U.S. patent application number 12/761292 was filed with the patent office on 2010-11-25 for prion free nanoparticle compositions and methods of making thereof.
Invention is credited to Neil P. Desai, Viktor Peykov, Patrick Soon-Shiong.
Application Number | 20100297243 12/761292 |
Document ID | / |
Family ID | 42982852 |
Filed Date | 2010-11-25 |
United States Patent
Application |
20100297243 |
Kind Code |
A1 |
Desai; Neil P. ; et
al. |
November 25, 2010 |
PRION FREE NANOPARTICLE COMPOSITIONS AND METHODS OF MAKING
THEREOF
Abstract
The present invention provides prion-free compositions
comprising nanoparticles comprising albumin and substantially water
insoluble drugs. Also provided are methods of making prion-free
compositions and methods of removing prion proteins from the
nanoparticle compositions. Methods of using the compositions, as
well as kits useful for carrying out the methods are also
provided.
Inventors: |
Desai; Neil P.; (Los
Angeles, CA) ; Peykov; Viktor; (Los Angeles, CA)
; Soon-Shiong; Patrick; (Los Angeles, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
755 PAGE MILL RD
PALO ALTO
CA
94304-1018
US
|
Family ID: |
42982852 |
Appl. No.: |
12/761292 |
Filed: |
April 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61169665 |
Apr 15, 2009 |
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61238052 |
Aug 28, 2009 |
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Current U.S.
Class: |
424/489 ;
514/449 |
Current CPC
Class: |
A61P 35/00 20180101;
G01N 33/6896 20130101; Y10S 210/905 20130101; A61K 9/1658 20130101;
A61P 31/12 20180101; A61K 31/337 20130101; A61K 9/5169 20130101;
B82Y 5/00 20130101; G01N 2800/2828 20130101; Y10S 977/906 20130101;
A61K 47/42 20130101; Y10S 977/773 20130101 |
Class at
Publication: |
424/489 ;
514/449 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 31/337 20060101 A61K031/337; A61P 31/12 20060101
A61P031/12 |
Claims
1. A composition comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent,
wherein the composition is substantially free of a prion
protein.
2. The composition of claim 1, wherein the composition has a prion
infectivity of less than about 100 fg/ml.
3. The composition of claim 1, wherein the composition has a prion
infectivity of less than about 10 IU-ic/ml.
4. The composition of claim 1, wherein the composition does not
show the presence of a prion protein based on a protein misfolding
cyclic amplification (PMCA) assay or based on an IPCR assay.
5. The composition of claim 1, further comprising a trace amount of
a ligand capable of binding to a prion protein.
6. A composition comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent,
wherein the albumin in the composition was obtained by a method
comprising a prion removal process, said prion removal process
comprising contacting an initial albumin composition with a ligand
capable of binding to a prion protein.
7. The composition of claim 6, wherein the prion removal process
further comprises removing said ligand and proteins bound thereto
from said albumin and composition.
8. The composition of claim 7, wherein the ligand is a peptide.
9. The composition of claim 7, wherein the ligand is a
trazine-based compound.
10. A method of producing a composition comprising nanoparticles
comprising albumin and a substantially water insoluble
pharmacologically active agent, said method comprising: a) removing
a prion protein from an initial albumin composition; b) subjecting
a mixture comprising a solution comprising the prion-removed
albumin and an organic phase comprising said substantially water
insoluble pharmacologically active agent dispersed in an organic
solvent to a high shear condition.
11. The method of claim 10, wherein step a) comprises: 1)
contacting the initial albumin solution with a ligand capable of
binding to a prion protein.
12. The method of claim 11, wherein step a) further comprises: 2)
removing the ligand and proteins bound thereto from the albumin
solution.
13. The method of claim 11, further comprising removing said
organic solvent from the mixture.
14. The method of claim 11, wherein said ligand is a peptide.
15. The method of claim 11, wherein the ligand is a triazine-based
compound.
16. The method of claim 10, wherein the initial albumin composition
is a blood derived product.
17. A method of producing a composition comprising nanoparticles
comprising albumin and a substantially water insoluble
pharmacologically active agent, comprising: a) subjecting a mixture
comprising an organic phase comprising said substantially water
insoluble pharmacologically active agent and an albumin solution to
a high shear condition, and b) removing a prion protein from said
mixture.
18. The method of claim 17, wherein step b) comprises: 1)
contacting the mixture with a ligand capable of binding to a prion
protein.
19. The method of claim 18, wherein step b) further comprises: 2)
removing the ligand and proteins bound thereto from said
mixture.
20. The method of claim 18, wherein said ligand is a peptide.
21. The method of claim 18, wherein the ligand is a triazine-based
compound.
22. A method of removing a prion protein from a composition
suspected of containing a prion protein comprising nanoparticles
comprising albumin and a substantially water insoluble
pharmacologically active agent, comprising: a) contacting the
composition with a ligand capable of binding to a prion protein, b)
removing the ligand and proteins bound thereto from the
composition.
Description
RELATED APPLICATIONS
[0001] The application claims priority benefit to U.S. Provisional
Patent Application No. 61/169,665, filed Apr. 15, 2009, and U.S.
Provisional Patent Application No. 61/238,052, filed Aug. 28, 2009,
the content of each of which is incorporated herein in their
entirety.
TECHNICAL FIELD
[0002] The present invention relates to prion-free compositions
comprising nanoparticles comprising albumin and substantially water
insoluble drugs, methods of making, and methods of using
thereof.
BACKGROUND
[0003] Prion diseases, also known as transmissible spongiform
encephalopathies (TSEs), are a group of fatal, transmittable
neurodegenerative diseases. Specific examples of TSE include
scrapie, which affects sheep and goats, bovine spongiform
encephalopathy (B SE), transmissible mink encephalopathy, feline
spongiform encephalopathy and chronic wasting disease (CWD). In
humans, TSE diseases may present themselves as kuru,
Creutzfeldt-Jekob disease (CJD), Gerstmann-Straussler-Scheinker
Syndrome (GSS), fatal insomnia and variant Creutzfeldt-Jekob
disease (vCJD). vCJD emerged in humans as a result of the BSE
epidemic in Britain and is most probably caused by the consumption
of food products derived from cattle infected with BSE or "mad cow
disease." (Will et al. (1996) Lancet 347:921-925) Because the
incubation period for the orally contracted disease may be more
than 20 years in human, the true incidence of vCJD may not become
apparent for many years.
[0004] In addition to ingestion of infected products of bovine
origin, blood transfusion and organ transplantation represent
another mode of transmission of vCJD among humans (Brown et al.
(1998) Transfusion 38:810-816; Diringer et al. (1984) Archives of
Virology 82:105-109; Manuelidis et al. (1978) Nature 271:778-779).
Major concerns were raised since the mid-1990s that vCJD can be
transmitted through blood transfusion or other blood products from
TSE-infected individuals. These individuals may be asymptomatic
during the long pre-clinical and incubation phase of vCJD, and
blood obtained from such donors may be able to transmit the disease
to persons receiving the blood or blood products derived from the
donor.
[0005] There are so far at least four reported human cases of blood
transfusion acquired vCJD in the United Kingdom. Of 64 people who
received whole blood from 22 donors, 4 people went on to develop
vCJD. In the first incidence, the recipient became ill 7 years
after receiving red cells from the donor who remained asymptomatic
and only showed signs of vCJD until 3 years after the donation
(Llewely et al. (2004) Lancet 363:417-421). In the second
incidence, the donor died of vCJD two years after donation, and the
recipient died of an aneurysm (not vCJD) 5 years after donation
(Peden et al. (2004) Lancet 364:527-529). On autopsy of the
recipient, PrPsc was present in lymph node and spleen, but not the
brain. In the third incidence, the recipient died of vCJD seven and
half years after transfusion from a donor who developed vCJD 20
months after the donation (Wroe et al. (2006) Lancet
368:2061-2067). The fourth incident occurred in a recipient eight
and half years after a transfusion from the same donor in the third
case (Health Protection Agency-Health Protection Report, (2007) Vol
1, No 3, 26. Available at:
http://www.hpa.org.uk/hpdarchives/2007/news2007/news0307.htm).
[0006] A common feature of all prion diseases is the conversion of
the normal cellular prion protein (PrPc) into an abnormal isoform
(PrPsc). The difference between PrPc and PrPsc are believed to be
purely conformational, with PrPc having primarily alpha-helical
structures and PrPsc having primarily beta sheets that frequently
assemble to form aggregates. PrPsc acts as a template to induce
normal protein molecules to convert into the same abnormal isoform,
which then in turn covert more PrPc into PrPsc (Prusiner et al.
(1998) Proc. Natl. Acad. Sci. USA 95:13363-13383). This
autocatalytic process leads to exponential formation of neurotoxic
PrPsc aggregates (Aguzzi et al. (2007) Nat Rev Mol. Cell Biol.
8:552-561). Prion protein ligands and uses thereof have been
described in WO04/050851, WO06/010915, WO04/090102, and
WO06/044459.
[0007] Studies have shown that the earliest appearance of prion
infectivity in the blood may occur during the early stage of the
incubation period of the disease (Brown et al. (2006) Blood
infectivity in the transmissible spongiform encephalopathies.
Chapter 4 In: Turner M L, ed. 95-118). Because it can be a long
time before the onset of disease symptoms, silently infected
individuals may still be considered as healthy active blood donors.
Furthermore, some individuals may be permanently or transiently
infected without developing the disease. It is thus difficult if
not impossible to ensure that sources of blood for blood derived
products are prion free.
[0008] Albumin-based nanoparticle compositions have been developed
as a drug delivery system for delivering substantially water
insoluble drugs. See, for example, U.S. Pat. Nos. 5,916,596;
6,506,405; 6,749,868, and 6,537,579 and also in U.S. Pat. Pub. Nos.
2005/0004002 and 2007/0082838. The albumin-based nanoparticle
technology utilizes the unique natural properties of the protein
albumin to transport and deliver substantially water insoluble
drugs to the site of disease. These nanoparticles are readily
incorporated into the body's own transport processes and are able
to exploit the tumors' attraction to albumin, enabling the delivery
of higher concentrations of the active drug to the target site. In
addition, the albumin-based nanoparticle technology offers the
ability to improve a drug's solubility by avoiding the need for
toxic chemicals, such as solvents, in the administration process,
thus potentially improving safety through the elimination of
solvent-related side effects.
[0009] The disclosures of all publications, patents, patent
applications and published patent applications referred to herein
are hereby incorporated herein by reference in their entirety.
BRIEF SUMMARY OF THE INVENTION
[0010] The present invention in one aspect provides prion-free
nanoparticle compositions (such as pharmaceutical compositions). In
some embodiments, there is provided a composition comprising
nanoparticles comprising albumin and a substantially water
insoluble pharmacologically active agent, wherein the composition
is substantially free of a prion protein. In some embodiments, the
composition is sterile. In some embodiments, the composition is
sterile filterable. In some embodiments, the composition further
comprises a pharmaceutically acceptable carrier.
[0011] In some embodiments, the composition has less than about 100
fg/ml prion protein. In some embodiments, the composition has a
prion infectivity of less than about 10 IU-ic/ml. In some
embodiments, the composition has a prion infectivity of less than
about 1 LD.sub.50/ml.
[0012] In some embodiments, the composition does not show the
presence of a prion protein based on a protein misfolding cyclic
amplification (PMCA) assay. In some embodiments, the composition
does not show the presence of a prion protein based on an IPCR
assay. In some embodiments, the composition has a prion infectivity
of less than about 10 IU-ic/ml and does not show the presence of a
prion protein based on a PMCA assay. In some embodiments, the
composition has a prion infectivity of less than about 10 IU-ic/ml
and does not show the presence of a prion protein based on an IPRC
assay.
[0013] The compositions described herein are generally
substantially free of PrPsc. In some embodiments, the composition
is also substantially free of PrPc. In some embodiments, the molar
ratio of PrPsc and PrPc in the composition is no greater than about
1:1, such as no great than about any one of 1:10, 1:100, 1:1000,
1:10000, or 1:100000.
[0014] The composition described herein in some embodiments
contains an amount (for example, a trace amount) of substances
introduced during a prion-removal process. For example, in some
embodiments, there is provided a composition comprising
nanoparticles comprising albumin and a substantially water
insoluble pharmacologically active agent, wherein the composition
is substantially free of a prion protein, and wherein the
composition comprises an amount (for example, a trace amount) of a
ligand capable of binding to a prion protein. In some embodiments,
there is provided a composition comprising nanoparticles comprising
albumin and a substantially water insoluble pharmacologically
active agent, wherein the composition is substantially free of a
prion protein, and wherein the composition comprises an amount (for
example, a trace amount) of a supporting material (such as
supporting material described herein, including a resin). In some
embodiments, there is provided a composition comprising
nanoparticles comprising albumin and a substantially water
insoluble pharmacologically active agent, wherein the composition
is substantially free of a prion protein, and wherein the
composition comprises an amount of a PRDT resin (for example, a
trace amount of a PRDT resin). In some embodiments, there is
provided a composition comprising nanoparticles comprising albumin
and a substantially water insoluble pharmacologically active agent,
wherein the composition is substantially free of a prion protein,
and wherein the composition comprises an amount of a DVR resin (for
example, a trace amount of a DVR resin).
[0015] In some embodiments, the level of an albumin stabilizer in
the composition is less than that of a composition wherein the
albumin has not been cleared by a prion-removal process. These
albumin stabilizers include, for example, N-acetyl tryptophanate
and sodium caprylate.
[0016] In some embodiments, the composition is bioequivalent to a
composition wherein the albumin has not been cleared by a
prion-removal process.
[0017] In some embodiments, there is provided a composition
comprising nanoparticles comprising albumin and a substantially
water insoluble pharmacologically active agent, wherein the albumin
in the composition was obtained by a method comprising a
prion-removal process, said prion removal process comprising
contacting an initial albumin composition with a ligand capable of
binding to a prion protein. In some embodiments, the prion-removal
process further comprises removing said ligand and proteins bound
thereto from said albumin composition.
[0018] In some embodiments, there is provided a composition
comprising nanoparticles comprising albumin and a substantially
water insoluble pharmacologically active agent, wherein the albumin
in the composition was obtained by a method comprising: a)
contacting an initial albumin composition with a ligand capable of
binding to a prion protein to cause formation of a complex between
the ligand and a prion protein, and b) removing the complex from
the initial composition.
[0019] In some embodiments, the initial albumin composition is a
blood derived product. In some embodiments, the initial albumin
composition is an albumin composition prepared from a body fluid
(such as blood). In some embodiments, the albumin is human serum
albumin
[0020] In some embodiments, the ligand is a peptide (such as any
peptides provided in Table 1). In some embodiments, the ligand is
an antibody recognizing a prion protein. In some embodiments, the
ligand is a chemical compound (such as triazine-based compounds).
In some embodiments, the ligand comprises an amino group, such as
an amino group on an amino resin.
[0021] The ligand can be attached to a supporting material,
including, for example, column, bead, matrix, filter, and
membrane.
[0022] In another aspect, there are provided methods of producing
prion-free nanoparticle compositions. For example, in some
embodiments, there is provided a method of producing a composition
comprising nanoparticles comprising albumin and a substantially
water insoluble pharmacologically active agent, said method
comprising subjecting a mixture comprising an albumin solution and
an organic phase containing said substantially water insoluble
pharmacologically active agent dispersed in an organic solvent to a
high shear condition, wherein the albumin was obtained by a method
comprising removing a prion protein from an initial albumin
composition. In some embodiments, there is provided a method of
producing a composition comprising nanoparticles comprising albumin
and a substantially water insoluble pharmacologically active agent,
said method comprising subjecting a mixture comprising an albumin
solution and an organic phase containing said substantially water
insoluble pharmacologically active agent dispersed in an organic
solvent to a high shear condition, wherein the albumin was obtained
by a method comprising contacting an initial albumin composition
with a ligand capable of binding to a prion protein. In some
embodiments, there is provided a method of producing a composition
comprising nanoparticles comprising albumin and a substantially
water insoluble pharmacologically active agent, said method
comprising subjecting a mixture comprising an albumin solution and
an organic phase containing said substantially water insoluble
pharmacologically active agent dispersed in an organic solvent to a
high shear condition, wherein the albumin was obtained by a method
comprising: a) contacting an initial albumin composition with a
ligand capable of binding to a prion protein, and b) removing the
ligand and protein bound thereto from the initial composition.
[0023] In some embodiments, there is provided a method of producing
a composition comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent, said
method comprising: a) removing a prion protein from an initial
albumin composition; b) subjecting a mixture comprising a solution
comprising the prion-removed albumin and an organic phase
comprising said substantially water insoluble pharmacologically
active agent dispersed in an organic solvent to a high shear
condition. In some embodiments, there is provided a method of
producing a composition comprising nanoparticles comprising albumin
and a substantially water insoluble pharmacologically active agent,
said method comprising: a) contacting an initial albumin
composition with a ligand capable of binding to a prion protein to
cause formation of a complex between the ligand and a prion
protein, b) removing the complex from the albumin initial
composition; and c) subjecting a mixture comprising a solution
comprising the prion-removed albumin and an organic phase
comprising said substantially water insoluble pharmacologically
active agent dispersed in an organic solvent to a high shear
condition.
[0024] In some embodiments, there is provided a method of producing
a composition comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent,
comprising: a) contacting an albumin solution with a ligand capable
of binding to a prion protein, b) removing the ligand and proteins
bound thereto from the albumin solution, and c) subjecting a
mixture comprising said albumin solution and an organic phase
comprising said substantially water insoluble pharmacologically
active agent dispersed in an organic solvent to a high shear
condition. In some embodiments, the mixture contains substantially
no surfactants.
[0025] The prions can be removed during the formation of the
nanoparticles. For example, in some embodiments, there is provided
a method of producing a composition comprising nanoparticles
comprising albumin and a substantially water insoluble
pharmacologically active agent, comprising: a) contacting a mixture
comprising an albumin solution and an organic phase comprising said
substantially water insoluble pharmacologically active agent
dispersed in an organic solvent with a ligand capable of binding to
a prion protein. In some embodiments, the method further comprises:
b) removing the ligand and proteins bound thereto from the mixture.
In some embodiments, the method further comprises c) subjecting the
mixture to a high shear condition. In some embodiments, the mixture
contains substantially no surfactants.
[0026] The prion proteins can also be removed after the formation
of the nanoparticle composition. For example, in some embodiments,
there is provided a method of producing a composition comprising
nanoparticles comprising albumin and a substantially water
insoluble pharmacologically active agent, comprising contacting a
mixture comprising an organic phase comprising said substantially
water insoluble pharmacologically active agent dispersed in an
organic solvent and an albumin solution with a ligand capable of
binding to a prion protein, wherein the mixture has been subjected
to a high shear condition prior to contacting with the ligand. In
some embodiments, there is provided a method of producing a
composition comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent,
comprising: a) subjecting a mixture comprising an organic phase
comprising said substantially water insoluble pharmacologically
active agent dispersed in an organic solvent and an albumin
solution to a high shear condition, and b) contacting the mixture
with a ligand capable of binding to a prion protein. In some
embodiments, the method further comprises: c) removing the ligand
and proteins bound thereto from the mixture. In some embodiments,
the mixture is substantially free of surfactants.
[0027] In some embodiments, there is provided a method of removing
a prion protein from a composition suspected of containing a prion
protein comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent, said
method comprising a) contacting the nanoparticle composition with a
ligand capable of binding to a prion protein, b) removing the
ligand and proteins bound thereto from the nanoparticle
composition. In some embodiments, there is provided a method of
removing a prion protein from an albumin composition suspected of
containing an abnormal prion protein, comprising: a) contacting the
composition comprising albumin with a ligand capable of binding to
a prion protein, b) removing the ligand and proteins bound thereto
from the albumin composition, wherein said albumin composition is
used to produce a composition comprising nanoparticles comprising
nanoparticles comprising albumin and a substantially water
insoluble pharmacologically active agent.
[0028] In some embodiments, there is provided a method of removing
a prion protein from a composition comprising nanoparticles
comprising albumin and a substantially water insoluble
pharmacologically active agent, comprising: a) determining the
presence or absence of a prion protein in the composition, b)
contacting the composition with a ligand capable of binding to a
prion protein, and c) removing the ligand and proteins bound
thereto from the composition.
[0029] Also provided are compositions made during the prion removal
process. For example, in some embodiments, there is provided a
composition comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent,
further comprising a ligand capable of binding to a prion protein.
In some embodiments, there is provided a mixture comprising
nanoparticle comprising albumin and a substantially water insoluble
pharmacologically active agent, and a ligand capable of binding to
a prion protein attached to a supporting material, such as one or
more supporting materials described herein. In some embodiments,
there is provided a column loaded with a composition comprising
nanoparticles comprising albumin and a substantially water
insoluble pharmacologically active agent, wherein the column
comprises a ligand capable of binding to a prion protein.
[0030] Also provided are compositions made by methods described
herein. Also provided are methods of using the prion-free
compositions described herein. For example, in some embodiments,
there is provided a method of administering a composition
comprising nanoparticles comprising albumin and a substantially
water insoluble pharmacologically active agent, wherein the
composition is substantially free of a prion protein. In some
embodiments, there is provided a method of treating a disease (such
as cancer) comprising administering a composition comprising
nanoparticles comprising albumin and a substantially water
insoluble pharmacologically active agent, wherein the composition
is substantially free of a prion protein.
[0031] In some embodiments, there is provided a method of
administering a composition comprising nanoparticles comprising
albumin and a substantially water insoluble pharmacologically
active agent, wherein the albumin in the composition was obtained
by a method comprising a prion-removal process, said prion-removal
process comprising contacting an initial albumin composition with a
ligand capable of binding to a prion protein. In some embodiments,
the prion removal process further comprises removing said ligand
and proteins bound thereto from said albumin composition. In some
embodiments, there is provided a method of treating a disease (such
as cancer) comprising administering a composition comprising
nanoparticles comprising albumin and a substantially water
insoluble pharmacologically active agent, wherein the albumin in
the composition was obtained by a method comprising a prion-removal
process, said prion removal process comprising contacting an
initial albumin composition with a ligand capable of binding to a
prion protein.
[0032] Also provided are kits and dosage forms (such as vials for
example sealed vials) comprising the prion-free nanoparticle
compositions described herein and kits useful for methods described
herein. Further provided are systems (including apparatus) for
carrying out one or more methods described herein.
[0033] These and other aspects and advantages of the present
invention will become apparent from the subsequent detailed
description and the appended claims. It is to be understood that
one, some, or all of the properties of the various embodiments
described herein may be combined to form other embodiments of the
present invention.
BRIEF DESCRIPTION OF FIGURES
[0034] FIG. 1 provides western blots and SDS-PAGE gels of DVR resin
challenged with 0.01% and 0.005% scrapie hamster brain homogenate
in 20% or 25% albumin AMN31 was the positive control resin. The
signal observed was the bound fraction. "-PK" and "+PK" denotes
absence or presence of Proteinase K digestion.
[0035] FIG. 2 provides western blots and SDS-PAGE gels of YVHEA and
SYA resins challenged with 0.01% and 0.005% scrapie hamster brain
homogenate in 20% or 25% albumin AMN31 was the positive control
resin. The signal observed was the bound fraction. "-PK" and "+PK"
denotes absence or presence of Proteinase K digestion.
[0036] FIG. 3 provides western blots and SDS-PAGE gels of D4 resin
challenged with 0.01% and 0.005% scrapie hamster brain homogenate
in 20% or 25% albumin AMN31 was the positive control resin. The
signal observed was the bound fraction. "-PK" and "+PK" denotes
absence or presence of Proteinase K digestion.
[0037] FIG. 4 depicts the process flow scheme for the TSE removal
by the prion reduction resin column (PRDT (Pathogen Removal and
Diagnostic Technologies) column) for 20% albumin.
[0038] FIG. 5 shows a chromatography profile from the spiked run in
the study of TSE removal by the PRDT column for 20% albumin.
[0039] FIG. 6 shows the Western blot interference testing of 20%
albumin solution with centrifugation (with and without 10-fold
concentration).
[0040] FIG. 7 depicts the process flow scheme for the TSE removal
by the prion reduction resin column (PRDT column) for 25%
albumin
[0041] FIG. 8 shows a chromatography profile from the spiked run in
the study of TSE removal by the PRDT column for 25% albumin.
[0042] FIG. 9 shows the Western blot interference testing of 25%
albumin solution with centrifugation (with and without 10-fold
concentration).
DETAILED DESCRIPTION OF THE INVENTION
[0043] The present invention provides prion-free compositions (such
as pharmaceutical compositions) comprising nanoparticles comprising
albumin and substantially water insoluble pharmacologically active
agents and methods of making prion-free compositions.
[0044] The present invention in one aspect provides compositions
(such as pharmaceutical compositions) comprising nanoparticles
comprising albumin and substantially water insoluble
pharmacologically active agents, wherein the composition is
substantially free of a prion protein.
[0045] In another aspect, there is provided a method of making a
prion-free composition (such as pharmaceutical compositions)
comprising nanoparticles comprising albumin and substantially water
insoluble pharmacologically active agent. Compositions made during
the method of making process are also provided.
[0046] In another aspect, there is provided a method of using a
prion-free composition (such as pharmaceutical compositions)
comprising nanoparticles comprising albumin and substantially water
insoluble pharmacologically active agent.
[0047] Also provided are kits and dosage forms (such as vials for
example sealed vials) comprising the prion-free nanoparticle
compositions described herein and kits and systems (including
apparatus) useful for methods described herein.
[0048] "Prion free" is used herein for convenience and generally to
describe the inventive compositions and is meant to encompass all
embodiments described herein.
[0049] Reference to "about" a value or parameter herein includes
(and describes) embodiments that are directed to that value or
parameter per se. For example, description referring to "about X"
includes description of "X."
[0050] It is understood that aspect and embodiments of the
invention described herein include "consisting" and/or "consisting
essentially of aspects and embodiments.
Prion-Free Nanoparticle Compositions
[0051] The present invention provides a composition (such as a
pharmaceutical composition) comprising nanoparticles comprising
albumin and a substantially water insoluble pharmacologically
active agent, wherein the composition is substantially free of a
prion protein. In some embodiments, the composition is sterile. In
some embodiments, the composition is sterile filterable. In some
embodiments, the composition further comprises a pharmaceutically
acceptable carrier.
[0052] In some embodiments, a prion protein cannot be detected in
the composition by a detection method having a detection
sensitivity of about 100 fg/ml or higher, that is, the test result
is negative in an assay using a method having a detection
sensitivity of about 100 fg/ml or higher (for example a detection
sensitivity of about 50 fg/ml, about 10 fg/ml, about 1 fg/ml, about
0.1 fg/ml). The prion content in the composition can be determined
directly from the composition. Alternatively, the composition can
be processed prior to the determination, for example, concentrated
or enriched in order to facilitate the detection and quantitation
of the prion protein in the composition.
[0053] One way of determining whether or not a composition is
substantially free of a prion protein is in vivo infectivity assay.
For example, in vivo infectivity can be demonstrated by inoculation
of the testing composition in mouse, mink, hamster, or goat models.
Infectivity can be determined by lethal dose (LD50), i.e., the dose
which when administered by a given route (such as by intracerebral
route) induces disease in 50% of exposed animals. Alternatively,
infectivity can be determined by infectious units, i.e., the
minimal infectious dose capable of transmitting the disease to one
experimental animal to another by a given route. Generally, 100
IU-ic/ml (infectious unit determined by intracerebral route)
corresponds to about 10 LD50/ml and 1 pg/ml of prion protein.
[0054] Another method for determining a prion protein in the
composition is immuno-polymerase chain reaction (IPCR), a technique
whereby the exponential amplification ability of PCR is coupled to
the detection of proteins by antibodies in an ELISA format and is
applied in a modified real-time IPCR method to detect ultra-low
levels of prion protein. See Barletta et al., J. Virology Method,
127 (2005):154-104. Using IPCR, recombinant hamster PrPc was
consistently detected at 1 fg/ml and proteinase K (PK)-digested
scrapie infected hamster brain homogenates diluted to 10.sup.-8
(approximately 10-100 infectious units) was detected with a
semi-quantitative dose response.
[0055] In some embodiments, the prion protein in the composition is
determined by Protein Misfolding Cyclic Amplification (PMCA). This
method has been used to detect PrPsc in the blood. Other methods
suitable for determining a prion protein in the composition
include, but not limited to: quantitative sandwich ELISA using
time-resolved dissociation-enhanced fluorescence technology;
dual-color fluorescent confocal scanning; conformation dependent
immunoassay (CDI). Western blotting, bead blot, gel-mobility shift
assays, fluorescent in situ hybridization analysis (FISH), tracking
of radioactive or bioluminescent markers, nuclear magnetic
resonance, electron paramagnetic resonance, stopped-flow
spectroscopy, column chromatography, capillary electrophoresis, or
other methods can also be developed to detect prion proteins in the
composition.
[0056] In some embodiments, the composition is substantially free
of a prion protein based on one of the assays for detecting prion
proteins (such as any one of the assays described above). In some
embodiments, two or more assays are used to analyze the
composition, and the correlation between these different assays is
used to determine whether or not the composition is free of a prion
protein.
[0057] Thus, in some embodiments, there is provided a composition
comprising nanoparticles comprising albumin and a substantially
water insoluble pharmacologically active agent, wherein the
composition is substantially free of a prion protein. In some
embodiments, the composition has a prion infectivity of less than
about 100 fg/ml. In some embodiments, the composition has a prion
infectivity of less than about 10 IU-ic/ml. In some embodiments,
the composition has a prion infectivity of less than about 1
LD50/ml. In some embodiments, the composition does not show the
presence of a prion protein based on the protein misfolding cyclic
amplification (PMCA) assay. In some embodiments, the composition
does not show the presence of a prion protein based on the IPCR
assay. In some embodiments, the composition has a prion infectivity
of less than about 10 IU-ic/ml and does not show the presence of a
prion protein based on the PMCA assay. In some embodiments, the In
some embodiments, the composition has a prion infectivity of less
than about 10 IU-ic/ml and does not show the presence of a prion
protein based on the IPRC assay. In some embodiments, the
composition is free of prion protein based on the standard provided
in the Guideline for the Investigation of Manufacturing Processes
for Plasma-Derived Medicinal Products with Regard to vCJD Risk
(CPMP5136/03), available at
http://www.emea.europa.eu/pdfs/human/bwp/513603en.pdf, the content
of which is incorporated herein in its entirety.
[0058] The compositions described herein are generally
substantially free of PrPsc. In some embodiments, the composition
is also substantially free of PrPc. In some embodiments, the molar
ratio of PrPsc and PrPc in the composition is no greater than about
1:1, such as no great than about any one of 1:10, 1:100, 1:1000,
1:10000, or 1:100000.
[0059] In some embodiments, the composition is substantially free
of a prion protein from human, bovine, sheep, and rodent (such as
hamster, mice, and mink) In some embodiments, the composition is
substantially free of the H-type prion protein, the L-type prion
protein, or both. In some embodiments, the composition is
substantially free of cell-bound of prion protein, the free prion
protein, or both.
[0060] The term "PrPc" used herein refers to the native prion
protein molecule which is naturally expressed within the body of
the mammalian. The term "PrPsc" used herein refers to the
conformationally altered form of the PrPc molecule that is thought
to be infectious.
[0061] "Albumin" used herein refers to naturally occurring albumin
and does not encompass albumin produced recombinantly. Naturally
occurring albumin is advantageous over recombinant albumin because
it is the natural ligand for albumin receptors in vivo. The albumin
used in the methods described herein generally retains the
post-translational modifications of albumin and thus has reduced
risks of immunogenicity. In some embodiments, the albumin is
obtained from a blood-derived composition. "Blood derived
composition" used herein include whole blood, red blood cell
concentrate, plasma, serum, platelet rich and platelet poor
fraction, platelet concentrate, while blood cell, blood plasma
precipitate, blood plasma fractionation precipitate and
supernatant, plasma fractionation intermediate, various other
substances which are derived from blood, and the like. In some
embodiments, the albumin is from human. In some embodiments, the
albumin is from an animal such as bovine, sheep, and rodent (such
as mouse, hamster, and mink) In some embodiments, the albumin is
obtained from a population of individuals (such as human) at least
some of which have been infected with prions. In some embodiments,
the albumin is obtained from a population of individuals (such as
human) at least some of which are suspected of having been infected
with prions. In some embodiments, the albumin is obtained from a
large batch-size pool of individuals.
[0062] The composition described herein in some embodiments
contains a trace amount of substances introduced during the
prion-removal process. For example, in some embodiments, there is
provided a composition comprising nanoparticles comprising albumin
and a substantially water insoluble pharmacologically active agent,
wherein the composition is substantially free of a prion protein,
and wherein the composition comprises a trace amount of a ligand
capable of binding to a prion protein. In some embodiments, there
is provided a composition comprising nanoparticles comprising
albumin and a substantially water insoluble pharmacologically
active agent, wherein the composition is substantially free of a
prion protein, and wherein the composition comprises a trace amount
of a supporting material (such as material from a supporting
material described herein, including a resin). In some embodiments,
there is provided a composition comprising nanoparticles comprising
albumin and a substantially water insoluble pharmacologically
active agent, wherein the composition is substantially free of a
prion protein, and wherein the composition comprises a trace amount
of a ligand capable of binding to a prion protein and a trace
amount of a supporting material (such as material from a supporting
material described herein, including a resin).
[0063] "Trace amount" refers to a detectable amount that does not
affect the property of the composition, for example in terms of
bioavailability and/or bioequivalency.
[0064] In some embodiments, the composition is bioequivalent to a
composition wherein the albumin has not been cleared by a
prion-removal process. Bioequivalence can be established, for
example, by a 90% confidence interval of between 0.80 and 1.25 for
both Cmax and AUC, or a 90% confidence interval of between 0.80 and
1.25 of AUC and a 90% confidence interval of between 0.70 and 1.43
for Cmax.
[0065] In some embodiments, the level of an albumin stabilizer in
the composition is less than that of a composition wherein the
albumin has not been cleared by a prion-removal process. These
albumin stabilizers include, for example, N-acetyl tryptophanate
and sodium caprylate.
[0066] The compositions described herein generally encompass
nanoparticles comprising a substantially water insoluble
pharmaceutically active agent and an albumin In some embodiments,
the nanoparticle composition comprises nanoparticles comprising a
substantially water insoluble pharmacologically active agent and an
albumin In some embodiments, the nanoparticles in the composition
described herein have an average diameter of no greater than about
1000 nm, including for example no greater than about any one of
900, 800, 700, 600, 500, 400, 300, 200, 190, 180, 170, 160, 150,
140, 130, 120, 110, 100, 90, 80, 70, or 60 nm. In some embodiments,
at least about 50% (for example at least about any one of 60%, 70%,
80%, 90%, 95%, or 99%) of all the nanoparticles in the composition
have a diameter of no greater than about 1000 nm, including for
example no greater than about any one of 900, 800, 700, 600, 500,
400, 300, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100,
90, 80, 70, or 60 nm. In some embodiments, at least about 50% (for
example at least any one of 60%, 70%, 80%, 90%, 95%, or 99%) of all
the nanoparticles in the composition fall within the range of about
20 to about 200 nm, including for example any one of about 30 to
about 180 nm, and any one of about 40 to about 150, about 50 to
about 120, and about 60 to about 100 nm.
[0067] In some embodiments, at least about 5% (including for
example at least about any one of 10%, 15%, 20%, 25%, 30%, 40%,
50%, 60%, 70%, 80%, or 90%) of the albumin in the nanoparticle
portion of the composition are crosslinked (for example crosslinked
through one or more disulfide bonds).
[0068] In some embodiments, the nanoparticles comprise the
substantially water insoluble pharmacologically active agent (such
as paclitaxel) coated with an albumin, (e.g., human serum albumin).
In some embodiments, the composition comprises substantially water
insoluble pharmacologically active agent in both nanoparticle and
non-nanoparticle forms, wherein at least about any one of 50%, 60%,
70%, 80%, 90%, 95%, or 99% of the substantially water insoluble
pharmacologically active agent in the composition are in
nanoparticle form. In some embodiments, the substantially water
insoluble pharmacologically active agent in the nanoparticles
constitutes more than about any one of 50%, 60%, 70%, 80%, 90%,
95%, or 99% of the nanoparticles by weight. In some embodiments,
the nanoparticles have a non-polymeric matrix. In some embodiments,
the nanoparticles comprise a core of substantially water insoluble
pharmacologically active agent that is substantially free of
polymeric materials (such as polymeric matrix).
[0069] In some embodiments, the nanoparticle composition is
substantially free (such as free) of surfactants or organic solvent
(such as Cremophor.RTM., Tween 80, or any other organic solvents
used for the administration of substantially water insoluble
pharmacologically active agents). In some embodiments, the
nanoparticle composition contains less than about any one of 20%,
15%, 10%, 7.5%, 5%, 2.5%, 1% or less organic solvent.
[0070] The removal of prion from the albumin-containing composition
makes it possible to administer higher amounts of albumin without
being concerned about prions. The present invention thus also
contemplates compositions (such as pharmaceutical compositions)
comprising nanoparticles comprising albumin and substantially water
insoluble pharmacologically active agent, wherein the weight ratio
of albumin to the substantially water insoluble pharmaceutical
agent is about 20:1 or more, such as about any of about 30:1 or
more, about 40:1 or more, or about 50:1 or more. Exemplary ratios
include, for example, about 20:1 to about 40:1, about 40:1 to about
60:1, about 60:1 to about 80:1, or about 90:1 to about 100:1. In
some embodiments, the weight ratio of albumin and substantially
water insoluble pharmacologically active agent in the nanoparticle
composition is about 18:1 or less, such as about 15:1 or less, for
example about 10:1 or less. In some embodiments, the weight ratio
of albumin and substantially water insoluble pharmacologically
active agent in the composition falls within the range of any one
of about 1:1 to about 18:1, about 2:1 to about 15:1, about 3:1 to
about 13:1, about 4:1 to about 12:1, about 5:1 to about 10:1. In
some embodiments, the weight ratio of albumin and substantially
water insoluble pharmacologically active agent in the nanoparticle
portion of the composition is about any one of 1:2, 1:3, 1:4, 1:5,
1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, or
less.
[0071] In some embodiments, the particle composition comprises one
or more of the above characteristics.
[0072] In some embodiments, the nanoparticle composition is
Abraxane.TM.. Nanoparticle compositions comprising other
substantially water insoluble pharmacologically active agents (such
as docetaxel and ortataxel) may also comprise one or more of the
above characteristics.
[0073] In some embodiments, there is provided a composition
comprising nanoparticles comprising albumin and a substantially
water insoluble pharmacologically active agent, wherein the albumin
in the composition was obtained by a method comprising a
prion-removal process, said prion removal process comprising
contacting an initial albumin composition with a ligand capable of
binding to a prion protein. In some embodiments, the prion-removal
process further comprises removing said ligand and proteins bound
thereto from said albumin composition.
[0074] In some embodiments, there is provided a composition
comprising nanoparticles comprising albumin and a substantially
water insoluble pharmacologically active agent, wherein the albumin
in the composition was obtained by a method comprising: a)
contacting an initial composition comprising albumin with a ligand
capable of binding to a prion protein to cause formation of a
complex between the ligand and a prion protein, and b) removing the
complex from the initial composition.
[0075] Nanoparticles comprising albumins and substantially water
insoluble drugs are further described below in more detail. The
method of removing prions from a composition (such as an initial
albumin composition, a nanoparticle composition comprising albumin
and a substantially water insoluble pharmacologically active agent,
or an intermediate composition formed during the process of making
the nanoparticles) are further described below in more detail. The
present invention encompasses compositions produced by any of the
methods described herein.
[0076] The compositions described herein generally have reduced
prion protein level as compared to compositions wherein the albumin
has not been cleared by a prion-removal process. For example, in
some embodiments, the composition has less than about any of 50%,
40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, or less of prion protein
than a composition wherein the albumin has not been cleared by a
prion-removal process. In some embodiments, the composition has any
of about 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, or 8 logs less prion
protein than a composition wherein the albumin has not been cleared
by a prion-removal process. In some embodiments, the composition
has any of about 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, or 8 logs less
infectivity than a composition wherein the albumin has not been
cleared by a prion-removal process. In some embodiments, the
composition of the present invention is bioequivalent to a
composition wherein the albumin has not been cleared by a
prion-removal process.
[0077] Although the present application focuses on albumin, it is
to be understood that other proteins normally found in blood or
plasma, which include, but are not limited to, immunoglobulin
(including IgA and IgG), lipoproteins, apolipoprotein B, alpha-acid
glycoprotein, beta-2-macroglobulin, thyroglobulin, transferin,
fibronectin, factor VII, factor VIII, factor IX, factor X, and the
like, are also contemplated. All relevant descriptions about
albumin provided herein are equally applicable to these other
proteins to the extent they are utilized in the formation of
nanoparticles.
Methods of Making Prion-Free Nanoparticle Compositions
[0078] In another aspect, there are provided methods of producing
prion-free nanoparticle compositions. For example, in some
embodiments, there is provided a method of producing a composition
comprising nanoparticles comprising albumin and a substantially
water insoluble pharmacologically active agent, said method
comprising subjecting a mixture comprising an albumin solution and
an organic phase containing said substantially water insoluble
pharmacologically active agent dispersed in an organic solvent to a
high shear condition, wherein the albumin was obtained by a method
comprising removing a prion protein from an initial albumin
composition. In some embodiments, there is provided a method of
producing a composition comprising nanoparticles comprising albumin
and a substantially water insoluble pharmacologically active agent,
said method comprising subjecting a mixture comprising an albumin
solution and an organic phase containing said substantially water
insoluble pharmacologically active agent dispersed in an organic
solvent to a high shear condition, wherein the albumin was obtained
by a method comprising contacting an initial albumin composition
with a ligand capable of binding to a prion protein. In some
embodiments, there is provided a method of producing a composition
comprising nanoparticles comprising albumin and a substantially
water insoluble pharmacologically active agent, said method
comprising subjecting a mixture comprising an albumin solution and
an organic phase containing said substantially water insoluble
pharmacologically active agent dispersed in an organic solvent to a
high shear condition, wherein the albumin was obtained by a method
comprising: a) contacting an initial albumin composition with a
ligand capable of binding to a prion protein, and b) removing the
ligand and protein bound thereto from the initial composition.
[0079] In some embodiments, there is provided a method of producing
a composition comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent, said
method comprising: a) removing a prion protein from an initial
albumin composition; b) subjecting a mixture comprising a solution
comprising the prion-removed albumin and an organic phase
comprising said substantially water insoluble pharmacologically
active agent dispersed in an organic solvent to a high shear
condition. In some embodiments, there is provided a method of
producing a composition comprising nanoparticles comprising albumin
and a substantially water insoluble pharmacologically active agent,
said method comprising: a) contacting an initial albumin
composition with a ligand capable of binding to a prion protein to
cause formation of a complex between the ligand and a prion
protein, and b) removing the complex from the albumin initial
composition; c) subjecting a mixture comprising a solution
comprising the prion-removed albumin and an organic phase
comprising said substantially water insoluble pharmacologically
active agent dispersed in an organic solvent to a high shear
condition.
[0080] In some embodiments, there is provided a method of producing
a composition comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent,
comprising: a) contacting an albumin solution with a ligand capable
of binding to a prion protein, b) removing the ligand and proteins
bound thereto from the albumin solution, c) subjecting a mixture
comprising said albumin solution and an organic phase comprising
said substantially water insoluble pharmacologically active agent
dispersed in an organic solvent to a high shear condition. In some
embodiments, the mixture contains substantially no surfactants.
[0081] The prions can be removed during the formation of the
nanoparticles. For example, in some embodiments, there is provided
a method of producing a composition comprising nanoparticles
comprising albumin and a substantially water insoluble
pharmacologically active agent, comprising: a) contacting a mixture
comprising an albumin solution and an organic phase comprising said
substantially water insoluble pharmacologically active agent
dispersed in an organic solvent with a ligand capable of binding to
a prion protein. In some embodiments, the method further comprises:
b) removing the ligand and proteins bound thereto from the mixture.
In some embodiments, the method further comprises c) subjecting the
mixture to a high shear condition. In some embodiments, the method
further comprises removing the organic solvent from the mixture. In
some embodiments, the mixture contains substantially no
surfactants.
[0082] In some embodiments, there is provided a method of producing
a composition comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent,
comprising: a) subjecting a mixture comprising an albumin solution
and an organic phase comprising a substantially water insoluble
pharmacologically active agent dispersed in an organic solvent to a
high shear condition; and b) contacting the mixture with a ligand
capable of binding to a prion protein. In some embodiments, the
method further comprises: c) removing the ligand and proteins bound
thereto from the mixture. In some embodiments, the method further
comprises removing the organic solvent from the mixture. In some
embodiments, the mixture contains substantially no surfactants.
[0083] In some embodiments, there is provided a method of producing
a composition comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent,
comprising contacting a mixture comprising an organic phase
comprising said substantially water insoluble pharmacologically
active agent dispersed in an organic solvent and an albumin
solution with a ligand capable of binding to a prion protein,
wherein the mixture has been subjected to a high shear condition
prior to contacting with the ligand. In some embodiments, there is
provided a method of producing a composition comprising
nanoparticles comprising albumin and a substantially water
insoluble pharmacologically active agent, comprising: a) subjecting
a mixture comprising an organic phase comprising said substantially
water insoluble pharmacologically active agent dispersed in an
organic solvent and an albumin solution to a high shear condition,
and b) contacting the mixture with a ligand capable of binding to a
prion protein. In some embodiments, the method further comprises:
c) removing the ligand and proteins bound thereto from the mixture.
In some embodiments, the method further comprises: d) removing the
aqueous phase from the mixture. In some embodiments, the mixture is
substantially free of surfactants.
[0084] In some embodiments, there is provided a method of producing
a composition comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent,
comprising: a) subjecting a mixture comprising an organic phase
comprising said substantially water insoluble pharmacologically
active agent dispersed in an organic solvent and an albumin
solution to a high shear condition, b) removing said organic
solvent, and c) contacting the mixture with organic solvent removed
with a ligand capable of binding to a prion protein. In some
embodiments, the method further comprises: d) removing the ligand
and proteins bound thereto from the mixture. In some embodiments,
the method further comprises: d) removing the aqueous phase from
the mixture. In some embodiments, the mixture is substantially free
of surfactants.
[0085] In some embodiments, there is provided a method of producing
a composition comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent,
comprising: a) subjecting a mixture comprising an organic phase
comprising said substantially water insoluble pharmacologically
active agent dispersed in an organic solvent and an albumin
solution to a high shear condition, b) removing said organic
solvent, c) adding albumin to the mixture, and d) contacting the
mixture with a ligand capable of binding to a prion protein. In
some embodiments, the method further comprises: e) removing the
ligand and proteins bound thereto from the mixture. In some
embodiments, the method further comprises: f) removing the aqueous
phase from the mixture. In some embodiments, the mixture is
substantially free of surfactants.
[0086] The methods described herein generally include the step of
subjecting a mixture comprising an organic phase comprising the
substantially water insoluble pharmacologically active agent
dispersed in an organic solvent and an albumin solution to a high
shear condition. In some embodiments, the high shear condition is
high pressure homogenization, for example at a pressure in the
range of about 3000 to about 30,000 psi, including for example
about 6000 to about 25,000 psi, about 9000 to about 18,000 psi,
about 10,000 to about 25,000 psi, about 15,000 to about 25,000 psi.
In some embodiments, organic solvent is a mixture of a
substantially water immiscible organic solvent (such as chloroform
or methylene chloride) and a water soluble organic solvent (such as
a water soluble alcohol, including ethanol and t-butanol). In some
embodiments, the ratio (v/v) of the substantially water immiscible
organic solvent and the water soluble organic solvent (for example
the ratio of chloroform/ethanol or chloroform/butanol) is about any
of 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1,
6:1, 7:1, 8:1, or 9:1, or with a ratio of about any of 3:7, 5:7,
4:6. 6:4, 5:5, 6:5, 8:5, 9:5, 9.5:5, 5:3, 7:3, 6:4, or 9.5:0.5.
[0087] In some embodiments, the method further comprises removing
the organic phase from the mixture (such as removal by evaporation
under reduced pressure). In some embodiments, the method further
comprises removing the aqueous phase from the mixture. In some
embodiments, the method further comprises sterile filtering the
nanoparticles formed by the method described above.
[0088] In some embodiments, there is provided a method of removing
a prion protein from a composition suspected of containing a prion
protein comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent, said
method comprising: a) contacting the nanoparticle composition with
a ligand capable of binding to a prion protein, b) removing the
ligand and proteins bound thereto from the nanoparticle
composition. In some embodiments, there is provided a method of
removing a prion protein from an albumin composition suspected of
containing an abnormal prion protein, comprising: a) contacting the
composition comprising albumin with a ligand capable of binding to
a prion protein, b) removing the ligand and proteins bound thereto
from the albumin composition, wherein said albumin composition is
used to produce a composition comprising nanoparticles comprising
nanoparticles comprising albumin and a substantially water
insoluble pharmacologically active agent.
[0089] In some embodiments, there is provided a method of removing
a prion protein from a composition comprising nanoparticles
comprising albumin and a substantially water insoluble
pharmacologically active agent, comprising: a) determining the
presence or absence of a prion protein in the composition, b)
contacting the composition with a ligand capable of binding to a
prion protein, and c) removing the ligand and proteins bound
thereto from the composition.
[0090] In some embodiments, one or more steps of the methods
described herein are carried out in batch mode. In some
embodiments, one of more steps of the methods described herein are
carried out in continuous mode.
Removing Prion Prior to the Formation of Nanoparticles
[0091] The prion protein can be removed from an initial albumin
composition before the albumin-containing nanoparticle compositions
are made. Generally, the method comprises contacting an initial
albumin composition with a ligand capable of binding to a prion
protein, and removing the ligand and protein bound thereto from the
albumin composition. This process can be repeated one or more
times, with the same or a different ligand. Two or more ligands can
also be used simultaneously during the prion removal process.
[0092] In some embodiments, the initial albumin composition is a
blood derived composition. For example, in some embodiments, the
initial albumin composition is whole blood, red blood cell
concentrate, plasma, serum, platelet rich and platelet poor
fraction, platelet concentrate, while blood cell, blood plasma
precipitate, blood plasma fractionation precipitate and
supernatant, or plasma fractionation intermediate. In some
embodiments, the initial albumin composition is obtained from
human. In some embodiments, the initial albumin composition is from
an animal such as bovine, sheep, and rodent (such as mouse,
hamster, and mink) In some embodiments, the initial albumin
composition is obtained from a population of individuals (such as
human) at least some of which have been infected with prions. In
some embodiments, the initial albumin composition is obtained from
a population of individuals (such as human) at least some of which
are suspected of having been infected with prions.
[0093] In some embodiments, the initial albumin composition is an
albumin composition prepared from a body fluid (such as blood) by
any of various methods common in the art including ion exchange,
affinity, gel permeation, and/or hydrophobic chromatography and/or
by differential precipitation. In some embodiments, the initial
composition is an albumin composition purified from the blood (such
as human blood). In some embodiments, the initial albumin
composition is an albumin composition purified from serum (such as
human serum). In some embodiments, the initial albumin composition
has a prion infectivity of about 100 IU-ic/ml, 90IU-ic/ml,
50IU-ic/ml, or 10IU-ic/ml. In some embodiments, the concentration
of albumin in the initial albumin composition is about 1% (w/v),
including for example about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
15%, 20%, 25%, or 30%.
[0094] During the prion-removal process, the ligand is brought into
contact with the initial albumin composition and allowed to bind to
prion proteins in the initial albumin composition. Conditions
suitable for the binding can be determined and optimized to
facilitate binding of the ligand to a prion protein based on the
nature of the ligand and its binding specificity to the prion
protein. The binding in some embodiments is carried out at a
temperature of about 0.degree. C. to about 39.degree. C., including
for example about 20.degree. C. to about 25.degree. C. The binding
can be carried out at pH of about 4 to about 10, including for
example about 5 to about 9, about 6 to about 8, about 6.8 to about
7.5, about 6.9 to about 7.4, or about 7. Optionally, blocking
agents can be used to reduce non-specific binding to the
ligand.
[0095] After the contacting step, the ligand and proteins bound
thereto are removed from the rest of the composition. The term
"removing" as used herein refers to the separation of the ligand
and protein bound thereto from the albumin-containing composition.
The separation can be carried out in a variety of ways, depending
on the nature of the ligand and the supporting material (if any)
used to facilitate the separation. For example, the ligand and
proteins bound thereto can be separated out by chromatography, such
as, but not limited to, thin-layer, column and batch
chromatography; solid support and membrane separation; reactor
separation; magnetic separation, immunoseparation; colloidal
separation; sedimentation; precipitation; or centrifugation.
[0096] In some embodiments, ligand may be attached to a supporting
material such as a bead or a membrane, which in turn is allowed to
contact the initial albumin composition. Ligand-immobilized support
is allowed to contact the initial albumin composition under a
condition sufficient to cause formation of a prion-ligand complex.
The solid phase is then separated from the composition, thereby
removing the prion protein bound to the ligand from the sample. For
example, in one exemplary embodiment, the ligands are immobilized
in a column, such as a chromatography column, a sample (such as the
initial albumin composition) is then passed through the column
either due to the force of gravity or under pressure, such as in a
high pressure liquid chromatography column. Prion proteins in the
sample will bind to the ligand immobilized on the column, and the
sample passing through can be collected. This process can repeat
several times to achieve the desired result, using the same or
different ligands.
[0097] The flow rate of a sample (such as the initial albumin
composition) in a column can be adjusted to maximize the binding of
the ligand and the prion proteins in a sample. In some embodiments,
the binding is carried out at a flow rate of about 0.1 ml per
minute to about 5.0 ml per minute, about 0.1 ml per minute to about
2.5 ml per minute, about 0.1 ml per minute to about 0.25 ml per
minute, about 0.25 per minute to about 0.5 ml per minute, about 0.5
ml per minute to about 1.0 ml per minute, about 1.0 ml per minute
to about 1.5 ml per minute, about 1.5 ml per minute to about 2.0 ml
per minute, about 2.0 ml per minute to about 2.5 ml per minute,
about 2.5 ml per minute to about 3.0 ml per minute, about 3.0 ml
per minute to about 3.5 ml per minute, about 3.5 ml per minute to
about 4.0 ml per minute, about 4.0 ml per minute to about 4.5 ml
per minute, or about 4.5 ml per minute to about 5.0 ml per minute,
including for example about 0.1 ml per minute, 0.25 ml per minute,
0.5 ml per minute, 1.0 ml per minute, 1.5 ml per minute, 1.7 ml per
minute, 1.8 ml per minute, 1.9 ml per minute, 2.0 ml per minute,
2.1 ml per minute, 2.3 ml per minute, 2.5 ml per minute, 2.7 ml per
minute, 3.0 ml per minute, 3.5 ml per minute, 4.0 ml per minute,
4.5 ml per minute, or 5.0 ml per minute. In some embodiments, the
flow rate is at least about 10 ml per minute, such as at least
about any of 20 ml per minute, 30 ml per minute, 40 ml per minute,
50 ml per minute.
[0098] The total flow-through volume or total flow-through time
during a binding process can also be adjusted to maximize the
binding of the ligand and the prion proteins in a sample (such as
the initial albumin composition). In some embodiments, the total
flow-through volume is about 1 time to about 1000 times of the
column volume, including for example about 2 times to about 10
times of the column volume, about 10 times to about 20 times of the
column volume, about 20 times to about 30 times of the column
volume, about 30 times to about 40 times of the column volume,
about 40 times to about 50 times of the column volume, about 50
times to about 1000 times of the column volume, about 50 times to
about 500 times of the column volume, about 100 times to about 600
times of the column volume, or about 200 times to about 800 times
of the column volume. In some embodiments, the total flow-through
volume is about 100 times of the column volume. In other
embodiments, the total flow-through volume is about 500 times of
the column volume. In some embodiments, the total flow-through time
is about 1 hour to about 30 hours, including for example about 2
hours to about 25 hours, about 3 hours to about 20 hours, or about
3 hours to about 17 hours. In some embodiments, the total
flow-through time is any of about 3 hours, 4 hours, 6 hours, 8
hours, 10 hours, 12 hours, 14 hours, 16 hours, 17 hours, 18 hours,
19 hours, or 20 hours. In some embodiments, the total flow through
time is more than about 24 hours. In some embodiments, the total
flow through time is less than about 8, including for example any
of 7, 6, 5, 4, 3, 2, 1, or 0.5 hours.
[0099] Alternatively, the ligand can first be brought into contact
with the initial albumin composition, under a condition sufficient
to cause formation of a prion-ligand complex. The prion-ligand
complex is then subsequently removed by using a column, such as an
affinity-based chromatography. To facilitate the separation, the
ligand may be conjugated to a binding partner so that the
ligand/prion complex can be removed via the binding partner, for
example by using an affinity column containing a molecule that
recognizes the binding partner.
[0100] In addition to batch or column chromatography, a variety of
other configurations, modifications and variations of the use of
the ligands for binding prion proteins are also envisioned. Such
variations and modifications include, but are not limited to: batch
processes, continuous processes, moving bed chromatography
processes; low, medium, or high pressure processes; or small,
medium or large scale processes. In some embodiments, the ligands
are on a membrane, fibers bead, impregnated into a non-woven mesh,
or coating fibers contained within a filter housing.
[0101] In some embodiments, the removal step does not significantly
result in yield loss and/or change in the property and/or stability
of the albumin In some embodiments, the recovery of the albumin in
its original biological state is substantially maintained at least
to a level in excess of 50%, including for example 80%, or 90%, or
more. In some embodiments, the recovery rate of albumin from the
prion removal process is higher than any of about 80%, 90%, 95%, or
99%. In some embodiments, the concentration of albumin in the
initial albumin composition is adjusted or controlled prior to the
prion removal step in order to minimize non-specific binding and
loss of albumin during the process. For example, the concentration
of albumin can be in the range of about 1% to about 50%, about 5%
to about 25%, about 5% to about 30%, about 5% to about 40%, about
5% to about 10%, about 10% to about 15%, about 15% to about 20%,
about 20% to about 25%, about 25% to about 30% etc., including for
example about 5%, 10%, 15%, 20%, 25%, or 30% albumin
[0102] The resulting albumin composition can be analyzed to
determine the clearance rate of the prion removal process. The
ligand with bound prion proteins may also be analyzed (directly or
after elution) to determine the clearance rate.
[0103] The removal of prion proteins can be evaluated based on
reduction of prion protein or reduction of infectivity. In some
embodiments, at least about 50%, including for example at least
about 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the prion proteins
are removed from the initial albumin composition. In some
embodiments, the infectivity of the post-removal albumin
composition is at least about 10.times., 20.times., 30.times.,
40.times., 50.times., 80.times., 100.times., 200.times.,
500.times., 1000.times., 10.sup.4.times., 10.sup.5.times.,
10.sup.6.times., 10.sup.7.times., 10.sup.8.times., 10.sup.9.times.
less than that of the initial albumin composition.
[0104] In some embodiments, serial infectivity is used to determine
the clearance rate of the prion removal process. Serial dilutions
of a samples are made and dilutions are examined for infectious
activity, for example in an assay animal. The dilution at which
half of the animals become infected is the infectious titer. For
example, if a 5 fold dilution is required, the sample may be
defined as having 5 logs of infectivity. By comparing the log
infectivity of the initial albumin composition and that of the
post-removal albumin composition, one can determine the clearance
rate of the prion removal process. In some embodiments, the prion
removal method results in a reduction of any one of 1, 1.1, 1.2,
1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, or 4, 5, 6, 7, 8, 9, or 10
logs of infectivity.
[0105] In some embodiments, the clearance rate of the prion-removal
process is determined based on spiking experiments with infectious
materials by following steps described herein for the prion-removal
method. Suitable spiking agents include, but are not limited to,
brain homogenates, microsomes, caveolae-like domains, purified
PrPsc, and prion fibrils. In some embodiments, the spiking agent is
detergent solublized (such as sarkosyl solubilized). In some
embodiments, the spiking ratio in the composition is in the range
of about 0.001% to about 5%, about 0.001% to about 0.25%, about
0.001% to about 0.1%, about 0.001% to about 0.005%, about 0.005% to
about 0.075%, about 0.075% to about 0.01%, about 0.01% to about
0.1%, about 0.1% to about 0.5%, about 0.5% to about 0.75%, about
0.75% to about 1%, about 1% to about 2%, about 2% to about 3%, or
about 3% to about 5%, including for example about 0.001%, 0.005%,
0.075%, 0.01%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, or 5%.
[0106] In some embodiments, the reduction factors (RF) is used to
determine the clearance rate of the prion removal process. The RF
can be calculated using the formula:
RF=(V.sub.1.times.T.sub.1)/(V.sub.2.times.T.sub.2)
or
Log.sub.10[RF]=[ Log.sub.10(V.sub.1)+Log.sub.10(T.sub.1)]-[
Log.sub.10(V.sub.2)+Log.sub.10((T.sub.2)].
Wherein V.sub.1 and T.sub.1 are the volume and titre of the initial
albumin composition, respectively, and V.sub.2 and T.sub.2 are the
volume and titre of the post-removal albumin composition. Reduction
factors can be rounded to 1 decimal place after the final
calculation. In some embodiments, a reduction factor of at least
about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,
7.0, 7.5, or 8.0 log.sub.10) infectivity of the prion proteins is
removed from the initial albumin composition. For example, the
prion protein can be removed by a reduction factor of greater than
or equal to 2.5 log.sub.10 in a 0.5% sarkosyl solubilized fraction
spiked into a 20% albumin composition. As another example, the
prion protein can also be removed by a reduction factor of greater
than or equal to 2.0 log.sub.10 in a 0.5% sarkosyl solubilized
fraction spiked into a 25% albumin composition.
[0107] The removal of prions can be evaluated by standard Western
blot analysis. For example, the post-binding ligands can first be
treated with Proteinase K, which digests all PrPc but not PrPsc.
The digest is then run on SDS gel and transblotted to a sheet of
nitrocellulose or PVDF membrane. The separated PrPsc bands are then
visualized using 3F4 or 6H4. 3F4 reacts with amino acid residues
109-112 PrP from humans, hamsters, and felines. In one exemplary
embodiment, incubation was carried out at a concentration of 0.6
ug/ml for a minimum of one hour, after which excess antibody was
washed away and the membranes incubated with a rabbit anti-mouse
horse-radish peroxidase conjugate (1:1000 dilution) for a minimum
of one hour. After extensive washing with TTBS, the membranes were
developed using enhanced chemiluminescence. In some embodiments,
the removal of prion proteins is evaluated according to the
Guideline for the Investigation of Manufacturing Processes for
Plasma-Derived Medicinal Products with Regard to vCJD Risk
(CPMP5136/03).
Ligands Capable of Binding to a Prion Protein and Supporting
Material
[0108] "Ligand" used herein refers to a molecule to which a prion
protein or peptide binds. A "ligand capable of binding to a prion
protein" refers to a ligand that specifically binds to a prion
protein under suitable conditions. In some embodiments, the ligand
specifically binds to a human prion protein. In some embodiments,
the ligand specifically binds to a hamster prion protein. In some
embodiments, the ligand specifically binds to a mouse protein. In
some embodiments, the ligand has binds to prion proteins from
multiple species. For example, in some embodiments, the ligand
binds to human prion protein, hamster prion protein, and mouse
prion protein.
[0109] In some embodiments, the ligand binds to the prion protein
(such as a human prion protein, hamster prion protein, and/or mouse
prion protein) with a high binding affinity. For example, in some
embodiments, the ligand has a binding Kassociation of more than
about any of 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, or
10.sup.11.
[0110] A number of ligands have been identified that bind to prion
protein and thus can be used in methods of the present invention.
See, e.g., WO04/090102, WO04/050851, WO06/010915, and WO06/044459.
These include, for example, peptides, chemical compounds, and
antibodies that specifically recognize a prion protein. The ligand
can be used to remove all forms of prion proteins from a
composition or can be selectively chosen to detect or remove a
single form of prion protein.
[0111] In some embodiments, the ligand for removing prion is a
peptide, such as peptides described in PCT published application
No. WO04/05051. For example, in some embodiments, the ligand is a
peptide having an amino acid of any of SEQ ID NOs: 1-232 as shown
in Table 1. In some embodiments, the ligand is a tripeptide, such
as peptides having an amino acid sequence of any one of SEQ ID
NO:48, 102, 105, 108, 109, 110, 111, 143, 148, 193, 194, 195, 202,
203, 204, or 210. In some embodiments, the ligand is a peptide with
six amino acids, such as 6-mers having an amino acid sequence of
any one of SEQ ID NO: 152, 153, 180, 181, 182, 183, 185, 186, 187,
188, 189, or 190. In some embodiments, the ligand has an amino acid
sequence of SEQ ID NO:150 or 151.
TABLE-US-00001 TABLE 1 Amino acid sequences binding to prion
sequences KIHKFLA (SEQ ID NO: 1) GTHDFQA (SEQ ID NO: 2) KFGSTHA
(SEQ ID NO: 3) FVNEIEA (SEQ ID NO: 4) GLHFKSA (SEQ ID NO: 5)
GRVLHHA (SEQ ID NO: 6) QKNSEWA (SEQ ID NO: 7) HAYFTHA (SEQ ID NO:
8) WPKGAVA (SEQ ID NO: 9) RPWKKAA (SEQ ID NO: 10) PKHIWPA (SEQ ID
NO: 11) HKLWGVA (SEQ ID NO: 12) GGYKPYA (SEQ ID NO: 13) ENVSQNA
(SEQ ID NO: 14) HTYYNGA (SEQ ID NO: 15) KKKSDHA (SEQ ID NO: 16)
HHLKGTA (SEQ ID NO: 17) KKHGVWA (SEQ ID NO: 18) DGTQAHA (SEQ ID NO:
19) APHRNNA (SEQ ID NO: 20) HHGHNIA (SEQ ID NO: 21) HTWHGQA (SEQ ID
NO: 22) HVFVTWA (SEQ ID NO: 23) THHFYIA (SEQ ID NO: 24) KLGWG(A/G)A
(SEQ ID NO: 25) GSKKKEA (SEQ ID NO: 26) PLLVVWA (SEQ ID NO: 27)
WLLVGGA (SEQ ID NO: 28) (W/G)QVLVYA (SEQ ID NO: 29) RRHQRQA (SEQ ID
NO: 30) LPWTFGA (SEQ ID NO: 31) IFIIITA (SEQ ID NO: 32) P(X)IEPHA
(SEQ ID NO: 33) EWGIIWA (SEQ ID NO: 34) GWYIYFA (SEQ ID NO: 35)
TLILFHA (SEQ ID NO: 36) FLLSNHA (SEQ ID NO: 37) WQIRFFA (SEQ ID NO:
38) VLLVFEA (SEQ ID NO: 39) GWVLEIA (SEQ ID NO: 40) FLLIDTA (SEQ ID
NO: 41) GFLFKFA (SEQ ID NO: 42) PWTIYIA (SEQ ID NO: 43) WH (SEQ ID
NO: 44) WW (SEQ ID NO: 45) LW (SEQ ID NO: 46) WNA (SEQ ID NO: 47)
EFW (SEQ ID NO: 48) LPW (SEQ ID NO: 49) YEY (SEQ ID NO: 50) WPA
(SEQ ID NO: 51) FNQ (SEQ ID NO: 52) YHE (SEQ ID NO: 53) LFA (SEQ ID
NO: 54) NHY (SEQ ID NO: 55) TLG (SEQ ID NO: 56) WVD (SEQ ID NO: 57)
YWDQA (SEQ ID NO: 58) YVHEA (SEQ ID NO: 59) WFDEA (SEQ ID NO: 60)
LQWYDA (SEQ ID NO: 61) YTHSEA (SEQ ID NO: 62) WIDYEA (SEQ ID NO:
63) VWIDAA (SEQ ID NO: 64) WDEAEEA (SEQ ID NO: 65) YDSYDDA (SEQ ID
NO: 66) NDFIDFA (SEQ ID NO: 67) YEPWGSA (SEQ ID NO: 68) EYGDWWA
(SEQ ID NO: 69) WDYDQEA (SEQ ID NO: 70) DWGDPFA (SEQ ID NO: 71)
DWPEVWA (SEQ ID NO: 72) FHDFSEA (SEQ ID NO: 73) DTFWDYA (SEQ ID NO:
74) WNDLDNA (SEQ ID NO: 75) ASALVYA (SEQ ID NO: 76) LINAGGA (SEQ ID
NO: 77) WESYVTA (SEQ ID NO: 78) WSDEGYA (SEQ ID NO: 79) YRWTGPA
(SEQ ID NO: 80) YEDQWQA (SEQ ID NO: 81) EWADDNA (SEQ ID NO: 82)
YEIDYGA (SEQ ID NO: 83) EFGYFDA (SEQ ID NO: 84) WGDEQDA (SEQ ID NO:
85) HEEDWAA (SEQ ID NO: 86) FEDFELA (SEQ ID NO: 87) TWGIDEA (SEQ ID
NO: 88) WDPTDYA (SEQ ID NO: 89) NDKIHTA (SEQ ID NO: 90) FEDFFSA
(SEQ ID NO: 91) YEWAEQA (SEQ ID NO: 92) THVYFLA (SEQ ID NO: 93)
(S/T/W)XDFSDA (SEQ ID NO: 94) YRTPNEA (SEQ ID NO: 95) (GIL)RSETA
(SEQ ID NO: 96) IHN (SEQ ID NO: 97) WEY (SEQ ID NO: 98) DYW (SEQ ID
NO: 99) WDW (SEQ ID NO: 100) WQD (SEQ ID NO: 101) YFE (SEQ ID NO:
102) NYE (SEQ ID NO: 103) SYA (SEQ ID NO: 104) WDL (SEQ ID NO: 105)
WLE (SEQ ID NO: 106) VQR (SEQ ID NO: 107) YID (SEQ ID NO: 108) RWD
(SEQ ID NO: 109) DVR (SEQ ID NO: 110) WSD (SEQ ID NO: 111) HWD (SEQ
ID NO: 112) WQD (SEQ ID NO: 113) WDD (SEQ ID NO: 114) WED (SEQ ID
NO: 115) ITN (SEQ ID NO: 116) YED (SEQ ID NO: 117) VADEEA (SEQ ID
NO: 118) YYVDAA (SEQ ID NO: 119) QDFNLA (SEQ ID NO: 120) DNPIDA
(SEQ ID NO: 121) FNEHEA (SEQ ID NO: 122) WGADGA (SEQ ID NO:
123)
VIYSHA (SEQ ID NO: 124) HILEEA (SEQ ID NO: 125) PHENFA (SEQ ID NO:
126) EDNGGA (SEQ ID NO: 127) DSEGPA (SEQ ID NO: 128) FQEFTA (SEQ ID
NO: 129) EGDEIA (SEQ ID NO: 130) IYAETA (SEQ ID NO: 131) RVRETA
(SEQ ID NO: 132) EEPQWA (SEQ ID NO: 133) EGEEFA (SEQ ID NO: 134)
(T/L)FNIHA (SEQ ID NO: 135) YDW (SEQ ID NO: 136) NYT (SEQ ID NO:
137) SYT (SEQ ID NO: 138) WAD (SEQ ID NO: 139) QWG (SEQ ID NO: 140)
WGD (SEQ ID NO: 141) EYF (SEQ ID NO: 142) WEH (SEQ ID NO: 143) LYD
(SEQ ID NO: 144) DYY (SEQ ID NO: 145) FYE (SEQ ID NO: 146) EYY (SEQ
ID NO: 147) YDY (SEQ ID NO: 148) WDH (SEQ ID NO: 149) RES(na)NVA
(SEQ ID NO: 150) ES(na)PRQA (SEQ ID NO: 151) VARENIA (SEQ ID NO:
152) RWEREDA (SEQ ID NO: 153) EWWETV (SEQ ID NO: 154) SVYQLDA (SEQ
ID NO: 155) (na)HEFYGA (SEQ ID NO: 156) HE(na)(na)LVA (SEQ ID NO:
157) A(na)VPV(na)A (SEQ ID NO: 158) YFDYWLA (SEQ ID NO: 159)
FE(na)HRQA (SEQ ID NO: 160) WRHEPAA (SEQ ID NO: 161) SS(na)KKDA
(SEQ ID NO: 162) R(na)DKEAA (SEQ ID NO: 163) (na)HEIFPA (SEQ ID NO:
164) aKWYHHRA (SEQ ID NO: 165) HWWPHNA (SEQ ID NO: 166) HWQVFYA
(SEQ ID NO: 167) FHE(na)EIA (SEQ ID NO: 168) HADF(na)QA (SEQ ID NO:
169) ALHFETA (SEQ ID NO: 170) DDPTGFA (SEQ ID NO: 171) VAPGLGA (SEQ
ID NO: 172) IFRLIEA (SEQ ID NO: 173) GLERPEA (SEQ ID NO: 174)
IVVRLWA (SEQ ID NO: 175) WHNPHYA (SEQ ID NO: 176) LIYKSDA (SEQ ID
NO: 177) EKPIFNA (SEQ ID NO: 178) HWSEPAA (SEQ ID NO: 179) GHNWKEA
(SEQ ID NO: 180) YWHHDDA (SEQ ID NO: 181) GYPKENA (SEQ ID NO: 182)
PVYWLYA (SEQ ID NO: 183) FGEHTPA (SEQ ID NO: 184) FQGTREA (SEQ ID
NO: 185) TGTNRYA (SEQ ID NO: 186) KWATRYA (SEQ ID NO: 187) NSTKFDA
(SEQ ID NO: 188) LIYKEEA (SEQ ID NO: 189) EHATYRA (SEQ ID NO: 190)
HND (SEQ ID NO: 191) HER (SEQ ID NO: 192) HGD (SEQ ID NO: 193) HSD
(SEQ ID NO: 194) HFD (SEQ ID NO: 195) WND (SEQ ID NO: 196) YEH (SEQ
ID NO: 197) HWD (SEQ ID NO: 198) YHD (SEQ ID NO: 199) YDW (SEQ ID
NO: 200) WDY (SEQ ID NO: 201) HYD (SEQ ID NO: 202) HWD (SEQ ID NO:
203) WTD (SEQ ID NO: 204) FPK (SEQ ID NO: 205) HWK (SEQ ID NO: 206)
WEE (SEQ ID NO: 207) LLR (SEQ ID NO: 208) SYF (SEQ ID NO: 209) EYY
(SEQ ID NO: 210) DRDLTFA (SEQ ID NO: 211) HNWWIIA (SEQ ID NO: 212)
EVKIGNA (SEQ ID NO: 213) SIV (SEQ ID NO: 214) AYP (SEQ ID NO: 215)
EVADEEA (SEQ ID NO: 216) EYYVDAA (SEQ ID NO: 217) YDNPIDA (SEQ ID
NO: 218) YFNEHEA (SEQ ID NO: 219) EWGADGA (SEQ ID NO: 220) DVIYSHA
(SEQ ID NO: 221) WHILEEA (SEQ ID NO: 222) NPHENFA (SEQ ID NO: 223)
HEDNGGA (SEQ ID NO: 224) SDSEGPA (SEQ ID NO: 225) EFQEFTA (SEQ ID
NO: 226) QEGDEIA (SEQ ID NO: 227) DIYAETA (SEQ ID NO: 228) DRVRETA
(SEQ ID NO: 229) FEEPQWA (SEQ ID NO: 230) FEGEEFA (SEQ ID NO: 231)
(T/L)FNIHA (SEQ ID NO: 232)
[0112] In some embodiments, the ligand is a peptide of the amino
acid sequence of DVR, SYA, AMN31, D4, or YVHEA. In some
embodiments, the ligand is a peptide that binds to a prion protein
at an affinity that is similar to or higher than that of DVR, SYA,
AMN31, D4, or YVHEA. In some embodiments, the ligand is DVR. In
other embodiments, the ligand is AMN31. In some embodiments, the
peptide ligands are provided in the form of resins (such as bound
to resins).
[0113] In some embodiments, the ligand is an antibody recognizing a
prion protein. Antibodies recognizing prion proteins are known in
the art. These include, but are not limited to, monoclonal
antibodies 3F4, 6H4, or 16A18. In some embodiments, the antibody is
a glycoform specific antibody, such as ICSM-4 and ICSM-10. Suitable
antibodies useful for the methods described herein include
polyclonal and monoclonal antibodies, single chain antibodies, Fab
fragments, and Fv fragments.
[0114] In some embodiments, the ligand binds to a specific sequence
of a prion protein. For example, in some embodiments, the ligand
(such as a peptide ligand or an antibody ligand) binds to any one
of the prion protein sequences SEQ ID NOs: 133-146 listed on Table
2.
TABLE-US-00002 TABLE 2 Prion amino acid sequences RYPxQ, x is G, P,
or N (SEQ ID NO: 233) xxYYux, x is G, P, or N, u is R (SEQ ID NO:
234) or Q RYPGQ (SEQ ID NO: 235) DRYYRD (SEQ ID NO: 236) QAYYQR
(SEQ ID NO: 237) QVYYRP (SEQ ID NO: 238) PHGGGWGQ (SEQ ID NO: 239)
PHGGSWGQ (SEQ ID NO: 240) PHGGGWSQ (SEQ ID NO: 241) PHGGGGWSQ (SEQ
ID NO: 242) PHGGGSNWGQ (SEQ ID NO: 243) PHNPGY (SEQ ID NO: 244)
PHNPSY (SEQ ID NO: 245) PHNPGY (SEQ ID NO: 246)
[0115] In some embodiments, the ligand is a chemical compound.
Compounds capable of binding to a prion protein can be found, for
example, in PCT Application publication No. WO06/010915, which is
incorporated herein in its entirety. In some embodiments, the
ligand is a substituted triazine. In some embodiments, the ligand
is a compound having formula (I):
##STR00001##
[0116] wherein R.sup.1 and R.sup.2 are the same or different and
are each optionally substituted alkyl, optionally substituted
cycloalkyl, optionally substituted aryl or optionally substituted
heteroaryl groups; R.sup.3 is hydrogen or an aryl group substituent
or R.sup.3 is a solid support optionally attached via a spacer; Z
represents an oxygen atom, a sulphur atom or NR.sup.4; Y represents
an oxygen atom, a sulphur atom or NR.sup.5; in which R.sup.4 and
R.sup.5, which may be the same or different, represent hydrogen,
optionally substituted alkyl containing 1 to 6 carbon atoms,
optionally substituted phenyl, optionally substituted benzyl or
optionally substituted .beta.-phenylethyl; and one of X.sup.1 and
X.sup.2 represents a nitrogen atom and the other of X.sup.1 and
X.sup.2 represents a nitrogen atom or CR.sup.6, in which R.sup.6
represents hydrogen or an aryl group substituent; for the affinity
binding of a prion protein.
[0117] In some embodiments, the ligand is a compound of formula
(II),
##STR00002##
wherein R1 represents a group --(CH.sub.2).sub.m-Q.sup.1, wherein m
is from 0 to 7, and Q.sup.1 represents --CR.sup.11R.sup.12R.sup.13
or --NR.sup.11R.sup.12, in which R.sup.11, R.sup.12 and R.sup.13
independently represent hydrogen, alkyl, cycloalkyl or
heterocycloalkyl, or two of R.sup.11, R.sup.12 and R.sup.13,
together with the carbon or nitrogen atom to which they are
attached, form an optionally substituted cycloalkyl or optionally
substituted heterocycloalkyl group; R.sup.2 represents a group
--(CH.sub.2).sub.n-Q.sup.2, wherein n is from 0 to 7, and Q.sup.2
represents --CR.sup.21R.sup.22R.sup.23 or --NR.sup.21R.sup.22, in
which R.sup.21, R.sup.22 and R.sup.23 independently represent
hydrogen, alkyl, cycloalkyl or heterocycloalkyl, or two of
R.sup.11, R.sup.12 and R.sup.13, together with the carbon or
nitrogen atom to which they are attached, form an optionally
substituted cycloalkyl or optionally substituted heterocycloalkyl
group, and R.sup.3 is hydrogen or an aryl group substituent or
R.sup.3 is a solid support optionally attached via a spacer; Z
represents an oxygen atom, a sulphur atom or NR.sup.4; Y represents
an oxygen atom, a sulphur atom or NR.sup.5; in which R.sup.4 and
R.sup.5, which may be the same or different, represent hydrogen,
optionally substituted alkyl containing 1 to 6 carbon atoms,
optionally substituted phenyl, optionally substituted benzyl or
optionally substituted .beta.-phenylethyl; and one of X.sup.1 and
X.sup.2 represents a nitrogen atom and the other of X.sup.1 and
X.sup.2 represents a nitrogen atom or CR.sup.6, in which R.sup.6
represents hydrogen or an aryl group substituent; for the affinity
binding of a prion protein.
[0118] In some embodiments, a) X.sup.1 and X.sup.2 are both
nitrogen; b) both Z and Y represent NR.sup.4, in particular where
R.sup.4 is hydrogen; c) m is from 0 to 4, more preferably from 0 to
3, and most preferably from 1 to 3; d) Q.sup.1 is
--NR.sup.11R.sup.12; and R.sup.H and R.sup.12 preferably form,
together with the nitrogen atom to which they are attached, a
heterocycloalkyl group; e) n is from 0 to 4, more preferably from 0
to 2, and most preferably is 0; and f) R.sup.21 and R.sup.22 form,
together with the carbon atom or nitrogen atom to which they are
attached, a cycloalkyl or heterocycloalkyl group.
[0119] In some embodiments, Q.sup.2 represents a hydrophobic
cycloalkyl or heterocycloalkyl group, especially with ring systems
that comprise at least six atoms.
[0120] In some embodiments, Q.sup.1 represents a heterocycloalkyl
group, especially piperidyl, particularly 1-piperidyl, or
piperazinyl, particularly 1-piperazinyl.
[0121] In some embodiments, the ligand is a compound of formula
(III),
##STR00003##
wherein R1 represents an alkylene chain
--(CH.sub.2).sub.p--CH.sub.3, wherein p is from 0 to 6, substituted
by one or more carboxyl groups and optionally substituted by one or
more further alkyl group substituents; R.sup.2 represents a group
--(CH.sub.2).sub.q--Ar, wherein q is from 0 to 7, and Ar represents
an optionally substituted aryl group; and R.sup.3 is hydrogen or an
aryl group substituent or R.sup.3 is a solid support optionally
attached via a spacer; Z represents an oxygen atom, a sulphur atom
or NR.sup.4; Y represents an oxygen atom, a sulphur atom or
NR.sup.5; in which R.sup.4 and R.sup.5, which may be the same or
different, represent hydrogen, optionally substituted alkyl
containing 1 to 6 carbon atoms, optionally substituted phenyl,
optionally substituted benzyl or optionally substituted
.beta.-phenylethyl; and one of X.sup.1 and X.sup.2 represents a
nitrogen atom and the other of X.sup.1 and X.sup.2 represents a
nitrogen atom or CR.sup.6, in which R.sup.6 represents hydrogen or
an aryl group substituent; for the affinity binding of a prion
protein.
[0122] In some embodiments, a) X.sup.1 and X.sup.2 are both
nitrogen; b) both Z and Y represent NR.sup.4, in particular where
R.sup.4 is hydrogen; c) p is from 0 to 4, more preferably from 0 to
3; d) R.sup.1 is substituted with one or two carboxyl groups, at
least one of those carboxyl groups being carried by the terminal
carbon atom of the alkylene chain --(CH.sub.2).sub.p--CH.sub.3; e)
q is from 0 to 4, more preferably from 0 to 3, and most preferably
is 1 or 2; and f) Ar is a monocyclic carbocyclic or heterocyclic
aromatic group, optionally substituted by one or more substituents,
selected from the group consisting of phenyl, phenoxy, tolyl,
chlorobenzyl, methoxybenzyl, fluorobenzyl, pyridyl and indoyl.
[0123] In some embodiments, R.sup.1 is carboxymethyl,
4-carboxybutyl or 1-(1,3-dicarboxy)propyl.
[0124] In some embodiments, Ar is phenyl, 4-hydroxyphenyl or
pyridyl, particularly 2-pyridyl.
[0125] In some embodiments, R.sup.3 is hydrogen or an aryl group
substituent or R.sup.3 is a solid support optionally attached via a
spacer; X.sup.1 and X.sup.2 are both N; Y and Z both represent NH;
m represents 2; Q.sup.1 represents piperidyl or piperazinyl; n
represents 0 or 2; and Q.sup.2 represents 1-piperidyl or
adamantyl.
[0126] In some embodiments, R.sup.3 is hydrogen or an aryl group
substituent or R.sup.3 is a solid support optionally attached via a
spacer; X.sup.1 and X.sup.2 are both N; Y and Z both represent NH;
R.sup.1 represents carboxymethyl, 4-carboxybutyl and
1-(1,3-dicarboxy)propyl; q represents 2; and Ar represents phenyl,
2-pyridyl or 4-hydroxyphenyl.
[0127] In some embodiments, the ligand is an inorganic compound or
component, such as, but not limited to, aluminum (such as aluminum
oxide) or silica (such as fused silica). In some embodiments, the
inorganic compound is Al203 or SiO2.
[0128] In some embodiments, the ligand comprises one or more
functional groups. The term "functional group" is used herein to
denote chemical groups, subgroups, or substructures that impart
characteristic chemical, physical, or physicochemical behaviors to
a molecule or a material. Functional groups described herein
include, but are not limited to, hydrophilic, such as positively,
negatively or uncharged or neutral, or hydrophobic. Amphiphilic or
multifunctional functional groups are also envisioned and fall
within the scope of the present invention. Functional groups
include organic and inorganic functional groups. Preferred
functional groups contain amine, phenyl or sulfite groups. A
preferred amine group is a primary, secondary, tertiary, or
quaternary ammonium ion such as dimethylaminoethyl (DMAE) or
trimethylaminoethyl (TMAE).
[0129] Other exemplary functional groups include, but are not
limited to: --CH2--CHOH--CH2NH2; --C6H5; --(CH2)3-CH3;
--CH2-CH2-NH(C2H5)2; --SO2-CH2-CH3'-CH2-CH2-H(CH3)2;
--CH2-CH2-(CH3)3; --SO32-. Additionally useful functional groups
include sulfonyl groups and tresyl groups. It is to be understood
that functional groups can be inherently present in a ligand, or
can be added to the ligand by chemical modification.
[0130] In some embodiments, the ligand contains an amino group.
These include, for example, amino resin, such as Toyopearl.TM.
Amino-650M, Toyopearl.TM. Amino-AMN31, TSK-GEL.TM.-Amino 750C, or
functional equivalents thereof. In some embodiments, the ligand
comprises a phenyl group. These include, for example, TSK-GEL.TM.
Phenyl-5PW or functional equivalent thereof.
[0131] In some embodiments, the ligand is a polymeric material,
such as chromatographic resins (for example amino resins), that
bind with selectivity and specificity to prion proteins. An example
of chromatographic resin is PRDT Prion Reduction Resins (ProMetic
Biosciences, Ltd, 211 Cambridge Science Park, Milton Road,
Cambridge, CB4 0WA, UK). In some embodiments, the polymeric
material contains one or more functional groups, such as functional
groups described above. In some embodiments, the ligand is a
polymeric material having a methacrylate backbone, such as, but not
limited to, a commercially available TSK, TOYOPEARL, or FACTOGEL
resin (Tosoh Bioscience, Montgomeryville, Pa.).
[0132] Other ligands that can be used in methods of the present
invention include, for example, ligands that interact with amyloid
plaque e.g., Congo:E;Led (Ingrosso, L., et al., Congo Red: Prolongs
the Incubation Period in Scrapie-infected Hamsters. Virology
69:506-508 (1995)); 1,4-iodo, 4-deoxy doxorubicin (Tagliavini, F.,
et al., Effectiveness of Anthracycline Against Experimental Prion
Diseases in Syrian Hamsters. Science 276:1119-1122 (1997));
amphotericin 13, porphyrins and phthalocyanines (Priola, S. A., et
al., Porphyrin and Phthalocyanine Antiscrapie Compounds, Science
287:1503-1506 (2000)); metals (Stocker et al., Biochemistry, 37,
7185-7193 (1998)); peptides that interact with PrP to form
complexes (see U.S. Pat. No. 5,750,361 to Prusiner et al. and Solo,
C. et al., Reversion of Prion Protein Conformational Changes in
Synthetic p-sheet Breaker Peptides, Lances, 355:192-197 (2000));
heparin and other polysulphated polyanions (Caughey, B., et al.,
Binding of the Protease-sensitive Form of Prion Protein PrP to
Sulphated Glycosaminoglycan and Congo Red, J. Virology
68:2135-2141(1994)); antibodies (Kascsak, R. J., et al.,
Immunodiagnosis of Prion Disease, Immunological Invest. 26:259-268
(1997)); and other proteins, e.g. plasminogen (Fischer, M. B. et
al., Binding of Disease-associated Prion Protein to: Plasminogen.,
Nature 408:479-483 (2000)).
[0133] The ligands may be attached to any supporting materials.
"Support material" used herein refer to any compound or material
which may provide a physical or chemical means of separating the
ligand and proteins bound thereto from the rest of the composition.
The supporting material may be particulate or non-particulate,
soluble or insoluble, porous or non-porous.
[0134] Examples of support materials include, but not limited to,
naturally occurring polymers, e.g., a polysaccharide such as
agarose, alginate, carrageenan, chitin, cellulose, dextran or
starch; synthetic polymers such as polyacrylamide, polystyrene,
polyacrolein, polyvinyl alcohol, polymethylacrylate,
perfluorocarbon; inorganic compounds such as silica, glass,
kieselquhr, alumina, iron oxide or other metal oxides, or
copolymers consisting of any combination of two or more naturally
occurring polymers, synthetic polymers or inorganic compounds. Also
contemplated are soluble support materials comprising polymers such
as dextran, polyethylene glycol, polyvinyl alcohol or hydrolysed
starch which provide affinity-ligand matrix conjugates for use in
liquid partitioning
[0135] In some embodiments, the supporting material is a solid
support such as a column, a bead, a membrane, a cartridge, a
filter, a dipstick, a microtiter plate, a test tube, solid powder,
a cast or extrusion molded module, a mesh, a magnetic particle
composite, or any other solid materials. The solid materials may be
coated with a substance such as polyethylene, polypropylene,
poly(4-methylbutene), polystyrene, polyacrylate, polyethylene
terephthalate, rayon, nylon, poly(vinyl butyrate), polyvinylidene
difluoride (PVDF), silicones, polyformaldehyde, cellulose,
cellulose acetate, nitrocellulose, and the like. Alternatively,
substances that form gels, such as proteins (e.g. gelatins),
lipopolysaccharides, silicates, agarose and polyacrylamides are
used. Polymers such as dextrans, polyalkylene glycols or
surfactants, such as phospholipids, long chain (12-24 carbon atoms)
alkyl ammonium salts and the like can also be used. The ligands are
attached to or dispersed throughout the support materials.
[0136] In some embodiments, the supporting material is activated
agarose, silica, cellulose, glass, methacrylate,
hydroxyethylmethacrylate, polyacrylamide, styrenedivinylbenzene,
Hyper D or perfluorocarbons. In some embodiments, the supporting
material is a methacrylate material, of the type sold under the
trade name Toyopearl (available from Tosoh Bioscience LLC, 156
Keystone Drive, Montgomeryville, Pa. I 18936, USA). WO 97/10887
describes methods of attaching affinity ligands to support
matrices, e.g. the use of activating methods, and methods of
attaching the affinity ligand to a matrix via a spacer, e.g. by
condensation reactions, to form affinity ligand-matrix
conjugates.
[0137] In some embodiments, the ligands and/or supporting materials
are reusable. In some embodiments, the ligands and/or supporting
materials for single use only.
[0138] The prion binding capacity of a ligand can be evaluated by
determining the infectious titre in a ligand. For example, a
dilution series of a reference stock is prepared and tested in the
standard Western blot assay. The titres observed from the reference
stock are compared with the corresponding titres observed in a
bioassay. The Western blot titres can then be converted into
infectious titres using the formula:
Titre.sub.[Bioassay]=Titre.sub.[WesternBlot]+(intercept of a linear
regression analysis)/(slope of a linear regression analysis). Once
the infectious titre per ml is calculated, the total prion protein
bound to the column can be determined. The capacity of the prion
binding of a ligand is determined using the amount of prion protein
observed directly bound to the ligand. In some embodiments, the
prion binding capacity of a ligand is at least about 2.0, 2.5, 3.0,
3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5,
7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,
8.9, 9.0, 9.5, or 10.0 log.sub.10 ID.sub.50 per ml of ligand. In
some embodiments, the prion binding capacity of a ligand is at
least about 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6,
10.sup.7, or 10.sup.8 ID.sub.50 per ml of ligand.
Method of Making Nanoparticles
[0139] Methods of making compositions containing albumins and
substantially water insoluble pharmacologically active agents are
known in the art. For example, nanoparticles containing
substantially water insoluble pharmacologically active agents and
albumins can be prepared under conditions of high shear forces
(e.g., sonication, high pressure homogenization, or the like).
These methods are disclosed in, for example, U.S. Pat. Nos.
5,916,596; 6,506,405; 6,749,868, and 6,537,579 and also in U.S.
Pat. Pub. Nos. 2005/0004002 and 2007/0082838, and PCT Publication
W099/00113, which are each hereby incorporated by reference in
their entireties.
[0140] In one exemplary embodiment, the substantially water
insoluble pharmacologically active agent (e.g., paclitaxel) is
dissolved in an organic solvent. Suitable organic solvents include,
for example, ketones, esters, ethers, chlorinated solvents, and
other solvents known in the art. For example, the organic solvent
can be methylene chloride. In some embodiments, the organic solvent
can be a mixture of a water immiscible solvent (such as chloroform)
and a water miscible solvent (such as a water miscible alcohol
solvent, such as chloroform/methanol, chloroform/ethanol,
chloroform/propanol, or chloroform/t-butanol (for example with a
ratio (v/v) of about any of 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2,
1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1 or with a ratio
(v/v) of about any of 3:7, 5:7, 4:6, 5:5, 6:5, 8:5, 9:5, 9.5:5,
5:3, 7:3, 6:4, or 9.5:0.5). The solution is added to an albumin
(e.g., human serum albumin) The mixture is subjected to high
pressure homogenization (e.g., using standard homogenization
devices). The emulsion may be cycled through the high pressure
homogenizer for between about 2 to about 100 cycles, such as about
5 to about 50 cycles or about 8 to about 20 cycles (e.g., about any
of 8, 10, 12, 14, 16, 18 or 20 cycles). The organic solvent can
then be removed by evaporation utilizing suitable equipment known
for this purpose, including, but not limited to, rotary
evaporators, thin file evaporators, falling film evaporators, wiped
film evaporators, spray driers, and the like. The solvent may be
removed, for example, at reduced pressure (such as at about any of
5 mm Hg, 10 mm Hg, 15 mm Hg, 20 mm Hg, 25 mm Hg, 30 mm Hg, 40 mm
Hg, 50 mm Hg, 100 mm Hg, 200 mm Hg, or 300 mm Hg). The amount of
time used to remove the solvent under reduced pressure may be
adjusted based on the volume of the formulation. For example, for a
formulation produced on a 300 mL scale, the solvent can be removed
at about 1 to about 300 mm Hg (e.g., about any of 5-100 mm Hg,
10-50 mm Hg, 20-40 mm Hg, or 25 mm Hg) for about 1 to about 120
minutes, including about 5 to about 60 minutes (e.g., about any of
7, 8, 9, 10, 11, 12, 13, 14, 15 16, 18, 20, 25, or 30 minutes).
[0141] If desired, albumin solution (such as prion-removed albumin
solution) may be added to the dispersion to adjust the albumin to
drug (e.g., paclitaxel) ratio or to adjust the concentration of the
taxane (e.g., paclitaxel) in the dispersion. For example, albumin
solution (e.g., 25% w/v) can be added to adjust the albumin to
substantially water insoluble pharmacologically active agent (e.g.,
paclitaxel) ratio to about any of 18:1, 15,:1 14:1, 13:1, 12:1,
11:1, 10:1, 9:1, 8:1, 7.5:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1,
1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13,
1:14, 1:15, 1:16, 1:17, or 1:18. For example, albumin solution
(e.g., 25% w/v) or another solution is added to adjust the
concentration of the substantially water insoluble
pharmacologically active agent (e.g., paclitaxel) in the dispersion
to about any of 0.5 mg/ml, 1.3 mg/ml, 1.5 mg/ml, 2 mg/ml, 3 mg/ml,
4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 15
mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, or 50 mg/ml. The
dispersion may be individually or serially filtered through one or
more filters, such as 1.2 .mu.m, 0.8 .mu.m, 0.45 .mu.m, and 0.22
.mu.m filters; combinations of two or more thereof, or the
combination with any other filters known in the art.
[0142] If desired, a second therapy (e.g., one or more compounds
useful for treating cancer), an antimicrobial agent (such as
citrate or edetate), sugar (such as sucrose), and/or stabilizing
agent can also be included in the composition. This additional
agent can either be admixed with the substantially water insoluble
pharmacologically active agent (e.g., paclitaxel) and/or the
albumin during preparation of the composition, or added after the
nanoparticle composition is prepared. In some embodiments, the
agent is admixed with the nanoparticle composition prior to
lyophilization. In some embodiments, the agent is added to the
lyophilized composition. In some embodiments when the addition of
the agent changes the pH of the composition, the pH in the
composition are generally (but not necessarily) adjusted to a
desired pH. Exemplary pH values of the compositions include, for
example, in the range of about 5 to about 8.5. In some embodiments,
the pH of the composition is adjusted to no less than about 6,
including for example no less than any of about 6.5, 7, or 8 (e.g.,
about 8).
[0143] As discussed below in more detail, the prion-removal process
can be carried out concurrently with the manufacturing process. For
example, the prion-removal process can be carried out after the
mixture of albumin and substantially water insoluble
pharmacologically active agent is formed and prior to subjecting
the mixture to the high shear condition. In some embodiments, the
prion-removal process can be carried out after the mixture has been
subjected to the high shear condition and prior to the removal of
the organic solvent. In some embodiments, the prion-removal process
is carried out after the removal of the organic solvent. In some
embodiments, additional albumin is added to the post-evaporation
suspension prior to the prion-removal process. In some embodiments,
the prion-removal process is carried out under a sterile condition.
In some embodiments, the prion-removal process is carried out by
using a cartridge which simultaneously sterile filters the
composition.
Method of Removing Prions from Nanoparticle Compositions or
Intermediate Compositions
[0144] The present invention in one aspect provides methods
comprising removing prion proteins from a nanoparticle composition,
such as nanoparticle compositions formed by methods described
above. In another aspect there are provided methods of removing
prion proteins from intermediate compositions generated during the
nanoparticle manufacture process (hereinafter referred to as "the
intermediate composition").
[0145] Generally, the methods comprise contacting a composition
comprising albumin and a substantially water insoluble
pharmacologically active agent with a ligand capable of binding to
a prion protein, and removing the ligand and protein bound thereto
from the nanoparticle composition. This process can be repeated one
or more times, with the same or a different ligand. Two or more
ligands can also be used simultaneously during the prion removal
process.
[0146] In some embodiments, the nanoparticle composition or the
intermediate composition has a prion infectivity of about 100
IU-ic/ml, 90IU-ic/ml, 50IU-ic/ml, or 10IU-ic/ml.
[0147] During the prion-removal process, the ligand is brought into
contact with the nanoparticle composition or intermediate
composition and allowed to bind to prion proteins in the
nanoparticle composition or the intermediate composition.
Conditions suitable for the binding can be determined and optimized
to facilitate binding of the ligand to a prion protein based on the
nature of the ligand and its binding specificity to the prion
protein. In some embodiments, the binding is carried out at a
temperature of about 0.degree. C. to about 39.degree. C., including
for example about 20.degree. C. to about 25.degree. C. The binding
can be carried out at pH of about 4 to about 10, including for
example about 5 to about 9, about 6 to about 8, about 6.8 to about
7.5, about 6.9 to about 7.4,or about 7. Optionally, blocking agents
can be used to reduce non-specific binding to the ligand.
[0148] After the contacting step, the ligand and proteins bound
thereto are removed from the rest of the composition. The removal
can be carried out in a variety of ways, depending on the nature of
the ligand and the supporting material (if any) used to facilitate
the separation. For example, the ligand and proteins bound thereto
can be separated out by chromatography, such as, but not limited
to, thin-layer, column and batch chromatography; solid support and
membrane separation; reactor separation; magnetic separation,
immunoseparation; and colloidal separation.
[0149] In some embodiments, ligand may be immobilized on a support
such as a bead or a membrane, which in turn is allowed to contact
the nanoparticle composition or the intermediate composition.
Ligand-immobilized support is allowed to contact the nanoparticle
composition or the intermediate composition under a condition
sufficient to cause formation of a prion-ligand complex. The solid
phase is then separated from the composition, thereby removing the
prion protein bound to the ligand from the sample. For example, in
one exemplary embodiment, the ligands are immobilized to a column,
such as a chromatography column, a sample (such as the nanoparticle
composition) is then passed through the column either due to the
force of gravity or under pressure, such as in a high pressure
liquid chromatography column. Prion proteins in the sample will
bind to the ligand immobilized on the column, and the sample
passing through can be collected. This process can repeat several
times to achieve the desired result.
[0150] The flow rate of a sample (such as nanoparticle composition
or intermediate composition) in a column can be adjusted to
maximize the binding of the ligand and the prion proteins in a
sample. In some embodiments, the binding is carried out at a flow
rate of about 0.1 ml per minute to about 5.0 ml per minute, about
0.1 ml per minute to about 2.5 ml per minute, about 0.1 ml per
minute to about 0.25 ml per minute, about 0.25 per minute to about
0.5 ml per minute, about 0.5 ml per minute to about 1.0 ml per
minute, about 1.0 ml per minute to about 1.5 ml per minute, about
1.5 ml per minute to about 2.0 ml per minute, about 2.0 ml per
minute to about 2.5 ml per minute, about 2.5 ml per minute to about
3.0 ml per minute, about 3.0 ml per minute to about 3.5 ml per
minute, about 3.5 ml per minute to about 4.0 ml per minute, about
4.0 ml per minute to about 4.5 ml per minute, or about 4.5 ml per
minute to about 5.0 ml per minute, including for example about 0.1
ml per minute, 0.25 ml per minute, 0.5 ml per minute, 1.0 ml per
minute, 1.5 ml per minute, 1.7 ml per minute, 1.8 ml per minute,
1.9 ml per minute, 2.0 ml per minute, 2.1 ml per minute, 2.3 ml per
minute, 2.5 ml per minute, 2.7 ml per minute, 3.0 ml per minute,
3.5 ml per minute, 4.0 ml per minute, 4.5 ml per minute, or 5.0 ml
per minute. In some embodiments, the flow rate is at least about 10
ml per minute, such as at least about any of 20 ml per minute, 30
ml per minute, 40 ml per minute, 50 ml per minute.
[0151] The total flow-through volume or total flow-through time
during a binding process can also be adjusted to maximize the
binding of the ligand and the prion proteins in a sample (such as
nanoparticle composition or the intermediate composition). In some
embodiments, the total flow-through volume is about 50 times to
about 1000 times of the column volume, including for example about
50 times to about 500 times of the column volume, about 100 times
to about 600 times of the column volume, or about 200 times to
about 800 times of the column volume. In some embodiments, the
total flow-through volume is about 100 times of the column volume.
In other embodiments, the total flow-through volume is about 500
times of the column volume. In some embodiments, the total
flow-through time is about 1 hour to about 30 hours, including for
example about 2 hours to about 25 hours, about 3 hours to about 20
hours, or about 3 hours to about 17 hours. In some embodiments, the
total flow-through time is any of about 3 hours, 4 hours, 6 hours,
8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 17 hours, 18
hours, 19 hours, or 20 hours. In some embodiments, the total flow
through time is more than about 24 hours. In some embodiments, the
total flow through time is less than about 8, including for example
any of 7, 6, 5, 4, 3, 2, 1, or 0.5 hours.
[0152] Alternatively, the ligand can first be brought into contact
with the nanoparticle composition or the intermediate composition,
under a condition sufficient to cause formation of a prion-ligand
complex. The prion-ligand complex is then subsequently removed by
using a column, such as an affinity-based chromatography. To
facilitate the separation, the ligand may be conjugated to a
binding partner so that the ligand/prion complex can be removed by
using an affinity column containing a molecule that recognizes the
binding partner.
[0153] In addition to batch or column chromatography, a variety of
configurations, modifications and variations of the use of the
ligands for binding prion proteins are also envisioned. Such
variations and modifications include, but are not limited to: batch
processes, continuous processes, moving bed chromatography
processes; low, medium, or high pressure processes; or small,
medium or large scale processes. In some embodiments, the ligands
are on a membrane, fibers bead, impregnated into a non-woven mesh,
or coating fibers contained within a filter housing.
[0154] In some embodiments, the removal step does not significantly
result in yield loss and/or change in the property and/or stability
of the albumin For practical purposes, the recovery of the albumin
in its original biological state should be substantially maintained
at least to a level in excess of 50%, including for example 80%, or
90%, or more. In some embodiments, the recovery rate of albumin
from the prion process is higher than any of about 80%, 90%, 95%,
or 99%. In some embodiments, the concentration of albumin in the
nanoparticle composition is adjusted or controlled prior to the
prion removal step in order to minimize non-specific binding and
loss of albumin during the process. For example, the concentration
of albumin can be in the range of about 1% to about 50%, about 5%
to about 25%, about 5% to about 30%, about 5% to about 40%, about
5% to about 10%, about 10% to about 15%, about 15% to about 20%,
about 20% to about 25%, about 25% to about 30% etc., including for
example about 5%, 10%, 15%, 20%, 25%, or 30% albumin.
[0155] In some embodiments, the removal step does not significantly
result in yield loss and/or change in the property and/or stability
of the nanoparticles in the composition.
[0156] In some embodiments, the removal step does not significantly
result in yield loss and/or change in the property or loading of
the substantially water insoluble pharmacologically active agent in
the composition.
[0157] In some embodiments, the removal step does not significantly
result in change in the ratio of albumin to the substantially water
insoluble pharmacological agent in the composition.
[0158] The prion-removed composition can be analyzed to determine
the clearance rate of the prion removal process. The ligand with
bound prion proteins may also be analyzed (directly or after
elution) to determine the clearance rate.
[0159] The removal of prion protein can be determined based on
reduction in the prion protein level and/or a reduction in
infectivity. In some embodiments, at least about 50%, including for
example at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the
prion proteins are removed from the nanoparticle composition. In
some embodiments, the infectivity of the post-removal albumin
composition is at least about 10.times., 20.times., 30.times.,
40.times., 50.times., 80.times., 100.times., 200.times.,
500.times., 1000.times., 10.sup.4.times., 10.sup.5.times.,
10.sup.6.times. less than that of the nanoparticle composition.
[0160] In some embodiments, serial infectivity is used to determine
the clearance rate of the prion removal process. Serial dilutions
of a samples are made and dilutions are examined for infectious
activity, for example in an assay animal The dilution at which half
of the animals become infected is the infectious titer. For
example, if a 5 fold dilution is required, the sample may be
defined as having 5 logs of infectivity. By comparing the log
infectivity of the nanoparticle composition and that of the
post-removal nanoparticle composition, one can determine the
clearance rate of the prion removal process. In some embodiments,
the prion removal method results in a reduction of any one of 1,
1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, or 4, 5, 6, 7,
8, 9, or 10 logs of infectivity.
[0161] In some embodiments, the clearance rate of the prion-removal
process is determined based on spiking experiments with infectious
materials by following steps described herein for the prion-removal
method. Suitable spiking agents include, but are not limited to,
brain homogenates, microsomes, caveolae-like domains, purified
PrPsc, and prion fibrils. In some embodiments, the spiking agent is
detergent solubilized (such as sarkosyl solubilized). In some
embodiments, the spiking ratio in the composition is in the range
of about 0.001% to about 5%, about 0.001% to about 0.25%, about
0.001% to about 0.1%, about 0.001% to about 0.005%, about 0.005% to
about 0.075%, about 0.075% to about 0.01%, about 0.01% to about
0.1%, about 0.1% to about 0.5%, about 0.5% to about 0.75%, about
0.75% to about 1%, about 1% to about 2%, about 2% to about 3%, or
about 3% to about 5%, including for example about 0.001%, 0.005%,
0.075%, 0.01%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, or 5%.
[0162] In some embodiments, the reduction factors (RF) is used to
determine the clearance rate of the prion removal process. The RF
can be calculated using the formula:
RF=(V.sub.1.times.T.sub.1)/(V.sub.2.times.T.sub.2)
or
Log.sub.10[RF]=[ Log.sub.10(V.sub.1)+Log.sub.10(T.sub.1)]-[
Log.sub.10(V.sub.2)+Log.sub.10(T.sub.2)].
Wherein V.sub.1 and T.sub.1 are the volume and titre of the initial
albumin composition, respectively, and V.sub.2 and T.sub.2 are the
volume and titre of the post-removal albumin composition. Reduction
factors can be rounded to 1 decimal place after the final
calculation. In some embodiments, a reduction factor of at least
about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,
7.0, 7.5, or 8.0 log.sub.10 infectivity of the prion proteins is
removed from the initial albumin composition. For example, the
prion protein can be removed by a reduction factor of greater than
or equal to 2.5 log.sub.10 in a 0.5% sarkosyl solubilized fraction
spiked into a 20% albumin composition. As another example, the
prion protein can also be removed by a reduction factor of greater
than or equal to 2.0 log.sub.10 in a 0.5% sarkosyl solubilized
fraction spiked into a 25% albumin composition.
[0163] The removal of prions can be evaluated by standard Western
blot analysis. For example, the post-binding ligands can first be
treated with Proteinase K, which digests all PrPc but not PrPsc.
The digest is then run on SDS gel and transblotted to a sheet of
nitrocellulose or PVDF membrane. The separated PrPsc bands are then
visualized using 3F4 or 6H4. 3F4 reacts with residues 109-112 PrP
from humans, hamsters, and felines. In one exemplary embodiment,
incubation was carried out at a concentration of 0.6 ug/ml for a
minimum of one hour, after which excess antibody was washed away
and the membranes incubated with a rabbit anti-mouse horse-radish
peroxidase conjugate (1:1000 dilution) for a minimum of one hour.
After extensive washing with TTBS, the membranes were developed
using enhanced chemiluminescence. In some embodiments, the removal
of prion proteins is evaluated according to the Guideline for the
Investigation of Manufacturing Processes for Plasma-Derived
Medicinal Products with Regard to vCJD Risk (CPMP5136/03).
[0164] Also contemplated herein are compositions made during the
prion removal process. Thus, in some embodiments, there is provided
a composition comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent,
further comprising a ligand capable of binding to a prion protein.
In some embodiments, there is provided a mixture comprising
nanoparticle comprising albumin and a substantially water insoluble
pharmacologically active agent, and a ligand capable of binding to
a prion protein attached to a support material, such as one or more
supporting materials described herein. In some embodiments, there
is provided a column loaded with a composition comprising
nanoparticles comprising albumin and a substantially water
insoluble pharmacologically active agent, wherein the column
comprises a ligand capable of binding to a prion protein. In some
embodiments, there is provided a composition comprising
nanoparticles comprising albumin and a substantially water
insoluble pharmacologically active agent and a resin comprising a
ligand capable of binding to a prior protein. For example, in some
embodiments, there is provided a composition comprising
nanoparticles comprising albumin and a substantially water
insoluble pharmacologically active agent and a DVR resin. In some
embodiments, there is provided a composition comprising
nanoparticles comprising albumin and a substantially water
insoluble pharmacologically active agent and a PRDT resin.
[0165] In some embodiments, there is provided a composition
comprising a mixture of an aqueous albumin solution and a
substantially water insoluble pharmacologically active agent
dispersed in an organic solvent, further comprising a ligand
capable of binding to a prion protein. In some embodiments, there
is provided a composition comprising a mixture of an aqueous
albumin solution and a substantially water insoluble
pharmacologically active agent dispersed in an organic solvent, and
a ligand capable of binding to a prion protein attached to a
support material, such as one or more supporting materials
described herein. In some embodiments, there is provided a column
loaded with a composition comprising a mixture of an aqueous
albumin solution and a substantially water insoluble
pharmacologically active agent dispersed in an organic solvent,
wherein the column comprises a ligand capable of binding to a prion
protein.
[0166] Also provided are compositions made by methods described
herein. The composition in some embodiments is bioequivalent to a
composition not subject to prion-removal process.
Nanoparticle Compositions
[0167] The nanoparticle compositions described herein comprise
nanoparticles comprising (in various embodiments consisting
essentially of) albumin and a substantially water insoluble
pharmacologically active agent (such as paclitaxel). Nanoparticles
of poorly water soluble drugs (such as taxane) have been disclosed
in, for example, U.S. Pat. Nos. 5,916,596; 6,506,405; 6,749,868,
and 6,537,579 and also in U.S. Pat. Pub. Nos. 2005/0004002 and
2007/0082838, and PCT Patent Application WO08/137148, the content
of each of which is incorporated herein in their entirety.
[0168] In some embodiments, the composition comprises nanoparticles
with an average or mean diameter of no greater than about 1000
nanometers (nm), such as no greater than about any of 900, 800,
700, 600, 500, 400, 300, 200, and 100 nm. In some embodiments, the
average or mean diameters of the nanoparticles is no greater than
about 200 nm. In some embodiments, the average or mean diameters of
the nanoparticles is no greater than about 150 nm. In some
embodiments, the average or mean diameters of the nanoparticles is
no greater than about 100 nm. In some embodiments, the average or
mean diameter of the nanoparticles is about 20 to about 400 nm. In
some embodiments, the average or mean diameter of the nanoparticles
is about 40 to about 200 nm In some embodiments, the nanoparticles
are sterile-filterable.
[0169] In some embodiments, the nanoparticles in the composition
described herein have an average diameter of no greater than about
200 nm, including for example no greater than about any one of 190,
180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, or 60 nm.
In some embodiments, at least about 50% (for example at least about
any one of 60%, 70%, 80%, 90%, 95%, or 99%) of all the
nanoparticles in the composition have a diameter of no greater than
about 200 nm, including for example no greater than about any one
of 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, or
60 nm. In some embodiments, at least about 50% (for example at
least any one of 60%, 70%, 80%, 90%, 95%, or 99%) of all the
nanoparticles in the composition fall within the range of about 20
to about 200 nm, including for example any one of about 30 to about
180 nm, and any one of about 40 to about 150, about 50 to about
120, and about 60 to about 100 nm
[0170] In some embodiments, at least about 5% (including for
example at least about any one of 10%, 15%, 20%, 25%, 30%, 40%,
50%, 60%, 70%, 80%, or 90%) of the album in the nanoparticle
portion of the composition are crosslinked (for example crosslinked
through one or more disulfide bonds).
[0171] In some embodiments, the nanoparticles comprise the
substantially water insoluble pharmacologically active agent (such
as paclitaxel) coated with an albumin (e.g., human serum albumin).
In some embodiments, the composition comprises substantially water
insoluble pharmacologically active agent in non-nanoparticle form,
wherein at least about any one of 50%, 60%, 70%, 80%, 90%, 95%, or
99% of the substantially water insoluble pharmacologically active
agent in the composition are in nanoparticle form. In some
embodiments, the substantially water insoluble pharmacologically
active agent in the nanoparticles constitutes more than about any
one of 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the nanoparticles by
weight. In some embodiments, the nanoparticles have a non-polymeric
matrix. In some embodiments, the nanoparticles comprise a core of
substantially water insoluble pharmacologically active agent that
is substantially free of polymeric materials (such as polymeric
matrix).
[0172] In some embodiments, the composition comprises albumin in
both nanoparticle and non-nanoparticle portions of the composition,
wherein at least about any one of 50%, 60%, 70%, 80%, 90%, 95%, or
99% of the albumin in the composition are in the non-nanoparticle
portion of the composition.
[0173] In some embodiments, the nanoparticle composition is
substantially free (such as free) of surfactants (such as
Cremophor.RTM., Tween 80, or other organic solvents used for the
administration of substantially water insoluble pharmacologically
active agents). In some embodiments, the nanoparticle composition
contains less than about any one of 20%, 15%, 10%, 7.5%, 5%, 2.5%,
1% or less organic solvent. In some embodiments, the weight ratio
of albumin and substantially water insoluble pharmacologically
active agent in the nanoparticle composition is about 18:1 or less,
such as about 15:1 or less, for example about 10:1 or less. In some
embodiments, the weight ratio of albumin and substantially water
insoluble pharmacologically active agent in the composition falls
within the range of any one of about 1:1 to about 18:1, about 2:1
to about 15:1, about 3:1 to about 13:1, about 4:1 to about 12:1,
about 5:1 to about 10:1. In some embodiments, the weight ratio of
albumin and substantially water insoluble pharmacologically active
agent in the nanoparticle portion of the composition is about any
one of 1:2, 1:3, 1:4, 1:5, 1:10, 1:15, or less. In some
embodiments, the weight ratio of the albumin (such as human serum
albumin) and the substantially water insoluble pharmacologically
active agent in the composition is any one of the following: about
1:1 to about 18:1, about 1:1 to about 15:1, about 1:1 to about
12:1, about 1:1 to about 10:1, about 1:1 to about 9:1, about 1:1 to
about 8:1, about 1:1 to about 7:1, about 1:1 to about 6:1, about
1:1 to about 5:1, about 1:1 to about 4.1, about 1:1 to about 3:1,
about 1:1 to about 2:1, or about 1:1.
[0174] In some embodiments, the nanoparticle composition comprises
one or more of the above characteristics.
[0175] The nanoparticles described herein may be present in a dry
formulation (such as lyophilized composition) or suspended in a
biocompatible medium. Suitable biocompatible media include, but are
not limited to, water, buffered aqueous media, saline, buffered
saline, optionally buffered solutions of amino acids, optionally
buffered solutions of proteins, optionally buffered solutions of
sugars, optionally buffered solutions of vitamins, optionally
buffered solutions of synthetic polymers, lipid-containing
emulsions, and the like.
[0176] In some embodiments, the pharmaceutically acceptable carrier
comprises human serum albumin Human serum albumin (HSA) is a highly
soluble globular protein of Mr 65K and consists of 585 amino acids.
HSA is the most abundant protein in the plasma and accounts for
70-80% of the colloid osmotic pressure of human plasma. The amino
acid sequence of HSA contains a total of 17 disulphide bridges, one
free thiol (Cys 34), and a single tryptophan (Trp 214). Intravenous
use of HSA solution has been indicated for the prevention and
treatment of hypovolumic shock and in conjunction with exchange
transfusion in the treatment of neonatal hyperbilirubinemia. Other
albumins are contemplated, such as bovine serum albumin Use of such
non-human albumins could be appropriate, for example, in the
context of use of these compositions in non-human mammals, such as
the veterinary (including domestic pets and agricultural
context).
[0177] Human serum albumin (HSA) has multiple hydrophobic binding
sites (a total of eight for fatty acids, an endogenous ligand of
HSA) and binds a diverse set of substantially water insoluble
pharmacologically active agents, especially neutral and negatively
charged hydrophobic compounds. Two high affinity binding sites have
been proposed in subdomains IIA and IIIA of HSA, which are highly
elongated hydrophobic pockets with charged lysine and arginine
residues near the surface which function as attachment points for
polar ligand features.
[0178] The albumin in the composition generally serves as a carrier
for the substantially water insoluble pharmacologically active
agent, i.e., the albumin in the composition makes the substantially
water insoluble pharmacologically active agent more readily
suspendable in an aqueous medium or helps maintain the suspension
as compared to compositions not comprising an albumin This can
avoid the use of toxic solvents (or surfactants) for solubilizing
the substantially water insoluble pharmacologically active agent,
and thereby can reduce one or more side effects of administration
of the substantially water insoluble pharmacologically active agent
into an individual (such as a human). Thus, in some embodiments,
the composition described herein is substantially free (such as
free) of surfactants, such as Cremophor (including Cremophor
EL.RTM. (BASF)). In some embodiments, the nanoparticle composition
is substantially free (such as free) of surfactants. A composition
is "substantially free of Cremophor" or "substantially free of
surfactant" if the amount of Cremophor or surfactant in the
composition is not sufficient to cause one or more side effect(s)
in an individual when the nanoparticle composition is administered
to the individual.
[0179] The amount of albumin in the composition described herein
will vary depending on other components in the composition. In some
embodiments, the composition comprises an albumin in an amount that
is sufficient to stabilize the substantially water insoluble
pharmacologically active agent in an aqueous suspension, for
example, in the form of a stable colloidal suspension (such as a
stable suspension of nanoparticles). In some embodiments, the
albumin is in an amount that reduces the sedimentation rate of the
substantially water insoluble pharmacologically active agent in an
aqueous medium. For particle-containing compositions, the amount of
the albumin also depends on the size and density of nanoparticles
of the substantially water insoluble pharmacologically active
agent.
[0180] A substantially water insoluble pharmacologically active
agent is "stabilized" in an aqueous suspension if it remains
suspended in an aqueous medium (such as without visible
precipitation or sedimentation) for an extended period of time,
such as for at least about any of 0.1, 0.2, 0.25, 0.5, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, 60, or 72 hours. The
suspension is generally, but not necessarily, suitable for
administration to an individual (such as human). Stability of the
suspension is generally (but not necessarily) evaluated at a
storage temperature (such as room temperature (such as
20-25.degree. C.) or refrigerated conditions (such as 4.degree.
C.)). For example, a suspension is stable at a storage temperature
if it exhibits no flocculation or particle agglomeration visible to
the naked eye or when viewed under the optical microscope at 1000
times, at about fifteen minutes after preparation of the
suspension. Stability can also be evaluated under accelerated
testing conditions, such as at a temperature that is higher than
about 40.degree. C.
[0181] In some embodiments, the albumin is present in an amount
that is sufficient to stabilize the substantially water insoluble
pharmacologically active agent in an aqueous suspension at a
certain concentration. For example, the concentration of the
substantially water insoluble pharmacologically active agent in the
composition is about 0.1 to about 100 mg/ml, including for example
any of about 0.1 to about 50 mg/ml, about 0.1 to about 20 mg/ml,
about 1 to about 10 mg/ml, about 2 mg/ml to about 8 mg/ml, about 4
to about 6 mg/ml, about 5 mg /ml. In some embodiments, the
concentration of the substantially water insoluble
pharmacologically active agent is at least about any of 1.3 mg/ml,
1.5 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8
mg/ml, 9 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml,
40 mg/ml, and 50 mg/ml. In some embodiments, the albumin is present
in an amount that avoids use of surfactants (such as Cremophor), so
that the composition is free or substantially free of surfactant
(such as Cremophor).
[0182] In some embodiments, the composition, in liquid form,
comprises from about 0.1% to about 50% (w/v) (e.g. about 0.5%
(w/v), about 5% (w/v), about 10% (w/v), about 15% (w/v), about 20%
(w/v), about 25% (w/v), about 30% (w/v), about 40% (w/v), or about
50% (w/v)) of albumin In some embodiments, the composition, in
liquid form, comprises about 0.5% to about 5% (w/v) of albumin
[0183] In some embodiments, the weight ratio of albumin, e.g.,
albumin, to the substantially water insoluble pharmacologically
active agent in the nanoparticle composition is such that a
sufficient amount of substantially water insoluble
pharmacologically active agent binds to, or is transported by, the
cell. While the weight ratio of albumin to substantially water
insoluble pharmacologically active agent will have to be optimized
for different albumin and substantially water insoluble
pharmacologically active agent combinations, generally the weight
ratio of albumin, e.g., albumin, to substantially water insoluble
pharmacologically active agent (w/w) is about 0.01:1 to about
100:1, about 0.02:1 to about 50:1, about 0.05:1 to about 20:1,
about 0.1:1 to about 20:1, about 1:1 to about 18:1, about 2:1 to
about 15:1, about 3:1 to about 12:1, about 4:1 to about 10:1, about
5:1 to about 9:1, or about 9:1. In some embodiments, the albumin to
substantially water insoluble pharmacologically active agent weight
ratio is about any of 18:1 or less, 15:1 or less, 14:1 or less,
13:1 or less, 12:1 or less, 11:1 or less, 10:1 or less, 9:1 or
less, 8:1 or less, 7:1 or less, 6:1 or less, 5:1 or less, 4:1 or
less, and 3:1 or less.
[0184] In some embodiments, the albumin allows the composition to
be administered to an individual (such as human) without
significant side effects. In some embodiments, the albumin is in an
amount that is effective to reduce one or more side effects of
administration of the substantially water insoluble
pharmacologically active agent to a human. The term "reducing one
or more side effects of administration of the substantially water
insoluble pharmacologically active agent" refers to reduction,
alleviation, elimination, or avoidance of one or more undesirable
effects caused by the substantially water insoluble
pharmacologically active agent, as well as side effects caused by
delivery vehicles (such as solvents that render the substantially
water insoluble pharmacologically active agents suitable for
injection) used to deliver the substantially water insoluble
pharmacologically active agent. Such side effects include, for
example, myelosuppression, neurotoxicity, hypersensitivity,
inflammation, venous irritation, phlebitis, pain, skin irritation,
peripheral neuropathy, neutropenic fever, anaphylactic reaction,
venous thrombosis, extravasation, and combinations thereof. These
side effects, however, are merely exemplary and other side effects,
or combination of side effects, associated with substantially water
insoluble pharmacologically active agents can be reduced.
[0185] In some embodiments, the composition comprises Abraxane.RTM.
(or Nab-paclitaxel). In some embodiments, the composition is
Abraxane.RTM. (or Nab-paclitaxel). Abraxane.degree. is a
formulation of paclitaxel stabilized by human albumin USP, which
can be dispersed in directly injectable physiological solution.
When dispersed in a suitable aqueous medium such as 0.9% sodium
chloride injection or 5% dextrose injection, Abraxane.TM. forms a
stable colloidal suspension of paclitaxel. The mean particle size
of the nanoparticles in the colloidal suspension is about 130
nanometers. Since HSA is freely soluble in water, Abraxane.TM. can
be reconstituted in a wide range of concentrations ranging from
dilute (0.1 mg/ml paclitaxel) to concentrated (20 mg/ml
paclitaxel), including for example about 2 mg/ml to about 8 mg/ml,
about 5 mg/ml.
Substantially Water Insoluble Pharmacologically Active Agent
[0186] The compositions described herein comprise substantially
water insoluble pharmacologically active agents. For example, the
solubility in water of the poorly water soluble agent at about
20-25.degree. C. may be less than about 10 mg/ml, including for
example less than about any of 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02,
or 0.01 mg/ml. In some embodiments, the substantially water
insoluble pharmacologically active agent is a solid. In some
embodiments, the substantially water insoluble pharmacologically
active agent is a liquid. Substantially water insoluble
pharmacologically active agents described herein can be, for
example, pharmaceutical agent, diagnostic agent, or an agent of
nutritional value.
[0187] Suitable pharmaceutical agents include, but are not limited
to, anticancer or antineoplastic agents, antimicrotubule agents,
immunosuppressive agents, anesthetics, hormones, agents for use in
cardiovascular disorders, antiarrhythmics, antibiotics,
antifungals, antihypertensives, antiasthmatics, anti-inflammatory
agents, anti-arthritic agents, vasoactive agents,
analgesics/antipyretics, antidepressants, antidiabetics, antifungal
agents, anti-inflammatories, antianxiety agents, immunosuppressive
agents, antimigraine agents, sedatives, antianginal agents,
antipsychotic agents, antimanic agents, antiarthritic agents,
antigout agents, anticoagulants, thrombolytic agents,
antifibrinolytic agents, hemorheologic agents, antiplatelet agents,
anticonvulsants, antiparkinson agents,
antihistamines/antipruritics, agents useful for calcium regulation,
antiviral agents, antimicrobials, anti-infectives,
bronchodialators, hormones, hypoglycemic agents, hypolipidemic
agents, antiulcer/antireflux agents, antinauseants/antiemetics, and
oil-soluble vitamins (e.g., vitamins A, D, E, K, and the like).
[0188] In some embodiments, the substantially water insoluble
pharmacologically active agent is any one of the following: a
tyrosine kinase inhibitor, a series/threonine kinase inhibitor, a
hedgehog inhibitor, a topoisomerase inhibitor, an in inhibitor of
microtubule assembly, an inhibitor of the AKT kinase pathway, a
proteasome inhibitor, an antimetabolite, and a platinum-based
agent.
[0189] In some embodiments, the substantially water insoluble
pharmacologically active agent is an antineoplastic agent. In some
embodiments, the substantially water insoluble pharmacologically
active agent is a chemotherapeutic agent.
[0190] Suitable substantially water insoluble pharmacologically
active agents include, but are not limited to, taxanes (such as
paclitaxel, docetaxel, ortataxel and other taxanes), epothilones,
camptothecins, colchicines, geladanamycins, amiodarones, thyroid
hormones, amphotericin, corticosteroids, propofol, melatonin,
cyclosporine, rapamycin (sirolimus) and derivatives, tacrolimus,
mycophenolic acids, ifosfamide, vinorelbine, vancomycin,
gemcitabine, SU5416, thiotepa, bleomycin, diagnostic radiocontrast
agents, and derivatives thereof. Other substantially water
insoluble pharmacologically active agents that are useful in the
inventive compositions are described in, for example, U.S. Pat.
Nos. 5,916,596, 6,096,331, 6,749,868, and 6,537,539. Additional
examples of substantially water insoluble pharmacologically active
agents include those compounds which are poorly water soluble and
which are listed in the "Therapeutic Category and Biological
Activity Index" of The Merck Index (12.sup.th Edition, 1996).
[0191] In some embodiments, the substantially water insoluble
pharmacologically active agent is any of (and in some embodiments
selected from the group consisting of) paclitaxel, docetaxel,
CY196, ortataxel or other taxane or taxane analog, 17-allyl amino
geldanamycin (17-AAG), 18-derivatized geldanamycin, camptothecin,
propofol, amiodarone, cyclosporine, epothilone, radicicol,
combretastatin, rapamycin, amphotericin, liothyronine, epothilone,
colchicine, thiocolchicine and its dimers, thyroid hormone,
vasoactive intestinal peptide, corticosteroids, melatonin,
tacrolimus, mycophenolic acids, epothilones, radicicols,
combretastatins, and analog or derivative thereof. In some
embodiments, the substantially water insoluble pharmacologically
active agent is any of (and in some embodiments selected from the
group consisting of) paclitaxel, docetaxel, CY196, ortataxel or
other taxanes, geldanamycin, 17-allyl amino geldanamycin,
thiocolchicine and its dimers, rapamycin, cyclosporine, epothilone,
radicicol, and combretastatin. In some embodiments, the
substantially water insoluble pharmacologically active agent is
rapamycin. In some embodiments, the substantially water insoluble
pharmacologically active agent is 17-AAG. In some embodiments, the
substantially water insoluble pharmacologically active agent is a
thiocolchicine dimer (such as IDN5404). In some embodiments, the
substantially water insoluble pharmacologically active agent is a
taxane. In some embodiments, the substantially water insoluble
pharmacologically active agent is paclitaxel. In some embodiments,
the substantially water insoluble pharmacologically active agent is
docetaxel. In some embodiments, the substantially water insoluble
pharmacologically active agent is CY196.
[0192] In some embodiments, the substantially water insoluble
pharmacologically active agent is a taxane or derivative thereof,
which includes, but is not limited to, paclitaxel, docetaxel and
IDN5109 (ortataxel), or a derivative thereof. In some embodiments,
the composition comprises a non-crystalline and/or amorphous taxane
(such as paclitaxel or a derivative thereof). In some embodiments,
the composition is prepared by using an anhydrous taxane (such as
anhydrous docetaxel or a derivative thereof). Anhydrous docetaxel
has been shown to produce more stable formulation than can be made
with a hydrated docetaxel such as docetaxel trihydrate or
hemi-hydrate.
Other Components in the Nanoparticle Compositions
[0193] The nanoparticles described herein can be present in a
composition that includes other agents, excipients, or stabilizers.
For example, to increase stability by increasing the negative zeta
potential of nanoparticles, certain negatively charged components
may be added. Such negatively charged components include, but are
not limited to bile salts of bile acids consisting of glycocholic
acid, cholic acid, chenodeoxycholic acid, taurocholic acid,
glycochenodeoxycholic acid, taurochenodeoxycholic acid, litocholic
acid, ursodeoxycholic acid, dehydrocholic acid and others;
phospholipids including lecithin (egg yolk) based phospholipids
which include the following phosphatidylcholines:
palmitoyloleoylphosphatidylcholine,
palmitoyllinoleoylphosphatidylcholine,
stearoyllinoleoylphosphatidylcholine
stearoyloleoylphosphatidylcholine,
stearoylarachidoylphosphatidylcholine, and
dipalmitoylphosphatidylcholine. Other phospholipids including
L-.alpha.-dimyristoylphosphatidylcholine (DMPC),
dioleoylphosphatidylcholine (DOPC), distearyolphosphatidylcholine
(DSPC), hydrogenated soy phosphatidylcholine (HSPC), and other
related compounds. Negatively charged surfactants or emulsifiers
are also suitable as additives, e.g., sodium cholesteryl sulfate
and the like.
[0194] In some embodiments, the composition is suitable for
administration to a human. In some embodiments, the composition is
suitable for administration to a mammal such as, in the veterinary
context, domestic pets and agricultural animals. There are a wide
variety of suitable formulations of the nanoparticle composition
(see, e.g., U.S. Pat. Nos. 5,916,596 and 6,096,331). The following
formulations and methods are merely exemplary and are in no way
limiting. Formulations suitable for oral administration can consist
of (a) liquid solutions, such as an effective amount of the
compound dissolved in diluents, such as water, saline, or orange
juice, (b) capsules, sachets or tablets, each containing a
predetermined amount of the active ingredient, as solids or
granules, (c) suspensions in an appropriate liquid, and (d)
suitable emulsions. Tablet forms can include one or more of
lactose, mannitol, corn starch, potato starch, microcrystalline
cellulose, acacia, gelatin, colloidal silicon dioxide,
croscarmellose sodium, talc, magnesium stearate, stearic acid, and
other excipients, colorants, diluents, buffering agents, moistening
agents, preservatives, flavoring agents, and pharmacologically
compatible excipients. Lozenge forms can comprise the active
ingredient in a flavor, usually sucrose and acacia or tragacanth,
as well as pastilles comprising the active ingredient in an inert
base, such as gelatin and glycerin, or sucrose and acacia,
emulsions, gels, and the like containing, in addition to the active
ingredient, such excipients as are known in the art.
[0195] Examples of suitable carriers, excipients, and diluents
include, but are not limited to, lactose, dextrose, sucrose,
sorbitol, mannitol, starches, gum acacia, calcium phosphate,
alginates, tragacanth, gelatin, calcium silicate, microcrystalline
cellulose, polyvinylpyrrolidone, cellulose, water, saline solution,
syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc,
magnesium stearate, and mineral oil. The formulations can
additionally include lubricating agents, wetting agents,
emulsifying and suspending agents, preserving agents, sweetening
agents or flavoring agents.
[0196] Formulations suitable for parenteral administration include
aqueous and non-aqueous, isotonic sterile injection solutions,
which can contain anti-oxidants, buffers, bacteriostats, and
solutes that render the formulation compatible with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. The formulations
can be presented in unit-dose or multi-dose sealed containers, such
as ampules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid excipient, for example, water, for injections, immediately
prior to use. Extemporaneous injection solutions and suspensions
can be prepared from sterile powders, granules, and tablets of the
kind previously described. Injectable formulations are
preferred.
[0197] In some embodiments, the composition is formulated to have a
pH range of about 4.5 to about 9.0, including for example pH ranges
of any of about 5.0 to about 8.0, about 6.5 to about 7.5, and about
6.5 to about 7.0. In some embodiments, the pH of the composition is
formulated to no less than about 6, including for example no less
than about any of 6.5, 7, or 8 (such as about 8). The composition
can also be made to be isotonic with blood by the addition of a
suitable tonicity modifier, such as glycerol.
Method of Using Prion-Free Nanoparticle Compositions
[0198] Also provided are methods of using the prion-free
compositions described herein. For example, in some embodiments,
there is provided a method of administering a composition
comprising nanoparticles comprising albumin and a substantially
water insoluble pharmacologically active agent, wherein the
composition is substantially free of a prion protein. In some
embodiments, there is provided a method of treating a disease (such
as cancer) comprising administering an effective amount of a
composition comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent,
wherein the composition is substantially free of a prion
protein.
[0199] In some embodiments, there is provided a method of
administering a composition comprising nanoparticles comprising
albumin and a substantially water insoluble pharmacologically
active agent, wherein the albumin in the composition was obtained
by a method comprising a prion-removal process, said prion-removal
process comprising contacting an initial albumin composition with a
ligand capable of binding to a prion protein. In some embodiments,
the prion removal process further comprises removing said ligand
and proteins bound thereto from said albumin composition. In some
embodiments, there is provided a method of treating a disease (such
as cancer) in an individual comprising administering to the
individual an effective amount of composition comprising
nanoparticles comprising albumin and a substantially water
insoluble pharmacologically active agent, wherein the albumin in
the composition was obtained by a method comprising a prion-removal
process, said prion removal process comprising contacting an
initial albumin composition with a ligand capable of binding to a
prion protein.
[0200] In some embodiments, the individual has vCJD. In some
embodiments, the individual has already been infected with a prion
protein. In some embodiments, the individual is suspected of having
vCJD or being infected with a prion protein. In some embodiments,
the individual is an asymptomic carrier of a prion protein. In some
embodiments, the individual has received blood transfusion at least
once. In some embodiments, the individual is at least 60 years old,
such as at least about 65, 70, or 75 years old. In some
embodiments, the individual is immunity compromised. In some
embodiments, the individual is a cancer patient.
[0201] The term "effective amount" used herein refers to an amount
of a compound or composition sufficient to treat a specified
disorder, condition or disease such as ameliorate, palliate,
lessen, and/or delay one or more of its symptoms. In reference to
cancers or other unwanted cell proliferation, an effective amount
comprises an amount sufficient to cause a tumor to shrink and/or to
decrease the growth rate of the tumor (such as to suppress tumor
growth). In some embodiments, an effective amount is an amount
sufficient to delay development. In some embodiments, an effective
amount is an amount sufficient to prevent occurrence and/or
recurrence. An effective amount can be administered in one or more
administrations.
[0202] Cancers to be treated by compositions described herein (such
as a composition comprising an antineoplastic agent such as taxane,
rapamycin, and 17-AAG) include, but are not limited to, carcinoma,
lymphoma, blastoma, sarcoma, and leukemia. Examples of cancers that
can be treated by compositions described herein include, but are
not limited to, squamous cell cancer, lung cancer (including small
cell lung cancer, non-small cell lung cancer, adenocarcinoma of the
lung, and squamous carcinoma of the lung, including squamous
NSCLC), cancer of the peritoneum, hepatocellular cancer, gastric or
stomach cancer (including gastrointestinal cancer), pancreatic
cancer (such as advanced pancreatic cancer), glioblastoma, cervical
cancer, ovarian cancer, liver cancer (such as hepatocellular
carcinoma), bladder cancer, heptoma, breast cancer, colon cancer,
melanoma, endometrical or uterine carcinoma, salivary gland
carcinoma, kidney or renal cancer, liver cancer, prostate cancer
(such as advanced prostate cancer), vulval cancer, thyroid cancer,
hepatic carcinoma, head and neck cancer, colorectal cancer, rectal
cancer, soft-tissue sarcoma, Kaposi's sarcoma, B-cell lymphoma
(including low grade/follicular non-Hodgkin's lymphoma (NHL), small
lymphocytic (SL) NHL, intermediate grade/follicular NHL,
intermediate grade diffuse NHL, high grade immunoblastic NHL, high
grade lymphoblastic NHL, high grade small non-cleaved cell NHL,
bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and
Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia
(CLL), acute lymphoblastic leukemia (ALL), myeloma, Hairy cell
leukemia, chronic myeloblastic leukemia, and post-transplant
lymphoproliferative disorder (PTLD), as well as abnormal vascular
proliferation associated with phakomatoses, edema (such as that
associated with brain tumors), and Meigs' syndrome. In some
embodiments, there is provided a method of treating metastatic
cancer (that is, cancer that has metastasized from the primary
tumor). In some embodiments, there is provided a method of reducing
cell proliferation and/or cell migration. In some embodiments,
there is provided a method of treating hyperplasia.
[0203] In some embodiments, there are provided methods of treating
cancer at advanced stage(s). In some embodiments, there are
provided methods of treating breast cancer (which may be HER2
positive or HER2 negative), including, for example, advanced breast
cancer, stage IV breast cancer, locally advanced breast cancer, and
metastatic breast cancer. In some embodiments, the cancer is lung
cancer, including, for example, non-small cell lung cancer (NSCLC,
such as advanced NSCLC), small cell lung cancer (SCLC, such as
advanced SCLC), and advanced solid tumor malignancy in the lung. In
some embodiments, the cancer is ovarian cancer, head and neck
cancer, gastric malignancies, melanoma (including metastatic
melanoma), colorectal cancer, pancreatic cancer, and solid tumors
(such as advanced solid tumors). In some embodiments, the cancer is
any of (and in some embodiments selected from the group consisting
of) breast cancer, colorectal cancer, rectal cancer, non-small cell
lung cancer, non-Hodgkins lymphoma (NHL), renal cell cancer,
prostate cancer, liver cancer, pancreatic cancer, soft-tissue
sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck
cancer, melanoma, ovarian cancer, mesothelioma, gliomas,
glioblastomas, neuroblastomas, and multiple myeloma. In some
embodiments, the cancer is a solid tumor.
[0204] Individual suitable for receiving these compositions depend
on the nature of the poorly water soluble pharmaceutical agent, as
well as the disease/condition/disorder to be treated and/or
prevented. Accordingly, the term individual includes any of
vertebrates, mammals, and humans. In some embodiments, the
individual is a mammal, including, but not limited to, human,
bovine, equine, feline, canine, rodent, or primate. In some
embodiments, the individual is human.
[0205] The dose of the inventive composition administered to an
individual (such as human) will vary with the particular
composition, the method of administration, and the particular
disease being treated. The dose should be sufficient to effect a
desirable response, such as a therapeutic or prophylactic response
against a particular disease. For example, the dosage of paclitaxel
in the composition can be in the range of 100-400 mg/m.sup.2 when
given on a 3 week schedule, or 50-250 mg/m.sup.2 when given on a
weekly schedule. In addition, if given in a metronomic regimen
(e.g., daily or a few times per week), the dosage may be in the
range of about 5-75 mg/m.sup.2.
[0206] The compositions described herein can be administered to an
individual (such as human) via various routes, including, for
example, intravenous, intra-arterial, intrapulmonary, intraportal,
intrahepatic, oral, inhalation, intravesicular, intramuscular,
intra-tracheal, subcutaneous, intraocular, intrathecal,
transmucosal, and transdermal. For example, the inventive
composition can be administered by inhalation to treat conditions
of the respiratory tract. The composition can be used to treat
respiratory conditions such as pulmonary fibrosis, broncheolitis
obliterans, lung cancer, bronchoalveolar carcinoma, and the
like.
[0207] In some embodiments, the administration of the composition
is conducted in conjunction with a prion-removal filter.
[0208] Also provided herein are methods of reducing side effects
associated with administration of the nanoparticle composition. For
example, the invention provides methods of reducing various side
effects associated with administration of the poorly water soluble
pharmaceutical agent, including, but not limited to,
myelosuppression, neurotoxicity, hypersensitivity, inflammation,
venous irritation, phlebitis, pain, skin irritation, peripheral
neuropathy, neutropenic fever, anaphylactic reaction, hematologic
toxicity, and cerebral or neurologic toxicity, and combinations
thereof. In some embodiments, there is provided a method of
reducing hypersensitivity reactions associated with administration
of the poorly water soluble pharmaceutical agent, including, for
example, severe skin rashes, hives, flushing, dyspnea, tachycardia,
and others.
Kits and Systems
[0209] The invention also provides kits for use in the instant
methods. Kits of the invention include one or more containers
comprising the prion-free nanoparticle compositions, and in some
embodiments, further comprise instructions for use in accordance
with any of the methods described herein. The kit may further
comprise a description of selection an individual suitable or
treatment. Instructions supplied in the kits of the invention are
typically written instructions on a label or package insert (e.g.,
a paper sheet included in the kit), but machine-readable
instructions (e.g., instructions carried on a magnetic or optical
storage disk) are also acceptable.
[0210] The kits of the invention are in suitable packaging.
Suitable packaging include, but is not limited to, vials, bottles,
jars, flexible packaging (e.g., seled Mylar or plastic bags), and
the like. Kits may optionally provide additional components such as
buffers and interpretative information.
[0211] The instructions relating to the use of the nanoparticle
compositions generally include information as to dosage, dosing
schedule, and route of administration for the intended treatment.
The containers may be unit doses, bulk packages (e.g., multi-dose
packages) or sub-unit doses. For example, kits may be provided that
contain sufficient dosages of the substantially water insoluble
pharmacologically active agent (such as substantially water
insoluble pharmacologically active agent) as disclosed herein to
provide effective treatment of an individual for an extended
period, such as any of a week, 2 weeks, 3 weeks, 4 weeks, 6 weeks,
8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9
months, or more. Kits may also include multiple unit doses of the
substantially water insoluble pharmacologically active agent and
pharmaceutical compositions and instructions for use and packaged
in quantities sufficient for storage and use in pharmacies, for
example, hospital pharmacies and compounding pharmacies.
[0212] In some embodiments, there is provided a kit for removing a
prion protein from a nanoparticle composition comprising albumin
and substantially water insoluble pharmacologically active agent,
comprising a ligand capable of binding to a prion protein. In some
embodiments, the kit further comprises a supporting material. In
some embodiments, the kit further comprises an instruction for
using the ligand for removing prion from the nanoparticle
composition.
[0213] Also provided are systems for carrying out methods described
herein. For example, in some embodiments, there is provided a
system for manufacturing prion-free nanoparticle composition
comprising albumin and a substantially water insoluble
pharmacologically active agent, said system comprising 1) an
apparatus for making the nanoparticle composition; and 2) an
apparatus for removing prion proteins from the albumin used for
making the nanoparticle composition. In some embodiments, the
apparatus for removing prion proteins from said albumin used for
making the nanoparticle composition is integrated into the
apparatus for making the nanoparticle composition. In some
embodiments, the apparatus for making the nanoparticle composition
is separated from the apparatus for removing the prion proteins
from the albumin used for making the nanoparticle composition.
[0214] in some embodiments, there is provided a system for
manufacturing prion-free nanoparticle composition comprising
albumin and a substantially water insoluble pharmacologically
active agent, said system comprising 1) an apparatus for making the
nanoparticle composition; and 2) an apparatus for removing prion
proteins (for example from an intermediate composition generated
during the making of the nanoparticles or from the generated
nanoparticle compositions). In some embodiments, the apparatus for
removing prion proteins is integrated into the apparatus for making
the nanoparticle composition. In some embodiments, the apparatus
for making the nanoparticle composition is separated from the
apparatus for removing the prion proteins.
[0215] Those skilled in the art will recognize that several
embodiments are possible within the scope and spirit of this
invention.
[0216] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0217] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Embodiments of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such embodiments as appropriate, and the inventors intend
for the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
embodiments thereof is encompassed by the invention unless
otherwise indicated herein or otherwise clearly contradicted by
context.
Exemplary Embodiments of the Present Application
[0218] In one aspect, there is provided a composition comprising
nanoparticles comprising albumin and a substantially water
insoluble pharmacologically active agent, wherein the composition
is substantially free of a prion protein. In some embodiments, the
composition has a prion infectivity of less than about 100 fg/ml.
In some embodiments, the composition is any one of the compositions
described above, wherein the composition has a prion infectivity of
less than about 10 IU-ic/ml. In some embodiments, the composition
is any one of the compositions described above, wherein the
composition does not show the presence of a prion protein based on
a protein misfolding cyclic amplification (PMCA) assay or based on
an IPCR assay. In some embodiments, the composition is any one of
the compositions described above, further comprising a trace amount
of a ligand capable of binding to a prion protein. In some
embodiments, the composition is any one of the compositions
described above, further comprising a trace amount of a supporting
material.
[0219] In another aspect, there is provided a composition
comprising nanoparticles comprising albumin and a substantially
water insoluble pharmacologically active agent, wherein the albumin
in the composition was obtained by a method comprising a prion
removal process, said prion removal process comprising contacting
an initial albumin composition with a ligand capable of binding to
a prion protein. In some embodiments, the prion removal process
further comprises removing said ligand and proteins bound thereto
from said albumin and composition. In some embodiments, the ligand
is a peptide. In some embodiments, the ligand is a trazine-based
compound.
[0220] In another aspect, there is provided a method of producing a
composition comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent, said
method comprising: a) removing a prion protein from an initial
albumin composition; b) subjecting a mixture comprising a solution
comprising the prion-removed albumin and an organic phase
comprising said substantially water insoluble pharmacologically
active agent dispersed in an organic solvent to a high shear
condition. In some embodiments, step a) comprises: 1) contacting
the initial albumin solution with a ligand capable of binding to a
prion protein. In some embodiments, step a) further comprises: 2)
removing the ligand and proteins bound thereto from the albumin
solution. In some embodiments, the method is any of the methods
described above, further comprising removing said organic solvent
from the mixture. In some embodiments, said removing of the organic
solvent is by evaporation. In some embodiments, the method is any
one of the methods described above, wherein said ligand is a
peptide. In some embodiments, the method is any one of the methods
described above, wherein the ligand is a triazine-based compound.
In some embodiments, the method is any one of the methods described
above, wherein the initial albumin composition is a blood derived
product.
[0221] In another aspect, there is provided a method of producing a
composition comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent,
comprising: a) subjecting a mixture comprising an organic phase
comprising said substantially water insoluble pharmacologically
active agent and an albumin solution to a high shear condition, and
b) removing a prion protein from said mixture. In some embodiments,
step b) comprises: 1) contacting the mixture with a ligand capable
of binding to a prion protein. In some embodiments, step b) further
comprises: 2) removing the ligand and proteins bound thereto from
said mixture. In some embodiments, the ligand is a peptide. In some
embodiments, the ligand is a triazine-based compound.
[0222] Also provided are composition produced by a method of any
one of claims 11-23. Also provided are uses of any one of the
compositions described above for treating a disease, such as
cancer.
[0223] In another aspect, there is provide a method of removing a
prion protein from a composition suspected of containing a prion
protein comprising nanoparticles comprising albumin and a
substantially water insoluble pharmacologically active agent,
comprising: a) contacting the composition with a ligand capable of
binding to a prion protein, b) removing the ligand and proteins
bound thereto from the composition. In some embodiments, the ligand
is a peptide. In some embodiments, the ligand is a triazine-based
compound. Also provided are compositions obtained after the
method.
[0224] In another aspect, there is provided a composition
comprising nanoparticles comprising albumin and a substantially
water insoluble pharmacologically active agent, further comprising
a ligand capable of binding to a prion protein. In some
embodiments, the ligand is a peptide. In some embodiments, the
ligand is a triazine-based compound.
[0225] The following examples are provided to illustrate, but not
to limit, the invention. It is understood that the examples
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application.
Examples
Example 1
Development of an Affinity Adsorbent for Removal of Prion Protein
from Albumin Preparations
[0226] This study screened a four-resin panel of prion binding
ligands by challenging the four-resin panel with two different
commercially available albumin preparations (containing 20% w/v or
25% w/v albumin), spiked with scrapie hamster brain homogenate at
two different concentrations (0.01% or 0.005%). The four-resin
panel was previously identified by PRDT (Pathogen Removal and
Diagnostic Technologies Inc; ProMetic Biosciences Ltd., Cambridge,
UK) as good prion binders in the presence of 25% albumin Selection
of an optimum resin can optimize the incorporation of a
prion-reduction step in the production of albumin
nanoparticles.
Methodology
[0227] Six Protein Isolation Kit for Sorbent Identification
(PIKSI.TM., ProMetic Biosciences Ltd) kits were packed with twelve
columns (at about 0.5 mL) of each of the four PRDT resins and the
control resin (Toyopearl Amino AMN31). Each resin was challenged
with solutions containing 20% and 25% albumin spiked with 0.01% or
0.005% scrapie hamster brain homogenate (SBH) in a three-column
series format in an effort to evaluate the binding capacity of each
resin to prion proteins. Comparison of resin performance was based
on prion protein binding as determined by Western blot and
densitometry. Total protein binding profile was determined by
SDS-PAGE gels. The bound proteins were stripped from the resins for
both the prion protein binding and the total protein binding
detection. Albumin binding was determined using NanoDrop.RTM.
ND-1000 spectrophotometer to measure absorbance at 280 nm. The
signals observed in the Western blots and SDS-PAGE gels correspond
to the bound fraction of prion protein and total protein,
respectively.
[0228] The commercial albumin preparations used in this invention
were Albumin (Human) U.S.P. Human Albumin Grifols.RTM. 20%, Lot No.
IBAB8MJ001, and Albumin (Human), USP, 25% solution Baxter, Lot No.
LA06D04AA.
Results
Western Blot and SDS-PAGE
[0229] The results obtained show that all four resins bound PrPsc
spiked into 20% or 25% human albumin solutions. The PrPsc signal
intensity in Western blot (FIGS. 1-3) suggests that prion binding
was strongest for DVR, followed by YVHEA, SYA, and D4 resins. The
control resin, AMN31, had an expected strong signal. The level of
signal obtained by using DVR resin suggests that high
concentrations of albumin did not interfere in prion binding.
[0230] No detectable signal was observed in the second and third
columns when using DVR (FIG. 1) at various conditions tested, even
when longer exposure times were tested, which indicates that all
the detectable PrPsc was captured by the first DVR column.
[0231] Signal intensity was weaker for the other resins in the
first column, with detection of prion protein in the second column
of the series (FIGS. 2 and 3), suggesting a possible interference
of albumin to prion protein binding. All three ligands showed no
prion signal in the third column, indicating that the resins are
capable of selectively removing prions from the SBH spiked albumin
solutions. The concentration of PrPsc in hamster brain was about 50
.mu.g/g, equivalent to about 5 ng/mL of PrPsc in 0.01% SBII, spiked
into a 250 mg/mL albumin solution, generating a 50,000,000-fold
excess of albumin
[0232] The total protein pattern observed in the Coomassie-stained
SDS-PAGE gels (FIGS. 1 to 3) shows that DVR had a much lower level
of total protein binding than the remaining prion-binding resins,
which is considered an advantage, despite the fact that most of the
observed bands in the total protein gel came from the brain
homogenate spike. As expected, the one visible protein band in the
DVR gel has an apparent molecular weight similar to albumin (66.5
KDa).
Densitometry
[0233] Prion removal was also assessed indirectly by calculating
the ratio of densitometric signal of PrPsc bound to the resin
versus the signal present in the albumin solutions spiked with SBH.
The ability of DVR resin to bind prion was comparably to the
positive control resin, adsorbing approximately all available PrPsc
in the first column Using infectious doses as the measurement for
prion binding, the 0.5 mL DVR column was able to remove prion in
the 10 mL SBH-spiked albumin solutions, which is equivalent to
about 10.sup.6 ID.sub.50, considering that 0.1% SBH contains about
10.sup.6 ID.sub.50/mL. Similarly to what was observed in the
Western blots, DVR had the best performance in prion binding than
YVHEA, D4, and SYA. All of three resins required the second column
of each series to further bind any detectable prion protein present
in SBH-spiked albumin solutions.
Albumin Binding
[0234] Albumin concentration was determined using a NanoDrop.RTM.
ND-1000 spectrophotometer at absorbance 280 nm for protein
quantitation. Each flow-through was measured for the concentration
of albumin after passing the SBH-spiked albumin solutions through
each of the four resins (DVR, YVHEA, SYA, and D4), and the control
resin (AMN31). The protein concentration of commercial albumin
solutions at 20% and 25% in the absence or presence of 0.01% or
0.005% SBH spikes was measured before flowing through the resin
columns The results obtained are shown in Table 3.
TABLE-US-00003 TABLE 3 Measurement of albumin concentration of
commercial albumin solutions (20% w/v or 25% w/v) spiked with or
without 0.01% and 0.005% scrapie hamster brain homogenate. Measured
Measured Concentration Concentration Average (%) (mg/mL) (%) 20%
albumin 24.8 248.0 24.6 20% albumin + 0.01% spike 24.7 247.1 20%
albumin + 0.005% spike 24.3 243.3 25% albumin 26.8 268.1 27.6 25%
albumin + 0.01% spike 28.3 282.5 25% albumin + 0.005% spike 27.8
278.3
[0235] The concentrations of the commercial albumin solutions were
measured higher than the commercially labeled value, especially for
the preparation containing the 20% w/v albumin solution. However,
since the study dealt with comparative values, this was not a
concern. The average of three values was obtained, and the amount
of the spike was considered negligible when compared to the amount
of albumin present in the solutions in determine protein
concentration. The concentration of albumin was obtained after
flowing SBH-spiked albumin solutions through the resins as shown in
Tables 4 and 5.
TABLE-US-00004 TABLE 4 Measurement of albumin concentration after
flowing commercial albumin solution (20% w/v) spiked with or
without 0.01% and 0.005% scrapie hamster brain homogenate through
different resin columns. Concentration Albumin (mg/mL) Loss (%) DVR
0.01% spike 260.4 ND 0.005% spike 260.4 ND YVHEA 0.01% spike 264.3
ND 0.005% spike 259.5 ND SYA 0.01% spike 245.5 0.2 0.005% spike
241.7 1.7 D4 0.01% spike 263.0 ND 0.005% spike 265.8 ND AMN31 0.01%
spike 249.8 ND 0.005% spike 246.9 ND ND = not detected.
TABLE-US-00005 TABLE 5 Measurement of albumin concentration after
flowing commercial albumin solution (25% w/v) spiked with or
without 0.01% and 0.005% scrapie hamster brain homogenate through
four different resin columns. Concentration Albumin (mg/mL) Loss
(%) DVR 0.01% spike 268.8 2.6 0.005% spike 284.2 ND YVHEA 0.01%
spike 285.7 ND 0.005% spike 294.2 ND SYA 0.01% spike 280.5 ND
0.005% spike 262.9 4.7 D4 0.01% spike 284.0 ND 0.005% spike 261.1
5.4 AMN31 0.01% spike 272.4 1.3 0.005% spike 282.4 ND ND = not
detected.
[0236] None of the resins tested showed a significant loss of
albumin In fact, no loss was detected for most of the conditions.
The values found indicate a variation of about .+-.5%. The data
suggest that albumin loss is unlikely a factor for choosing one of
the resins at this scale, within the range of tested
conditions.
Conclusions
[0237] Based on the results obtained, DVR is the best resin among
the four tested resins for removing prion from commercial albumin
solutions. The resin was able to remove around 10.sup.6 ID.sub.50
in a 0.5-mL column. This value is similar to the ones obtained in
previous trials for other challenges. There was little loss of
albumin detected at the tested ratio of challenge with 20 or 25%
albumin/resin of 20. Albumin binding should be lower at process
conditions, since this ratio is likely to increase.
Example 2
Prion-Removal Feasibility Study of Formulated Suspensions
Containing Paclitaxel and of Human Albumin Solution
[0238] Application of the prion-removal technology in the
nanoparticle composition was evaluated using formulated suspensions
containing paclitaxel (e.g., Abraxane.TM.) and using human albumin
solution containing various percentages of albumin (% w/v).
Experimental Conditions
Study Systems
[0239] Formulated Suspensions for Abraxane.TM. and sugar-paclitaxel
formulation were evaluated. Formulated Suspension (FS) for
Abraxane.TM. was formulated with post-evaporated suspension (PE)
obtained from the production and human albumin solution (25%,
Baxter, Deerfield, Ill.), containing approximately 7 mg/mL
paclitaxel and 56 mg/mL human albumin Formulated Suspension (FS)
for sugar-paclitaxel formulation was formulated with
post-evaporated suspension (PE) obtained from the production, human
albumin solution (25%, Baxter), sucrose, sodium chloride and
edetate disodium dihydrate, containing approximately 7 mg/ml
paclitaxel, 56 mg/ml human albumin; 32 mg/ml sucrose, 8.4 NaCl
mg/ml, and 0.07 mg/ml EDTA (ethylenediaminetetraacetic acid).
[0240] Human Albumin Solutions in 25%, 20%, and 5% were also
evaluated. Human albumin solution in 25% or 250 mg/ml was obtained
from Baxter; human albumin solution in 20% or 200 mg/ml (e.g.,
Grifols.RTM.) was obtained from Grifols Biologicals, Inc. (Los
Angeles, Calif.). Human albumin solution 5% was made by dilution of
25% human albumin (Baxter) with sterile water for injection.
Prion-Removal Columns
[0241] The prion removal columns (1-ml) used in this study were
commercially available PIKSI.RTM. (Protein isolation Kit for
Sorbent Identification) kit columns containing Toyopearl Amino
650CU resin (sample AMN31) supplied by ProMetic Biosciences, Ltd
(Cambridge, UK).
Flow-Through Conditions
[0242] For the study on formulation suspension, the total
flow-through volume was about 500 mL, which was 500 times the
column volume (1-ml). The flow rate was about 0.5 ml/min Total
flow-through time was more than 16 hours. Samples were taken every
two hours. No clogging of the column was observed.
[0243] For the study on albumin solution, the total flow-through
volume was at least 100 mL, which was 100 times the column volume
(1-ml). The flow rate was about 0.5 ml/min Total flow-through time
was more than 3 hours. Samples were taken every hour. No clogging
of the column was observed.
Results and Discussion
[0244] The results from the prion-removal feasibility study on the
formulated suspension are summarized in Table 6 and Table 7. The
physical and chemical testing results for the formulated suspension
for Abraxane.TM. show no significant differences between the pre
and post-column suspension, in terms of particle size, pH,
paclitaxel assay and impurities, human albumin assay, and human
albumin composition. Likewise, the physical and chemical testing
results for the formulated suspension for sugar-paclitaxel
formulation show no significant differences between the pre and
post-column suspension, in terms of particle size, pH, paclitaxel
assay and impurities, human albumin assay, human albumin
composition, sucrose, and EDTA.
TABLE-US-00006 TABLE 6 Prion-Removal Feasibility Study of Abraxane
.TM. Formulated Suspension Sample I.D. Physical and Chemical Pre-
Post Column Properties Column #1 #2 #3 #4 #5 #6 #7 Particle Size
Mean 131 132 132 133 133 133 131 130 (nm) <5% 82 86 85 86 85 87
84 82 <95% 193 190 191 192 192 191 191 190 <99.9% 254 250 251
252 253 250 251 250 pH 7.0 7.0 7.1 7.0 7.0 7.0 7.0 7.0 Paclitaxel
(mg/mL) 6.9 6.9 6.9 6.8 6.8 6.8 6.8 6.8 Human Polymer 3.99 3.86
3.93 3.95 3.95 3.92 3.88 3.76 Albumin Oligomer 1.32 1.36 1.32 1.34
1.31 1.32 1.34 1.39 Composition Dimer 5.95 5.96 6.00 5.98 6.00 5.99
5.98 5.96 (%) Monomer 88.29 88.37 88.24 88.27 88.27 88.28 88.29
88.38 Total Human Albumin 55 56 56 56 56 56 56 56 (mg/mL) Impurity
7-Epi 0.08 0.08 NA NA 0.09 NA NA 0.09 (%) Total 0.25 0.25 NA NA
0.26 NA NA 0.26 * NA: Data not Available.
TABLE-US-00007 TABLE 7 Prion-Removal Feasibility Study of
Sugar-EDTA-Paclitaxel Formulated Suspension Sample I.D. Physical
and Chemical Pre- Post Column Properties Column #1 #2 #3 #4 #5 #6
#7 Particle Size Mean 129 130 133 131 132 132 130 132 (nm) <5%
80 81 85 82 84 85 81 85 <95% 191 192 193 193 192 191 192 191
<99.9% 252 253 253 254 253 251 253 250 pH 6.8 6.8 6.8 6.9 6.8
6.8 6.9 6.9 Paclitaxel (mg/mL) 6.5 6.5 6.5 6.5 6.5 6.6 6.6 6.5
Human Polymer 3.90 3.90 3.91 3.89 3.91 3.93 3.94 3.93 Albumin
Oligomer 1.92 1.89 1.92 1.90 1.91 1.89 1.44 1.41 Composition Dimer
6.14 6.11 6.11 6.10 6.10 6.09 6.09 6.10 (%) Monomer 88.02 88.10
88.07 88.09 88.06 88.09 88.03 88.04 Total Human Albumin 55 55 54 54
55 55 54 54 (mg/mL) Impurity 7-Epi 0.08 0.08 NA NA 0.08 NA NA 0.09
(%) Total 0.25 0.25 NA NA 0.25 NA NA 0.26 Sucrose (mg/mL) 31.8 31.8
NA NA 31.6 NA NA 31.9 EDTA (mg/mL) 0.071 0.071 NA NA 0.070 NA NA
0.070 * NA: Data not Available.
[0245] The results from the prion-removal feasibility study on the
albumin solutions are summarized in Table 8. There are no
significant differences between the pre and post-column solution,
in terms of human albumin assay and human albumin composition.
TABLE-US-00008 TABLE 8 Prion-Removal Feasibility Study of Human
Albumin Solution HA (%) HA Total Sample I.D. Polymer Oligomer Dimer
Monomer (mg/mL) Lot 28203-37A 25% Pre column 3.66 0.28 2.38 93.69
248 Human Post column #1 3.60 0.25 2.41 93.74 248 Albumin Post
column #2 3.63 0.28 2.38 93.71 250 (Baxter) Post column #3 3.64
0.24 2.41 93.71 247 Post column #4 3.64 0.26 2.40 93.70 248 Lot
28203-37B 20% Pre column 4.85 0.51 3.25 91.39 199 Human Post column
#1 4.78 0.49 3.25 91.48 199 Albumin Post column #2 4.78 0.51 3.25
91.45 200 (Grifols) Post column #3 4.86 0.51 3.25 91.38 200 Lot
28203-37C 5% Human Pre column 3.66 0.27 2.32 93.75 52 Albumin Post
column #1 3.33 0.25 2.31 94.10 51 (Baxter) Post column #2 3.48 0.27
2.30 93.94 51 Post column #3 3.54 0.27 2.31 93.89 51 Post column #4
3.50 0.27 2.31 93.92 52
[0246] This study demonstrates that the prion-removal column
treatment has no adverse impact on the physical and chemical
properties of human albumin solution and formulated suspensions for
both Abraxane.TM. and sugar-paclitaxel formulation.
Example 3
TSE Removal by Prion Reduction Resins for 20% Albumin
[0247] In this study, potential TSE removal by prion reduction
resins (PRDT column; ProMetic Biosciences, Ltd) in 20% (w/v)
albumin (Grifols.RTM.) was evaluated. Starting material for the TSE
removal process step was spiked with a model TSE agent. The process
step was performed in the VirusSure laboratories (Virusure
Forschung und Entwicklung GmbH, Vienna, Austria). Various fractions
were collected during performance of the process step, and the TSE
removal capacity of the process was calculated based on a
determination of levels of TSE agent using a Western Blot assay for
the detection of PrPsc.
[0248] This study followed and referenced the following guidelines,
including 1) CPMP/BWP/268/95 (revised in 1996), Note for Guidance
on Virus Validation Studies: The Design, Contribution, and
Interpretation of Studies Validating the Inactivation and Removal
of Viruses; 2) CPMP/BWP/5136/03 Guideline on the Investigation of
Manufacturing Processes for Plasma-Derived Medicinal Products with
Regards to vCJD Risk; 3) OECD Principles of Good Laboratory
Practice as outlined in ENV/MC/CHEM(98)17, revised in 1997; 4) 21
CFR part 58, Good Laboratory Practice, US FDA; 5) EU directive
2004/9/EG, Inspection und Uberprufung der Guten Laborpraxis (GLP);
6) EU Directive 2004/10/EG, Anwendung der Grundsatze der guten
Laborpraxis und zur Kontrolle ihrer Anwendung bei Versunchen mit
chemischen Stoffen; 7) Austrian BGBI. II, 211. Verordnung,
Chemikalien-GLP-Inspektionsverordnung, Jahrgang 2000; and 8)
Austrian BGBI. II, 450. Verordnung, gute Laborpraxis 2006, Jahrgang
2006.
Materials and Methods
[0249] The following test articles, reagents, and materials were
used during the course of this study for the investigation of TSE
removal by the prion reduction resins (PRDT column) for 20% albumin
(Grifols.RTM.).
[0250] Grifols.RTM. Albumin (human) (USP 20% solution (Lot:
IBAB7GX001/TA09/0122)) was used for spiked run and interference
testing.
[0251] Disposable SepFast.TM. Column (ProMetic Biosciences Ltd.)
containing 5 ml of prion removal resin packed in 9% saline was used
for process run.
[0252] Various buffers were prepared. They include the following:
[0253] 1) 0.9% NaCL (9 g/l) was prepared as equilibration buffer
for the prion reduction resins; [0254] 2) Tris Buffered Saline
(TBS) was prepared as re-suspension of the resins following
chromatography; [0255] 3) 2M NaCl (116.88 g/l) was prepared as
prion reduction resin regeneration buffer; [0256] 4) NaOH (0.1 M,
0.5M, and 1.0 M) buffers were prepared for pH adjustments of spiked
study samples and process intermediates inactivation of infectious
material; and [0257] 5) HCL (0.1 M and 1.0 M) buffers were prepared
for pH adjustments of spiked study samples and process
intermediates.
263 Scrapie
[0258] Strain 263K Hamster Adapted scrapie (0.5% sarkosyl treated)
was used in this study. The 263K strain of hamster adapted scrapie
provides the advantages of high titres in the brains of hamsters.
Typical titres for a 10% brain homogenate are in the range of
10.sup.8-10.sup.10 ID.sub.50 units per ml. The PrPsc protein
deposited by this agent is relatively resistant to Proteinase K
digestion, allowing the possibility of distinguishing between the
non-disease associated form of the protein PrPc. The seed 263K
strain of hamster adapted scrapie was supplied as a 10% homogenate
by the laboratory of Dr. Robert Rohwer (Baltimore Research and
Education Foundation, Mail Stop 151-A, 10 North Greene Street,
Baltimore, Md. 21201, USA). A 0.5% sarkosyl-treated fraction was
selected for this experiment. This fraction was prepared from a
crude brain homogenate (from which the microsomal/cytosolic 263K
fraction had already been removed) by treatment with 0.5% sarkosyl
followed by differential centrifugation to remove larger
aggregates, leaving only the detergent solubilized fragments in the
supernatant. The 0.5% sarkosyl-treated fraction was prepared to
mimic detergent solubilized contamination (i.e., as found in
solvent detergent containing processes), and this type of fraction
has been widely used in prion clearance studies for human plasma
and recombinant products.
Western Blot Assay for the Detection of PrPsc
[0259] The Western blot assay for detection of PrPsc was used for
the semi-quantitative determination of TSE levels (PrPsc) in the
various samples. The dynamic range of the Western blot assay is
normally in the region of 4-5 log.sub.10 dilutions before signal is
lost, and thus the assay is less sensitive than the hamster
bioassay. However, the Western blot assay is a useful tool in
assessing prion removal by biopharmaceutical manufacturing
processes.
[0260] In the Western blot assay, the sample was first submitted to
digestion with Proteinase K to remove the normal form of the
protein, PrPc (all process samples were digested using a Proteinase
K concentration of 83 pg/ml). After blocking of the proteolytic
reaction, the sample was mixed with SDS-buffer and boiled to
denature the PrPsc from its aggregated form. 0.5 log.sub.10
dilutions of the sample are then prepared and loaded onto a
SDS-PAGE gel along with a molecular weight marker. Following
electrophoresis, the gel was Western blotted onto a PVDF membrane,
followed by blocking and probing with antibodies allowing the
detection of bound PrPsc protein with the antibody 3F4. The 263K
strain of scrapie results in a characteristic banding pattern in
the region of 25-33 KDa, which assists in confirming the presence
of the PrPsc protein in samples. The end point of titre of the
sample was defined as the first dilution where no signal was
observed on the Western blot.
Calculation of Reduction Factors
[0261] Reduction factors (RF) were calculated as follows:
RF=(V.sub.1.times.T.sub.1)/(V.sub.2.times.T.sub.2)
Where:
[0262] V.sub.1 and T.sub.1 are the volume and titre of the starting
material respectively, and
[0263] V.sub.2 and T.sub.2 are the volume and titre of the product
fraction respectively
[0264] In logarithmic terms, this equation can be expressed as:
Log.sub.10[RF]=[ Log.sub.10(V.sub.1)+Log.sub.10(T.sub.1)]-[
Log.sub.10(V.sub.2)+Log .sub.10(T.sub.2)].
[0265] Reduction factors were rounded to 1 decimal place only after
the final calculation.
Interference Testing
[0266] The starting material was tested undiluted and followed by a
1.0 log.sub.10 pre-dilution in TBSA. Following spiking with 263K to
a final concentration equivalent to a titre within approximately 2
log.sub.10 of the end point of the Scrapie stock used for spiking,
the undiluted and pre-diluted starting material samples were
centrifuged at 15.558.times.g for 60 min at room temperature.
Following centrifugation, the supernatant was carefully decanted
and the pellets re-suspended with TBSA in 1/10 of the original
centrifuged sample volume (equivalent to no effective concentration
for 1.0 log.sub.10 pre-diluted sample and equivalent to a 10-fold
concentration for the undiluted sample). Protease K digestion and
Western blotting was then performed following the standard
protocol. The regeneration and column samples were diluted 0.5
log.sub.10 or tested undiluted respectively prior to analysis by
Western blotting (i.e., without centrifugation).
Adjustment of pH
[0267] Prior to aliquotting and storage at .ltoreq.-60.degree. C.,
samples were checked to be at pH 6-8 (pH adjustment was not
required for any of the samples).
Equipment
[0268] The following main equipment items were used for performance
of this study. Sterile Class II Biohazard Safety Cabinet, Sanyo
& Angelantoni .ltoreq.-60.degree. C. freezers, Angelantoni
2-8.degree. C. fridges and .ltoreq.-15.degree. C. freezers,
Sartorius or Kern (analytical) balance and printer, Hanna
electronic thermometer, Mettler Toledo pH meter, Grant or Selecta
water baths, Oregon Laboratory Timer, Hettich microfuge, BioRad
Criterion Electrophoresis Cell, BioRad Criterion Blotter, AKTA
Chromatography system, Wealtec Power Supply, Agfa Film Developer,
and Biotoolomics column packed with PRDT resin.
Process Flow
[0269] The process flow scheme along with the samples collected was
depicted in FIG. 4. The volumes of the respective samples can be
found in Table 9 in the Results Section.
[0270] In preparation of the process flow, the Laminar Flow (LF)
safety cabinet was cleaned and turned on for at least 10 to 15
minutes to equilibrate. The water bath was equilibrated to
30.+-.2.degree. C. and the starting material equilibrated until a
temperature of 29.5.degree. C. was reached. The 0.5% Sarkosyl
solubilized spike was thawed in the same water bath and as soon as
it was thawed, placed on ice until use. The PRDT column was
equilibrated to ambient temperature (23.0.+-.5.0.degree. C.)
overnight.
[0271] The column inlet tubing (top) was connected to the outlet
from the AKTA. The tubing from the column outlet (bottom) was fed
to an appropriate collection vessel (beaker or 50 ml disposable
centrifuge tube). All tubing was already primed with WFI (Water for
Injection) such that no air bubbles were introduced into the
system.
[0272] The column was first equilibrated with 5 column volumes (CV)
of WFI, and then with 10 CV of Equilibration Buffer. The target
flow rate throughout was 2.0.+-.0.1 ml per minute.
Sample Preparation and Prion Reduction Resin Chromatography
[0273] 50.7 ml of the equilibrated starting material was spiked
with 0.51 ml of 0.5% sarkosyl solubilized 263K homogenate. The pH
was then be checked and found to be within the target range of
6.9-7.4. Subsequently, a 0.5 ml aliquot was removed (sample SSM)
and aliquotted and stored at .ltoreq.-60.degree. C.
[0274] The spiked sample was then applied to the above equilibrated
PRDT column at a flow rate of 1.8.+-.0.1 ml per min and the flow
through collected as the following fractions:
TABLE-US-00009 Sample ID Volume Sample Description EI 2.1 ml Eluate
1 (-0-2 ml) E2 3.2 ml Eluate 2 (-2-5 ml) E3 5.4 ml Eluate 3 (-5-10
ml) E4 16.1 ml Eluate 4 (-10-25 ml) E5 30.3 ml Eluate 5 (-25-44
ml)
[0275] Collection of E1 began once the absorbance had reached 80%
of the full scale deflection. For each run, the flow through
fraction was collected (the volume of each Eluate sample collected
was determined by weighing) After loading of the spiked Albumin
solution, the column was washed with 10.0 ml of Equilibration
Buffer. Collection of the E5 sample was stopped once the absorbance
had dropped below 80% of full scale deflection. Following the
Equilibration Buffer wash, the column was regenerated using
>20.0 ml of 2M NaCl (sample REG), and following regeneration the
resin removed and resuspended in 5 ml of TBS (sample COL).
Results
[0276] Samples from Spiked Runs
[0277] Table 9 below lists the samples that were collected from the
spiked run along with the volume of each sample. Where the sample
size was determined by weight, then a density of 1.0 g/ml was
assumed to allow a calculation of the volume for each sample. All
samples were stored aliquotted at -60.degree. C. until analysis. A
scanned reproduction of the chromatography profile from the spiked
run is shown in FIG. 5.
TABLE-US-00010 TABLE 9 Summary of volumes collection during the
process run Actual volume of sample collected at Sample Description
Sample Code point of collection (ml) Spiked Start Material
SP0913-SSM 51.2 (Sarkosyl-treated spike) Eluate 1: 0-2 ml SP0913-E1
2.1 Eluate 2: 2-5 ml SP0913-E2 3.2 Eluate 3: 5-10 ml SP0913-E3 5.4
Eluate 4: 10-25 ml SP0913-E4 16.1 Eluate 5: 25--44 ml SP0913-E5
30.3 Regeneration fraction SP0913-Reg 27.5 Resin sample SP0913-Col
10.0
Interference Results
[0278] To overcome interference all samples except the regeneration
and resin samples were diluted by 1.0 log.sub.10 with TBS
containing 0.1% BSA followed by a centrifugation and a 1/10
concentration. The undiluted sample tested for interference at a
10.times. concentration displayed strong interference. For the
regeneration samples a 0.5 log.sub.10 dilution was prepared prior
to testing to reduce the concentration of NaCl. For the resin
samples, as the resin was resuspended in TBS buffer, these samples
were tested without pre-dilution.
[0279] The dilution of sample required to overcome interference
with albumin was made using 1.0 Log10 predilution with
centrifugation and resuspension in 1/10.sup.th original volume. See
FIG. 6.
Prion Titration Data and Calculation of Reduction Factors
[0280] The calculation of the prion reduction factors for the
process runs is shown in Table 10. The dilution of sample used in
order to overcome interference is also shown in Table 10.
TABLE-US-00011 TABLE 10 Summary of Sample Titration Data and Prion
Reduction Factors Log Volume of dilution Sample Correction Volume
of Samples pH Sample for End Volume Volume Factor for Samples After
Adjustment Description interf- point at Further Volume Before pH pH
Correction Log Total Sample ID Spiked erence titre collection
Processed processed* Adjustment Adjustment Factor volume Load
SP0913- Spiked start 0.0 2.5 51.2 ml 50.7 ml 0.99 -- 1.7 4.205 SSM
material SP0913-E1 Eluate Fraction 1 0.0 0.0 50.7 ml -- -- 1.7
.ltoreq.1.705 SP0913-E2 Eluate Fraction 2 0.0 1.0 50.7 ml -- -- 1.7
2.705 SP0913-E3 Eluate Fraction 3 0.0 1.0 50.7 ml -- -- 1.7 2.705
SP0913-E4 Eluate Fraction 4 0.0 1.5 50.7 ml -- -- 1.7 3.205
SP0913-E5 Eluate Fraction 5 0.0 1.0 50.7 ml -- -- 1.7 2.705
SP0913-Reg Regeneration 0.5 0.0 27.5 ml -- -- 1.4 .ltoreq.1.939
Fraction SP0913-Col Column Resin 0.0 3.0 10.0 ml -- -- 1.0 4.000
*Correction factors are applied in the final log volume
calculation. The correction factors applicable for each sample are
the correction factors of the respective sample itself along with
all correction factors for samples listed below that sample. **A
volume of 50 ml was used for the purpose of calculating the
reduction factor for each eluate sample, to ensure a direct
comparison with the volume loaded onto the column. Reduction Factor
1: Sample # Sample ID Sample Description Log Titre RF Sample 1
SP0913-SSM Spiked start material 4.205 Sample 2 SP0913-E1 Eluate
Fraction 1 1.705 .gtoreq.2.50 Comments: RF for eluate 1 relative to
spiked start material Reduction Factor 2: Sample # Sample ID Sample
Description Log Titre RF Sample 1 SP0913-SSM Spiked start material
4.205 Sample 6 SP0913-E5 Eluate Fraction 5 2.705 1.50 Comments: RF
for eluate 5 relative to spiked start material
[0281] A reduction factor of 2.5 log.sub.10 relative to the spiked
start material was calculated for E1 sample, which represented the
first 2.0 ml passed over the PRDT column. In samples E2 to E5, a
breakthrough of PrPsc into the flow through fraction was observed
(reduction factor 1.5 log.sub.10 for sample E5). No PrPsc was
detected in the regeneration sample and the equivalents of PrPsc
bound to the PRDT resin was discussed in more detail in the section
of calculation of the prion binding capacity of the PRDT resin
below.
Calculation of the Prion Binding Capacity of the Prion Reduction
Resins
[0282] In order to estimate the binding capacity for the prion
reduction resins (PRDT resin) for infectious prion protein, the
titres observed in the Western blot testing were related to
infectious titres. The Western blot assay used a 263K stock of
known titre. A dilution series of the reference stock was prepared
and tested in the Western blot assay. After plotting the obtained
titres versus the respective titres observed in the hamster
bioassay, a linear regression analysis was performed to assess the
relation between the two test systems. The slope and intercept for
the regression line were calculated to be 1.0667 and -4.5867,
respectively. The regression parameters were used to convert
Western blot titres into infectious titres using the following
formula:
Titre.sub.[Bioassay]=(Titre.sub.[WesternBlot]+4.5867)/(1.0667).
[0283] Once the infectious titre per ml was calculated, the total
prion bound to the column could be determined. The calculated prion
protein binding capacity of the prion reduction resin is shown in
the Table 11 below. The capacity was determined using the amount of
PrPsc observed directly bound to the prion reduction resin (samples
Reg and Col).
TABLE-US-00012 TABLE 11 Prion binding capacity of Prion Reduction
Resin (PRDT Resin) Binding capacity Infectious PrPsc per Sample ID
Sample Description Total PrPsc (log.sub.10 ID.sub.50 total) = ml
Albumin spiked with a sarkosyl solubilised spike
(log.sub.10.sup.WB).sup.# Total PrPsc + 4.1 (ID.sub.50/ml)*
SP0913-SSM 50 ml albumin spiked with 0.5 ml 4 2 8.3 -- of a
sarkosyl solubilised spike SP0912-Reg 2M NaCl salt wash (sample
.ltoreq.1.9 .ltoreq.6.0 .ltoreq.5.3 log.sub.10/ml volume 25.1 ml)
5.0 ml PRDT Resin; 50 ml of SP0912-SSM sample loaded (Target flow
rate: 1.8 ml/min) SP0912-Col Column resin (sample volume 4.0 8.1
7.4 log.sub.10/ml 8.7 ml) .sup.#Total PrPSc includes corrections
for volume of sample as well as concentration/dilution prior to
testing Total load based on a final volume of 50 ml loaded onto the
PRDT column *Based on a 5.0 ml column size
[0284] The total binding of PrPsc to the PRDT resin following a 2M
salt wash was 7.4 log.sub.10 ID.sub.50/ml. No PrPsc was detected in
the salt wash fractions by western blot. Significant PrPsc removal
(.gtoreq.2.5 log.sub.10) was also observed.
[0285] When performing interference testing, the starting material
was tested undiluted, with centrifugation and 10-fold
concentration. This was performed to allow an evaluation of the
possibility for concentrating the samples and thus achieving higher
reduction factors.
Example 4
TSE Removal by the Prion Reduction Resins for 25% Albumin
[0286] In this study, potential TSE removal by the prion reduction
resins (PRDT column) in 25% albumin (Baxter, Deerfield, Ill.) was
evaluated. Materials and Methods used were the same as described in
Example 3, with the exception that Albumin (Human) USP 25% solution
from Baxter (Lot: LA07D051AB/TA09/0123) was used as the starting
material for spiked run and interference testing.
Process Flow
[0287] The downscale process was established and performed at the
ViruSure facilities. The process flow scheme along with the samples
collected was depicted in FIG. 7. The volumes of the respective
samples can be found in Table 12 in the Results Section. The
parameters and procedures used in preparation of the process flow,
including connecting the AKTA and column, column equilibration are
the same as described in Example 3.
Sample Preparation and Prion Reduction Resin Chromatography
[0288] 50.1 ml of the equilibrated starting material was spiked
with 0.51 ml of 0.5% sarkosyl solubilized 263K homogenate. The pH
was then be checked and found to be within the target range of
6.9-7.4 with 0.1 M HCL or 0.5 M NaOH. The pH of the spiked starting
material (6.85) was outside the target range of 6.9-7.4 (see
Deviations Section). After the addition of 65 .mu.L 0.1 M NaOH and
50 .mu.l 1 M NaOH, the pH value remained unchanged, so the decision
was taken to load the starting material at this pH. Subsequently, a
0.5 ml aliquot was removed (sample SSM) and aliquotted and stored
at .ltoreq.-60.degree. C.
[0289] The spiked sample was then applied to the above equilibrated
PRDT column at a flow rate of 1.8.+-.0.1 ml per min, and the flow
through were collected as the following fractions:
TABLE-US-00013 Sample ID Volume Sample Description EI 2.3 ml Eluate
1 (-0-2 ml) E2 3.3 ml Eluate 2 (-2-5 ml) E3 5.6 ml Eluate 3 (-5-10
ml) E4 16.1 ml Eluate 4 (-10-25 ml) E5 32.7 ml Eluate 5 (-25-44
ml)
[0290] Collection of E1 began once the absorbance had reached 80%
of the full scale deflection. For each run, the flow through
fraction was collected (the volume of each Eluate sample collected
was determined by weighing) After loading of the spiked albumin
solution, the column was washed with 10.0 ml of Equilibration
Buffer. Collection of the E5 sample was stopped once the absorbance
had dropped below 80% of full scale deflection. Following the
Equilibration Buffer wash, the column was regenerated using
>20.0 ml of 2M NaCl (sample REG), and following regeneration the
resin removed and resuspended in 5 ml of TBS (sample COL).
Results
[0291] Samples from Spiked Runs
[0292] Table 12 below lists the samples that were collected from
the spiked run along with the volume of each sample. Where the
sample size was determined by weight, then a density of 1.0 g/ml
was assumed to allow a calculation of the volume for each sample.
All samples were stored aliquotted at .ltoreq.-60.degree. C. until
analysis. A scanned reproduction of the chromatography profile from
the spiked run is shown in FIG. 8.
TABLE-US-00014 TABLE 12 Summary of volumes collection during the
process run Actual volume of sample collected at Sample Description
Sample Code point of collection (ml) Spiked Start Material
SP0912-SSM 50.6 (Sarkosyl-treated spike) Eluate 1: 0-2 ml SP0912-E1
2.3 Eluate 2: 2-5 ml SP0912-E2 3.3 Eluate 3: 5-10 ml SP0912-E3 5.6
Eluate 4: 10-25 ml SP0912-E4 16.1 Eluate 5: 25--44 ml SP0912-E5
32.7 Regeneration fraction SP0912-Reg 25.1 Resin sample SP0912-Col
8.7
Interference Results
[0293] To overcome interference with albumin, all samples except
the regeneration and resin samples were diluted by 1.0 log.sub.10
with TBS containing 0.1% BSA followed by a centrifugation and a
1/10 concentration. The undiluted sample tested for interference at
a 10-fold concentration displayed strong interference. For the
regeneration samples, a 0.5 log.sub.10 dilution was prepared prior
to testing to reduce the concentration of NaCl. For the resin
samples, as the resin was resuspended in TBS buffer, these samples
were tested without pre-dilution.
[0294] The dilution of sample required to overcome interference was
made using 1.0 Log10 predilution with centrifugation and
resuspension in 1/10.sup.th of the original volume. See FIG. 9.
Prion Titration Data and Calculation of Reduction Factors
[0295] The calculation of the prion reduction factors for the
process runs is shown in Table 13. The dilution of sample used in
order to overcome interference is also shown in Table 13.
TABLE-US-00015 TABLE 13 Summary of Sample Titration Data and Prion
Reduction Factors Log Volume of dilution Sample Correction Volume
of Samples pH Sample for End Volume Volume Factor for Samples After
Adjustment Description interf- point at Further Volume Before pH pH
Correction Log Total Sample ID Spiked erence titre collection
Processed processed* Adjustment Adjustment Factor volume Load
SP0912- start material 0.0 2.0 50.6 ml 50.2 ml 0.99 50.6 ml 50.7 ml
1.00 1.7 3.702 SSM SP0912-E1 Eluate Fraction 1 0.0 0.0 50.2 ml --
-- 1.7 .ltoreq.1.701 SP0912-E2 Eluate Fraction 2 0.0 0.5 50.2 ml --
-- 1.7 2.201 SP0912-E3 Eluate Fraction 3 0.0 1.0 50.2 ml -- -- 1.7
2.701 SP0912-E4 Eluate Fraction 4 0.0 1.0 50.2 ml -- -- 1.7 2.701
SP0912-E5 Eluate Fraction 5 0.0 1.0 50.2 ml -- -- 1.7 2.701
SP0912-Reg Regeneration 0.5 0.0 25.1 ml -- -- 1.4 .ltoreq.1.900
Fraction SP0912-Col Column Resin 0.0 3.0 8.7 ml -- -- 0.9 3.940
*Correction factors are applied in the final log volume
calculation. The correction factors applicable for each sample are
the correction factors of the respective sample itself along with
all correction factors for samples listed below that sample. **A
volume of 50 ml was used for the purpose of calculating the
reduction factor for each eluate sample to ensure a direct
comparison with the volume loaded onto the column. Reduction Factor
1: Sample # Sample ID Sample Description Log Titre RF Sample 1
SP0912-SSM Spiked start material 3.702 Sample 2 SP0912-E1 Eluate
Fraction 1 1.701 .gtoreq.2.00 Comments: RF for eluate 1 relative to
spiked start material Reduction Factor 2: Sample # Sample ID Sample
Description Log Titre RF Sample 1 SP0912-SSM Spiked start material
3.702 Sample 6 SP0912-E5 Eluate Fraction 5 2.701 1.00 Comments: RF
for eluate 5 relative to spiked start material
[0296] A reduction factor of .gtoreq.2.5 log.sub.10 relative to the
spiked start material was calculated for E1 sample, which
represented the first 2.0 ml passed over the PRDT column In samples
E2 to E5, a breakthrough of PrPsc into the flow through fraction
was observed (reduction factor 1.5 log.sub.10 for sample E5). No
PrPsc was detected in the regeneration sample and the equivalents
of PrPsc bound to the PRDT resin was discussed in more detail in
the section of calculation of the prion binding capacity of the
PRDT resin below.
Calculation of the Prion Binding Capacity of the Prion Reduction
Resins
[0297] In order to estimate the binding capacity for the prion
reduction resins (PRDT resin) for infectious prion protein, the
titres observed in the Western blot testing were related to
infectious titres. The Western blot assay used a 263K stock of
known titre. A dilution series of the reference stock was prepared
and tested in the Western blot assay. After plotting the obtained
titres versus the respective titres observed in the hamster
bioassay, a linear regression analysis was performed to assess the
relation between the two test systems. The slope and intercept for
the regression line were calculated to be 1.0667 and -4.5867,
respectively. The regression parameters were used to convert
Western blot titres into infectious titres using the following
formula:
Titre.sub.[Bioassay]=(Titre.sub.[WesternBlot]+4.58671/(1.0667).
[0298] Once the infectious titre per ml was calculated, the total
prion bound to the column was determined. The calculated prion
protein binding capacity of the PRDT resin is shown in the Table 14
below. The capacity was determined using the amount of PrPsc
observed directly bound to the PRDT resin (samples Reg and
Col).
TABLE-US-00016 TABLE 14 Prion binding capacity of PRDT Resin
Infectious Binding PrPsc capacity Sample ID Sample Description
(log.sub.10 ID.sub.50 total) = per Albumin spiked with a sarkosyl
solubilized Total PrP.sup.sc Total PrPsc + ml spike
(log.sub.10.sup.WB).sup.# 4.1 (ID.sub.50/ml)* SP0912-SSM 50 ml
plasma spiked with 0.5 ml 3.7 7.8 -- of a sarkosyl solubilized
spike 5.0 ml PRDT Resin; 50 ml of SP0912-SSM sample loaded (Target
flow rate: 1.8 ml/min) SP0912-Reg 2M NaCl salt wash (sample
.ltoreq.1.9 .ltoreq.6.0 .ltoreq.5.3 log.sub.10/ml volume 25.1 ml)
SP0912-Col Column resin (sample volume 3.9 8.0 7.3 log.sub.10/ml
8.7 ml) .sup.#Total PrPSc includes corrections for volume of sample
as well as concentration/dilution prior to testing Total load based
on a final volume of 50 ml loaded onto the PRDT column *Based on a
5.0 ml column size
[0299] The total binding of PrPsc to the PRDT matrix following a 2M
salt wash was 7.4 log.sub.10 ID.sub.50/ml. No PrPsc was detected in
the salt wash fractions by Western blot. Significant PrPsc removal
(.gtoreq.2.0 log.sub.10) was also observed.
[0300] The pH of the spiked starting material was 6.85. After the
addition of 65 .mu.l 0.1 M NaOH and 50 .mu.l 1 M NaOH the pH value
remained unchanged. The effect probably resulted from the
significant buffering capacity of 25% albumin
[0301] When performing interference testing, the starting material
was tested undiluted, with centrifugation and 10-fold
concentration. This was in addition to the interference testing
described in the study plan, and was performed to allow an
evaluation of the possibility for concentrating the samples and
thus achieving higher reduction factors.
Example 5
In-Process and Stability Analyses for Human Albumin Treated with
Prion Removal
[0302] In this experiment, the effect of prion-removal column on
the concentration and composition of human albumin was evaluated.
In two separate experiments, one liter of albumin was treated with
the PRIOCLEAR.TM. B column (50 ml) (ProMetic Biosciences). The
untreated sample, sample collected at the beginning of the run, and
sample collected at the end of the run were then subject to
concentration and composition analyses. The results are shown in
Tables 15 and 16.
TABLE-US-00017 TABLE 15 Result for Human Albumin in Experiment One
HA Concentration, HA Composition (%) Sample Description mg/mL
Monomer Dimer Polymer Oligomer HA, Untreated 252 93.73 2.71 3.56 ND
HA Post Prion-removal Column, 253 93.93 2.71 3.36 ND Sample
Collected at the Beginning of Run HA Post Prion-removal Column, 255
93.76 2.72 3.52 ND Sample Collected at the End of Run (1 L)
TABLE-US-00018 TABLE 16 Result for Human Albumin in Experiment Two
HA Concentration, HA Composition (%) Sample Description mg/mL
Monomer Dimer Polymer Oligomer HA, Untreated 206 92.15 3.08 4.34
0.44 HA Post Prion-removal Column, 181 92.26 3.05 4.27 0.42 Sample
Collected at the Beginning of Run HA Post Prion-removal Column, 206
92.19 3.08 4.30 0.43 Sample Collected at the End of Run (1 L)
[0303] No significant differences on the albumin were observed
before and after the prion removal process.
[0304] We further evaluated the in-process stability of human
albumin treated for prion-removal and Abraxane.RTM. suspensions
manufactured using the human albumin treated for prion-removal. The
result is shown in Tables 17 and 18.
TABLE-US-00019 TABLE 17 In-process Stability of Treated HA
Solutions from Experiment One and Abraxane Suspensions Manufactured
using Treated HA HA Sample Storage Concentration, HA Composition
(%) Description Conditions mg/mL Monomer Dimer Polymer Oligomer
Concentrated 0-time 255 93.79 2.73 3.48 ND HA Solution 24 hrs at
5.degree. C. 257 93.76 2.74 3.50 ND 48 hrs at 5.degree. C. 255
93.74 2.77 3.49 ND 72 hrs at 5.degree. C. 255 93.76 2.73 3.50 ND
Diluted HA 0-time 51 93.79 2.73 3.47 ND Solution 24 hrs at
5.degree. C. 51 93.82 2.71 3.47 ND 48 hrs at 5.degree. C. 51 93.83
2.70 3.47 ND 72 hrs at 5.degree. C. 51 93.85 2.69 3.46 ND Abraxane
.RTM. 0-time 57 88.39 7.14 3.26 1.22 Suspension 24 hrs at
25.degree. C. 57 86.32 8.19 3.25 2.25 before 36 hrs at 25.degree.
C. 55 88.22 8.44 2.13 1.21 Lyophilization
TABLE-US-00020 TABLE 18 In-process Stability of Treated HA
Solutions from Experiment Two and Abraxane Suspensions Manufactured
using Treated HA HA Sample Storage Concentration, Description
Conditions mg/mL HA Composition (%) Sample Storage Concentration,
Monomer Dimer Polymer Oligomer Concentrated 0-time 206 92.22 3.09
4.25 0.44 HA Solution 24 hrs at 5.degree. C. 202.7 92.28 3.03 4.26
0.43 48 hrs at 5.degree. C. 204.1 92.32 2.99 4.25 0.44 72 hrs at
5.degree. C. 200.3 92.14 3.06 4.35 0.44 Diluted HA 0-time 51.4
92.28 3.04 4.26 0.42 Solution 24 hrs at 5.degree. C. 51.1 92.30
3.01 4.27 0.43 48 hrs at 5.degree. C. 52.1 92.38 2.95 4.24 0.43 72
hrs at 5.degree. C. 51.7 92.45 2.93 4.22 0.41 Abraxane .RTM. 0-time
57 85.78 8.30 3.97 1.94 Suspension 24 hrs at 25.degree. C. 57.1
85.28 8.67 3.95 2.10 before 36 hrs at 25.degree. C. 57.4 87.72 7.62
3.43 1.23 Lyophilization
[0305] We further compared the accelerated stability of human
albumin in finished Abraxane.RTM. products manufactured using human
albumin treated for prion-removal (Pilot plant batch using human
albumin treated for Prion removal) and finished Abraxane.RTM.
products manufactured using human albumin not treated for
prion-removal (Abraxane Exhibit lot, Abraxane Validation lot, and
Abraxane pilot plant batch). The result is shown in Table 19.
TABLE-US-00021 TABLE 19 Accelerated Stability of HA in Abraxane
Finished Products Manufactured using HA Treated for Prion-removal.
Comparison with Finished Products Manufacturing using Untreated HA
HA Composition Monomer Dimer Polymer Oligomer Change Change Change
Change Sample Storage During During During During Description
Experiments Conditions % Storage % Storage % Storage % Storage
Pilot Plant Experiment 1 0-time 84.66 N/A 9.33 N/A 2.76 N/A 3.25
N/A Batch using 2 W at 55.degree. C. 70.39 -14.27 17.16 7.83 8.77
6.01 3.67 0.42 HA Treated 1 M at 55.degree. C. 61.83 -22.83 19.34
10.01 14.55 11.79 4.27 1.02 for Prion 1 M at 40.degree. C. 76.22
-8.44 14.58 5.25 5.71 2.95 3.49 0.24 Removal Abraxane Experiment 1
0-time 86.70 N/A 9.40 N/A 2.1 N/A 2.50 N/A Exhibit Lot 3 M at
40.degree. C. 71.20 -15.50 17.20 7.80 8.9 6.80 2.70 0.20 3 M at
40.degree. C. 63.90 -22.80 19.30 9.90 13.9 11.80 3.00 0.50 Pilot
Plant Experiment 2 0-time 84.44 N/A 9.03 N/A 2.32 N/A 4.17 N/A
Batch using 2 W at 55.degree. C. 72.43 -12.01 16.02 6.99 7.12 4.80
4.39 0.22 HA Treated for Prion Removal Abraxane Experiment 2 0-time
86.70 N/A 7.00 N/A 1.5 N/A 4.80 N/A Process 3 M at 40.degree. C.
75.60 -11.10 14.30 7.30 5.4 3.90 4.70 -0.10 Validation Lot 6 M at
40.degree. C. 68.80 -17.90 16.80 9.80 9.2 7.70 5.20 0.40 Abraxane
Pilot Experiments 0-time 89.45 N/A 5.73 N/A 1.33 N/A 3.5 N/A Plant
Batch 1 and 2 2 W at 55.degree. C. 75.19 -14.26 14.92 9.19 6.21
4.88 3.68 0.18
[0306] Comparison of stability data shows that storage for 2 weeks
at 55.degree. C. is equivalent to storage for 3 months at
40.degree. C. No significant differences were observed during the
manufacturing process and during the in-process testing between
pilot plant batches manufactured using human albumin treated for
prion removal and pilot plant batches manufactured using untreated
human albumin There were no significant differences between the
properties of the finished products manufactured using human
albumin treated for prion removal and the properties of the
finished product manufactured using untreated human albumin
Example 6
Evaluation of the Effect of Prion-Removal Column on Abraxane.RTM.
in-Process Suspension
[0307] This experiment evaluates the effect of prion removal from
Abraxane.RTM. in-process suspension, namely, the suspension of
Abtaxane.RTM. prior to lyophilization. Commercially available PIKSI
kit columns (1 cc), containing Toyopearl Amino 650CU resin,
ProMetic Biosciences was used in this experiment.
[0308] In two separate experiments, 0.5 L Abraxane.RTM. in process
suspension containing human albumin were processed through the
column The analysis results are summarized in Table 20.
TABLE-US-00022 TABLE 20 Effect of Prion-Removal Column on Abraxane
In-Process Suspension HA Impurities, % Sample Conc., HA
Composition, % Paclitaxel, 7- Particle size, nm No. mg/mL Monomer
Dimer Polymer Oligomer mg/mL Epipaclitaxel Total Mean <5%
<95% <99.9% pH 1 55 88.29 5.95 3.99 1.32 6.9 0.08 0.25 131 82
193 254 7.0 2 56 88.37 5.96 3.86 1.36 6.9 0.08 0.25 132 86 190 250
7.0 3 56 88.38 5.96 3.76 1.39 6.8 0.09 0.26 130 82 190 250 7.0
[0309] Sample 1: Abraxane In-Process Suspension, Untreated
[0310] Sample 2: Abraxane In-Process Suspension, post Prion-removal
Column, sample Collected at the Beginning of Run
[0311] Sample 3: Abraxane In-Process Suspension, post Prion-removal
Column, sample Collected at the End of Run (0.5 L).
[0312] As shown in Table 20, the physical and chemical testing of
Abraxane.RTM. in-process suspension treated for prion removal show
no significant differences between the untreated and treated
suspension, in terms of particle size, pH, paclitaxel assay and
impurities, human albumin (HA) assay, and HA composition.
Sequence CWU 1
1
24617PRTArtificial SequenceSynthetic Construct 1Lys Ile His Lys Phe
Leu Ala1 527PRTArtificial SequenceSynthetic Construct 2Gly Thr His
Asp Phe Gln Ala1 537PRTArtificial SequenceSynthetic Construct 3Lys
Phe Gly Ser Thr His Ala1 547PRTArtificial SequenceSynthetic
Construct 4Phe Val Asn Glu Ile Glu Ala1 557PRTArtificial
SequenceSynthetic Construct 5Gly Leu His Phe Lys Ser Ala1
567PRTArtificial SequenceSynthetic Construct 6Gly Arg Val Leu His
His Ala1 577PRTArtificial SequenceSynthetic Construct 7Gln Lys Asn
Ser Glu Trp Ala1 587PRTArtificial SequenceSynthetic Construct 8His
Ala Tyr Phe Thr His Ala1 597PRTArtificial SequenceSynthetic
Construct 9Trp Pro Lys Gly Ala Val Ala1 5107PRTArtificial
SequenceSynthetic Construct 10Arg Pro Trp Lys Lys Ala Ala1
5117PRTArtificial SequenceSynthetic Construct 11Pro Lys His Ile Trp
Pro Ala1 5127PRTArtificial SequenceSynthetic Construct 12His Lys
Leu Trp Gly Val Ala1 5137PRTArtificial SequenceSynthetic Construct
13Gly Gly Tyr Lys Pro Tyr Ala1 5147PRTArtificial SequenceSynthetic
Construct 14Glu Asn Val Ser Gln Asn Ala1 5157PRTArtificial
SequenceSynthetic Construct 15His Thr Tyr Tyr Asn Gly Ala1
5167PRTArtificial SequenceSynthetic Construct 16Lys Lys Lys Ser Asp
His Ala1 5177PRTArtificial SequenceSynthetic Construct 17His His
Leu Lys Gly Thr Ala1 5187PRTArtificial SequenceSynthetic Construct
18Lys Lys His Gly Val Trp Ala1 5197PRTArtificial SequenceSynthetic
Construct 19Asp Gly Thr Gln Ala His Ala1 5207PRTArtificial
SequenceSynthetic Construct 20Ala Pro His Arg Asn Asn Ala1
5217PRTArtificial SequenceSynthetic Construct 21His His Gly His Asn
Ile Ala1 5227PRTArtificial SequenceSynthetic Construct 22His Thr
Trp His Gly Gln Ala1 5237PRTArtificial SequenceSynthetic Construct
23His Val Phe Val Thr Trp Ala1 5247PRTArtificial SequenceSynthetic
Construct 24Thr His His Phe Tyr Ile Ala1 5257PRTArtificial
SequenceSynthetic Construct 25Lys Leu Gly Trp Gly Xaa Ala1
5267PRTArtificial SequenceSynthetic Construct 26Gly Ser Lys Lys Lys
Glu Ala1 5277PRTArtificial SequenceSynthetic Construct 27Pro Leu
Leu Val Val Trp Ala1 5287PRTArtificial SequenceSynthetic Construct
28Trp Leu Leu Val Gly Gly Ala1 5297PRTArtificial SequenceSynthetic
Construct 29Xaa Gln Val Leu Val Tyr Ala1 5307PRTArtificial
SequenceSynthetic Construct 30Arg Arg His Gln Arg Gln Ala1
5317PRTArtificial SequenceSynthetic Construct 31Leu Pro Trp Thr Phe
Gly Ala1 5327PRTArtificial SequenceSynthetic Construct 32Ile Phe
Ile Ile Ile Thr Ala1 5337PRTArtificial SequenceSynthetic Construct
33Pro Xaa Ile Glu Pro His Ala1 5347PRTArtificial SequenceSynthetic
Construct 34Glu Trp Gly Ile Ile Trp Ala1 5357PRTArtificial
SequenceSynthetic Construct 35Gly Trp Tyr Ile Tyr Phe Ala1
5367PRTArtificial SequenceSynthetic Construct 36Thr Leu Ile Leu Phe
His Ala1 5377PRTArtificial SequenceSynthetic Construct 37Phe Leu
Leu Ser Asn His Ala1 5387PRTArtificial SequenceSynthetic Construct
38Trp Gln Ile Arg Phe Phe Ala1 5397PRTArtificial SequenceSynthetic
Construct 39Val Leu Leu Val Phe Glu Ala1 5407PRTArtificial
SequenceSynthetic Construct 40Gly Trp Val Leu Glu Ile Ala1
5417PRTArtificial SequenceSynthetic Construct 41Phe Leu Leu Ile Asp
Thr Ala1 5427PRTArtificial SequenceSynthetic Construct 42Gly Phe
Leu Phe Lys Phe Ala1 5437PRTArtificial SequenceSynthetic Construct
43Pro Trp Thr Ile Tyr Ile Ala1 5442PRTArtificial SequenceSynthetic
Construct 44Trp His1452PRTArtificial SequenceSynthetic Construct
45Trp Trp1462PRTArtificial SequenceSynthetic Construct 46Leu
Trp1473PRTArtificial SequenceSynthetic Construct 47Trp Asn
Ala1483PRTArtificial SequenceSynthetic Construct 48Glu Phe
Trp1493PRTArtificial SequenceSynthetic Construct 49Leu Pro
Trp1503PRTArtificial SequenceSynthetic Construct 50Tyr Glu
Tyr1513PRTArtificial SequenceSynthetic Construct 51Trp Pro
Ala1523PRTArtificial SequenceSynthetic Construct 52Phe Asn
Gln1533PRTArtificial SequenceSynthetic Construct 53Tyr His
Glu1543PRTArtificial SequenceSynthetic Construct 54Leu Phe
Ala1553PRTArtificial SequenceSynthetic Construct 55Asn His
Tyr1563PRTArtificial SequenceSynthetic Construct 56Thr Leu
Gly1573PRTArtificial SequenceSynthetic Construct 57Trp Val
Asp1585PRTArtificial SequenceSynthetic Construct 58Tyr Trp Asp Gln
Ala1 5595PRTArtificial SequenceSynthetic Construct 59Tyr Val His
Glu Ala1 5605PRTArtificial SequenceSynthetic Construct 60Trp Phe
Asp Glu Ala1 5616PRTArtificial SequenceSynthetic Construct 61Leu
Gln Trp Tyr Asp Ala1 5626PRTArtificial SequenceSynthetic Construct
62Tyr Thr His Ser Glu Ala1 5636PRTArtificial SequenceSynthetic
Construct 63Trp Ile Asp Tyr Glu Ala1 5646PRTArtificial
SequenceSynthetic Construct 64Val Trp Ile Asp Ala Ala1
5657PRTArtificial SequenceSynthetic Construct 65Trp Asp Glu Ala Glu
Glu Ala1 5667PRTArtificial SequenceSynthetic Construct 66Tyr Asp
Ser Tyr Asp Asp Ala1 5677PRTArtificial SequenceSynthetic Construct
67Asn Asp Phe Ile Asp Phe Ala1 5687PRTArtificial SequenceSynthetic
Construct 68Tyr Glu Pro Trp Gly Ser Ala1 5697PRTArtificial
SequenceSynthetic Construct 69Glu Tyr Gly Asp Trp Trp Ala1
5707PRTArtificial SequenceSynthetic Construct 70Trp Asp Tyr Asp Gln
Glu Ala1 5717PRTArtificial SequenceSynthetic Construct 71Asp Trp
Gly Asp Pro Phe Ala1 5727PRTArtificial SequenceSynthetic Construct
72Asp Trp Pro Glu Val Trp Ala1 5737PRTArtificial SequenceSynthetic
Construct 73Phe His Asp Phe Ser Glu Ala1 5747PRTArtificial
SequenceSynthetic Construct 74Asp Thr Phe Trp Asp Tyr Ala1
5757PRTArtificial SequenceSynthetic Construct 75Trp Asn Asp Leu Asp
Asn Ala1 5767PRTArtificial SequenceSynthetic Construct 76Ala Ser
Ala Leu Val Tyr Ala1 5777PRTArtificial SequenceSynthetic Construct
77Leu Ile Asn Ala Gly Gly Ala1 5787PRTArtificial SequenceSynthetic
Construct 78Trp Glu Ser Tyr Val Thr Ala1 5797PRTArtificial
SequenceSynthetic Construct 79Trp Ser Asp Glu Gly Tyr Ala1
5807PRTArtificial SequenceSynthetic Construct 80Tyr Arg Trp Thr Gly
Pro Ala1 5817PRTArtificial SequenceSynthetic Construct 81Tyr Glu
Asp Gln Trp Gln Ala1 5827PRTArtificial SequenceSynthetic Construct
82Glu Trp Ala Asp Asp Asn Ala1 5837PRTArtificial SequenceSynthetic
Construct 83Tyr Glu Ile Asp Tyr Gly Ala1 5847PRTArtificial
SequenceSynthetic Construct 84Glu Phe Gly Tyr Phe Asp Ala1
5857PRTArtificial SequenceSynthetic Construct 85Trp Gly Asp Glu Gln
Asp Ala1 5867PRTArtificial SequenceSynthetic Construct 86His Glu
Glu Asp Trp Ala Ala1 5877PRTArtificial SequenceSynthetic Construct
87Phe Glu Asp Phe Glu Leu Ala1 5887PRTArtificial SequenceSynthetic
Construct 88Thr Trp Gly Ile Asp Glu Ala1 5897PRTArtificial
SequenceSynthetic Construct 89Trp Asp Pro Thr Asp Tyr Ala1
5907PRTArtificial SequenceSynthetic Construct 90Asn Asp Lys Ile His
Thr Ala1 5917PRTArtificial SequenceSynthetic Construct 91Phe Glu
Asp Phe Phe Ser Ala1 5927PRTArtificial SequenceSynthetic Construct
92Tyr Glu Trp Ala Glu Gln Ala1 5937PRTArtificial SequenceSynthetic
Construct 93Thr His Val Tyr Phe Leu Ala1 5947PRTArtificial
SequenceSynthetic Construct 94Xaa Xaa Asp Phe Ser Asp Ala1
5957PRTArtificial SequenceSynthetic Construct 95Tyr Arg Thr Pro Asn
Glu Ala1 5966PRTArtificial SequenceSynthetic Construct 96Xaa Arg
Ser Glu Thr Ala1 5973PRTArtificial SequenceSynthetic Construct
97Ile His Asn1983PRTArtificial SequenceSynthetic Construct 98Trp
Glu Tyr1993PRTArtificial SequenceSynthetic Construct 99Asp Tyr
Trp11003PRTArtificial SequenceSynthetic Construct 100Trp Asp
Trp11013PRTArtificial SequenceSynthetic Construct 101Trp Gln
Asp11023PRTArtificial SequenceSynthetic Construct 102Tyr Phe
Glu11033PRTArtificial SequenceSynthetic Construct 103Asn Tyr
Glu11043PRTArtificial SequenceSynthetic Construct 104Ser Tyr
Ala11053PRTArtificial SequenceSynthetic Construct 105Trp Asp
Leu11063PRTArtificial SequenceSynthetic Construct 106Trp Leu
Glu11073PRTArtificial SequenceSynthetic Construct 107Val Gln
Arg11083PRTArtificial SequenceSynthetic Construct 108Tyr Ile
Asp11093PRTArtificial SequenceSynthetic Construct 109Arg Trp
Asp11103PRTArtificial SequenceSynthetic Construct 110Asp Val
Arg11113PRTArtificial SequenceSynthetic Construct 111Trp Ser
Asp11123PRTArtificial SequenceSynthetic Construct 112His Trp
Asp11133PRTArtificial SequenceSynthetic Construct 113Trp Gln
Asp11143PRTArtificial SequenceSynthetic Construct 114Trp Asp
Asp11153PRTArtificial SequenceSynthetic Construct 115Trp Glu
Asp11163PRTArtificial SequenceSynthetic Construct 116Ile Thr
Asn11173PRTArtificial SequenceSynthetic Construct 117Tyr Glu
Asp11186PRTArtificial SequenceSynthetic Construct 118Val Ala Asp
Glu Glu Ala1 51196PRTArtificial SequenceSynthetic Construct 119Tyr
Tyr Val Asp Ala Ala1 51206PRTArtificial SequenceSynthetic Construct
120Gln Asp Phe Asn Leu Ala1 51216PRTArtificial SequenceSynthetic
Construct 121Asp Asn Pro Ile Asp Ala1 51226PRTArtificial
SequenceSynthetic Construct 122Phe Asn Glu His Glu Ala1
51236PRTArtificial SequenceSynthetic Construct 123Trp Gly Ala Asp
Gly Ala1 51246PRTArtificial SequenceSynthetic Construct 124Val Ile
Tyr Ser His Ala1 51256PRTArtificial SequenceSynthetic Construct
125His Ile Leu Glu Glu Ala1 51266PRTArtificial SequenceSynthetic
Construct 126Pro His Glu Asn Phe Ala1 51276PRTArtificial
SequenceSynthetic Construct 127Glu Asp Asn Gly Gly Ala1
51286PRTArtificial SequenceSynthetic Construct 128Asp Ser Glu Gly
Pro Ala1 51296PRTArtificial SequenceSynthetic Construct 129Phe Gln
Glu Phe Thr Ala1 51306PRTArtificial SequenceSynthetic Construct
130Glu Gly Asp Glu Ile Ala1 51316PRTArtificial SequenceSynthetic
Construct 131Ile Tyr Ala Glu Thr Ala1 51326PRTArtificial
SequenceSynthetic Construct 132Arg Val Arg Glu Thr Ala1
51336PRTArtificial SequenceSynthetic Construct 133Glu Glu Pro Gln
Trp Ala1 51346PRTArtificial SequenceSynthetic Construct 134Glu Gly
Glu Glu Phe Ala1 51356PRTArtificial SequenceSynthetic Construct
135Xaa Phe Asn Ile His Ala1 51363PRTArtificial SequenceSynthetic
Construct 136Tyr Asp Trp11373PRTArtificial SequenceSynthetic
Construct 137Asn Tyr Thr11383PRTArtificial SequenceSynthetic
Construct 138Ser Tyr Thr11393PRTArtificial SequenceSynthetic
Construct 139Trp Ala Asp11403PRTArtificial SequenceSynthetic
Construct 140Gln Trp Gly11413PRTArtificial SequenceSynthetic
Construct 141Trp Gly Asp11423PRTArtificial SequenceSynthetic
Construct 142Glu Tyr Phe11433PRTArtificial SequenceSynthetic
Construct 143Trp Glu His11443PRTArtificial SequenceSynthetic
Construct 144Leu Tyr Asp11453PRTArtificial SequenceSynthetic
Construct 145Asp Tyr Tyr11463PRTArtificial SequenceSynthetic
Construct 146Phe Tyr Glu11473PRTArtificial SequenceSynthetic
Construct 147Glu Tyr Tyr11483PRTArtificial SequenceSynthetic
Construct 148Tyr Asp Tyr11493PRTArtificial SequenceSynthetic
Construct 149Trp Asp His11507PRTArtificial SequenceSynthetic
Construct 150Arg Glu Ser Xaa Asn Val Ala1 51517PRTArtificial
SequenceSynthetic Construct 151Glu Ser Xaa Pro Arg Gln Ala1
51527PRTArtificial SequenceSynthetic Construct 152Val Ala Arg Glu
Asn Ile Ala1 51537PRTArtificial SequenceSynthetic Construct 153Arg
Trp Glu Arg Glu Asp Ala1 51546PRTArtificial SequenceSynthetic
Construct 154Glu Trp Trp Glu Thr Val1 51557PRTArtificial
SequenceSynthetic Construct 155Ser Val Tyr Gln Leu Asp Ala1
51567PRTArtificial SequenceSynthetic Construct 156Xaa His Glu Phe
Tyr Gly Ala1 51577PRTArtificial SequenceSynthetic Construct 157His
Glu Xaa Xaa Leu Val Ala1 51587PRTArtificial SequenceSynthetic
Construct 158Ala Xaa Val Pro Val Xaa Ala1 51597PRTArtificial
SequenceSynthetic Construct 159Tyr Phe Asp Tyr Trp Leu Ala1
51607PRTArtificial SequenceSynthetic Construct 160Phe Glu Xaa His
Arg Gln Ala1 51617PRTArtificial SequenceSynthetic Construct 161Trp
Arg His Glu Pro Ala Ala1 51627PRTArtificial SequenceSynthetic
Construct 162Ser Ser Xaa Lys Lys Asp Ala1 51637PRTArtificial
SequenceSynthetic Construct 163Arg Xaa Asp Lys Glu Ala Ala1
51647PRTArtificial SequenceSynthetic Construct 164Xaa His Glu Ile
Phe Pro Ala1 51657PRTArtificial SequenceSynthetic Construct 165Lys
Trp Tyr His His Arg Ala1 51667PRTArtificial SequenceSynthetic
Construct 166His Trp Trp Pro His Asn Ala1 51677PRTArtificial
SequenceSynthetic Construct 167His Trp Gln Val Phe Tyr Ala1
51687PRTArtificial SequenceSynthetic Construct 168Phe His Glu Xaa
Glu Ile Ala1 51697PRTArtificial SequenceSynthetic Construct 169His
Ala Asp Phe Xaa Gln Ala1 51707PRTArtificial SequenceSynthetic
Construct 170Ala Leu His Phe Glu Thr Ala1 51717PRTArtificial
SequenceSynthetic Construct 171Asp Asp Pro Thr Gly Phe Ala1
51727PRTArtificial SequenceSynthetic Construct 172Val Ala Pro Gly
Leu Gly Ala1 51737PRTArtificial SequenceSynthetic Construct 173Ile
Phe Arg Leu Ile Glu Ala1 51747PRTArtificial SequenceSynthetic
Construct 174Gly Leu Glu Arg Pro Glu Ala1 51757PRTArtificial
SequenceSynthetic Construct 175Ile Val Val Arg Leu Trp Ala1
51767PRTArtificial SequenceSynthetic Construct 176Trp His Asn Pro
His Tyr Ala1 51777PRTArtificial SequenceSynthetic Construct 177Leu
Ile Tyr Lys Ser Asp Ala1 51787PRTArtificial SequenceSynthetic
Construct 178Glu Lys Pro Ile Phe Asn Ala1 51797PRTArtificial
SequenceSynthetic Construct 179His Trp Ser Glu Pro Ala Ala1
51807PRTArtificial SequenceSynthetic Construct 180Gly His Asn Trp
Lys Glu Ala1 51817PRTArtificial SequenceSynthetic Construct 181Tyr
Trp His His Asp Asp Ala1 51827PRTArtificial SequenceSynthetic
Construct 182Gly Tyr Pro Lys Glu Asn Ala1 51837PRTArtificial
SequenceSynthetic Construct 183Pro Val Tyr Trp Leu Tyr Ala1
51847PRTArtificial SequenceSynthetic Construct 184Phe Gly Glu His
Thr Pro Ala1 51857PRTArtificial SequenceSynthetic Construct 185Phe
Gln Gly Thr Arg Glu Ala1 51867PRTArtificial SequenceSynthetic
Construct 186Thr Gly Thr Asn Arg Tyr Ala1 51877PRTArtificial
SequenceSynthetic Construct 187Lys Trp Ala Thr Arg Tyr Ala1
51887PRTArtificial SequenceSynthetic Construct 188Asn Ser Thr Lys
Phe Asp Ala1 51897PRTArtificial SequenceSynthetic Construct 189Leu
Ile Tyr Lys Glu Glu Ala1 51907PRTArtificial SequenceSynthetic
Construct 190Glu His Ala Thr Tyr Arg Ala1 51913PRTArtificial
SequenceSynthetic Construct 191His Asn Asp11923PRTArtificial
SequenceSynthetic Construct 192His Glu Arg11933PRTArtificial
SequenceSynthetic Construct 193His Gly Asp11943PRTArtificial
SequenceSynthetic Construct 194His Ser Asp11953PRTArtificial
SequenceSynthetic Construct 195His Phe Asp11963PRTArtificial
SequenceSynthetic Construct 196Trp Asn Asp11973PRTArtificial
SequenceSynthetic Construct 197Tyr Glu His11983PRTArtificial
SequenceSynthetic Construct 198His Trp Asp11993PRTArtificial
SequenceSynthetic Construct 199Tyr His Asp12003PRTArtificial
SequenceSynthetic Construct 200Tyr Asp Trp12013PRTArtificial
SequenceSynthetic Construct 201Trp Asp Tyr12023PRTArtificial
SequenceSynthetic Construct 202His Tyr Asp12033PRTArtificial
SequenceSynthetic Construct 203His Trp Asp12043PRTArtificial
SequenceSynthetic Construct 204Trp Thr Asp12053PRTArtificial
SequenceSynthetic Construct 205Phe Pro Lys12063PRTArtificial
SequenceSynthetic Construct 206His Trp Lys12073PRTArtificial
SequenceSynthetic Construct 207Trp Glu Glu12083PRTArtificial
SequenceSynthetic Construct 208Leu Leu Arg12093PRTArtificial
SequenceSynthetic Construct 209Ser Tyr Phe12103PRTArtificial
SequenceSynthetic Construct 210Glu Tyr Tyr12117PRTArtificial
SequenceSynthetic Construct 211Asp Arg Asp Leu Thr Phe Ala1
52127PRTArtificial SequenceSynthetic Construct 212His Asn Trp Trp
Ile Ile Ala1 52137PRTArtificial SequenceSynthetic Construct 213Glu
Val Lys Ile Gly Asn Ala1 52143PRTArtificial SequenceSynthetic
Construct 214Ser Ile Val12153PRTArtificial SequenceSynthetic
Construct 215Ala Tyr Pro12167PRTArtificial SequenceSynthetic
Construct 216Glu Val Ala Asp Glu Glu Ala1 52177PRTArtificial
SequenceSynthetic Construct 217Glu Tyr Tyr Val Asp Ala Ala1
52187PRTArtificial SequenceSynthetic Construct 218Tyr Asp Asn Pro
Ile Asp Ala1 52197PRTArtificial SequenceSynthetic Construct 219Tyr
Phe Asn Glu His Glu Ala1 52207PRTArtificial SequenceSynthetic
Construct 220Glu Trp Gly Ala Asp Gly Ala1 52217PRTArtificial
SequenceSynthetic Construct 221Asp Val Ile Tyr Ser His Ala1
52227PRTArtificial SequenceSynthetic Construct 222Trp His Ile Leu
Glu Glu Ala1 52237PRTArtificial SequenceSynthetic Construct 223Asn
Pro His Glu Asn Phe Ala1 52247PRTArtificial SequenceSynthetic
Construct 224His Glu Asp Asn Gly Gly Ala1 52257PRTArtificial
SequenceSynthetic Construct 225Ser Asp Ser Glu Gly Pro Ala1
52267PRTArtificial SequenceSynthetic Construct 226Glu Phe Gln Glu
Phe Thr Ala1 52277PRTArtificial SequenceSynthetic Construct 227Gln
Glu Gly Asp Glu Ile Ala1 52287PRTArtificial SequenceSynthetic
Construct 228Asp Ile Tyr Ala Glu Thr Ala1 52297PRTArtificial
SequenceSynthetic Construct 229Asp Arg Val Arg Glu Thr Ala1
52307PRTArtificial SequenceSynthetic Construct 230Phe Glu Glu Pro
Gln Trp Ala1 52317PRTArtificial SequenceSynthetic Construct 231Phe
Glu Gly Glu Glu Phe Ala1 52326PRTArtificial SequenceSynthetic
Construct 232Xaa Phe Asn Ile His Ala1 52335PRTArtificial
SequenceSynthetic Construct 233Arg Tyr Pro Xaa Gln1
52346PRTArtificial SequenceSynthetic Construct 234Xaa Xaa Tyr Tyr
Xaa Xaa1 52355PRTArtificial SequenceSynthetic Construct 235Arg Tyr
Pro Gly Gln1 52366PRTArtificial SequenceSynthetic Construct 236Asp
Arg Tyr Tyr Arg Asp1 52376PRTArtificial SequenceSynthetic Construct
237Gln Ala Tyr Tyr Gln Arg1 52386PRTArtificial SequenceSynthetic
Construct 238Gln Val Tyr Tyr Arg Pro1 52398PRTArtificial
SequenceSynthetic Construct 239Pro His Gly Gly Gly Trp Gly Gln1
52408PRTArtificial SequenceSynthetic Construct 240Pro His Gly Gly
Ser Trp Gly Gln1 52418PRTArtificial SequenceSynthetic Construct
241Pro His Gly Gly Gly Trp Ser Gln1 52429PRTArtificial
SequenceSynthetic Construct 242Pro His Gly Gly Gly Gly Trp Ser Gln1
524310PRTArtificial SequenceSynthetic Construct 243Pro His Gly Gly
Gly Ser Asn Trp Gly Gln1 5 102446PRTArtificial SequenceSynthetic
Construct 244Pro His Asn Pro Gly Tyr1 52456PRTArtificial
SequenceSynthetic Construct 245Pro His Asn Pro Ser Tyr1
52466PRTArtificial SequenceSynthetic Construct 246Pro His Asn Pro
Gly Tyr1 5
* * * * *
References