield was higher than Example 23, likely due to the elimination of
clogging for this batch. Laser diffraction (Beckman Coulter LS 13
320) was used to analyze the particle size distribution of the
resulting emulsion. The volumetric median particle size was found
to be 47.9 .mu.m with a d10 of 38.2 .mu.m and d90 of 57.5 .mu.m.
The volume percent of particles in the desired range of 25-65 .mu.m
was 97.6%.
Example 29 (Scaled Up)
[0133] To demonstrate scalability, Example 22 was repeated at a
larger scale, using three helices in parallel instead of a single
helix. An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 51 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1321, Evonik
Corp.) in 528 grams of dichloromethane (DCM. The solution was
allowed to stir overnight at room temperature (.about.19.degree.
C.). A second phase, the Water Phase, was prepared by dissolving 32
grams of poly(vinyl alcohol) (PVA) in 3210 grams of deionized
water. The solution was stirred for one hour at 60.degree. C. then
stirred overnight at room temperature. 110 grams of DCM was then
added to the Water phase and allowed to stir in a sealed bottle
overnight. A dilution water phase was made by setting the
temperature of a vessel containing deionized water to 19.degree. C.
Three helical mixers were packed with borosilicate glass spheres
(MO-SCI Health Care, GL0179B5/300-355) and 100 mesh screens (140
.mu.m approximate retention) were placed on each inlet and outlet.
Approximately 232 ml of the Oil Phase and 783 ml of the Water Phase
were pumped simultaneously upwards, against gravity, through the
packed helical apparatus at rates of 8 ml/min and 27 ml/min,
respectively. The resulting Reynolds number through the apparatus
was laminar, falling between 0.02 and 0.32. The outgoing emulsion
was met with the dilution water phase at a flowrate of 166 ml/min.
Maximum pressure observed was 30psi and no reduction in flow rate
was observed during the emulsion step. Laser diffraction (Beckman
Coulter LS 13 320) was used to analyze the particle size
distribution of the resulting emulsion. The volumetric median
particle size was found to be 45.3 .mu.m with a d10 of 32.0 .mu.m
and d90 of 55.8 .mu.m. The volume percent of particles in the
desired range of 25-65 .mu.m was 91.85%.
[0134] This example demonstrated that the emulsification process
could be scaled up to make an emulsion sufficient to make 30 grams
of microspheres, using the same process as Example 22, which made
an emulsion sufficient to make 10 grams of microspheres. This
example showed that a triple helix could be used to scale up the
amount of emulsion produced without increasing the space occupied
by the emulsifier. The three helices occupy roughly the same space
as a single helix. The particle size distribution was similar for
this example and Example 22, which suggests that the emulsification
is scalable.
Example 30 (Screens for Classification)
[0135] In this example, the emulsion was performed at a reduced
temperature (.about.4.degree. C.) compared to the other examples,
to determine how this could affect the particle size distribution.
In addition, a 200.times.1150 mesh screen (10 .mu.m approximate
retention) with recirculating flow from the secondary stirred tank
was used, instead of a ceramic 5 .mu.m cross-flow filtration
membrane, for both the concentration step and the diafiltration
step. After concentration and diafiltration, the microspheres, with
recirculating flow from the stirred tank, were passed through a 150
mesh screen (100 .mu.m approximate retention) and into the filter
dryer. This step eliminates any microspheres or aggregates that are
larger than 100 .mu.m and collects only the desired microsphere
product. This example demonstrated that a plurality of screens with
recirculating flow from a stirred tank, can be used to classify the
microspheres based on their particle size distribution and result
in better control of the microparticles produced.
[0136] An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was
prepared by dissolving 8.5 grams of 50:50 poly(lactic-co-glycolic
acid) (PLGA) (Resomer Select 502H, Lot number 577, Evonik Corp.) in
88 grams of dichloromethane (DCM). The solution was allowed to stir
overnight at room temperature (.about.19.degree. C.) until
dissolved, then stored overnight at 2-8.degree. C. A second phase,
the Water Phase, was prepared by dissolving 2.67 grams of
poly(vinyl alcohol) (PVA) in 267 grams of deionized water. The
solution was stirred for one hour at 60.degree. C. then stirred at
room temperature until dissolved and stored overnight at
2-8.degree. C. A dilution water phase was made by setting the
temperature of a vessel containing deionized water to 19.degree. C.
One of the three helical mixers was packed with 39.47 g of 1 mm
borosilicate glass beads (MO-SCI Health Care, GL01915B/1000) then
the remaining volume within the mixer was filled with 327 .mu.m
borosilicate glass beads (MO-SCI Health Care, GL0179B5/300-355),
100 mesh screens (140 .mu.m approximate retention) were placed on
both the inlet and outlet, and the packed helical mixer was stored
overnight at 2-8.degree. C. The Oil Phase and Water Phase were
pumped simultaneously upwards, against gravity, through the packed
bed apparatus at rates of 9 ml/min and 21 ml/min, respectively. The
outgoing emulsion was met with the dilution water phase at a
flowrate of 168 ml/min. The maximum pressure through the mixer was
20 psi.
[0137] Once the entirety of the Oil Phase had been passed through
the mixer, the emulsion was held in the primary tank while stirring
for 30 minutes. After the 30-minute hold, the contents of the
primary tank were transferred to a secondary tank at a flowrate of
24 ml/min. Dilution water, in line with the emulsion transfer, was
pumped at a rate of 115 ml/min. Once the volume in the secondary
tank reached 0.5 liters, the cross-flow filtration (CFF) step was
started. The microspheres were recirculated through the straight
run of a tee at a rate of 1.9 L/min. A 200.times.1150 mesh screen
(10 .mu.m approximate retention) was placed on the branch of the
tee and the waste permeate flow rate was controlled via diaphragm
valve at a rate of 139 ml/min in order to maintain the fluid volume
in the secondary tank at 0.5 L. Once the entirety of the primary
tank was transferred to the secondary tank, the temperature of the
tank jackets was increased to 25.degree. C. and the dilution water
flow rate was reduced to 25 ml/min. Additionally, the permeate
flowrate was reduced to 25 ml/min for the entirety of the
diafiltration step. A total of six diavolumes were completed and
then the jacket of the secondary tank was increased to 35.degree.
C. and held for 2 hours.
[0138] At the end of the temperature hold, the microspheres were
cooled to 4.degree. C. and then classified by size, by
recirculating through the straight run of a tee at a rate of 1.9
L/min. A 150 mesh screen (100 .mu.m approximate retention) was
placed on the branch of the tee and the product permeate flow rate
was controlled via diaphragm valve and allowed to flow at a rate of
500 ml/min onto the 25 .mu.m screen of the filter dryer. Four
liters of water was concurrently added to the tank at a rate of 500
ml/min. Once microspheres were no longer observed in the secondary
tank, the material in the filter dryer was rinsed with 600 ml of
chilled 0.5% sodium bicarbonate. The filter dryer vibrated in the
forward direction while loading and the liquid was drained off
leaving the microsphere product on the screen. An air sweep of 5
sLpm was applied to the filter dryer overnight to facilitate in
drying of the microspheres. After approximately 24 hours, the
microspheres were harvested from the screen. The total harvested
mass was 4.5 grams of product. Laser diffraction (Beckman Coulter
LS 13 320) was used to analyze the particle size distribution of
the resulting emulsion. The volumetric median particle size was
44.6 .mu.m with a d10 of 27.9 .mu.m and d90 of 56.3 .mu.m. The
volume percent of particles in the desired range of 25-65 .mu.m was
92%.
[0139] The particle size distribution of the emulsion was not
significantly different than when the emulsion was performed at
room temperature, so the cold emulsion was not observed to
significantly affect the particle size distribution. The volumetric
median particle size after classification with the screens was 44.8
.mu.m with a d10 of 32.9 .mu.m and d90 of 54.5 .mu.m. The volume
percent of particles in the desired range of 25-65 .mu.m was 96%.
This example demonstrated that screens can be used for cross-flow
filtration instead of the ceramic membrane and have the added
advantage that they can be used to eliminate undersized particles,
oversized particles, aggregates, or unwanted foreign material at
the same time. Different screens sizes could be used to select a
desired size distribution of the final product.
E. Examples 31-33
[0140] Examples 31-33 use a helical mixer. The helical mixer, shown
in FIG. 6, for the preparation of polymer microspheres was created
by wrapping a 6-inch piece of 1/4'' PTFE tubing ( 3/16'' inner
diameter) around a 0.62-inch cylinder for a total of 1 complete
coil. The resulting helix has a mean diameter of 0.9 inches and a
helix angle of 47 degrees. The helix was then packed with
borosilicate beads with an average diameter of 327 microns. Each
end of the helix was capped with a 100 mesh screen. The helical
apparatus was connected to a peristaltic pump and oriented
vertically in such a way that the net fluid flow was up, against
gravity, then used during the following experiments.
Example 31 (GLP-1 Microspheres)
[0141] A 10% w/v polymer-in-oil phase (Oil Phase) was prepared by
dissolving 1.275 grams of 50:50 poly(D,L-lactide-co-glycolide)
(PLGA) (Resomer RG 502, Lot number D140800505, Evonik Corp.)+0.425
grams of 50:50 PLGA (Resomer RG 504, Lot number DBCBS4537V, Sigma
Aldrich)+0.3 grams of GLP-1 protein PEGylated with a 5K PEG, in
26.6 grams of dichloromethane (DCM) and allowed to stir until
dissolved at room temperature (.about.19.degree. C.). Next, 360
microliters of a 50 mg/ml pamoic acid solution, prepared in
N-Dimethylformamide (DMF), was added and stirred for 20 more
minutes. A second solution (Water Phase) was made by dissolving 10
grams of poly(vinyl alcohol) (PVA) in 1000 milliliters of deionized
water. Twenty-five milliliters of the PVA solution was added to 10
ml of the oil phase, stirred to form a course emulsion, and pumped
through the helical apparatus at a rate of 3.8 ml/min. The
resulting Reynolds number through the apparatus was laminar,
falling between 0.07 and 0.13. Microspheres were collected in 1000
ml of a 7.2% w/v NaCl solution stirring at 200 rpm. The microsphere
product was analyzed for drug loading and encapsulation efficiency
using HPLC. GLP-1 drug was found to be 11.9% by mass with an
encapsulation efficiency of 95%. The particle size distribution of
the microspheres was analyzed using laser diffraction (Beckman
Coulter LS 13 320). The volumetric median particle size of the
microspheres in the primary solution was 39 .mu.m with a d10 of 29
.mu.m and a d90 of 47 .mu.m.
Example 32 (GLP-1 and Insulin Microspheres)
[0142] A 10% w/v polymer-in-oil phase (Oil Phase) was prepared by
dissolving 0.17 grams of 50:50 poly(D,L-lactide-co-glycolide)
(PLGA) (Resomer RG 502, Lot number D140800505, Evonik Corp.)+0.015
grams of a 5K PEGylated insulin+0.015 grams of a 5K PEGylated
GLP-1, in 1.995 grams of dichloromethane (DCM)+0.52 grams of benzyl
alcohol, and allowed to stir until dissolved at room temperature
(.about.19.degree. C.). Next, 18 microliters of a 50 mg/ml pamoic
acid solution, prepared in N-Dimethylformamide (DMF), was added and
stirred for 20 more minutes. A second solution (Water Phase) was
made by dissolving 10 grams of poly(vinyl alcohol) (PVA) in 1000
milliliters of deionized water. Five milliliters of the PVA
solution was added to the oil phase, stirred to form a course
emulsion, and pumped through the helical apparatus at a rate of 3.8
ml/min. The resulting Reynolds number through the apparatus was
laminar, falling between 0.07 and 0.13. Microspheres were collected
in 200 milliliters of a 3.6% w/v NaCl solution stirring at 120 rpm.
After 2 hours, the microsphere suspension was diluted by adding 400
milliliters of 3.6% w/v NaCl and let stirring at 180 rpm for 1
hour. Next, the microspheres were centrifuged at 33 g for 5 minutes
and washed with Milli-Q water. The microsphere product was analyzed
for drug loading using HPLC. PEGylated GLP-1 loading was found to
be 4.9% by mass. PEGylated insulin loading was found to be 6.9% by
mass. The particle size distribution of the microspheres was
analyzed using laser diffraction (Beckman Coulter LS 13 320). The
volumetric median particle size of the microspheres was 21 .mu.m
with a d10 of 14 .mu.m and a d90 of 27 .mu.m.
Example 33 (Microspheres with GLP-1 and Two Different Polymers)
[0143] Two different oil phases were prepared, A and B. Oil phase A
was prepared by dissolving 0.85 grams of 50:50
poly(D,L-lactide-co-glycolide) (PLGA) (Resomer RG 502, Lot number
D140800505, Evonik Corp.)+0.15 grams of a 5K PEGylated GLP-1, in
13.3 grams of dichloromethane (DCM), and allowed to stir until
dissolved at room temperature (.about.19.degree. C.). Next, 180
microliters of a 50 mg/ml pamoic acid solution, prepared in
N-Dimethylformamide (DMF), was added and stirred for 20 more
minutes.
[0144] Oil phase B was prepared by dissolving 0.6375 grams of 50:50
poly(D,L-lactide-co-glycolide) (PLGA) (Resomer RG 502, Lot number
D140800505, Evonik Corp.)+0.2125 grams of 50:50
poly(D,L-lactide-co-glycolide) (PLGA) (Resomer RG 503, Lot number
BCBR7837V, Evonik Corp.)+0.15 grams of a 5K PEGylated GLP-1, in
13.3 grams of dichloromethane (DCM), and allowed to stir until
dissolved at room temperature. One hundred and eighty microliters
of the 50 mg/ml pamoic acid solution was added and stirred for 20
more minutes.
[0145] The Water Phase solution was made by dissolving 10 grams of
poly(vinyl alcohol) (PVA) in 1000 milliliters of deionized water.
Five milliliters of each Oil Phase were mixed with 12.5 ml of the
PVA solution to form a course emulsion. The course emulsion formed
with Oil Phase A was first pumped through the helical apparatus at
a rate of 3.8 ml/min and collected in 1000 milliliters of a 7.2%
NaCl solution stirring at 200 rpm. The resulting Reynolds number
through the apparatus was laminar, falling between 0.07 and 0.13.
Immediately after the first course emulsion had been pumped, the
second course emulsion, formed with the Oil Phase B, was pumped
through the helical apparatus at the same rate, and collected in
the same solution. After 2 hours, the microsphere suspension was
diluted by adding 2000 milliliters of 7.2% w/v NaCl and left
stirring for 1 hour. The microspheres were centrifuged at 33 g for
5 minutes and washed with Milli-Q water. The microsphere product
was analyzed for drug loading using HPLC. PEGylated GLP-1 loading
was found to be 12.8% by mass. The particle size distribution of
the microspheres was analyzed using laser diffraction (Beckman
Coulter LS 13 320). The volumetric median particle size of the
microspheres was 42 .mu.m with a d10 of 31 .mu.m and a d90 of 55
.mu.m.
Examples 31-33 Summary:
[0146] These examples demonstrated that the helical mixer can be
used for making microspheres with different APIs and polymers. The
resulting particle size distributions had a high percentage in the
targeted range, and the processes used laminar flow which is less
likely to cause shear stresses on fragile protein drugs and results
in better, more uniform emulsions.
F. Examples 34-37
[0147] Examples 34-36 show the viability of helical mixers packed
with a gradient of bead diameters. Example 37 shows the viability
of producing two sets of microspheres with two different
physiologically active substances.
Example 34 (Budesonide, Bead Gradient)
[0148] A batch of microspheres was made with budesonide, a small
molecule drug which is not water soluble. This example demonstrates
using the mixer to make microspheres with small molecule drugs as
well as using a gradient of bead sizes to fine tune the particle
size distribution.
[0149] The median bead sizes of the gradient were 4 mm, 2 mm, 1 mm,
and 0.327 mm.
[0150] An 10% w/w polymer-in-oil oil phase (Oil Phase) was prepared
by dissolving 4.5g grams of 50:50 poly(lactic-co-glycolic acid)
(PLGA) (Resomer RG 504H, Lot number 10H40700512, Evonik Corp.) and
0.5 grams of budesonide (Sigma-Aldrich PHR1178-lot LRAA8997) in 44
grams of dichloromethane (DCM). A second phase, the Water Phase,
was prepared by dissolving 2.67 grams of poly(vinyl alcohol) (PVA)
in 267 grams of deionized water. The solution was stirred for one
hour at 60.degree. C. then stirred overnight at room temperature. A
dilution water phase was made by setting the temperature of a
vessel containing deionized water to 19.degree. C. One of the three
helical mixers was packed, from bottom to top, with 10.47 g of 4 mm
borosilicate glass beads, 13.14 g of 2 mm borosilicate glass beads,
13.17 g of 1 mm borosilicate glass beads, and 71.6 g of 327 .mu.m
borosilicate glass beads (MO-SCI Health Care, GL0179B5/300-355) and
100 mesh screens (140 .mu.m approximate retention) were placed on
both the inlet and outlet.
[0151] The resulting Oil Phase and Water Phase were pumped
simultaneously upwards, against gravity, through the bottom of the
packed bed apparatus at rates of 9 ml/min and 21 ml/min,
respectively. The sequential decrease of packing size through the
helix enabled the gradual reduction of Reynolds number through the
mixer. The 4 mm beads placed at the inlet of the mixer produced a
laminar Reynolds number ranging from 0.58 to 10.25. The emulsion
then flowed through the 2 mm and 1 mm packing producing a Reynolds
number ranging from 0.28 to 5.12 followed a Reynolds number ranging
from 0.15 to 2.56. The final particle size was produced as the
emulsion flowed through the 327 .mu.m packing, resulting in a final
Reynolds number ranging from 0.05 to 0.84. The outgoing emulsion
was met with the dilution water phase at a flowrate of 168
ml/min.
[0152] Once the remaining Oil Phase had been passed through the
mixer, laser diffraction (Beckman Coulter LS 13 320) was used to
analyze the particle size distribution of the resulting emulsion.
The volumetric median particle size was found to be 47.22 .mu.m
with a d10 of 36.1 .mu.m and d90 of 57.2 .mu.m. The volume percent
of particles in the desired range of 25-65 .mu.m was 95.1%. The
resulting microspheres were stirred at room temperature in a beaker
to allow the methylene chloride to evaporate. After about 2 hours,
the microspheres were collected on a 25 .mu.m screen and allowed to
dry at room temperature. After drying, the microspheres were
harvested from the screen. The total harvested mass was 1.8 grams
of product.
[0153] The particle size distribution had a high percentage (95.1%)
in the targeted range of 25-65 .mu.m, which is slightly better than
the previous examples, suggesting that the gradient of beads might
help to fine tune the particle size distribution.
Example 35: (GLP-1, Double Emulsion, Bead Gradient)
[0154] A batch of microspheres was made with GLP-1 using an
water/oil/water (w/o/w) emulsion. This example demonstrates using
the mixer to make w/o/w microspheres with a water-soluble peptide
as well as using a gradient of bead sizes to fine tune the particle
size distribution. The median bead sizes of the gradient were 4 mm,
2 mm, 1 mm, and 0.327 mm.
[0155] A 10% w/w polymer-in-oil oil phase (Oil Phase) was prepared
by dissolving 2.38 grams of 50:50 poly(lactic-co-glycolic acid)
(PLGA) (Resomer RG 504H, Lot number 10H40700512, Evonik Corp.) in
22 grams of dichloromethane (DCM). Separately, 0.125 grams of GLP-1
(7-36) Chemleader (107444-51-9) was dissolved in 1.0 mL water. A
second phase, the Water Phase, was prepared by dissolving 2.67
grams of poly(vinyl alcohol) (PVA) in 267 grams of deionized water.
The solution was stirred for one hour at 60.degree. C. then stirred
overnight at room temperature. A dilution water phase was made by
setting the temperature of a vessel containing deionized water to
19.degree. C. One of the three helical mixers was packed, from
bottom to top, with 10.47 g of 4 mm borosilicate glass beads, 13.14
g of 2 mm borosilicate glass beads, 13.17 g of 1 mm borosilicate
glass beads, and 71.6 g of 327 .mu.m borosilicate glass beads
(MO-SCI Health Care, GL0179B5/300-355) and 100 mesh screens (140
.mu.m approximate retention) were placed on both the inlet and
outlet. The first emulsion (w/o) was made by adding the water/GLP-1
mixture to the PLGA/DCM mixture, followed by homogenization. The
resulting w/o phase and the Water Phase were pumped simultaneously
upwards, against gravity, through the bottom of the packed bed
apparatus at rates of 9 ml/min and 21 ml/min, respectively.
[0156] The sequential decrease of packing size through the helix
enabled the gradual reduction of Reynolds number through the mixer.
The 4 mm beads placed at the inlet of the mixer produced a laminar
Reynolds number ranging from 0.58 to 10.25. The emulsion then
flowed through the 2 mm and 1 mm packing producing a Reynolds
number ranging from 0.28 to 5.12 followed a Reynolds number ranging
from 0.15 to 2.56. The final particle size was produced as the
emulsion flowed through the 327 .mu.m packing, resulting in a final
Reynolds number ranging from 0.05 to 0.84. The outgoing emulsion
was met with the dilution water phase at a flowrate of 168
ml/min.
[0157] Once the entirety of the w/o phase had been passed through
the helical mixer, laser diffraction (Beckman Coulter LS 13 320)
was used to analyze the particle size distribution of the resulting
w/o/w emulsion. The volumetric median particle size was found to be
45.25 .mu.m with a d10 of 33.55 .mu.m and d90 of 55.37 .mu.m. The
volume percent of particles in the desired range of 25-65 .mu.m was
94.4%. The resulting microspheres were stirred at room temperature
in a beaker to allow the methylene chloride to evaporate. After
about 2 hours, the microspheres were collected on a 25 .mu.m screen
and allowed to dry at room temperature. After drying, the
microspheres were harvested from the screen. The total harvested
mass was 1.03 grams of product.
Example 36 (Albuterol Sulfate, Double Emulsion, Bead Gradient)
[0158] A batch of microspheres was made with albuterol sulfate
using a water/oil/water (w/o/w) emulsion. Albuterol sulfate is a
small molecule drug that is water soluble. This example
demonstrates using the mixer to make w/o/w microspheres with a
water-soluble small molecule drug, as well as using a gradient of
bead sizes to fine tune the particle size distribution. The median
bead sizes of the gradient were 4 mm, 2 mm, 1 mm, and 0.327 mm.
[0159] A 10% w/w polymer-in-oil oil phase (Oil Phase) was prepared
by dissolving 2.38 grams of 50:50 poly(lactic-co-glycolic acid)
(PLGA) (Resomer RG 504H, Lot number 10H40700512, Evonik Corp.) in
22 grams of dichloromethane (DCM). Separately, 0.125 grams of
Buterol Sulfate (Sigma-Aldrich PHR1053-lot LRAA7128) was dissolved
in 0.5 mL water. A second phase, the Water Phase, was prepared by
dissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 grams of
deionized water. The solution was stirred for one hour at
60.degree. C. then stirred overnight at room temperature. A
dilution water phase was made by setting the temperature of a
vessel containing deionized water to 19.degree. C. One of the three
helical mixers was packed, from bottom to top, with 10.47 g of 4 mm
borosilicate glass beads, 13.14 g of 2 mm borosilicate glass beads,
13.17 g of 1 mm borosilicate glass beads, and 71.6 g of 327 .mu.tm
borosilicate glass beads (MO-SCI Health Care, GL0179B5/300-355) and
100 mesh screens (140 .mu.m approximate retention) were placed on
both the inlet and outlet. The first emulsion (w/o) was made by
adding the water/Buterol Sulfate mixture to the PLGA/DCM mixture,
followed by homogenization. The resulting w/o phase and the Water
Phase were pumped simultaneously upwards, against gravity, through
the packed bed apparatus at rates of 9 ml/min and 21 ml/min,
respectively.
[0160] The sequential decrease of packing size through the helix
enabled the gradual reduction of Reynolds number through the mixer.
The 4 mm beads placed at the inlet of the mixer produced a laminar
Reynolds number ranging from 0.58 to 10.25. The emulsion then
flowed through the 2 mm and 1 mm packing producing a Reynolds
number ranging from 0.28 to 5.12 followed a Reynolds number ranging
from 0.15 to 2.56. The final particle size was produced as the
emulsion flowed through the 327 .mu.m packing, resulting in a final
Reynolds number ranging from 0.05 to 0.84. The outgoing emulsion
was met with the dilution water phase at a flowrate of 168
ml/min.
[0161] Once the entirety of the w/o phase had been passed through
the mixer, laser diffraction (Beckman Coulter LS 13 320) was used
to analyze the particle size distribution of the resulting
emulsion. The volumetric median particle size was found to be 47.54
.mu.m with a d10 of 25.76 p.mu.m and d90 of 59.49 .mu.m. The volume
percent of particles in the desired range of 25-65 .mu.m was 88.3%.
The resulting microspheres were stirred at room temperature in a
beaker to allow the methylene chloride to evaporate. After about 2
hours, the microspheres were collected on a 25 .mu.m screen and
allowed to dry at room temperature. After drying, the microspheres
were harvested from the screen. The total harvested mass was 1.18
grams of product.
Example 37 (Two Different Microspheres)
[0162] Two different oil phases were prepared. The first 15% w/v
polymer-in-oil phase (Oil Phase) was prepared by combining 0.255
grams of 50:50 poly(D,L-lactide-co-glycolide) (PLGA)
[0163] (Resomer RG 502H, Lot number LP1487, Evonik Corp.) and 0.045
grams of a 5K PEGylated insulin in a solution of 2 mL
dichloromethane (DCM) and 18 microliters of 50 mg/ml pamoic acid
prepared in N-Dimethylformamide (DMF). This oil phase was then
allowed to stir at room temperature (.about.19.degree. C.) until
completely dissolved. A second 10% w/v polymer-in-oil phase (Oil
Phase) was prepared by combining 0.170 grams of 50:50
poly(D,L-lactide-co-glycolide) (PLGA) (Resomer RG 502, Lot number
D140800505, Evonik Corp.) and 0.030 grams of a 5K PEGylated GLP-1
in a solution of 1.75 mL of dichloromethane (DCM), 250 microliters
of benzyl alcohol, and 18 microliters of 50 mg/ml pamoic acid
prepared in N-Dimethylformamide (DMF). This second oil phase was
then allowed to stir at room temperature (.about.19.degree. C.)
until completely dissolved. A Water Phase was made by dissolving 10
grams of poly(vinyl alcohol) (PVA) in 1000 milliliters of deionized
water. Five milliliters of the PVA solution was added to each oil
phase and the oil phases were stirred to form course emulsions.
[0164] The two course emulsions were pumped through two separate
helical mixers at 3.8 ml/min and microspheres were collected
together in 400 milliliters of a 20% w/v sucrose solution stirring
at 120 rpm. The resulting Reynolds number through the two helices
was laminar, falling between 0.07 and 0.13. After 1 hour, the
microspheres were centrifuged at 33 g for 5 minutes and washed
three times with Milli-Q water. The particle size distribution of
the microspheres was analyzed using laser diffraction (Beckman
Coulter LS 13 320). The volumetric median particle size of the
microspheres was 37.6 .mu.m with a d10 of 27.3 .mu.m and a d90 of
46.3 .mu.m.
[0165] The specific details of particular embodiments may be
combined in any suitable manner without departing from the spirit
and scope of embodiments of the invention. However, other
embodiments of the invention may be directed to specific
embodiments relating to each individual aspect, or specific
combinations of these individual aspects.
[0166] The above description of example embodiments of the
invention has been presented for the purposes of illustration and
description. It is not intended to be exhaustive or to limit the
invention to the precise form described, and many modifications and
variations are possible in light of the teaching above.
[0167] In the preceding description, for the purposes of
explanation, numerous details have been set forth in order to
provide an understanding of various embodiments of the present
technology. It will be apparent to one skilled in the art, however,
that certain embodiments may be practiced without some of these
details, or with additional details.
[0168] Having described several embodiments, it will be recognized
by those of skill in the art that various modifications,
alternative constructions, and equivalents may be used without
departing from the spirit of the invention. Additionally, a number
of well-known processes and elements have not been described in
order to avoid unnecessarily obscuring the present invention.
Additionally, details of any specific embodiment may not always be
present in variations of that embodiment or may be added to other
embodiments.
[0169] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed. The upper and lower limits of these
smaller ranges may independently be included or excluded in the
range, and each range where either, neither, or both limits are
included in the smaller ranges is also encompassed within the
invention, subject to any specifically excluded limit in the stated
range. Where the stated range includes one or both of the limits,
ranges excluding either or both of those included limits are also
included.
[0170] As used herein and in the appended claims, the singular
forms "a", "an", and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a method" includes a plurality of such methods and reference to
"the tube" includes reference to one or more tubes and equivalents
thereof known to those skilled in the art, and so forth. The
invention has now been described in detail for the purposes of
clarity and understanding. However, it will be appreciated that
certain changes and modifications may be practice within the scope
of the appended claims.
[0171] All publications, patents, and patent applications cited
herein are hereby incorporated by reference in their entirety for
all purposes. None is admitted to be prior art.
* * * * *