U.S. patent application number 13/680069 was filed with the patent office on 2013-05-23 for polymer protein microparticles.
This patent application is currently assigned to REGENERON PHARMACEUTICALS, INC.. The applicant listed for this patent is Regeneron Pharmaceuticals, Inc.. Invention is credited to Hunter CHEN, Scott Walsh.
Application Number | 20130129830 13/680069 |
Document ID | / |
Family ID | 47295198 |
Filed Date | 2013-05-23 |
United States Patent
Application |
20130129830 |
Kind Code |
A1 |
CHEN; Hunter ; et
al. |
May 23, 2013 |
Polymer Protein Microparticles
Abstract
Microparticles containing a core of therapeutic protein and a
cortex of a biocompatible and biodegradable polymer, and methods of
making and using the microparticles are provided. The extended
release of a therapeutic protein from the microparticles in a
physiological solution is demonstrated over an extended period of
time.
Inventors: |
CHEN; Hunter; (Tarrytown,
NY) ; Walsh; Scott; (Tarrytown, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Regeneron Pharmaceuticals, Inc.; |
Tarrytown |
NY |
US |
|
|
Assignee: |
REGENERON PHARMACEUTICALS,
INC.
Tarrytown
NY
|
Family ID: |
47295198 |
Appl. No.: |
13/680069 |
Filed: |
November 18, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61561525 |
Nov 18, 2011 |
|
|
|
Current U.S.
Class: |
424/495 ;
424/133.1; 424/134.1; 424/142.1; 424/490; 424/497; 427/2.14 |
Current CPC
Class: |
A61K 9/1617 20130101;
A61K 38/179 20130101; A61K 9/5047 20130101; A61K 9/14 20130101;
A61P 27/02 20180101; A61K 9/5089 20130101; A61K 9/1652 20130101;
A61K 9/1611 20130101; A61K 9/1623 20130101; A61K 9/1647 20130101;
A61K 9/5031 20130101; A61K 9/1641 20130101 |
Class at
Publication: |
424/495 ;
424/490; 424/133.1; 424/134.1; 424/142.1; 424/497; 427/2.14 |
International
Class: |
A61K 9/14 20060101
A61K009/14 |
Claims
1. A composition comprising a protein coated with a polymer,
wherein the composition is a particle with a diameter of from about
5 .mu.m to about 40 .mu.m.
2. The composition of claim 1, wherein the protein is an
antigen-binding protein.
3. The composition of claim 2, wherein the protein comprises an Fc
domain.
4. The composition of claim 3, wherein the protein is an
antibody.
5. The composition of claim 3, wherein the protein is a
receptor-Fc-fusion protein.
6. The composition of claim 2, wherein the protein is an antibody
fragment.
7. The composition of claim 5, wherein the protein is a
VEGF-Trap.
8. The composition of claim 4, wherein the antibody is a human
monoclonal antibody.
9. The composition of claim 1, wherein the polymer is a
biodegradable polymer.
10. The composition of claim 9, wherein the polymer is selected
from the group consisting of poly (lactic acid) (PLA),
polyanhydride poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH),
poly(hydroxbutyric acid-cohydroxyvaleric acid) (PHB-PVA),
polyethylene glycol-poly (lactic acid) copolymer (PEG-PLA),
poly-D,L-lactide-co-glycolide (PLGA), polyorthoester (POE), ethyl
cellulose (EC), and poly-.epsilon.-caprolactone (PCL).
11. The composition of claim 1, wherein the protein is either a
trap or an antibody, and the polymer is a POE.
12. The composition of claim 11, wherein the average diameter of
the particle is about 15 .mu.m.
13. The composition of claim 1, wherein the protein is either a
trap or an antibody, and the polymer is a PLGA.
14. A microparticle having a diameter of about 2 .mu.m to about 70
.mu.m comprising a protein particle core of about 2 .mu.m to about
12 .mu.m in diameter and a biodegradable polymer cortex.
15. The microparticle of claim 14, wherein the biodegradable
polymer is selected from the group consisting of poly (lactic acid)
(PLA), polyanhydride poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH),
poly(hydroxbutyric acid-cohydroxyvaleric acid) (PHB-PVA),
polyethylene glycol-poly (lactic acid) copolymer (PEG-PLA),
poly-D,L-lactide-co-glycolide (PLGA), polyorthoester (POE), ethyl
cellulose (EC), and poly-.epsilon.-caprolactone (PCL).
16. The microparticle of claim 15, wherein the protein particle
core comprises an antigen-binding protein.
17. The microparticle of claim 16, wherein the antigen binding
protein is a receptor-Fc-fusion protein.
18. The microparticle of claim 17, wherein the protein particle
core comprises a VEGF-Trap and the polymer cortex comprises POE,
PLA, or PLGA.
19. The microparticle of claim 16, wherein the antigen binding
protein is an antibody.
20. The microparticle of claim 19, wherein the antibody is a human
IgG molecule, and the polymer cortex comprises POE, PLA, or
PLGA.
21. A plurality of microparticles having diameters ranging from
about 2 .mu.m to about 70 .mu.m comprising a protein particle core
of about 2 .mu.m to about 12 .mu.m in diameter and a biodegradable
polymer cortex.
22. The plurality of microparticles of claim 21, wherein the
microparticles degrade at different rates and the protein is
released in an aqueous environment over an extended period of
time.
23. The plurality of microparticles of claim 22, wherein the period
of time is at least 3 days.
24. The plurality of microparticles of claim 23, wherein the period
of time is at least 60 days.
25. The plurality of microparticles of claim 24, wherein the
protein is an antigen-binding protein.
26. The plurality of microparticles of claim 25, wherein the
antigen-binding protein is a receptor-Fc-fusion protein.
27. The plurality of microparticles of claim 26, wherein the
receptor-Fc-fusion protein is a VEGF Trap and the polymer cortex
comprises POE, PLA, or PLGA.
28. The plurality of microparticles of claim 25, wherein the
antigen-binding protein is an antibody.
29. The plurality of microparticles of claim 28, wherein the
antibody is a human IgG molecule and the polymer cortex comprises
POE, PLA, or PLGA.
30. A method of treating a disease, comprising administering to a
patient a therapeutically effective amount of a plurality of
microparticles of claim 21, wherein a therapeutic protein is
released from the microparticles over an extended period of
time.
31. The method of claim 30, wherein the protein is a VEGF-Trap and
the polymer is POE, PLA, or PLGA.
32. The method of claim 31, wherein the disease is an ocular
disease and the microparticles are delivered to the vitreous of the
patient at no less than 60 day intervals.
33. A method of manufacturing a composition comprising a protein
and a biodegradable polymer, the method comprising the steps of: a.
obtaining a protein particle; b. suspending the protein particle in
a solution comprising the polymer and a solvent; and c. removing
the solvent, wherein a particle is formed comprising the protein
coated with the polymer.
34. The method of claim 33, wherein the protein particle is
obtained by spray drying a solution comprising the protein.
35. The method of claim 34, wherein the spray drying is performed
by dual-nozzle sonication, single-nozzle sonication, or
electrospray.
36. The method of claim 35, wherein the solvent is evaporated by
creating a dispersion of the protein suspension of step (b) by
spray drying.
37. The method of claim 36, wherein the solvent is removed by heat
evaporation, air evaporation, or extraction.
38. The method of claim 37, wherein the polymer is selected from
the group consisting of poly (lactic acid) (PLA), polyanhydride
poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxbutyric
acid-cohydroxyvaleric acid) (PHB-PVA), polyethylene glycol-poly
(lactic acid) copolymer (PEG-PLA), poly-D,L-lactide-co-glycolide
(PLGA), polyorthoester (POE), ethyl cellulose (EC), and
poly-.epsilon.-caprolactone (PCL).
39. The method of claim 38, wherein the protein is a
receptor-Fc-fusion protein or an antibody.
40. The method of claim 39, wherein the protein is a VEGF-Trap or
an IgG molecule, and the polymer is POE, PLA, or PLGA.
41. The method of claim 40, wherein the particle has an average
diameter of about 15 .mu.m.
42. A method for modulating the release of a protein, comprising
the step of (a) combining a protein with a biodegradable polymer to
form a protein-polymer complex; followed by the step of (b)
contacting the protein-polymer complex with a solvent, wherein the
polymer degrades over time and the protein is gradually released
into the solvent.
43. The method of claim 42, wherein step (a) is performed according
to claim 39.
44. The method of claim 43, wherein the protein is a VEGF-Trap or
an IgG molecule, and the polymer is POE, PLA, or PLGA.
45. The method of claim 44, wherein the particle has an average
diameter of about 15 .mu.m.
46. The method of claim 45, wherein the protein is released from
the protein-polymer complex over at least three days.
47. The method of claim 45, wherein the protein is released from
the protein-polymer complex over at least 60 days.
48. The method of claim 47, wherein the solvent is buffered
saline.
49. The method of claim 47, wherein the solvent is in vivo.
50. The method of claim 49, wherein the solvent is vitreous humour.
Description
FIELD
[0001] The invention relates to the manufacture, composition, and
use of an extended release protein therapeutic. Specifically, the
invention relates to the manufacture, composition, and use of a
plurality of polymer coated protein microspheres for the extended
and uniform release of protein in an aqueous-based or physiological
environment over time.
BACKGROUND
[0002] The extended release of a therapeutic protein administered
toward a biological target, such as e.g., the retina or a tumor, or
administered parenterally is desirable for the treatment of many
different conditions, including cancers, cardiovascular diseases,
vascular conditions, orthopedic disorders, dental disorders,
wounds, autoimmune diseases, gastrointestinal disorders, and ocular
diseases. Biocompatible and biodegradable polymers for the
controlled and extended delivery of drugs have been in use for
decades. As the polymer degrades over time, the therapeutic drug is
slowly released.
[0003] In the case of intraocular therapeutics, there is a
significant unmet medical need for extended release formulations to
deliver protein therapeutics effectively over time with as few
intraocular injections as possible. In the case of other diseases,
such as cancer, diseases of inflammation, and other diseases, there
is a need for improved implantable extended release formulations
containing protein therapeutics.
[0004] Applicants have discovered and herein disclose and claim
methods of manufacturing and using microparticles containing a
biodegradable polymer and a therapeutic protein, which is capable
of releasing a therapeutically effective amount of the therapeutic
protein uniformly over an extended period of time.
SUMMARY
[0005] In one aspect, the invention provides a microparticle
comprising a protein coated with a polymer. In one embodiment, the
microparticle has a diameter of from about 2 microns to about 70
microns. In one embodiment, the microparticle has a diameter of
about 15 microns.
[0006] In one embodiment, the protein is an antigen-binding
protein. In one embodiment, the protein comprises an Fc domain. In
one embodiment, the protein comprises a receptor domain. In one
embodiment, the protein is an antibody. In another embodiment, the
protein is a receptor-Fc-fusion protein. In another embodiment, the
protein is a trap-type protein, which comprises a cognate-receptor
fragment and an Fc domain. In one particular embodiment, the
protein is a VEGF-Trap protein. In one embodiment, the VEGF-Trap
protein comprises an amino acid sequence set forth in SEQ ID
NO:1.
[0007] In one embodiment, the polymer is a biodegradable polymer.
In some embodiments, the polymer is selected from the group
consisting of polylactic acid (PLA), polyglycolic acid (PGA),
polylactic-polyglycolic copolymer (PLGA),
poly-D,L-lactide-co-glycolide (PLGA), PLGA-ethylene oxide fumarate,
PLGA-alpha-tocopheryl succinate esterified to polyethylene glycol
1000 (PLGA-TGPS), polyanhydride
poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxbutyric
acid-cohydroxyvaleric acid) (PHB-PVA), polyethylene glycol-poly
(lactic acid) copolymer (PEG-PLA), poly-.epsilon.-caprolactone
(PCL), poly-alkyl-cyano-acrylate (PAC), poly(ethyl)cyanoacrylate
(PEC), polyisobutyl cyanoacrylate,
poly-N-(2-hydroxypropyl)methacrylamide (poly(HPMA)),
poly-.beta.-R-hydroxy butyrate (PHB), poly-.beta.-R-hydroxy
alkanoate (PHA), poly-.beta.-R-malic acid, phospholipid-cholesterol
polymers,
2-dioleoyl-sn-glycero-3-phosphatidylcholine/polyethyleneglycol--
distearoylphosphatidylehtanolamine (DOPC/PEG-DSPE)/Cholesterol,
polysaccharides, cellulose, ethyl cellulose, methyl cellulose,
alginates, dextran and dextran hydrogel polymers, amylose, inulin,
pectin and guar gum, chitosan, chitin, heparin, hyaluronic acid,
cyclodextrin (CD)-based polyrotaxanes and polypseudorotaxanes,
polyaspartates, polyglutamates, polylucine, leucine-glutamate
co-polymers, polybutylene succinate (PBS), gelatin, collagens,
fibrins, fibroin, polyorthoesters, polyorthoester-polyamidine
copolymer, polyorthoester-diamine copolymers, polyorthoesters
incorporating latent acids, poly(ethylene glycol)/poly(butylene
terephthalate) copolymer, and combinations and copolymers thereof.
In one embodiment, the polymer is poly-.epsilon.-caprolactone (PCL)
or a derivative or copolymer thereof. In one embodiment, the
polymer is PLGA or a derivative or copolymer thereof. In one
embodiment, the polymer is ethyl cellulose or a derivative or
copolymer thereof. In one embodiment, the polymer is polyorthoester
or a derivative or copolymer thereof.
[0008] In one embodiment, the microparticle comprises a micronized
protein core of less that ten microns and a polymer cortex. In one
embodiment, the micronized protein core is at least 50% coated with
polymer, which means that no more than 50% of the surface of the
micronized protein core is exposed. In one embodiment, at least
60%, at least 70%, at least 80%, at least 90%, at least 95%, at
least 99%, or 100% of the surface of the micronized protein core is
coated with polymer.
[0009] In one embodiment, the microparticle of greater than 10
microns in size comprises (a) a micronized protein core of less
that 10 microns, wherein the protein is any one or more of an
antibody or antibody fragment, a receptor or soluble fragment
thereof, a soluble T-cell receptor fragment, a soluble MHC
fragment, a receptor-Fc-fusion protein, a trap-type protein, and a
VEGF-Trap protein; and (b) a polymer coat, wherein the polymer is
any one or more of a biocompatible polymer, a biodegradable
polymer, a bio-erodible polymer, polylactic acid (PLA),
polyglycolic acid (PGA), polylactic-polyglycolic copolymer (PLGA),
poly-D,L-lactide-co-glycolide (PLGA), PLGA-ethylene oxide fumarate,
PLGA-alpha-tocopheryl succinate esterified to polyethylene glycol
1000 (PLGA-TGPS), polyanhydride
poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxbutyric
acid-cohydroxyvaleric acid) (PHB-PVA), polyethylene glycol-poly
(lactic acid) copolymer (PEG-PLA), poly-.epsilon.-caprolactone
(PCL), poly-alkyl-cyano-acrylate (PAC), poly(ethyl)cyanoacrylate
(PEC), polyisobutyl cyanoacrylate,
poly-N-(2-hydroxypropyl)methacrylamide (poly(HPMA)),
poly-.beta.-R-hydroxy butyrate (PHB), poly-.beta.-R-hydroxy
alkanoate (PHA), poly-.beta.-R-malic acid, phospholipid-cholesterol
polymers,
2-dioleoyl-sn-glycero-3-phosphatidylcholine/polyethyleneglycol-distearoyl-
phosphatidylehtanolamine (DOPC/PEG-DSPE)/Cholesterol,
polysaccharides, cellulose, ethyl cellulose, methyl cellulose,
alginates, dextran and dextran hydrogel polymers, amylose, inulin,
pectin and guar gum, chitosan, chitin, heparin, hyaluronic acid,
cyclodextrin (CD)-based polyrotaxanes and polypseudorotaxanes,
polyaspartates, polyglutamates, polylucine, leucine-glutamate
co-polymers, polybutylene succinate (PBS), gelatin, collagens,
fibrins, fibroin, polyorthoesters, polyorthoester-polyamidine
copolymer, polyorthoester-diamine copolymers, polyorthoesters
incorporating latent acids, poly(ethylene glycol)/poly(butylene
terephthalate) copolymer, and combinations and copolymers
thereof.
[0010] In one embodiment, the microparticle of an average diameter
of about 15 microns to about 30 microns comprises (a) a micronized
protein core of about 10 to about 12 microns, wherein the protein
is a VEGF-Trap protein; and (b) a polymer coat, wherein the polymer
is any one or more of PCL, PLGA, ethyl cellulose and
polyorthoester, and copolymers or derivatives thereof.
[0011] In one aspect, the invention provides a plurality of
microparticles, which range in size from about two microns to about
70 microns, and which comprise a micronized protein core of about
two microns to about 30 microns, and a polymer cortex.
[0012] In one embodiment, the protein is an antigen-binding
protein. In some embodiments, the antigern-binding protein is any
one or more of an antibody or antibody fragment, a receptor or
soluble fragment thereof, a soluble T-cell receptor fragment, a
soluble MHC fragment, a receptor-Fc-fusion protein, a trap-type
protein, and a VEGF-Trap protein. In one embodiment, the protein
comprises an Fc domain. In one embodiment, the protein is an
antibody. In another embodiment, the protein is a
receptor-Fc-fusion protein. In another embodiment, the protein is a
trap-type protein, which comprises a cognate-receptor fragment and
an Fc domain. In one particular embodiment, the protein is a
VEGF-Trap protein. In a specific embodiment, the VEGF-Trap protein
comprises the amino acid sequence set forth in SEQ ID NO:1.
[0013] In one embodiment, the polymer is a biocompatible polymer.
In one embodiment, the polymer is a bioerodible polymer. In one
embodiment, the polymer is a biodegradable polymer. In some
embodiments, the polymer is selected from the group consisting of
polylactic acid (PLA), polyglycolic acid (PGA),
polylactic-polyglycolic copolymer (PLGA),
poly-D,L-lactide-co-glycolide (PLGA), PLGA-ethylene oxide fumarate,
PLGA-alpha-tocopheryl succinate esterified to polyethylene glycol
1000 (PLGA-TGPS), polyanhydride
poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxbutyric
acid-cohydroxyvaleric acid) (PHB-PVA), polyethylene glycol-poly
(lactic acid) copolymer (PEG-PLA), poly-.epsilon.-caprolactone
(PCL), poly-alkyl-cyano-acrylate (PAC), poly(ethyl)cyanoacrylate
(PEC), polyisobutyl cyanoacrylate,
poly-N-(2-hydroxypropyl)methacrylamide (poly(HPMA)),
poly-.beta.-R-hydroxy butyrate (PHB), poly-.beta.-R-hydroxy
alkanoate (PHA), poly-.beta.-R-malic acid, phospholipid-cholesterol
polymers,
2-dioleoyl-sn-glycero-3-phosphatidylcholine/polyethyleneglycol-distearoyl-
phosphatidylehtanolamine (DOPC/PEG-DSPE)/Cholesterol,
polysaccharides, cellulose, ethyl cellulose, methyl cellulose,
alginates, dextran and dextran hydrogel polymers, amylose, inulin,
pectin and guar gum, chitosan, chitin, heparin, hyaluronic acid,
cyclodextrin (CD)-based polyrotaxanes and polypseudorotaxanes,
polyaspartates, polyglutamates, polylucine, leucine-glutamate
co-polymers, polybutylene succinate (PBS), gelatin, collagens,
fibrins, fibroin, polyorthoesters, polyorthoester-polyamidine
copolymer, polyorthoester-diamine copolymers, polyorthoesters
incorporating latent acids, poly(ethylene glycol)/poly(butylene
terephthalate) copolymer, and combinations and copolymers thereof.
In one embodiment, the polymer is poly-.epsilon.-caprolactone (PCL)
or a derivative or copolymer thereof. In one embodiment, the
polymer is PLGA or a derivative or copolymer thereof. In one
embodiment, the polymer is ethyl cellulose or a derivative or
copolymer thereof. In one embodiment, the polymer is a
polyorthoester incorporating a latent acid.
[0014] In one embodiment, the micronized protein core of most
microparticles of the plurality of microparticles is at least 50%
coated with polymer, which means that no more than 50% of the
surface of the micronized protein core is exposed. In one
embodiment, at least 60%, at least 70%, at least 80%, at least 90%,
at least 95%, at least 99%, or 100% of the surface of the
micronized protein core is coated with polymer.
[0015] In one embodiment, the plurality of microparticles, which
range in size from about two microns to about 70 microns, comprise
(a) a micronized protein core of from about two microns to about 30
microns, wherein the protein is any one or more of an antibody or
antibody fragment, a receptor or soluble fragment thereof, a
soluble T-cell receptor fragment, a soluble MHC fragment, a
receptor-Fc-fusion protein, a trap-type protein, and a VEGF-Trap
protein; and (b) a polymer cortex, wherein the polymer is any one
or more of a biocompatible polymer, a biodegradable polymer, a
bio-erodible polymer, polylactic acid (PLA), polyglycolic acid
(PGA), polylactic-polyglycolic copolymer (PLGA),
poly-D,L-lactide-co-glycolide (PLGA), PLGA-ethylene oxide fumarate,
PLGA-alpha-tocopheryl succinate esterified to polyethylene glycol
1000 (PLGA-TGPS), poly-.epsilon.-caprolactone (PCL),
poly-alkyl-cyano-acrylate (polyanhydride
poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxbutyric
acid-cohydroxyvaleric acid) (PHB-PVA), polyethylene glycol-poly
(lactic acid) copolymer (PEG-PLA),), poly(ethyl)cyanoacrylate
(PEC), polyisobutyl cyanoacrylate,
poly-N-(2-hydroxypropyl)methacrylamide (poly(HPMA)),
poly-.beta.-R-hydroxy butyrate (PHB), poly-.beta.-R-hydroxy
alkanoate (PHA), poly-.beta.-R-malic acid, phospholipid-cholesterol
polymers,
2-dioleoyl-sn-glycero-3-phosphatidylcholine/polyethyleneglycol--
distearoylphosphatidylehtanolamine (DOPC/PEG-DSPE)/Cholesterol,
polysaccharides, cellulose, ethyl cellulose, methyl cellulose,
alginates, dextran and dextran hydrogel polymers, amylose, inulin,
pectin and guar gum, chitosan, chitin, heparin, hyaluronic acid,
cyclodextrin (CD)-based polyrotaxanes and polypseudorotaxanes,
polyaspartates, polyglutamates, polylucine, leucine-glutamate
co-polymers, polybutylene succinate (PBS), gelatin, collagens,
fibrins, fibroin, polyorthoesters, polyorthoester-polyamidine
copolymer, polyorthoester-diamine copolymers, polyorthoesters
incorporating latent acids, poly(ethylene glycol)/poly(butylene
terephthalate) copolymer, and combinations and copolymers
thereof.
[0016] In one embodiment, the plurality of microparticles, which
range in size from about two microns to about 70 microns, with a
median size of from about 15 microns to about 30 microns, comprise
(a) a micronized protein core of from about two microns to about 30
microns, with a median size of about 10 microns to about 12
microns, wherein the protein is a VEGF-Trap protein; and (b) a
polymer cortex, wherein the polymer is any one or more of PLA, PCL,
PLGA, ethyl cellulose and polyorthoester, and copolymers or
derivatives thereof.
[0017] In one aspect, the invention provides a method of
manufacturing a microparticle, which comprises a protein core and a
polymer cortex. In one embodiment, the manufactured microparticle
has a diameter of about two microns to about 70 microns, or a
median diameter of about 15 microns to about 30 microns. In one
embodiment, the method of manufacturing the microparticle comprises
(1) obtaining a protein particle; (2) suspending the protein
particle in a solution comprising the polymer and a solvent; and
(3) removing the solvent, wherein a microparticle is formed
comprising the protein core coated with the polymer cortex.
[0018] In one embodiment, the protein particle of step (1) is a
micronized protein particle, which is obtained by spray drying a
solution comprising the protein. In some embodiments, the protein
solution is spray dried via dual-nozzle sonication, single-nozzle
sonication, or electrospray. In some embodiments, the resultant
micronized protein particle, which forms the core of the
manufactured microparticle, has a diameter of from about two
microns to about 30 microns, with a median diameter of about 10
microns to about 12 microns.
[0019] In some embodiments, the protein which forms the core is an
antigen-binding protein. In some embodiments, the antigen-binding
protein is any one or more of an antibody (e.g., IgG) or antibody
fragment, a receptor or soluble fragment thereof, a soluble T-cell
receptor fragment, a soluble MHC fragment, a receptor-Fc-fusion
protein, a trap-type protein, and a VEGF-Trap protein. In a
specific embodiment, the protein is a VEGF-Trap comprising the
amino acid sequence set forth in SEQ ID NO:1.
[0020] In one embodiment, the solvent is removed at step (3) by
creating a dispersion of the protein-polymer-solvent mixture of
step (2) and allowing the solvent to evaporate from the droplets
created by the dispersion. In one embodiment, the dispersion is
created by spray-drying, which may be performed by dual-nozzle
sonication, single-nozzle sonication, or electrospray. In one
embodiment, the solvent is removed from the droplets by applying
heat or air, or by chemical extraction.
[0021] In one embodiment, the polymer is biodegradable,
bioerodible, and/or biocompatible. In some embodiments, the polymer
is any one or more of polylactic acid (PLA), polyglycolic acid
(PGA), polylactic-polyglycolic copolymer (PLGA),
poly-D,L-lactide-co-glycolide (PLGA), PLGA-ethylene oxide fumarate,
PLGA-alpha-tocopheryl succinate esterified to polyethylene glycol
1000 (PLGA-TGPS), polyanhydride
poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxbutyric
acid-cohydroxyvaleric acid) (PHB-PVA), polyethylene glycol-poly
(lactic acid) copolymer (PEG-PLA), poly-.epsilon.-caprolactone
(PCL), poly-alkyl-cyano-acrylate (PAC), poly(ethyl)cyanoacrylate
(PEC), polyisobutyl cyanoacrylate,
poly-N-(2-hydroxypropyl)methacrylamide (poly(HPMA)),
poly-.beta.-R-hydroxy butyrate (PHB), poly-.beta.-R-hydroxy
alkanoate (PHA), poly-.beta.-R-malic acid, phospholipid-cholesterol
polymers,
2-dioleoyl-sn-glycero-3-phosphatidylcholine/polyethyleneglycol--
distearoylphosphatidylehtanolamine (DOPC/PEG-DSPE)/Cholesterol,
polysaccharides, cellulose, ethyl cellulose, methyl cellulose,
alginates, dextran and dextran hydrogel polymers, amylose, inulin,
pectin and guar gum, chitosan, chitin, heparin, hyaluronic acid,
cyclodextrin (CD)-based polyrotaxanes and polypseudorotaxanes,
polyaspartates, polyglutamates, polylucine, leucine-glutamate
co-polymers, polybutylene succinate (PBS), gelatin, collagens,
fibrins, fibroin, polyorthoesters, polyorthoester-polyamidine
copolymer, polyorthoester-diamine copolymers, polyorthoesters
incorporating latent acids, poly(ethylene glycol)/poly(butylene
terephthalate) copolymer, and combinations and copolymers thereof.
In one embodiment, the polymer is poly-.epsilon.-caprolactone (PCL)
or a derivative or copolymer thereof. In one embodiment, the
polymer is PLGA or a derivative or copolymer thereof. In one
embodiment, the polymer is ethyl cellulose or a derivative or
copolymer thereof. In one embodiment, the polymer is
polyorthoester, or a derivative thereof, which contains acid labile
elements. In another embodiment, the polymer is PLA.
[0022] In one aspect, the invention provides a method of
manufacturing a microparticle comprising the steps of (1) forming a
micronized protein particle having a diameter of from about two
microns to about 30 microns, with a median diameter of from about
10 microns to 12 microns, by spray-drying a solution containing a
protein, wherein the protein is an antigen-binding protein. In some
embodiments, the antigen-binding protein is any one or more of an
antibody or antibody fragment, a receptor or soluble fragment
thereof, a soluble T-cell receptor fragment, a soluble MHC
fragment, a receptor-Fc-fusion protein, a trap-type protein, and a
VEGF-Trap protein (e.g., one having the sequence of SEQ ID NO:1);
(2) suspending the micronized protein particle in a solution
comprising the polymer and a solvent, wherein the polymer is any
one or more of a biodegradable polymer, a bioerodible polymer, a
biocompatible polymer, polylactic acid (PLA), polyglycolic acid
(PGA), polylactic-polyglycolic copolymer (PLGA),
poly-D,L-lactide-co-glycolide (PLGA), PLGA-ethylene oxide fumarate,
PLGA-alpha-tocopheryl succinate esterified to polyethylene glycol
1000 (PLGA-TGPS), polyanhydride
poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxbutyric
acid-cohydroxyvaleric acid) (PHB-PVA), polyethylene glycol-poly
(lactic acid) copolymer (PEG-PLA), poly-.epsilon.-caprolactone
(PCL), poly-alkyl-cyano-acrylate (PAC), poly(ethyl)cyanoacrylate
(PEC), polyisobutyl cyanoacrylate,
poly-N-(2-hydroxypropyl)methacrylamide (poly(HPMA)),
poly-.beta.-R-hydroxy butyrate (PHB), poly-.beta.-R-hydroxy
alkanoate (PHA), poly-.beta.-R-malic acid, phospholipid-cholesterol
polymers,
2-dioleoyl-sn-glycero-3-phosphatidylcholine/polyethyleneglycol--
distearoylphosphatidylehtanolamine (DOPC/PEG-DSPE)/Cholesterol,
polysaccharides, cellulose, ethyl cellulose, methyl cellulose,
alginates, dextran and dextran hydrogel polymers, amylose, inulin,
pectin and guar gum, chitosan, chitin, heparin, hyaluronic acid,
cyclodextrin (CD)-based polyrotaxanes and polypseudorotaxanes,
polyaspartates, polyglutamates, polylucine, leucine-glutamate
co-polymers, polybutylene succinate (PBS), gelatin, collagens,
fibrins, fibroin, polyorthoesters, polyorthoester-polyamidine
copolymer, polyorthoester-diamine copolymers, polyorthoesters
incorporating latent acids, poly(ethylene glycol)/poly(butylene
terephthalate) copolymer, and combinations and copolymers thereof;
and (3) removing the solvent by spray-drying micronized protein
particle-polymer-solvent suspension and driving off the solvent by
applying heat or air, or by extracting the solvent, wherein a
microparticle is formed having a diameter of about two microns to
about 70 microns, with a median diameter of from about 15 microns
to about 30 microns, and comprising a protein core and a polymer
cortex.
[0023] In some embodiments, the spray-drying of step (1) or step
(3) is performed via dual-nozzle sonication, single-nozzle
sonication, or electrospray.
[0024] In one embodiment, the method of manufacturing the
microparticle comprises the steps of (1) forming a micronized
VEGF-Trap particle having a diameter of from about 10 microns to 12
microns by spray-drying a solution containing a VEGF Trap protein;
(2) suspending the micronized VEGF Trap particle in a solution
comprising polyorthoester incorporating a latent acid and a
compatible solvent, or ethylcellulose and a compatible solvent; and
(3) removing the solvent by (a) spray-drying the micronized VEGF
Trap particle-polyorthoester-latent acid-solvent suspension or the
micronized VEGF Trap particle-ethyl cellulose-solvent suspension
and (b) driving off the solvent by applying heat or air, or by
extracting the solvent, wherein a microparticle is formed having a
diameter of about 15 microns to about 30 microns, and comprising a
VEGF-Trap core and a polymer cortex of polyorthoester, and
copolymers or derivatives thereof.
[0025] In one aspect, the invention provides an extended release
formulation of a therapeutic protein for the release or delivery of
a steady level of the therapeutic protein over time. The extended
release formulation comprises a plurality of microparticles, which
range in size from about two microns to about 70 microns, each of
which comprises a micronized protein core of about two microns to
about 30 microns, and a polymer cortex.
[0026] In one embodiment, the therapeutic protein is an
antigen-binding protein. In some embodiments, the antigen-binding
protein is any one or more of an antibody (e.g., IgG) or antibody
fragment, a receptor or soluble fragment thereof, a soluble T-cell
receptor fragment, a soluble MHC fragment, a receptor-Fc-fusion
protein, a trap-type protein, and a VEGF-Trap protein (e.g., one of
which has a primary structure of SEQ ID NO:1). In one embodiment,
the therapeutic protein comprises an Fc domain. In one embodiment,
the protein is an antibody. In another embodiment, the protein is
an IgG. In another embodiment, the therapeutic protein is a
receptor-Fc-fusion protein. In another embodiment, the therapeutic
protein is a trap-type protein, which comprises a cognate-receptor
fragment and an Fc domain. In one particular embodiment, the
therapeutic protein is a VEGF-Trap protein. In yet another
embodiment, the VEGF-Trap comprises the amino acid sequence set
forth in SEQ ID NO:1.
[0027] In one embodiment, the polymer cortex comprises a
biocompatible polymer. In one embodiment, the polymer cortex
comprises a bioerodible polymer. In one embodiment, the polymer
cortex comprises a biodegradable polymer. In some embodiments, the
polymer cortex comprises a polymer selected from the group
consisting of polylactic acid (PLA), polyglycolic acid (PGA),
polylactic-polyglycolic copolymer (PLGA),
poly-D,L-lactide-co-glycolide (PLGA), PLGA-ethylene oxide fumarate,
PLGA-alpha-tocopheryl succinate esterified to polyethylene glycol
1000 (PLGA-TGPS), polyanhydride
poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxbutyric
acid-cohydroxyvaleric acid) (PHB-PVA), polyethylene glycol-poly
(lactic acid) copolymer (PEG-PLA), poly-.epsilon.-caprolactone
(PCL), poly-alkyl-cyano-acrylate (PAC), poly(ethyl)cyanoacrylate
(PEC), polyisobutyl cyanoacrylate,
poly-N-(2-hydroxypropyl)methacrylamide (poly(HPMA)),
poly-.beta.-R-hydroxy butyrate (PHB), poly-.beta.-R-hydroxy
alkanoate (PHA), poly-.beta.-R-malic acid, phospholipid-cholesterol
polymers,
2-dioleoyl-sn-glycero-3-phosphatidylcholine/polyethyleneglycol-distearoyl-
phosphatidylehtanolamine (DOPC/PEG-DSPE)/Cholesterol,
polysaccharides, cellulose, ethyl cellulose, methyl cellulose,
alginates, dextran and dextran hydrogel polymers, amylose, inulin,
pectin and guar gum, chitosan, chitin, heparin, hyaluronic acid,
cyclodextrin (CD)-based polyrotaxanes and polypseudorotaxanes,
polyaspartates, polyglutamates, polylucine, leucine-glutamate
co-polymers, polybutylene succinate (PBS), gelatin, collagens,
fibrins, fibroin, polyorthoesters, polyorthoester-polyamidine
copolymer, polyorthoester-diamine copolymers, polyorthoesters
incorporating latent acids, poly(ethylene glycol)/poly(butylene
terephthalate) copolymer, and combinations and copolymers thereof.
In one embodiment, the polymer is poly-.epsilon.-caprolactone (PCL)
or a derivative or copolymer thereof. In one embodiment, the
polymer cortex comprises a PLGA. In one embodiment, the polymer
cortex comprises an ethyl cellulose. In one embodiment, the polymer
cortex comprises any one or more of PLA, PLGA, ethyl cellulose, and
polyorthoester, and copolymers or derivatives thereof.
[0028] In one embodiment, plurality of microparticles comprises a
collection of microparticles having a range of thicknesses of the
polymer cortex, such that individual microparticles of the
collection of microparticles degrades at a different rate, which
allows for a uniform rate of release of the therapeutic
protein.
[0029] In one embodiment, the plurality of microparticles comprises
a mixture of uncoated micronized protein particles and
microparticles having a range of thicknesses of the polymer cortex,
which allows for the release of therapeutic protein at periodic
intervals based on cortex thickness.
[0030] In one embodiment, the plurality of microparticles comprises
a mixture of microparticles having polymer cortices of varying
levels of hydrophobicity to control the timing or duration of
degradation and subsequent release. In one embodiment, the
microparticles each comprise an inner polymer layer and an outer
polymer layer, wherein the outer polymer layer limits the hydration
of the inner polymer layer to control release of the therapeutic
protein.
[0031] In one embodiment, the therapeutic protein is released from
the plurality of microparticles at a rate of from about 0.01
mg/week to about 0.30 mg/week for a duration of at least 60 days,
when the microparticles are in an aqueous environment. In one
embodiment, the aqueous environment is in vitro buffer. In one
embodiment, the aqueous environment is in vivo. In one embodiment,
the aqueous environment is ex vivo. In one embodiment, the aqueous
environment is a vitreous humor.
[0032] In one embodiment, the extended release formulation
comprises a plurality of microparticles, which range in size from
about two microns to about 70 microns and which comprise (a) a core
of micronized therapeutic protein of from about two microns to
about 30 microns, wherein the therapeutic protein is an
antigen-binding protein, which in some cases can be any one or more
of an antibody or antibody fragment, a receptor or soluble fragment
thereof, a soluble T-cell receptor fragment, a soluble MHC
fragment, a receptor-Fc-fusion protein, a trap-type protein, and a
VEGF-Trap protein; and (b) a polymer cortex of a range of
thicknesses, wherein the polymer is any one or more of a
biocompatible polymer, a biodegradable polymer, a bio-erodible
polymer, polylactic acid (PLA), polyglycolic acid (PGA),
polylactic-polyglycolic copolymer (PLGA),
poly-D,L-lactide-co-glycolide (PLGA), PLGA-ethylene oxide fumarate,
PLGA-alpha-tocopheryl succinate esterified to polyethylene glycol
1000 (PLGA-TGPS), polyanhydride
poly[1,6-bis(p-carboxyphenoxy)hexane](pCPH), poly(hydroxbutyric
acid-cohydroxyvaleric acid) (PHB-PVA), polyethylene glycol-poly
(lactic acid) copolymer (PEG-PLA), poly-.epsilon.-caprolactone
(PCL), poly-alkyl-cyano-acrylate (PAC), poly(ethyl)cyanoacrylate
(PEC), polyisobutyl cyanoacrylate,
poly-N-(2-hydroxypropyl)methacrylamide (poly(HPMA)),
poly-.beta.-R-hydroxy butyrate (PHB), poly-.beta.-R-hydroxy
alkanoate (PHA), poly-.beta.-R-malic acid, phospholipid-cholesterol
polymers,
2-dioleoyl-sn-glycero-3-phosphatidylcholine/polyethyleneglycol-distearoyl-
phosphatidylehtanolamine (DOPC/PEG-DSPE)/Cholesterol,
polysaccharides, cellulose, ethyl cellulose, methyl cellulose,
alginates, dextran and dextran hydrogel polymers, amylose, inulin,
pectin and guar gum, chitosan, chitin, heparin, hyaluronic acid,
cyclodextrin (CD)-based polyrotaxanes and polypseudorotaxanes,
polyaspartates, polyglutamates, polylucine, leucine-glutamate
co-polymers, polybutylene succinate (PBS), gelatin, collagens,
fibrins, fibroin, polyorthoesters, polyorthoester-polyamidine
copolymer, polyorthoester-diamine copolymers, polyorthoesters
incorporating latent acids, poly(ethylene glycol)/poly(butylene
terephthalate) copolymer, and combinations and copolymers thereof,
wherein the microparticles release or deliver a steady level of the
therapeutic protein at a rate of from about 0.01 mg/week to about
0.30 mg/week for at least 60 days.
[0033] In one embodiment, the extended release formulation
comprises a plurality of microparticles, which range in size from
about two microns to about 70 microns, with a median size of from
about 15 microns to about 30 microns, and which comprise (a) a
micronized protein core of from about two microns to about 30
microns, with a median size of about 10 microns to about 12
microns, wherein the protein is a VEGF-Trap protein; and (b) a
polymer cortex of a range of thicknesses, wherein the polymer is
any one or more of PLGA, ethyl cellulose, and polyorthoester, and
copolymers or derivatives thereof, such that in an aqueous
environment the microparticles release or deliver a steady level of
VEGF Trap at a rate of about 0.06.+-.0.02 mg/week for at least 60
days.
[0034] In one aspect, the invention provides a method for
modulating the release of a protein. In one embodiment, the method
comprises the step of making a plurality of microparticles as
described in the previous aspect, followed by the step of placing
the microparticles into a solvent. The solvent in some embodiments
is aqueous. The solvent can be in vitro, such as in a phosphate
buffered solution. The solvent can be in vivo, such as e.g.
vitreous humour.
DRAWINGS
[0035] FIG. 1 depicts the relative amount (% volume) of protein
particles without a polymer cortex of a given diameter (ECD
(.mu.m)) in a population of protein particles manufactured from 50
mg/mL of VEGF Trap protein, 25 mg/mL of VEGF Trap protein, and 25
mg/mL of VEGF Trap protein plus 0.1% polysorbate 80.
[0036] FIG. 2 depicts the relative amount (% volume determined by
MFI) of microparticles of a given diameter (ECD (.mu.m)) in a
population of microparticles manufactured from 50 mg/mL of VEGF
Trap protein plus 50 mg/mL POE, 250 mg/mL POE, and 50 mg/mL EC.
[0037] FIG. 3 depicts the amount of VEGF Trap protein in milligrams
released from microparticles manufactured from 50 mg/mL POE, 250
mg/mL POE, or 50 mg/mL EC over approximately 60 days.
DETAILED DESCRIPTION
[0038] The micro particle and protein core particle of the subject
invention are roughly spherical in shape. Some microparticles and
protein cores will approach sphericity, while others will be more
irregular in shape. Thus, as used herein, the term "diameter" means
each and any of the following: (a) the diameter of a sphere which
circumscribes the microparticle or protein core, (b) the diameter
of the largest sphere that fits within the confines of the
microparticle or the protein core, (c) any measure between the
circumscribed sphere of (a) and the confined sphere of (b),
including the mean between the two, (d) the length of the longest
axis of the microparticle or protein core, (e) the length of the
shortest axis of the microparticle or protein core, (f) any measure
between the length of the long axis (d) and the length of the short
axis (e), including the mean between the two, and/or (g) equivalent
circular diameter ("ECD"), as determined by micro-flow imaging
(MFI), nanoparticle tracking analysis (NTA), or light obscuration
methods such as dynamic light scattering (DLS). See generally
Sharma et al., Micro-flow imaging: flow microscopy applied to
subvisible particulate analysis in protein formulations, AAPS J.
2010 September; 12(3): 455-64. Diameter is generally expressed in
micrometers (.mu.m or micron). Diameter can be determined by
optical measurement
[0039] "Micronized protein particle" or "protein particle" means a
particle containing multiple molecules of protein with low, very
low, or close to zero amounts of water (e.g., <3% water by
weight). As used herein, the micronized protein particle is
generally spherical in shape and has an ECD ranging from 2 microns
to about 35 microns. The micronized protein particle is not limited
to any particular protein entity, and is suited to the preparation
and delivery of a therapeutic protein. Common therapeutic proteins
include inter alia antigen-binding proteins, such as e.g., soluble
receptor fragments, antibodies (including IgGs) and derivatives or
fragments of antibodies, other Fc containing proteins, including Fc
fusion proteins, and receptor-Fc fusion proteins, including the
trap-type proteins (Huang, C., Curr. Opin. Biotechnol. 20: 692-99
(2009)) such as e.g. VEGF-Trap.
[0040] The micronized protein particle of the invention can be made
by any method known in the art for making micron-sized protein
particles. For example, the protein particle may be made by inter
alia spray-drying (infra), lyophilization, jet milling, hanging
drop crystallization (Ruth et al., Acta Crystallographica D56:
524-28 (2000)), gradual precipitation (U.S. Pat. No. 7,998,477
(2011)), lyophilyzation of a protein-PEG (polyethylene glycol)
aqueous mixture (Morita et al., Pharma. Res. 17: 1367-73 (2000)),
supercritical fluid precipitation (U.S. Pat. No. 6,063,910 (2000)),
or high pressure carbon dioxide induced particle formation (Bustami
et al., Pharma. Res. 17: 1360-66 (2000)).
[0041] As used herein, the term "protein" refers to a molecule
comprising two or more amino acid residues joined to each other by
peptide bonds. Peptides, polypeptides and proteins are also
inclusive of modifications including, but not limited to,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation.
Polypeptides can be of scientific or commercial interest, including
protein-based drugs. Polypeptides include, among other things,
antibodies and chimeric or fusion proteins. Polypeptides are
produced by recombinant animal cell lines using cell culture
methods.
[0042] An "antibody" is intended to refer to immunoglobulin
molecules consisting of four polypeptide chains, two heavy (H)
chains and two light (L) chains inter-connected by disulfide bonds.
Each heavy chain has a heavy chain variable region (HCVR or VH) and
a heavy chain constant region. The heavy chain constant region
contains three domains, CH1, CH2 and CH3. Each light chain has of a
light chain variable region and a light chain constant region. The
light chain constant region consists of one domain (CL). The VH and
VL regions can be further subdivided into regions of
hypervariability, termed complementarity determining regions (CDR),
interspersed with regions that are more conserved, termed framework
regions (FR). Each VH and VL is composed of three CDRs and four
FRs, arranged from amino-terminus to carboxy-terminus in the
following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term
"antibody" includes reference to both glycosylated and
non-glycosylated immunoglobulins of any isotype or subclass. The
term "antibody" is inclusive of, but not limited to, those that are
prepared, expressed, created or isolated by recombinant means, such
as antibodies isolated from a host cell transfected to express the
antibody. An IgG comprises a subset of antibodies.
[0043] "Fc fusion proteins" comprise part or all of two or more
proteins, one of which is an Fc portion of an immunoglobulin
molecule, that are not fused in their natural state. Preparation of
fusion proteins comprising certain heterologous polypeptides fused
to various portions of antibody-derived polypeptides (including the
Fc domain) has been described, e.g., by Ashkenazi et al., Proc.
Natl. Acad. ScL USA 88: 10535, 1991; Byrn et al., Nature 344:677,
1990; and Hollenbaugh et al., "Construction of Immunoglobulin
Fusion Proteins", in Current Protocols in Immunology, Suppl. 4,
pages 10.19.1-10.19.11, 1992. "Receptor Fc fusion proteins"
comprise one or more of one or more extracellular domain(s) of a
receptor coupled to an Fc moiety, which in some embodiments
comprises a hinge region followed by a CH2 and CH3 domain of an
immunoglobulin. In some embodiments, the Fc-fusion protein contains
two or more distinct receptor chains that bind to a single or more
than one ligand(s). For example, an Fc-fusion protein is a trap,
such as for example an IL-1 trap (e.g., Rilonacept, which contains
the IL-1 RAcP ligand binding region fused to the IL-1R1
extracellular region fused to Fc of hIgG1; see U.S. Pat. No.
6,927,004, which is herein incorporated by reference in its
entirety), or a VEGF Trap (e.g., Aflibercept, which contains the Ig
domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the
VEGF receptor Flk1 fused to Fc of hIgG1; e.g., SEQ ID NO:1; see
U.S. Pat. Nos. 7,087,411 and 7,279,159, which are herein
incorporated by reference in their entirety).
[0044] As used herein, the term "polymer" refers to a macromolecule
comprising repeating monomers connected by covalent chemical bonds.
Polymers used in the practice of this invention are biocompatible
and biodegradable. A biocompatible and biodegradable polymer can be
natural or synthetic. Natural polymers include polynucleotides,
polypeptides, such as naturally occurring proteins, recombinant
proteins, gelatin, collagens, fibrins, fibroin, polyaspartates,
polyglutamates, polyleucine, leucine-glutamate co-polymers; and
polysaccharides, such as cellulose alginates, dextran and dextran
hydrogel polymers, amylose, inulin, pectin and guar gum, chitosan,
chitin, heparin, and hyaluronic acid. Synthetic biocompatible or
biodegradable polymers include polylactic acid (PLA), polyglycolic
acid (PGA), polylactic-polyglycolic copolymer (PLGA),
poly-D,L-lactide-co-glycolide (PLGA), PLGA-ethylene oxide fumarate,
PLGA-alpha-tocopheryl succinate esterified to polyethylene glycol
1000 (PLGA-TGPS), polyanhydride
poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxbutyric
acid-cohydroxyvaleric acid) (PHB-PVA), polyethylene glycol-poly
(lactic acid) copolymer (PEG-PLA), poly-.epsilon.-caprolactone
(PCL), poly-alkyl-cyano-acrylate (PAC), poly(ethyl)cyanoacrylate
(PEC), polyisobutyl cyanoacrylate,
poly-N-(2-hydroxypropyl)methacrylamide (poly(HPMA)),
poly-.beta.-R-hydroxy butyrate (PHB), poly-.beta.-R-hydroxy
alkanoate (PHA), poly-.beta.-R-malic acid, phospholipid-cholesterol
polymers,
2-dioleoyl-sn-glycero-3-phosphatidylcholine/polyethyleneglycol-distearoyl-
phosphatidylehtanolamine (DOPC/PEG-DSPE)/Cholesterol, ethyl
cellulose, cyclodextrin (CD)-based polyrotaxanes and
polypseudorotaxanes, polybutylene succinate (PBS), polyorthoesters,
polyorthoester-polyamidine copolymers, polyorthoester-diamine
copolymers, polyorthoesters incorporating latent acids tom control
rates of degradation, and inter alia poly(ethylene
glycol)/poly(butylene terephthalate) copolymers.
[0045] Ethyl cellulose (EC) is a well-known and readily available
biomaterial used in the pharmaceutical and food sciences. It is a
cellulose derivative in which some of the glucose hydroxyl groups
are replaced with ethyl ether. See Martinac et al., J.
Microencapsulation, 22(5): 549-561 (2005) and references therein,
which describe methods of using ethyl cellulose as biocompatible
polymers in the manufacture of microspheres. See also U.S. Pat. No.
4,210,529 (1980) and references therein for a detailed description
of ethyl cellulose and methods of making derivatives of ethyl
cellulose.
[0046] Poly-D,L-lactide-co-glycolide (PLGA) is also a well-known
Food and Drug Administration (FDA) approved biocompatible and
biodegradable polymer used in tissue engineering and pharmaceutical
delivery systems. PLGA is a polyester comprising glycolic acid and
lactic acid monomers. For a description of the synthesis of PLGA
and manufacture of PLGA nanoparticles, see Astete and Sabliov,
Biomater. Sci. Polym. Ed., 17(3): 247-89 (2006) and references
therein.
[0047] Poly-.epsilon.-caprolactone (PCL) is another biocompatible
and biodegradable polymer approved by the FDA for use in humans as
a drug delivery device. PCL is a polyester of
.epsilon.-caprolactone, which hydrolyses rapidly in the body to
form a non-toxic or low toxicity hydroxycarboxylic acid. For a
description of the manufacture of PCL, see Labet and Thielemans,
Chemical Society Reviews 38: 3484-3504 (2009) and references
therein. For a description of the manufacture and use of PCL-based
microspheres and nanospheres as delivery systems, see Sinha et al.,
Int. J. Pharm., 278(1): 1-23 (2004) and references therein.
[0048] Polyorthoester (POE) is a bioerodible polymer designed for
drug delivery. It is generally a polymer of a ketene acetal,
preferably a cyclic diketene acetal, such as e.g.,
3,9-dimethylene-2,4,8,10-tetraoxa spiro[5.5]-undecane, which is
polymerized via glycol condensation to form the orthoester
linkages. A description of polyorthoester synthesis and various
types can be found e.g. in U.S. Pat. No. 4,304,767. Polyorthoesters
can be modified to control their drug release profile and
degradation rates by swapping in or out various hydrophobic diols
and polyols, such as e.g., replacing a hexanetriol with a
decanetriol.; as well as adding latent acids, such as e.g.,
octanedioic acid or the like, to the backbone to increase pH
sensitivity. Other modifications to the polyorthoester include the
integration of an amine to increase functionality. The formation,
description, and use of polyorthoesters are described in U.S. Pat.
No. 5,968,543; U.S. Pat. No. 4,764,364; Heller and Barr,
Biomacromolecules, 5(5): 1625-32 (2004); and Heller, Adv. Drug.
Deliv. Rev., 57: 2053-62 (2005).
[0049] As used herein, the phrase "spray-dry" means a method of
producing a dry powder comprising micron-sized particles from a
slurry or suspension by using a spray-dryer. Spray dryers employ an
atomizer or spray nozzle to disperse the suspension or slurry into
a controlled drop size spray. Drop sizes from 10 to 500 .mu.m can
be generated by spray-drying. As the solvent (water or organic
solvent) dries, the protein substance dries into a micron-sized
particle, forming a powder-like substance; or in the case of a
protein-polymer suspension, during drying, the polymer hardened
shell around the protein load.
[0050] The microparticles of the invention comprise a protein core
surrounded by a polymer cortex or coat. Briefly, a micronized
protein particle is formed, which is then dispersed in a polymer
solution (polymer dissolved in solvent) to form a protein-polymer
suspension. The protein-polymer suspension is then dispersed into
micronized (atomized) droplets, and the solvent is driven-off to
form the microparticle.
[0051] In one embodiment, the micronized protein particle is formed
by making a solution of the protein and then subjecting that
protein solution to dispersion and heat to form a dry powder
comprising the protein. One method to form the micronized protein
particles is by spray-drying. In one embodiment, the protein is a
therapeutic protein that is formulated to include buffers,
stabilizers and other pharmaceutically acceptable excipients to
make a pharmaceutical formulation of the therapeutic protein.
Exemplary pharmaceutical formulations are described in U.S. Pat.
No. 7,365,165, U.S. Pat. No. 7,572,893, U.S. Pat. No. 7,608,261,
U.S. Pat. No. 7,655,758, U.S. Pat. No. 7,807,164, US 2010-0279933,
US 2011-0171241, and PCT/US11/54856.
[0052] The amount of therapeutic protein contained within the
pharmaceutical formulations of the present invention may vary
depending on the specific properties desired of the formulations,
as well as the particular circumstances and purposes for which the
formulations are intended to be used. In certain embodiments, the
pharmaceutical formulations may contain about 1 mg/mL to about 500
mg/mL of protein; about 5 mg/mL to about 400 mg/mL of protein;
about 5 mg/mL to about 200 mg/mL of protein; about 25 mg/mL to
about 180 mg/mL of protein; about 25 mg/mL to about 150 mg/mL of
protein; or about 50 mg/mL to about 180 mg/mL of protein. For
example, the formulations of the present invention may comprise
about 1 mg/mL; about 2 mg/mL; about 5 mg/mL; about 10 mg/mL; about
15 mg/mL; about 20 mg/mL; about 25 mg/mL; about 30 mg/mL; about 35
mg/mL; about 40 mg/mL; about 45 mg/mL; about 50 mg/mL; about 55
mg/mL; about 60 mg/mL; about 65 mg/mL; about 70 mg/mL; about 75
mg/mL; about 80 mg/mL; about 85 mg/mL; about 86 mg/mL; about 87
mg/mL; about 88 mg/mL; about 89 mg/mL; about 90 mg/mL; about 95
mg/mL; about 100 mg/mL; about 105 mg/mL; about 110 mg/mL; about 115
mg/mL; about 120 mg/mL; about 125 mg/mL; about 130 mg/mL; about 131
mg/mL; about 132 mg/mL; about 133 mg/mL; about 134 mg/mL; about 135
mg/mL; about 140 mg/mL; about 145 mg/mL; about 150 mg/mL; about 155
mg/mL; about 160 mg/mL; about 165 mg/mL; about 170 mg/mL; about 175
mg/mL; about 180 mg/mL; about 185 mg/mL; about 190 mg/mL; about 195
mg/mL; about 200 mg/mL; about 205 mg/mL; about 210 mg/mL; about 215
mg/mL; about 220 mg/mL; about 225 mg/mL; about 230 mg/mL; about 235
mg/mL; about 240 mg/mL; about 245 mg/mL; about 250 mg/mL; about 255
mg/mL; about 260 mg/mL; about 265 mg/mL; about 270 mg/mL; about 275
mg/mL; about 280 mg/mL; about 285 mg/mL; about 200 mg/mL; about 200
mg/mL; or about 300 mg/mL of therapeutic protein.
[0053] The pharmaceutical formulations of the present invention
comprise one or more excipients. The term "excipient," as used
herein, means any non-therapeutic agent added to the formulation to
provide a desired consistency, viscosity or stabilizing effect.
[0054] The pharmaceutical formulations of the present invention may
also comprise one or more carbohydrate, e.g., one or more sugar.
The sugar can be a reducing sugar or a non-reducing sugar.
"Reducing sugars" include, e.g., sugars with a ketone or aldehyde
group and contain a reactive hemiacetal group, which allows the
sugar to act as a reducing agent. Specific examples of reducing
sugars include fructose, glucose, glyceraldehyde, lactose,
arabinose, mannose, xylose, ribose, rhamnose, galactose and
maltose. Non-reducing sugars can comprise an anomeric carbon that
is an acetal and is not substantially reactive with amino acids or
polypeptides to initiate a Maillard reaction. Specific examples of
non-reducing sugars include sucrose, trehalose, sorbose, sucralose,
melezitose and raffinose. Sugar acids include, for example,
saccharic acids, gluconate and other polyhydroxy sugars and salts
thereof.
[0055] The amount of sugar contained within the pharmaceutical
formulations of the present invention will vary depending on the
specific circumstances and intended purposes for which the
formulations are used. In certain embodiments, the formulations may
contain about 0.1% to about 20% sugar; about 0.5% to about 20%
sugar; about 1% to about 20% sugar; about 2% to about 15% sugar;
about 3% to about 10% sugar; about 4% to about 10% sugar; or about
5% to about 10% sugar. For example, the pharmaceutical formulations
of the present invention may comprise about 0.5%; about 1.0%; about
1.5%; about 2.0%; about 2.5%; about 3.0%; about 3.5%; about 4.0%;
about 4.5%; about 5.0%; about 5.5%; about 6.0%; 6.5%; about 7.0%;
about 7.5%; about 8.0%; about 8.5%; about 9.0%; about 9.5%; about
10.0%; about 10.5%; about 11.0%; about 11.5%; about 12.0%; about
12.5%; about 13.0%; about 13.5%; about 14.0%; about 14.5%; about
15.0%; about 15.5%; about 16.0%; 16.5%; about 17.0%; about 17.5%;
about 18.0%; about 18.5%; about 19.0%; about 19.5%; or about 20.0%
sugar (e.g., sucrose).
[0056] The pharmaceutical formulations of the present invention may
also comprise one or more surfactant. As used herein, the term
"surfactant" means a substance which reduces the surface tension of
a fluid in which it is dissolved and/or reduces the interfacial
tension between oil and water. Surfactants can be ionic or
non-ionic. Exemplary non-ionic surfactants that can be included in
the formulations of the present invention include, e.g., alkyl
poly(ethylene oxide), alkyl polyglucosides (e.g., octyl glucoside
and decyl maltoside), fatty alcohols such as cetyl alcohol and
oleyl alcohol, cocamide MEA, cocamide DEA, and cocamide TEA.
Specific non-ionic surfactants that can be included in the
formulations of the present invention include, e.g., polysorbates
such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate
60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate
85; poloxamers such as poloxamer 188, poloxamer 407;
polyethylene-polypropylene glycol; or polyethylene glycol (PEG).
Polysorbate 20 is also known as TWEEN 20, sorbitan monolaurate and
polyoxyethylenesorbitan monolaurate.
[0057] The amount of surfactant contained within the pharmaceutical
formulations of the present invention may vary depending on the
specific properties desired of the formulations, as well as the
particular circumstances and purposes for which the formulations
are intended to be used. In certain embodiments, the formulations
may contain about 0.05% to about 5% surfactant; or about 0.1% to
about 0.2% surfactant. For example, the formulations of the present
invention may comprise about 0.05%; about 0.06%; about 0.07%; about
0.08%; about 0.09%; about 0.10%; about 0.11%; about 0.12%; about
0.13%; about 0.14%; about 0.15%; about 0.16%; about 0.17%; about
0.18%; about 0.19%; about 0.20%; about 0.21%; about 0.22%; about
0.23%; about 0.24%; about 0.25%; about 0.26%; about 0.27%; about
0.28%; about 0.29%; or about 0.30% surfactant (e.g., polysorbate
20).
[0058] The pharmaceutical formulations of the present invention may
also comprise one or more buffers. In some embodiments, the buffer
has a buffering range that overlaps fully or in part the range of
pH 5.5-7.4. In one embodiment, the buffer has a pKa of about
6.0.+-.0.5. In certain embodiments, the buffer comprises a
phosphate buffer. In certain embodiments, the phosphate is present
at a concentration of 5 mM.+-.0.75 mM to 15 mM.+-.2.25 mM; 6
mM.+-.0.9 mM to 14 mM.+-.2.1 mM; 7 mM.+-.1.05 mM to 13 mM.+-.1.95
mM; 8 mM.+-.1.2 mM to 12 mM.+-.1.8 mM; 9 mM.+-.1.35 mM to 11
mM.+-.1.65 mM; 10 mM.+-.1.5 mM; or about 10 mM. In certain
embodiments, the buffer system comprises histidine at 10 mM.+-.1.5
mM, at a pH of 6.0.+-.0.5.
[0059] The pharmaceutical formulations of the present invention may
have a pH of from about 5.0 to about 8.0. For example, the
formulations of the present invention may have a pH of about 5.0;
about 5.2; about 5.4; about 5.6; about 5.8; about 6.0; about 6.2;
about 6.4; about 6.6; about 6.8; about 7.0; about 7.2; about 7.4;
about 7.6; about 7.8; or about 8.0.
[0060] In one particular embodiment, the therapeutic protein is a
VEGF Trap protein. Pharmaceutical formulations for the formation of
micronized VEGF Trap protein particles may contain from about 10
mg/mL to about 100 mg/mL VEGF Trap protein, about 10 mg/mL, about
15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35
mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55
mg/mL, about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 75
mg/mL, about 80 mg/mL, about 85 mg/mL, about 90 mg/mL, about 95
mg/mL, or about 100 mg/mL VEGF Trap protein. Solutions may contain
one or more buffers of from about 5 mM to about 50 mM. In one
embodiment, the buffer is about 10 mM phosphate at a pH of about
6.+-.0.5. Solutions may also contain sucrose at a concentration of
from about 1% to about 10%. In one embodiment, the solution
contains sucrose at about 2% w/w.
[0061] In some embodiments, the therapeutic protein solution
contains VEGF Trap protein at about 25 mg/mL or about 50 mg/mL in
10 mM phosphate, pH 6.2, 2% sucrose, and optionally 0.1%
polysorbate.
[0062] The therapeutic protein formulation is then subjected to
dispersion and drying to form micronized protein particles. One
method of making the micronized protein particles is to subject the
protein solution to spray-drying. Spray-drying is generally known
in the art and may be performed on equipment such as e.g., a BUCHI
Mini Spray Dryer B-290 (Buchi Labortechnik AG, Flawil, CH). In one
particular embodiment, the protein solution (e.g., but not limited
to any one of the VEGF Trap formulations described above) is pumped
into the spray dryer at a rate of about 2 mL/min to about 15
mL/min, or about 7 mL/min. The inlet temperature of the spray dryer
is set at a temperature above the boiling point of water, such as
e.g., at about 130.degree. C. The outlet temperature at a
temperature below the boiling point of water and above ambient
temperature, such as e.g., 55.degree. C. In one specific
embodiment, a protein solution (e.g., VEGF Trap solution or IgG
solution) is pumped into a BUCHI Mini Spray Dryer B-290 at about 7
mL/min, with an inlet temperature of about 130.degree. C. and an
outlet temperature of about 55.degree. C., with the aspirator set
at 33 m.sup.3/h and the spray gas at 530 L/h.
[0063] The resulting micronized protein particles range in size
from about 1 .mu.m to about 100 .mu.m in diameter, depending upon
the particular formulation and concentration of protein and
excipients. In some embodiments, the micronized protein particles
have a diameter of from about 1 .mu.m to about 100 .mu.m, from
about 1 .mu.m to about 40 .mu.m, from about 2 .mu.m to about 15
.mu.m, from about 2.5 .mu.m to about 13 .mu.m, from about 3 .mu.m
to about 10 .mu.m, about 5 .mu.m, about 6 .mu.m, about 7 .mu.m,
about 8 .mu.m, about 9 .mu.m, about 10 .mu.m, about 11 .mu.m, or
about 12 .mu.m.
[0064] The micronized protein particles are then coated with a
biocompatible and biodegradable polymer. This is can be
accomplished by suspending the micronized protein particles in a
polymer solution. A polymer solution is essentially a polymer
dissolved in a solvent. For example, the biocompatible and
biodegradable polymer may be dissolved in inter alia methylene
chloride, tetrahydrofuran, ethyl acetate, or or some other useful
solvent. Ethyl acetate is widely known as a safe solvent and is
often used in the preparation of drugs, implants and
foodstuffs.
[0065] In some embodiments, the polymer can be ethyl cellulose
("EC"), poly(lactic acid) ("PLA"), polyorthoester ("POE"),
poly-D,L-lactide-co-glycolide ("PLGA"), or
poly-.epsilon.-caprolactone ("PCL"). The polymer can be dissolved
in the solvent (e.g., ethyl acetate) at a concentration of from
about 10 mg/mL to about 300 mg/mL, from about 15 mg/mL to about 295
mg/mL, from about 20 mg/mL to about 290 mg/mL, from about 25 mg/mL
to about 280 mg/mL, from about 30 mg/mL to about 270 mg/mL, from
about 35 mg/mL to about 265 mg/mL, from about 40 mg/mL to about 260
mg/mL, from about 45 mg/mL to about 260 mg/mL, from about 50 mg/mL
to about 255 mg/mL, from about 55 mg/mL to about 250 mg/mL, about
20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40
mg/mL, about 45 mg/mL, about 50 mg/mL, about 75 mg/mL, about 100
mg/mL, about 125 mg/mL, about 150 mg/mL, about 175 mg/mL, about 200
mg/mL, about 225 mg/mL, or about 250 mg/mL.
[0066] The micronized protein particles are added to the polymer
solution at about 10 mg/mL to about 100 mg/mL, about 15 mg/mL to
about 95 mg/mL, about 20 mg/mL to about 90 mg/mL, about 25 mg/mL to
about 85 mg/mL, about 30 mg/mL to about 80 mg/mL, about 35 mg/mL to
about 75 mg/mL, about 40 mg/mL to about 70 mg/mL, about 45 mg/mL to
about 65 mg/mL, about 50 mg/mL to about 60 mg/mL, at about 25
mg/mL, at about 30 mg/mL, at about 35 mg/mL, at about 40 mg/mL, at
about 45 mg/mL, or at about 50 mg/mL. The particles are mixed to
form a slurry or suspension, which is then subjected to dispersion
and drying to form the polymer coated protein particle (i.e.,
microparticle).
[0067] In one embodiment, the protein particle-polymer solution
suspension is subjected the spray-drying, which is performed in a
manner similar to the method for manufacturing the micronized
protein particles, but with a reduced intake temperature to protect
against igniting the organic solvent or polymer. Briefly, the
protein particle-polymer solution suspension is pumped into the
spray dryer at a rate of about 5 mL/min to about 20 mL/min, or
about 12.5 mL/min. The suspension was pumped at 12.5 mL/min into
the spray dryer with an aspirator air and spray gas flow rate of
about 530 L/h and 35 m.sup.3/h (mm), respectively. The inlet
temperature was set at 90.degree. and the outlet temperature was
set at about 54.degree. C. The inlet temperature of the spray dryer
is set at a temperature above the flash point of the solvent, such
as e.g., at about 90.degree. C. The outlet temperature at a
temperature below the intake temperature and above ambient
temperature, such as e.g., about 54.degree. C. In one particular
embodiment, a suspension containing about 50 mg/mL of protein
particle (e.g., VEGF Trap) in about 50 mg/mL to about 250 mg/mL
polymer/ethyl acetate solution is pumped into a BUCHI Mini Spray
Dryer B-290 at about 12.5 mL/min, with an inlet temperature of
about 90.degree. C. and an outlet temperature of about 54.degree.
C., with the aspirator set at about 35 m.sup.3/h and the spray gas
at about 530 L/h.
[0068] The resulting microparticles, which contain a protein
particle core within a polymer cortex, have a range of diameters of
from about 2 .mu.m to about 70 .mu.m, about 5 .mu.m to about 65
.mu.m, about 10 .mu.m to about 60 .mu.m, about 15 .mu.m to about 55
.mu.m, about 20 .mu.m to about 50 .mu.m, about 15 .mu.m, about 20
.mu.m, about 25 .mu.m, or about 30 .mu.m. The size variation in
large part reflects the thickness of the polymer cortex, although
the diameter of the protein core could contribute to size variation
to some extent. Manipulating the starting concentration of the
polymer solution, and/or the polymer itself can control the
diameter of the microparticle. For example, those microparticles
which were manufactured using 50 mg/mL polymer have a median size
of about 15 .mu.m to 20 .mu.m, whereas those microparticles which
were manufactured using 250 mg/mL polymer had a median size of
about 30 .mu.m.
[0069] The microparticles of the instant invention are useful in
the time-release or extended release of protein therapeutics. For
example, it is envisioned that the VEGF Trap microparticles are
useful in the extended release of VEGF Trap therapeutic protein in,
for example, the vitreous for the treatment of vascular eye
disorders, or subcutaneous implantation for the extended release of
VEGF Trap to treat cancer or other disorders.
[0070] The microparticles of the instant invention release protein
in a physiological aqueous environment at about 37.degree. C. at a
relatively constant rate over an extended period of time, to at
least 60 days. In general, those microparticles manufactured with a
higher concentration of polymer (e.g., 250 mg/mL) tended to show a
relatively linear protein release profile; whereas those
microparticles manufactured with a lower concentration of polymer
(e.g., 50 mg/mL) tended to show an initial burst followed by an
onset of a delayed burst release. Furthermore, microparticles
formed from a higher concentration of polymer showed a slower rate
of release of protein than those formed from a lower concentration
of particles. The quality of protein released from the
microparticles over time was consistent with the quality of the
stating protein material. Little to no protein degradation
occurred.
EXAMPLES
[0071] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the methods and compositions of
the invention, and are not intended to limit the scope of what the
inventors regard as their invention. Efforts have been made to
ensure accuracy with respect to numbers used (e.g., amounts, sizes,
etc.) but some experimental errors and deviations should be
accounted for.
[0072] In the following examples, VEGF-Trap protein ("VGT"), which
is a dimer of the polypeptide comprising the amino acid sequence
SEQ ID NO:1, serves as an exemplar receptor-Fc-fusion protein.
Example 1
Micronized Proteins
[0073] Solutions containing 25 mg/mL VEGF Trap protein ("VGT"), 25
mg/mL VGT plus 0.1% polysorbate 80, and 50 mg/mL VGT in 10 mM
phosphate, 2% sucrose, pH 6.2 were each independently atomized in a
spray dry micronizer (BUCHI Mini Spray Dryer B-290, Buchi
Labortechnik AG, Flawil, CH) to form droplets containing VEGF Trap.
Heat was applied to evaporate the water from the droplets,
resulting in a powder containing VEGF Trap. The inlet temperature
was set at 130.degree. C. and outlet temperature at about
55.degree. C. The aspirator was set at 33 m.sup.3/h and spray gas
at 530 L/h. The VGT solution was pumped at about 7 mL/min.
[0074] The size of the resultant VGT particles was measured by
micro-flow imaging (MFI) and dynamic light imaging (DLS). FIG. 1
depicts the particle size distribution as determined by MFI for the
VGT particles derived from each of the 25 mg/mL VGT, 25 mg/mL VGT
plus 0.1% polysorbate 80, and 50 mg/mL VGT concentrations. For all
concentrations, the equivalent circular diameter (ECD) of VGT
particles ranged from about 1 .mu.m to about 39 .mu.m, with the
majority of particles ranging in size of from about 2 .mu.m to
about 14 .mu.m. For the 25 mg/mL VGT solution, the particles
clustered in the range of about 2.5 .mu.m to about 8.8 .mu.m, with
a mode of about 6 .mu.m. For the 25 mg/mL VGT plus 0.1% polysorbate
80 solution, the particles clustered in the range of about 2.5
.mu.m to about 9.7 .mu.m, with a mode of about 6 .mu.m. For the 50
mg/mL VGT solution, the particles clustered in the range of about
2.7 .mu.m to about 12.8 .mu.m, with a mode of about 7 .mu.m. Median
diameters for each formulation, as determined by both MFI and DLS
methods, are described in Table 1.
[0075] VGT particles were reconstituted in water for injection and
examined via size exclusion, i.e., size exclusion-ultra performance
liquid chromatography (SE-UPLC) to determine protein purity. No
change in purity was noted after micronization relative to starting
material (see Table 3).
TABLE-US-00001 TABLE 1 Median protein particle sizes (.mu.m) as
determined by MFI and DLS Median size Median size Formulation by
MFI (.mu.m) by DLS (.mu.m) 50 mg/mL VEGF Trap 7 7.6 25 mg/mL VEGF
Trap 6 5.9 25 mg/mL VEGF Trap, 0.1% polysorbate 80 6 7.1
Example 2
Micronized Protein Suspensions in Organic Polymer Solutions
[0076] Various polymers were used or are contemplated for use in
the manufacture of the polymer cortex of the microparticles. Those
polymers include inter alia ethyl cellulose ("EC"), polyorthoester
("POE"), poly-D,L-lactide-co-glycolide ("PLGA"), and
poly-.epsilon.-caprolactone ("PCL").
[0077] Ethyl Cellulose Coating
[0078] Micronized VEGF Trap particles were suspended in a solution
of 50 mg/mL ethyl cellulose in ethyl acetate at a concentration of
about 50 mg/mL VGT; herein designated "VGT-50-EC suspension".
[0079] Micronized VEGF Trap particles were suspended in a solution
of 100 mg/mL ethyl cellulose in ethyl acetate at a concentration of
about 50 mg/mL VGT; herein designated "VGT-100-EC suspension".
[0080] Micronized VEGF Trap particles are suspended in a solution
of 250 mg/mL ethyl cellulose in ethyl acetate at a concentration of
about 50 mg/mL VGT; herein designated "VGT-250-EC suspension".
[0081] Polyorthoester Coating
[0082] Micronized VEGF Trap particles were suspended in a solution
of 50 mg/mL polyorthoester containing about 5% latent acid in ethyl
acetate at a concentration of about 50 mg/mL VGT; herein designated
"VGT-50-POE suspension".
[0083] Micronized VEGF Trap particles were suspended in a solution
of 250 mg/mL polyorthoester containing about 5% latent acid in
ethyl acetate at a concentration of about 50 mg/mL VGT; herein
designated "VGT-250-POE suspension".
[0084] Poly-D,L-lactide-co-glycolide Coating
[0085] Micronized VEGF Trap particles were suspended in a solution
of 50 mg/mL PLGA in ethyl acetate at a concentration of about 50
mg/mL VGT; herein designated "VGT-50-PLGA suspension".
[0086] Micronized VEGF Trap particles were suspended in a solution
of 200 mg/mL PLGA in ethyl acetate at a concentration of about 50
mg/mL VGT; herein designated "VGT-200-PLGA suspension".
[0087] Micronized VEGF Trap particles were suspended in a solution
of 250 mg/mL PLGA in ethyl acetate at a concentration of about 50
mg/mL VGT; herein designated "VGT-250-PLGA suspension".
[0088] Poly-.epsilon.-caprolactone Coating
[0089] Micronized VEGF Trap particles are suspended in a solution
of 50 mg/mL PCL in ethyl acetate at a concentration of about 50
mg/mL VGT; herein designated "VGT-50-PCL suspension".
[0090] Micronized VEGF Trap particles are suspended in a solution
of 250 mg/mL PCL in ethyl acetate at a concentration of about 50
mg/mL VGT; herein designated "VGT-250-PCL suspension".
[0091] PCL has a low Tg and may not be suitable for heat-drying as
described below, but can be used for solvent extraction in an
aqueous bath with polyvinyl alcohol (PVA), for example.
Example 3
Dispersion of Protein-Polymer Fine Droplets and Solvent Removal
[0092] Each VGT polymer suspension, which was made according to
Example 2 (supra), was subjected to spray drying using a BUCHI Mini
Spray Dryer B-290 (Buchi Labortechnik AG, Flawil, CH). Briefly,
each suspension was atomized to form microdroplets, which were
subsequently heat dried to remove the solvent and form the
polymer-coated protein microparticles. The suspension was pumped at
12.5 mL/min into the spray dryer with an aspirator air and spray
gas flow rate of about 530 L/h and 35 m.sup.3/h, respectively. The
inlet temperature was set at 90.degree. and the outlet temperature
was set at about 54.degree. C.
Example 4
Characterization of Protein-Polymer Microparticles
[0093] Spray dried polymer coated protein particles manufactured
according to the exemplified process generate a plurality of
microparticles having a range of equivalent circular diameters of
from about 2.5 .mu.m to about 65 .mu.m (FIG. 2). The size variation
in large part reflects the thickness of the polymer cortex,
although the diameter of the protein core could contribute to size
variation to some extent.
[0094] The diameter of the microparticle correlates with the
starting concentration of the polymer solution (Table 2, FIG. 2).
Those microparticles which were manufactured using 50 mg/mL polymer
had a median size of about 17 .mu.m.+-.2.8 .mu.m. Those
microparticles which were manufactured using 250 mg/mL polymer had
a median size of about 29 .mu.m.
Example 5
Protein Stability Post Spray Dry
[0095] The stability of the VEGF-Trap protein was assessed using
quantitative size exclusion chromatography (SE-UPLC), which allows
for the quantification of smaller degradation products and larger
aggregation products relative to the intact monomer. The results
are described in Table 3. Essentially, the protein remained stable
throughout the spray drying and spray coating processes.
[0096] The average ratio of protein to polymer by weight was also
determined for the manufactured microparticles. A collection of
microparticles manufactured with varying polymers and polymer
concentration was extracted and subjected to quantitative reverse
phase chromatography (RP-HPLC). The results are presented in Table
3. The data may be interpreted to support the theory that a higher
starting concentration of polymer yields a thicker polymer cortex
on the microparticle.
TABLE-US-00002 TABLE 2 Equivalent circular diameter values Range
Median Material (.mu.m) (.mu.m) Mode (.mu.m) VEGF-Trap ("VGT") (50
mg/mL) 2.5-29.4 10-12 8.3 VGT (50 mg/mL) + POE (50 mg/mL) 2.5-64.5
15 9.4 VGT (50 mg/mL) + POE (250 mg/mL) 2.5-49.4 29 28.5 VGT (50
mg/mL) + EC (50 mg/mL) 2.5-49.6 19 16.5
TABLE-US-00003 TABLE 3 Protein stability and loading VGT Extracted
from VGT Coated Polymers.sup.1 starting % w/w material % VGT/
Material % Native Native.sup.2 polymer.sup.3 VGT starting material
97.7 -- -- Reconstituted VGT 97.6 -- -- VGT (50 mg/mL) + POE (50
mg/mL) -- 96.3 14.6 VGT (50 mg/mL) + POE (250 mg/mL) -- 97.7 1.8
VGT (50 mg/mL) + EC (50 mg/mL) -- 97.1 6.1 .sup.1Based on extracted
VEGF Trap after 1 hour reconstitution to remove uncoated VEGF Trap.
.sup.2Average of percent native by SE-UPLC (n = 4). .sup.3Average
of percent weight to weight loading of VGT to polymer by RP-HPLC (n
= 4).
Example 6
Protein Release from Microparticles
[0097] The release of protein from microparticles was determined by
suspending various batches of microparticles in buffer (10 mM
phosphate, 0.03% polysorbate 20, pH 7.0) and measuring the amount
and quality of protein released into solution over time while
incubated at 37.degree. C. At 1-2 week intervals, the
microparticles were pelleted by mild centrifugation and 80% of the
supernatant containing released protein was collected for
subsequent analysis. An equivalent amount of fresh buffer was
replaced and the microparticles were resuspended by mild vortexing
and returned to the 37.degree. C. incubation chamber. Protein
amount and quality in the supernatant was assessed by size
exclusion chromatography.
[0098] In general, those microparticles manufactured with a higher
concentration of polymer (e.g., 250 mg/mL) tended to show a
relatively linear protein release profile; whereas those
microparticles manufactured with a lower concentration of polymer
(e.g., 50 mg/mL) tended to show an initial burst followed by an
onset of a delayed burst release. The data showing the extended
release of protein, which remained stable, for up to about 60 days
is depicted in FIG. 3 (release data). Table 4 summarizes the linear
rate-of-release data.
TABLE-US-00004 TABLE 4 Protein release dynamics VEGF Trap protein
release Material (mg VGT/week) VGT (50 mg/mL) + POE (50 mg/mL) 0.14
.+-. 0.16 VGT (50 mg/mL) + POE (250 mg/mL) 0.06 .+-. 0.02 VGT (50
mg/mL) + EC (50 mg/mL) 0.031 .+-. 0.02
Example 7
Particle Size can be Manipulated by Polymer Concentration and Spray
Gas Flow
[0099] Particle size distributions were controlled by polymer
concentration and atomization spray gas flow. Increased polymer
concentration shifted the distribution towards larger particles
(200 mg/mL PLGA at 45 mm spray gas flow v. 100 mg/mL PLGA at 45 mm
spray gas flow; see Table 5). Similarly, a lower atomization spray
gas flow resulted in larger droplets and thus, larger particles
(100 mg/mL PLGA at 25 mm spray gas flow v. 100 mg/mL PLGA at 45 mm
spray gas flow; see Table 5).
TABLE-US-00005 TABLE 5 Particle Size (all metrics are approximate)
Particle Mode of Percent total volume [PLGA] Gas Flow size range
particle size of particles with 15 (mg/mL) Rate (m.sup.3/h)
(microns) (microns) micron particle size Protein NA 2.5-25 3.5 1.5%
alone 100 25 2.5-40 9.4 3.7% 100 45 2.5-30 9.4 3.7% 200 45 2.5-30
10.2-15.4 5.4%
Example 8
Particle Size and Protein Release Across Various Polymers
[0100] VEGF Trap or IgG was spray coated with low molecular weight
(202S) poly(lactic acid) (PLA-LMW), high molecular weight (203S)
poly(lactic acid) (PLA-HMW), polyanhydride
poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxbutyric
acid-cohydroxyvaleric acid) (PHB-PVA), PEG-poly(lactic acid) block
copolymer (PEG-PLA), and poly-D,L-lactide-co-glycolide (PLGA). 25
mg/mL of spray-dried protein was combined with 50-100 mg/mL
polymer. In vitro release assays were performed in 10 mM phosphate
buffer, pH7.2 at 37.degree. C. The results are depicted in Table
6.
TABLE-US-00006 TABLE 6 Polymer dependent particle size and protein
release (all metrics are approximate) Relative number of Time to
100% Polymer Protein particles at 15 microns protein release
PLA-LMW VEGF Trap 0.8 .times. 10.sup.2 3 days PLA-HMW VEGF Trap 0.8
.times. 10.sup.2 3 days pCPH VEGF Trap 1 .times. 10.sup.2 3 days
PHB-PVA VEGF Trap 5 .times. 10.sup.2 1 days PEG-PLA VEGF Trap 0.6
.times. 10.sup.2 6 hours PLGA IgG 1 .times. 10.sup.2 8 days
Example 9
Protein Stability in Various Polymers
[0101] VEGF Trap and IgG were extracted from their respective
polymer coats and measured for purity by SE-UPLC. The results are
summarized in Table 7. The proteins generally were compatible with
the spray coating process for the polymers tested. Protein remained
stable for at least 14 days for those polymers that continued to
release protein.
TABLE-US-00007 TABLE 7 % Purity by Size Exclusion Chromatography
After 1 day in spray vitro release 3 days 14 days Protein Polymer
coating (IVR) IVR IVR VEGF Trap POE (AP141) 97.7 98.3 98.2 96.7
VEGF Trap PLA-LMW 97.0 97.4 92.8 -- VEGF Trap PLA-HMW 93.9 97.3
95.4 -- VEGF Trap PEG-PLA 89.9 91.2 -- -- VEGF Trap pCPH 89.2 94.2
84.8 -- VEGF Trap PHB-PVA 97.4 96.2 -- -- VEGF Trap PLGA 96.6 97.8
-- 93.6 IgG PLGA 99.2 98.0 -- 92.0
Sequence CWU 1
1
11415PRTArtificial Sequencesynthetic 1Ile Ile His Met Thr Glu Gly
Arg Glu Leu Val Ile Pro Cys Arg Val 1 5 10 15 Thr Ser Pro Asn Ile
Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 20 25 30 Leu Ile Pro
Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 35 40 45 Ile
Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 50 55
60 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg
65 70 75 80 Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His
Gly Ile 85 90 95 Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys
Thr Ala Arg Thr 100 105 110 Glu Leu Asn Val Gly Ile Asp Phe Asn Trp
Glu Tyr Pro Ser Ser Lys 115 120 125 His Gln His Lys Lys Leu Val Asn
Arg Asp Leu Lys Thr Gln Ser Gly 130 135 140 Ser Glu Met Lys Lys Phe
Leu Ser Thr Leu Thr Ile Asp Gly Val Thr 145 150 155 160 Arg Ser Asp
Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met 165 170 175 Thr
Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr 180 185
190 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
195 200 205 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg 210 215 220 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro 225 230 235 240 Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 245 250 255 Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val 260 265 270 Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 275 280 285 Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 290 295 300 Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 305 310
315 320 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys 325 330 335 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser 340 345 350 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp 355 360 365 Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser 370 375 380 Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala 385 390 395 400 Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 405 410 415
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