U.S. patent application number 17/738362 was filed with the patent office on 2022-08-25 for c3 fusion protein and methods of making and using thereof.
The applicant listed for this patent is BioAxone BioSciences, Inc.. Invention is credited to Ricardo BORJAS, Mark FLEMING, Mei HUANG, Mayur JAIN, Lisa MCKERRACHER, Elizabeth RYU, Tapan SANGHVI, Kumkum SAXENA, Amaris TORRES-DELGADO, Ping YIN.
Application Number | 20220265765 17/738362 |
Document ID | / |
Family ID | 1000006320494 |
Filed Date | 2022-08-25 |
United States Patent
Application |
20220265765 |
Kind Code |
A1 |
BORJAS; Ricardo ; et
al. |
August 25, 2022 |
C3 FUSION PROTEIN AND METHODS OF MAKING AND USING THEREOF
Abstract
The present invention provides, among other things, improved
therapeutic compositions comprising a C3 fusion protein and methods
of making and using the same. In particular, the present invention
provides improved methods for the treatment of spinal cord injury
and other CNS trauma and/or facilitate axon growth or other tissue
repair.
Inventors: |
BORJAS; Ricardo; (Boston,
MA) ; FLEMING; Mark; (Boston, MA) ; HUANG;
Mei; (Boston, MA) ; JAIN; Mayur; (Boston,
MA) ; SANGHVI; Tapan; (Boston, MA) ; SAXENA;
Kumkum; (Boston, MA) ; TORRES-DELGADO; Amaris;
(Boston, MA) ; YIN; Ping; (Boston, MA) ;
MCKERRACHER; Lisa; (Boston, MA) ; RYU; Elizabeth;
(Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BioAxone BioSciences, Inc. |
Boston |
MA |
US |
|
|
Family ID: |
1000006320494 |
Appl. No.: |
17/738362 |
Filed: |
May 6, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16618021 |
Nov 27, 2019 |
11324802 |
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PCT/US2018/035144 |
May 30, 2018 |
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17738362 |
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62675659 |
May 23, 2018 |
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62675680 |
May 23, 2018 |
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62675714 |
May 23, 2018 |
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62512661 |
May 30, 2017 |
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62512673 |
May 30, 2017 |
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62512695 |
May 30, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/57 20130101;
A61K 38/38 20130101; A61K 38/1725 20130101; C07K 2319/70 20130101;
C07K 14/472 20130101; C12Y 204/02036 20130101; A61K 38/363
20130101; C12N 9/1077 20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 38/36 20060101 A61K038/36; A61K 38/38 20060101
A61K038/38; A61K 38/57 20060101 A61K038/57; C07K 14/47 20060101
C07K014/47; C12N 9/10 20060101 C12N009/10 |
Claims
1. A method for producing recombinant ADP-ribosyl transferase C3
(C3 fusion protein), comprising: cultivating host cells comprising
a nucleic acid encoding a recombinant C3 fusion protein having an
amino acid sequence of SEQ ID NO:2 in a large scale vessel under
conditions that: promote expression of the C3 fusion protein at a
titer concentration of greater than 1 g/L; and/or involve a
fermentation process to promote expression of the C3 fusion
protein.
2. The method of claim 1, wherein the fermentation process is a fed
batch culturing process.
3. The method of claim 2, wherein: the fed batch culturing process
includes a batch mode, a first stage of exponential feeding, and a
second stage of constant feeding; the carbon source is selected
from glycerol, glucose, sucrose, lactose, arabinose, maltotriose,
sorbitol, xylose, rhamnose, and/or mannose; and/or the method
further comprises a step of adding an inducing agent to trigger
expression of the C3 fusion protein.
4. The method of claim 3, wherein: the batch mode lasts for at
least about 7 hours, the first stage of exponential feeding lasts
for about 7-8 hours and the second stage of constant feeding lasts
for about 8 hours; the second stage of constant feeding is at a
feed rate at the end of the exponential feeding stage; the first
exponential feeding stage is maintained at a temperature between
34.degree. C. to 40.degree. C., and wherein the second and/or third
constant feeding stage is maintained at a temperature between
24.degree. C. to 32.degree. C.; and/or the method further comprises
a third stage of constant feeding.
5. The method of claim 4, wherein: the feed rate at the third stage
of constant feeding is the same as the second stage; and/or the
third stage of constant feeding lasts for at least about 4
hours.
6. The method of claim 3, wherein the carbon source at the first
stage is a first sugar and the carbon source at the second stage is
a second sugar; wherein the first sugar and the second sugar are
different sugars or wherein the first sugar and the second sugar
are identical sugars.
7. The method of claim 6, wherein the first sugar is selected from
the group consisting of glycerol, glucose, sucrose, and any
combination thereof and the second sugar is selected from the group
consisting of glycerol, glucose, sucrose, and any combination
thereof; or wherein the identical sugars comprise glycerol.
8. The method of claim 1, wherein: the large scale vessel is a
bioreactor; the host cells are bacterial cells; and/or the method
further comprises a step of recovering the expressed C3 fusion
protein from the host cells.
9. The method of claim 8, wherein: the bacterial cells are E. coli;
and/or the method further comprises purifying the expressed
recombinant C3 fusion protein.
10. The method of claim 9, wherein at least 80%, 85%, 90%, or 95%
of the purified recombinant C3 fusion protein recovered from the
host cells do not contain a methionine at the N-terminus.
11. The method of claim 1, further comprising purifying recombinant
ADP-ribosyl transferase C3 (C3 fusion protein), comprising:
providing a biological sample comprising a recombinant C3 fusion
protein having an amino acid sequence of SEQ ID NO: 1, and
subjecting the biological sample to a series of chromatography
steps comprising at least one cation exchange chromatography step
and at least one hydrophobic interaction chromatography step.
12. A method of purifying recombinant ADP-ribosyl transferase C3
(C3 fusion protein), comprising: providing a biological sample
comprising a recombinant C3 fusion protein having an amino acid
sequence of SEQ ID NO: 1, and subjecting the biological sample to a
series of chromatography steps comprising at least one cation
exchange chromatography step and at least one hydrophobic
interaction chromatography step.
13. The method of claim 12, wherein: the at least one cation
exchange chromatography step includes two or more cation exchange
chromatography steps; the at least one hydrophobic interaction
chromatography step follows the at least cation exchange
chromatography step; and/or the series of chromatography steps
comprise a first cation exchange chromatography step, a second
cation exchange chromatography step and a hydrophobic interaction
chromatography step, in that order.
14. The method of claim 12, wherein: the at least one cation
exchange chromatography step uses a column comprising SP Sepharose;
the at least one cation exchange chromatography step uses a column
with a bed height of 5 cm-30 cm; the at least one cation exchange
chromatography step uses a column with a loading velocity of 100
cm/hr-200 cm/hr; the at least one cation exchange chromatography
step uses a column with a load conductivity of 7 mS/cm-8 mS/cm; the
at least one hydrophobic interaction chromatography step uses a
column comprising Butyl 650M resin; the at least one hydrophobic
interaction chromatography step uses a column with a bed height of
10 cm-30 cm; the method further comprises one or more steps of
Ultrafiltration/Diafiltration I (UF/DF I) and membrane adsorption
before, between or after the chromatography steps; the method
further comprises a step of concentrating the purified recombinant
C3 fusion protein to a concentration of 0.1 mg/mL-40 mg/mL; and/or
the biological sample comprises a homogenate of cells expressing
the recombinant C3 fusion protein.
15. The method of claim 14, wherein the cells are bacterial cells;
and/or wherein the cells comprise a nucleic acid encoding a
recombinant C3 fusion protein having an amino acid sequence of SEQ
ID NO: 2.
16. The method of claim 15, wherein at least 80% of the purified
recombinant C3 fusion protein does not contain a methionine at the
N-terminus, or wherein substantially all purified recombinant C3
fusion proteins do not contain a methionine at the N-terminus.
17. The method of claim 15, wherein at least 85% of the purified
recombinant C3 fusion protein does not contain a methionine at the
N-terminus, or wherein substantially all purified recombinant C3
fusion proteins do not contain a methionine at the N-terminus.
18. The method of claim 15, wherein at least 95% of the purified
recombinant C3 fusion protein does not contain a methionine at the
N-terminus, or wherein substantially all purified recombinant C3
fusion proteins do not contain a methionine at the N-terminus.
19. The method of claim 16, wherein: the purified recombinant C3
fusion protein has a purity of equal to or greater than 85%
measured by main peak of IE-HPLC; the purified recombinant C3
fusion protein has a purity equal to or greater than 90% measured
by the main peak of IE-HPLC; and/or the purified recombinant C3
fusion protein has a concentration of about 31-37 mg/mL.
20. A C3 recombinant ADP-ribosyl transferase protein composition
purified according to a method of claim 12.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 16/618,021, filed Nov. 27, 2019, which is a
national stage of International Patent Application No.
PCT/US2018/035144, filed May 30, 2018, which claims priority to
U.S. Provisional Application No. 62/512,661, filed May 30, 2017;
U.S. Provisional Application No. 62/512,673, filed May 30, 2017;
U.S. Provisional Application No. 62/512,695, filed May 30, 2017;
U.S. Provisional Application No. 62/675,659, filed May 23, 2018;
U.S. Provisional Application No. 62/675,680, filed May 23, 2018;
and U.S. Provisional Application No. 62/675,714, filed May 23,
2018, each of which are hereby incorporated by reference in their
entirety.
REFERENCE TO SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. The ASCII copy, created
on Aug. 27, 2020 is named
51245-017008_Sequence_Listing_4_20_22_ST25 and is 23,058 bytes in
size.
BACKGROUND
[0003] Spinal cord injury is damage to any part of the spinal cord
or nerves at the end of the spinal canal and often causes permanent
changes in strength, sensation and other body functions below the
site of the injury. A patient's ability to control their limbs
after spinal cord injury depends on two factors: the place of the
injury along the spinal cord and the severity of injury to the
spinal cord. Spinal cord injuries of any kind may result in one or
more of the following signs and symptoms: loss of movement, loss of
sensation, including the ability to feel heat, cold and touch, loss
of bowel or bladder control, exaggerated reflex activities or
spasms, changes in sexual function, sexual sensitivity and
fertility, pain or an intense stinging sensation caused by damage
to the nerve fibers in the spinal cord, and difficulty breathing,
coughing or clearing secretions the lungs. Currently there is no
approved treatment that reverses damage to the spinal cord.
[0004] ADP-ribosyl transferase C3 fusion proteins are being
developed as therapy for spinal cord injury and other CNS
trauma.
SUMMARY
[0005] The present invention provides improved methods and
compositions for the effective treatment of spinal cord injury and
other CNS trauma and/or for facilitating axon growth or other
tissue repair based on an ADP-ribosyl transferase C3 fusion
protein. Among other things, the invention provides improved
processes for producing and purifying a composition comprising a C3
fusion protein, which is particularly robust, reproducible and
results in increased yield, increased protein purity, and/or
increased drug substance concentration. Thus, improved processes
described herein allow more efficient manufacturing of a drug
product based on an ADP-ribosyl transferase C3 fusion protein.
Furthermore, the present invention provides an improved method of
using a C3 fusion protein as a therapeutics for treating spinal
cord injury and other CNS trauma. In particular, the present
invention encompasses the surprising observation that combining a
C3 fusion protein first with a fibrinogen composition without
thrombin eliminates proteinase cleavage, providing a safer and more
efficacious therapeutic composition for neurite outgrowth.
[0006] In one aspect, the present disclosure provides a
pharmaceutical composition comprising a population of polypeptides,
each having an amino acid sequence at least 85% (e.g., at least
90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%%) identical to SEQ ID
NO:1, wherein the first amino acid of each polypeptide is not a
methionine and wherein the population of the polypeptides
constitutes greater than 85% (e.g., greater than 88%, 90%, 92%,
94%, 95%, 96%, 97%, 98%, or 99%) of the total amount of
polypeptides in the composition.
[0007] In some embodiments, the pharmaceutical composition
comprises a population of polypeptides, each having an amino acid
sequence at least 85% (e.g., at least 90%, 92%, 94%, 95%, 96%, 97%,
98%, or 99%%) identical to SEQ ID NO:1, wherein the amino acid
sequence is not SEQ ID NO:2 and wherein the population of the
polypeptides constitutes greater than 85% (e.g., greater than 88%,
90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) of the total amount of
polypeptides in the composition.
[0008] In some embodiments, the population of the polypeptides
constitutes greater than 90% of the total amount of polypeptides in
the composition.
[0009] In some embodiments, the population of the polypeptides
constitutes greater than 95% of the total amount of polypeptides in
the composition.
[0010] In some embodiments, the population of the polypeptides
constitutes greater than 98% of the total amount of polypeptides in
the composition.
[0011] In some embodiments, the population of the polypeptides
constitutes greater than 99% of the total amount of polypeptides in
the composition.
[0012] In some embodiments, the composition is substantially free
of other polypeptides.
[0013] In some embodiments, the amount of the polypeptides in the
composition is determined scanning densitometry or image
analysis.
[0014] In some embodiments, the amino acid sequence is at least 90%
identical to SEQ ID NO:1. In some embodiments, the amino acid
sequence is at least 95% identical to SEQ ID NO:1. In some
embodiments, the amino acid sequence is identical to SEQ ID
NO:1.
[0015] In some embodiments, each polypeptide has 213-231 amino
acids in total. In some embodiments, each polypeptide has 231 amino
acids in total.
[0016] In one aspect, the present disclosure provides a
pharmaceutical composition comprising: a first polypeptide and a
second polypeptide, wherein the first polypeptide comprises an
amino acid sequence at least 85% (e.g., at least 90%, 92%, 94%,
95%, 96%, 97%, 98%, or 99%%) identical to SEQ ID NO:1 but does not
contain a methionine at the N-terminus, the second polypeptide is
otherwise identical to the first polypeptide but contains a
methionine at the N-terminus, and the weight ratio of the first
polypeptide to the second polypeptide is at least 6:1.
[0017] In some embodiments, the weight ratio of the first
polypeptide to the second polypeptide is at least 7:1, 8:1, 9:1,
10:1, 12:1, 15:1, 20:1, 50:1, or 100:1.
[0018] In some embodiments, the amino acid sequence is at least
90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1.
[0019] In some embodiments, the amino acid sequence is identical to
SEQ ID NO:1.
[0020] In one aspect, the present disclosure provides a
pharmaceutical composition comprising a polypeptide having an amino
acid sequence at least 85% (e.g., at least 90%, 92%, 94%, 95%, 96%,
97%, 98%, or 99%%) identical to SEQ ID NO:1, wherein the
polypeptide does not contain a methionine at the N-terminus, and
wherein the polypeptide is present at a concentration ranging from
1.0 mg/mL-40 mg/mL, as determined by UV spectrometry at 280 nm.
[0021] In some embodiments, the concentration of the polypeptide is
in a range of about 5.0 mg/mL-40 mg/mL, as determined by UV
spectrometry at 280 nm. In some embodiments, the concentration of
the polypeptide is in a range of about 8.0 mg/mL-20 mg/mL, as
determined by UV spectrometry at 280 nm. In some embodiments, the
concentration of the polypeptide is in a range of about 10 mg/mL-15
mg/mL, as determined by UV spectrometry at 280 nm.
[0022] In some embodiments, the concentration of the polypeptide is
in a range of about 9.0 mg/mL-11 mg/mL, as determined by UV
spectrometry at 280 nm. In some embodiments, the concentration of
the polypeptide is in a range of about 27.0 mg/mL-33 mg/mL, as
determined by UV spectrometry at 280 nm.
[0023] In one aspect, the present disclosure provides a
pharmaceutical composition comprising a polypeptide having an amino
acid sequence at least 85% (e.g., at least 90%, 92%, 94%, 95%, 96%,
97%, 98%, or 99%%) identical to SEQ ID NO:1, wherein the
polypeptide does not contain a methionine at the N-terminus, and
wherein the composition contains less than 100 ng/mg host cell
protein (HCP).
[0024] In some embodiments, the composition contains less than 90
ng/mg, less than 80 ng/mg, less than 70 ng/mg, less than 60 ng/mg,
less than 50 ng/mg, less than 40 ng/mg, less than 30 ng/mg, less
than 20 ng/mg, less than 10 ng/mg, or less than 10 ng/mg, or below
the limit of detection of host cell protein (HCP).
[0025] In one aspect, the present disclosure provides a
pharmaceutical composition comprising a polypeptide having an amino
acid sequence at least 85% (e.g., at least 90%, 92%, 94%, 95%, 96%,
97%, 98%, or 99%%) identical to SEQ ID NO:1, wherein the
polypeptide does not contain a methionine at the N-terminus, and
wherein the composition contains less than 2.9.times.10.sup.-4
EU/mg Endotoxin.
[0026] In one aspect, the present disclosure provides a
pharmaceutical composition comprising a polypeptide having an amino
acid sequence at least 85% (e.g., at least 90%, 92%, 94%, 95%, 96%,
97%, 98%, or 99%%) identical to SEQ ID NO:1, wherein the
polypeptide does not contain a methionine at the N-terminus, and
wherein the pharmaceutical composition comprises a buffer and has a
pH ranging from 5.5-7.5 at 25.degree. C.
[0027] In some embodiments, the buffer is a citrate buffer.
[0028] In various embodiments, a composition described herein has a
purity of equal to or greater than 80% measured by main peak of
IE-HPLC. In various embodiments, the purity is equal to or greater
than 83% measured by the main peak of IE-HPLC. In various
embodiments, the purity is equal to or greater than 85% measured by
the main peak of IE-HPLC. In various embodiments, the purity is
equal to or greater than 90% measured by the main peak of
IE-HPLC.
[0029] In some embodiments, a composition described herein contains
less than or equal to 15% total acidic peaks measured by IE-HPLC.
In some embodiments, a composition described herein contains less
than or equal to 10% total acidic peaks measured by IE-HPLC.
[0030] In some embodiments, a composition described herein contains
less than or equal to 5% total basic peaks measured by IE-HPLC.
[0031] In one aspect, the present disclosure provides a
pharmaceutical composition comprising a purified recombinant C3
fusion protein having an amino acid sequence at least 85% (e.g., at
least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%%) identical to SEQ
ID NO:1, wherein the purified recombinant C3 fusion protein has a
purity of equal to or greater than 80% measured by main peak of
IE-HPLC. In some embodiments, the purity is equal to or greater
than 83% measured by the main peak of IE-HPLC. In some embodiments,
the purity is equal to or greater than 85% measured by the main
peak of IE-HPLC. In some embodiments, the purity is equal to or
greater than 90% measured by the main peak of IE-HPLC.
[0032] In some embodiments, the composition contains less than or
equal to 10% total acidic peaks measured by IE-HPLC. In some
embodiments, the composition contains less than or equal to 15%
total acidic peaks measured by IE-HPLC.
[0033] In some embodiments, the composition contains less than or
equal to 5% total basic peaks measured by IE-HPLC.
[0034] In some embodiments, the purified recombinant C3 fusion
protein does not have a methionine at the N-terminus.
[0035] In some embodiments, the pharmaceutical composition is
substantially free of polypeptides comprising methionine as the
first amino acid.
[0036] In some embodiments, a composition described herein further
comprises fibrinogen and does not contain thrombin.
[0037] In some embodiments, a composition described herein further
comprises albumin, one or more blood coagulation factors, globulin,
and/or one or more plasminogen-activator inhibitors or plasmin
inhibitors.
[0038] In some embodiments, the one or more plasminogen-activator
inhibitors or plasmin inhibitors comprise aprotinin.
[0039] In some embodiments, a composition described herein further
comprises thrombin. In some embodiments, a composition further
comprises a tissue adhesive.
[0040] In one aspect, the present invention provides a method for
producing recombinant ADP-ribosyl transferase C3 (C3 fusion
protein), comprising: cultivating host cells comprising a nucleic
acid encoding a recombinant C3 fusion protein having an amino acid
sequence of SEQ ID NO:2 in a large scale vessel under conditions
that promote expression of the C3 fusion protein at a titer
concentration of or greater than 1 g/L (e.g., of or greater than
1.5 g/L, 2 g/L, 2.5 g/L, 3 g/L, 3.5 g/L, 4 g/L, 4.5 g/L, or 5
g/L).
[0041] In some embodiments, the conditions for cultivating cells
involve a fermentation process.
[0042] In one aspect, the present invention provides a method for
producing recombinant ADP-ribosyl transferase C3 (C3 fusion
protein), comprising: cultivating host cells comprising a nucleic
acid encoding a recombinant C3 fusion protein having an amino acid
sequence of SEQ ID NO:2 in a large scale vessel under conditions
that involve a fermentation process to promote expression of the C3
fusion protein.
[0043] In some embodiments, the fermentation process is a fed batch
culturing process. In some embodiments, the fed batch culturing
process includes a batch mode, a first stage of exponential
feeding, and a second stage of constant feeding. In some
embodiments, the carbon source is selected from glycerol, glucose,
sucrose, lactose, arabinose, maltotriose, sorbitol, xylose,
rhamnose, and/or mannose.
[0044] In some embodiments, the batch mode lasts for at least about
7-8 hours, the first stage of exponential feeding lasts for about
7-13 hours, the second stage of constant feeding lasts for about
1-9 hours.
[0045] In some embodiments, the batch mode lasts for at least about
7 hours, the first stage of exponential feeding lasts for about 7-8
hours and the second stage of constant feeding lasts for about 8
hours.
[0046] In some embodiments, the second stage of constant feeding is
at a feed rate at the end of the exponential feeding stage.
[0047] In some embodiments, the method further comprises a third
stage of constant feeding. In some embodiments, the feed rate at
the third stage of constant feeding is the same as the second
stage. In some embodiments, the third stage of constant feeding
lasts for at least about 4 hours.
[0048] In some embodiments, the first exponential feeding stage is
maintained at a temperature between 34.degree. C. to 40.degree. C.,
and the second and/or third constant feeding stage is maintained at
a temperature between 24.degree. C. to 32.degree. C.
[0049] In some embodiments, the first exponential feeding stage is
maintained at 37.+-.2.degree. C. In some embodiments, the second
and/or third constant feeding stage is maintained at
28.+-.2.degree. C.
[0050] In some embodiments, the method comprises a step of adding
an inducing agent to trigger expression of the C3 fusion protein.
In some embodiments, the inducing agent is Isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG). In some embodiments, the
IPTG is added at a concentration range of 0.1 mM-10 mM. In some
embodiments, wherein the IPTG is added at a concentration of 5
mM.
[0051] In some embodiments, the carbon source at the first stage is
a first sugar and the carbon source at the second stage is a second
sugar. In certain embodiments, the first sugar and second sugar are
different sugars.
[0052] In some embodiments, the first sugar is selected from the
group consisting of glycerol, glucose, sucrose, and any combination
thereof and the second sugar is selected from the group consisting
of glycerol, glucose, sucrose, and any combination thereof. In some
embodiments, the first sugar and second sugar are identical sugars.
In some embodiments, the identical sugars comprise glycerol.
[0053] In some embodiments, the large scale vessel is a
bioreactor.
[0054] In some embodiments, the host cells are bacterial cells. In
some embodiments the bacterial cells are E. coli.
[0055] In some embodiments, the method further comprises a step of
recovering the expressed C3 fusion protein from the host cells. In
some embodiments, the method further comprises purifying the
expressed recombinant C3 fusion protein.
[0056] In some embodiments, the at least 80%, 85%, 90%, or 95% of
the purified recombinant C3 fusion protein recovered from the host
cells do not contain a methionine at the N-terminus.
[0057] In some embodiments, purifying recombinant ADP-ribosyl
transferase C3 (C3 fusion protein), comprises providing a
biological sample comprising a recombinant C3 fusion protein having
an amino acid sequence of SEQ ID NO:1, and subjecting the
biological sample to a series of chromatography steps comprising at
least one cation exchange chromatography step and at least one
hydrophobic interaction chromatography step.
[0058] In one aspect, the present invention provides a method of
purifying recombinant ADP-ribosyl transferase C3 (C3 fusion
protein), comprising providing a biological sample comprising a
recombinant C3 fusion protein having an amino acid sequence of SEQ
ID NO:1, and subjecting the biological sample to a series of
chromatography steps comprising at least one cation exchange
chromatography step and at least one hydrophobic interaction
chromatography step. In some embodiments, the at least one cation
exchange chromatography step includes two or more cation exchange
chromatography steps. In some embodiments, the at least one cation
exchange chromatography step includes a first cation exchange
chromatography step and a second cation exchange chromatography
step. In some embodiments, the at least one hydrophobic interaction
chromatography step follows the at least cation exchange
chromatography step. In some embodiments, the series of
chromatography steps comprise a first cation exchange
chromatography step, a second cation exchange chromatography step
and a hydrophobic interaction chromatography step, in that
order.
[0059] In some embodiments, the at least one cation exchange
chromatography step uses a column comprising SP Sepharose.
[0060] In some embodiments, the at least one cation exchange
chromatography step uses a column with a bed height of 5 cm-30 cm
(e.g., a bed height of 5 cm, 10 cm, 15 cm, 20 cm, 25 cm, or 30 cm).
In some embodiments, the at least one cation exchange
chromatography step uses a column with a loading velocity of 100
cm/hr-200 cm/hr. In some embodiments, the at least one cation
exchange chromatography step uses a column with a load conductivity
of 7 mS/cm-8 mS/cm.
[0061] In some embodiments, the first and second cation exchange
chromatography steps use identical columns. In some embodiments,
the first and second cation exchange chromatography steps use
different columns.
[0062] In some embodiments, the at least one hydrophobic
interaction chromatography step uses a column comprising Butyl 650M
resin.
[0063] In some embodiments, the at least one hydrophobic
interaction chromatography step uses a column with a bed height of
5 cm-30 cm (e.g., a bed height of 5 cm, 10 cm, 15 cm, 20 cm, 25 cm,
or 30 cm).
[0064] In some embodiments, the method further comprises one or
more steps of Ultrafiltration/Diafiltration I (UF/DF I) and
membrane adsorption before, between or after the chromatography
steps.
[0065] In some embodiments, the method further comprises a step of
concentrating the purified recombinant C3 fusion protein to a
concentration of 0.1 mg/mL-40 mg/mL.
[0066] In some embodiments, the biological sample comprises
homogenate of cells expressing the recombinant C3 fusion protein.
In some embodiments, the cells are bacterial cells. In some
embodiments, the cells comprise a nucleic acid encoding a
recombinant C3 fusion protein having an amino acid sequence of SEQ
ID NO: 2.
[0067] In some embodiments, at least 80%, 85%, 90%, 5%, 96%, 97%,
98%, or 99% of the purified recombinant C3 fusion protein do not
contain a methionine at the N-terminus. In some embodiments,
substantially all purified recombinant C3 fusion protein do not
contain a methionine at the N-terminus.
[0068] In some embodiments, the purified recombinant C3 fusion
protein has a purity of equal to or greater than 85% measured by
main peak of IE-HPLC. In some embodiments, the purity is equal to
or greater than 90% measured by the main peak of IE-HPLC.
[0069] In some embodiments, the purified recombinant C3 fusion
protein has a protein concentration of about 31-37 mg/mL.
[0070] In one aspect, the present invention provides a C3
recombinant ADP-ribosyl transferase protein composition purified
according to a method described herein.
[0071] In one aspect, the present invention provides a method of
preparing a therapeutic composition, comprising mixing a
therapeutically effective amount of a therapeutic protein with a
fibrinogen composition that does not contain a thrombin to generate
a therapeutic polypeptide-fibrinogen composition, wherein the
therapeutic protein comprises a transport domain covalently linked
to a therapeutic active domain, and wherein the transport domain is
selected from a Tat peptide, an antennapedia peptide, a fragment or
subdomain thereof, or a polypeptide having an amino acid sequence
having at least 80% sequence identity thereto; and combining the
therapeutic protein-fibrinogen composition with a thrombin
composition to generate a therapeutic composition.
[0072] In some embodiments, the transport domain comprises an amino
acid sequence of EFVMNPANAQGRHTPGTRL (SEQ ID NO: 10).
[0073] In some embodiments, the therapeutic active domain comprises
an ADP-ribosyl transferase C3 protein.
[0074] In one aspect, the present invention provides a method for
preparing a therapeutic composition to promote neuroregeneration or
neuroprotection, or to treat spinal cord injury or facilitate axon
growth, comprising: mixing a therapeutically effective amount of a
pharmaceutical composition comprising a polypeptide having an amino
acid sequence at least 85% (e.g., at least 90%, 92%, 94%, 95%, 96%,
97%, 98%, or 99%%) identical to SEQ ID NO:1 with a fibrinogen
composition that does not contain a thrombin to generate a
therapeutic protein-fibrinogen solution; and combining the
therapeutic protein-fibrinogen solution with a thrombin composition
to generate a therapeutic composition to promote neuroregeneration
and neuroprotection.
[0075] In some embodiments, the therapeutically effective amount of
the pharmaceutical composition is first added to a solution
comprising one or more plasminogen-activator inhibitors or plasmin
inhibitors before mixing with the fibrinogen composition.
[0076] In some embodiments, the fibrinogen composition comprises
fibrinogen and, albumin, one or more blood coagulation factors,
and/or globulin.
[0077] In some embodiments, the therapeutic protein-fibrinogen
solution and the thrombin composition is combined using a Duploject
Syringe.
[0078] In some embodiments, the therapeutic composition to promote
neuroregeneration and neuroprotection is a tissue adhesive.
[0079] In one aspect, the present invention provides a method of
treating spinal cord injury in a subject in need thereof,
comprising: mixing a therapeutically effective amount of a
pharmaceutical composition comprising a polypeptide having an amino
acid sequence at least 85% (e.g., at least 90%, 92%, 94%, 95%, 96%,
97%, 98%, or 99%%) identical to SEQ ID NO:1 with a fibrinogen
composition that does not contain a thrombin to generate a
therapeutic protein-fibrinogen solution; combining the therapeutic
protein-fibrinogen solution with a thrombin composition to generate
a therapeutic composition to promote neuroregeneration and
neuroprotection; and administering the therapeutic composition to
the subject in need of treatment of spinal cord injury.
[0080] In one aspect, the present invention provides a method of
facilitating axon growth in a subject in need thereof, comprising:
mixing a therapeutically effective amount of a pharmaceutical
composition comprising a polypeptide having an amino acid sequence
at least 85% (e.g., at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or
99%%) identical to SEQ ID NO:1 with a fibrinogen composition that
does not contain a thrombin to generate a therapeutic
protein-fibrinogen solution; combining the therapeutic
protein-fibrinogen solution with a thrombin composition to generate
a therapeutic composition to promote neuroregeneration and
neuroprotection; and administering the therapeutic composition to
the subject in need of axon growth treatment.
[0081] In one aspect, the present invention provides a method of
repairing tissue, comprising: mixing a therapeutically effective
amount of a pharmaceutical composition comprising a polypeptide
having an amino acid sequence at least 85% (e.g., at least 90%,
92%, 94%, 95%, 96%, 97%, 98%, or 99%%) identical to SEQ ID NO:1
with a fibrinogen composition that does not contain a thrombin to
generate a therapeutic protein-fibrinogen solution; combining the
therapeutic protein-fibrinogen solution with a thrombin composition
to generate a therapeutic composition to promote neuroregeneration
and neuroprotection; and administering the therapeutic composition
to the subject in need of tissue repairing.
[0082] In some embodiments, the pharmaceutical composition is first
added to a solution comprising one or more plasminogen-activator
inhibitors or plasmin inhibitors before mixing with the fibrinogen
composition.
[0083] In some embodiments, the one or more plasminogen-activator
inhibitors or plasmin inhibitors comprises aprotinin.
[0084] In some embodiments, the thrombin composition comprises
thrombin and calcium chloride.
[0085] In some embodiments, the therapeutic active-fibrinogen
solution and the thrombin composition are combined and administered
using a Duploject Syringe to the subject in need thereof.
[0086] In one aspect, the present invention provides a kit to
promote neuroregeneration or neuroprotection, treating spinal cord
injury, or facilitating axon growth comprising: a first container
containing a pharmaceutical composition comprising a polypeptide
having an amino acid sequence at least 85% (e.g., at least 90%,
92%, 94%, 95%, 96%, 97%, 98%, or 99%%) identical to SEQ ID NO:1; a
second container containing a fibrinogen composition; and a third
container containing a thrombin composition.
[0087] In some embodiments, the fibrinogen composition in the kit
comprises fibrinogen and, albumin, one or more blood coagulation
factors, and/or globulin.
[0088] In some embodiments, the kit further comprises an additional
container containing a solution comprising one or more
plasminogen-activator inhibitors or plasmin inhibitors. In some
embodiments, the one or more plasminogen-activator inhibitors or
plasmin inhibitors comprises aprotinin.
[0089] In some embodiments, wherein the kit further comprises a
solution comprising calcium chloride.
[0090] In some embodiments, the kit further comprises a syringe. In
some embodiments, the syringe is a Duploject Syringe.
[0091] In one aspect, the present invention provides a kit to
promote neuroregeneration or neuroprotection, treating spinal cord
injury, or facilitating axon growth, comprising: a first chamber
containing a pharmaceutical composition comprising a polypeptide
having an amino acid sequence at least 85% (e.g., at least 90%,
92%, 94%, 95%, 96%, 97%, 98%, or 99%%) identical to SEQ ID NO:1 and
a fibrinogen composition; and a second chamber containing a
thrombin composition.
[0092] In some embodiments, the first chamber further contains one
or more plasminogen-activator inhibitors or plasmin inhibitors.
[0093] In some embodiments, the one or more plasminogen-activator
inhibitors or plasmin inhibitors comprises aprotinin.
[0094] In some embodiments, the thrombin composition comprises
thrombin and calcium chloride.
[0095] In some embodiments, the method or the kit comprises a
polypeptide having an amino acid sequence at least 90% identical to
SEQ ID NO:1.
[0096] In some embodiments, the method or the kit comprises a
polypeptide having an amino acid sequence at least 95% identical to
SEQ ID NO:1.
[0097] In some embodiments, the method or the kit comprises a
polypeptide having an amino acid sequence identical to SEQ ID
NO:1.
[0098] In some embodiments, the first amino acid of the polypeptide
is not a methionine.
[0099] In some embodiments, the method or the kit comprises a
polypeptide having an amino acid sequence identical to SEQ ID NO:1.
In some embodiments, the method or the kit comprises a polypeptide
having an amino acid sequence identical to SEQ ID NO: 8. In some
embodiments, the method or the kit comprises a polypeptide having
an amino acid sequence identical to SEQ ID NO: 9.
[0100] In one aspect, the present invention provides a method for
preparing a therapeutic composition to promote neuroregeneration or
neuroprotection, or to treat spinal cord injury or facilitate axon
growth, comprising: mixing a therapeutically effective amount of a
pharmaceutical composition comprising a purified recombinant C3
fusion protein having an amino acid sequence at least 85% identical
to SEQ ID NO:1, wherein the purified recombinant C3 fusion protein
has a purity of equal to or greater than 80% measured by main peak
of IE-HPLC, with a fibrinogen composition that does not contain a
thrombin to generate a therapeutic protein-fibrinogen solution; and
combining the therapeutic protein-fibrinogen solution with a
thrombin composition to generate a therapeutic composition to
promote neuroregeneration and neuroprotection.
[0101] In some embodiments, the therapeutically effective amount of
the pharmaceutical composition is first added to a solution
comprising one or more plasminogen-activator inhibitors or plasmin
inhibitors before mixing with the fibrinogen composition.
[0102] In some embodiments, the fibrinogen composition comprises
fibrinogen and, albumin, one or more blood coagulation factors,
and/or globulin.
[0103] In some embodiments, the therapeutic protein-fibrinogen
solution and the thrombin composition is combined using a Duploject
Syringe.
[0104] In some embodiments, the therapeutic composition to promote
neuroregeneration and neuroprotection is a tissue adhesive.
[0105] In one aspect, the present invention provides, a method of
treating spinal cord injury, facilitating axon growth, or repairing
tissue, in a subject in need thereof, comprising: mixing a
therapeutically effective amount of a pharmaceutical composition
comprising a purified recombinant C3 fusion protein having an amino
acid sequence at least 85% identical to SEQ ID NO:1, wherein the
purified recombinant C3 fusion protein has a purity of equal to or
greater than 80% measured by main peak of IE-HPLC, with a
fibrinogen composition that does not contain a thrombin to generate
a therapeutic protein-fibrinogen solution; combining the
therapeutic protein-fibrinogen solution with a thrombin composition
to generate a therapeutic composition to promote neuroregeneration
and neuroprotection; and administering the therapeutic composition
to the subject in need of treatment of spinal cord injury.
[0106] In some embodiments, the pharmaceutical composition is first
added to a solution comprising one or more plasminogen-activator
inhibitors or plasmin inhibitors before mixing with the fibrinogen
composition.
[0107] In some embodiments, the one or more plasminogen-activator
inhibitors or plasmin inhibitors comprises aprotinin.
[0108] In some embodiments, the thrombin composition comprises
thrombin and calcium chloride.
[0109] In some embodiments, the therapeutic active-fibrinogen
solution and the thrombin composition are combined and administered
using a Duploject Syringe to the subject in need thereof.
[0110] In one aspect, the present invention provides a kit to
promote neuroregeneration or neuroprotection, treating spinal cord
injury, or facilitating axon growth comprising: (A) a first
container containing a pharmaceutical composition comprising a
purified recombinant C3 fusion protein having an amino acid
sequence at least 85% identical to SEQ ID NO:1, wherein the
purified recombinant C3 fusion protein has a purity of equal to or
greater than 80% measured by main peak of IE-HPLC; a second
container containing a fibrinogen composition; and a third
container containing a thrombin composition; or (B) a first chamber
containing a pharmaceutical composition comprising a purified
recombinant C3 fusion protein having an amino acid sequence at
least 85% identical to SEQ ID NO:1, wherein the purified
recombinant C3 fusion protein has a purity of equal to or greater
than 80% measured by main peak of IE-HPLC; and a second chamber
containing a thrombin composition.
[0111] In some embodiments, the fibrinogen composition comprises
fibrinogen and, albumin, one or more blood coagulation factors,
and/or globulin.
[0112] In some embodiments, the kit further comprises an additional
container containing a solution comprising one or more
plasminogen-activator inhibitors or plasmin inhibitors. In some
embodiments, the one or more plasminogen-activator inhibitors or
plasmin inhibitors comprises aprotinin.
[0113] In some embodiments, the kit further comprises a solution
comprising calcium chloride.
[0114] In some embodiments, the kit further comprises a syringe. In
some embodiments, the syringe is a Duploject Syringe.
[0115] In some embodiments, pharmaceutical composition comprises
the purified recombinant C3 protein having an amino acid sequence
at least 90% identical to SEQ ID NO:1. In some embodiments, the
purified recombinant C3 protein having an amino acid sequence at
least 95% identical to SEQ ID NO:1. In some embodiments, the
purified recombinant C3 protein having an amino acid sequence
identical to SEQ ID NO:1. In some embodiments, the purified
recombinant C3 protein has 213-231 amino acids in total. In some
embodiments, the purified recombinant C3 protein has 213 231 amino
acids in total. In some embodiments, the purified recombinant C3
protein has 213 the purified recombinant C3 protein having an amino
acid sequence at least 90% identical to SEQ ID NO:1.
[0116] In some embodiments, the purified recombinant C3 protein
having an amino acid sequence at least 95% identical to SEQ ID
NO:1. In some embodiments, the purified recombinant C3 protein
having an amino acid sequence identical to SEQ ID NO:1. In some
embodiments, the purified recombinant C3 protein has 213-231 amino
acids in total. In some embodiments, the purified recombinant C3
has 231 amino acids in total.
[0117] Other features, objects, and advantages of the present
invention are apparent in the detailed description, drawings and
claims that follow. It should be understood, however, that the
detailed description, the drawings, and the claims, while
indicating embodiments of the present invention, are given by way
of illustration only, not limitation. Various changes and
modifications within the scope of the invention will become
apparent to those skilled in the art.
BRIEF DESCRIPTION OF THE DRAWING
[0118] The drawings are for illustration purposes only not for
limitation.
[0119] FIG. 1 shows a graphical representation of an exemplary
exponential fermentation feed strategy that is carbon source
(c-source) limiting with controlled growth.
[0120] FIG. 2 shows an example of C3 fusion protein activity
dependence on .epsilon.-NAD concentration.
[0121] FIG. 3 shows exemplary C3 fusion protein (Compound X)
production titer at 30 liter development scale.
[0122] FIGS. 4A and 4B show exemplary cell growth profiles using
the HCD fermentation method (FIG. 4A) and the standard fermentation
method (FIG. 4B).
[0123] FIG. 5 shows exemplary stability profiles of exemplary C3
fusion protein drug substance before and after stress measured by
RP-HPLC and IE-HPLC methods.
[0124] FIG. 6 shows characterization of exemplary C3 fusion protein
drug substance produced by Process 1 using IE-HPLC.
[0125] FIG. 7A shows characterization of exemplary C3 fusion
protein drug substance produced by Process 1 after the fermentation
and downstream processes and Process 2 after the fermentation but
prior to the downstream processes.
[0126] FIG. 7B shows characterization of exemplary C3 fusion
protein drug substance produced by Process 1 and Process 2 after
the fermentation and downstream processes for both.
[0127] FIG. 8 shows exemplary chromatograms from two exemplary
batches of C3 fusion protein drug substance purified with Process 2
including an intermediate cation exchange column.
[0128] FIG. 9 shows biological activity of exemplary C3 fusion
protein produced by Process 1 and Process 2.
[0129] FIG. 10 depicts an exemplary illustration of combining
fibrinogen, aprotinin and a C3 fusion protein to create a C3 fusion
protein-fibrinogen solution and separately combining thrombin and
CaCl.sub.2 to create a thrombin solution and then putting each
solution in a separate chamber of a Duploject Syringe.
[0130] FIG. 11 depicts an exemplary illustration of combining
fibrinogen and aprotinin fibrinogen solution and separately
combining thrombin, CaCl.sub.2 and a C3 fusion protein to create a
C3 fusion protein-thrombin solution and then putting each solution
in a separate chamber of a Duploject Syringe.
[0131] FIG. 12 depicts an exemplary comparison of neurite outgrowth
assay results for a C3 fusion protein and a C3 fusion protein with
a truncated transport sequence using dissociated primary human
neurons at 24 and 48 hours in vitro.
[0132] FIG. 13 depicts an exemplary graph comparing the effect of
premixing a C3 fusion protein with thrombin vs. premixing a C3
fusion protein with fibrinogen as measured using high-performance
liquid chromatography-mass spectrometry (HPLC-MS).
[0133] FIG. 14 depicts an exemplary graph comparing neurite
outgrowth of dissociated human neurons 24 hours after being treated
with a C3 fusion protein that was mixed with either thrombin or
fibrinogen for 4 hours before treatment.
DEFINITIONS
[0134] In order for the present invention to be more readily
understood, certain terms are first defined below. Additional
definitions for the following terms and other terms are set forth
throughout the specification. The publications and other reference
materials referenced herein to describe the background of the
invention and to provide additional detail regarding its practice
are hereby incorporated by reference.
[0135] Amino acid: As used herein, term "amino acid," in its
broadest sense, refers to any compound and/or substance that can be
incorporated into a polypeptide chain. In some embodiments, an
amino acid has the general structure H.sub.2N--C(H)(R)--COOH. In
some embodiments, an amino acid is a naturally occurring amino
acid. In some embodiments, an amino acid is a synthetic amino acid;
in some embodiments, an amino acid is a d-amino acid; in some
embodiments, an amino acid is an 1-amino acid. "Standard amino
acid" refers to any of the twenty standard 1-amino acids commonly
found in naturally occurring peptides. "Nonstandard amino acid"
refers to any amino acid, other than the standard amino acids,
regardless of whether it is prepared synthetically or obtained from
a natural source. As used herein, "synthetic amino acid"
encompasses chemically modified amino acids, including but not
limited to salts, amino acid derivatives (such as amides), and/or
substitutions. Amino acids, including carboxy- and/or
amino-terminal amino acids in peptides, can be modified by
methylation, amidation, acetylation, protecting groups, and/or
substitution with other chemical groups that can change the
peptide's circulating half-life without adversely affecting their
activity. Amino acids may participate in a disulfide bond. Amino
acids may comprise one or posttranslational modifications, such as
association with one or more chemical entities (e.g., methyl
groups, acetate groups, acetyl groups, phosphate groups, formyl
moieties, isoprenoid groups, sulfate groups, polyethylene glycol
moieties, lipid moieties, carbohydrate moieties, biotin moieties,
etc.). The term "amino acid" is used interchangeably with "amino
acid residue," and may refer to a free amino acid and/or to an
amino acid residue of a peptide. It will be apparent from the
context in which the term is used whether it refers to a free amino
acid or a residue of a peptide.
[0136] Animal: As used herein, the term "animal" refers to any
member of the animal kingdom. In some embodiments, "animal" refers
to humans, at any stage of development. In some embodiments,
"animal" refers to non-human animals, at any stage of development.
In certain embodiments, the non-human animal is a mammal (e.g., a
rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep,
cattle, a primate, and/or a pig). In some embodiments, animals
include, but are not limited to, mammals, birds, reptiles,
amphibians, fish, insects, and/or worms. In some embodiments, an
animal may be a transgenic animal, genetically-engineered animal,
and/or a clone.
[0137] Approximately or about: As used herein, the term
"approximately" or "about," as applied to one or more values of
interest, refers to a value that is similar to a stated reference
value. In certain embodiments, the term "approximately" or "about"
refers to a range of values that fall within 25%, 20%, 19%, 18%,
17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,
2%, 1%, or less in either direction (greater than or less than) of
the stated reference value unless otherwise stated or otherwise
evident from the context (except where such number would exceed
100% of a possible value).
[0138] Batch culture or batch mode: The term "batch culture" or
"batch mode" as used herein refers to a method of culturing cells
in which the components that will ultimately be used in culturing
the cells, including the medium (see definition of "medium" below)
as well as the cells themselves, are provided at the beginning of
the culturing process. Thus, a batch culture or batch mode
typically refers to a culture allowed to progress from inoculation
to conclusion of either the culture process or a density without
refeeding the cultured cells with fresh medium. A batch culture is
typically stopped at some point and the cells and/or components in
the medium are harvested and optionally purified.
[0139] Biologically active: As used herein, the phrase
"biologically active" refers to a characteristic of any agent that
has activity in a biological system, and particularly in an
organism. For instance, an agent that, when administered to an
organism, has a biological effect on that organism, is considered
to be biologically active. In particular embodiments, where a
protein or polypeptide is biologically active, a portion of that
protein or polypeptide that shares at least one biological activity
of the protein or polypeptide is typically referred to as a
"biologically active" portion.
[0140] Bioreactor: The term "bioreactor" as used herein refers to a
vessel used for the growth of microorganisms and mammalian cell
culture. A bioreactor can be of any size so long as it is useful
for the culturing cells. Typically, a bioreactor will be at least 1
liter and may be 5, 10, 15, 20, 30 100, 250, 300, 500, 1000, 2500,
5000, 8000, 10,000, 12,0000 liters or more, or any volume in
between. Internal conditions of a bioreactor, including, but not
limited to pH, osmolarity, CO2 saturation, O2 saturation,
temperature and combinations thereof, are typically controlled
during the culturing period. A bioreactor can be composed of any
material that suitable for holding cells in media under the culture
conditions of the present invention, including glass, plastic or
metal. In some embodiments, a bioreactor may be used for performing
animal cell culture. In some embodiments, a bioreactor may be used
for performing mammalian cell culture. In some embodiments, a
bioreactor may be used with cells and/or cell lines derived from
such organisms as, but not limited to, mammalian cell, insect
cells, bacterial cells, yeast cells and human cells. In some
embodiments, a bioreactor is used for large-scale cell culture
production and is typically at least 30 liters and may be 100, 200,
300, 500, 1000, 2500, 5000, 8000, 10,000, 12,0000 liters or more,
or any volume in between. In some embodiments, a bioreactor may
comprise a fermentor. One of ordinary skill in the art will be
aware of and will be able to choose suitable bioreactors for use in
practicing the present invention. As used herein, the term
"bioreactor" and "fermentor" may be used interchangeably.
[0141] Cell density: The term "cell density" as used herein refers
to that number of cells present in a given volume of medium.
[0142] Cell culture or culture: These terms as used herein refer to
a cell population that is gown in a medium under conditions
suitable to survival and/or growth of the cell population. As will
be clear to those of ordinary skill in the art, these terms as used
herein may refer to the combination comprising the cell population
and the medium in which the population is grown. As used herein,
the expressions "cell," "cell line," and "cell culture" are used
interchangeably and all such designations include progeny. Thus,
the words "transformants" and "transformed cells" include the
primary subject cell and cultures derived therefrom without regard
for the number of transfers. It is also understood that all progeny
may not be precisely identical in DNA content, due to deliberate or
inadvertent mutations. Mutant progeny that have the same function
or biological activity as screened for in the originally
transformed cell are included.
[0143] Chromatography: As used herein, the term "chromatography"
refers to a technique for separation of mixtures. Typically, the
mixture is dissolved in a fluid called the "mobile phase," which
carries it through a structure holding another material called the
"stationary phase." Column chromatography is a separation technique
in which the stationary bed is within a tube, i.e., column
[0144] Cultivation: As used herein, the term "cultivation" or
grammatical equivalents refers to a process of maintaining cells
under conditions favoring growth or survival. The terms
"cultivation" and "cell culture" or any synonyms are used
inter-changeably in this application.
[0145] Culture vessel: As used herein, the term "culture vessel"
refers to any container that can provide an aseptic environment for
culturing cells. Exemplary culture vessels include, but are not
limited to, glass, plastic, or metal containers.
[0146] Delivery: As used herein, the term "delivery" encompasses
both local and systemic delivery. For example, delivery of mRNA
encompasses situations in which an mRNA is delivered to a target
tissue and the encoded protein is expressed and retained within the
target tissue (also referred to as "local distribution" or "local
delivery"), and situations in which an mRNA is delivered to a
target tissue and the encoded protein is expressed and secreted
into patient's circulation system (e.g., serum) and systematically
distributed and taken up by other tissues (also referred to as
"systemic distribution" or "systemic delivery).
[0147] Elution: As used herein, the term "elution" refers to the
process of extracting one material from another by washing with a
solvent. For example, in ion-exchange chromatography, elution is a
process to wash loaded resins to remove captured ions.
[0148] Eluate: As used herein, the term "eluate" refers to a
combination of mobile phase "carrier" and the analyte material that
emerge from the chromatography, typically as a result of
eluting.
[0149] Equilibrate or Equilibration: As used herein, the terms
"equilibrate" or "equilibration" in relation to chromatography
refer to the process of bringing a first liquid (e.g., buffer) into
balance with another, generally to achieve a stable and equal
distribution of components of the liquid (e.g., buffer). For
example, in some embodiments, a chromatographic column may be
equilibrated by passing one or more column volumes of a desired
liquid (e.g., buffer) through the column.
[0150] Expression: As used herein, "expression" of a nucleic acid
sequence refers to translation of an mRNA into a polypeptide,
assemble multiple polypeptides into an intact protein (e.g.,
enzyme) and/or post-translational modification of a polypeptide or
fully assembled protein (e.g., enzyme). In some embodiments,
protein expression encompasses the process of transcription of the
DNA encoding a polypeptide of interest (e.g., a recombinant C3
fusion protein) into an mRNA transcript and translation of an mRNA
transcript into a polypeptide, the assembly into an intact protein
(e.g., enzyme) and/or post-translational modification of a
polypeptide or fully assembled protein (e.g., enzyme). In this
application, the terms "expression" and "production," and
grammatical equivalent, are used inter-changeably.
[0151] Fed-batch culture or Fed-batch mode: The term "fed-batch
culture" or "fed-batch mode" as used herein refers to a method of
culturing cells in which additional components are provided to the
culture at some time subsequent to the beginning of the culture
process. The provided components typically comprise nutritional
supplements for the cells which have been depleted during the
culturing process. A fed-batch culture is typically stopped at some
point and the cells and/or components in the medium are harvested
and optionally purified.
[0152] Fermentor or Fermenter: As used herein, "fermentor" or
"fermenter" refers to an apparatus that maintains optimal
conditions for the growth of microorganisms. In some embodiments, a
fermentor may be used in large-scale fermentation and in the
commercial production of recombinant proteins. In some embodiments
a bioreactor may also comprise a fermentor. Bioreactors such as
steel fermentors can accommodate very large culture volumes.
Bioreactors also typically allow for the control of culture
conditions such as temperature, pH, oxygen tension, and carbon
dioxide levels. For example, bioreactors are typically
configurable, for example, using ports attached to tubing, to allow
gaseous components, like oxygen or nitrogen, to be bubbled through
a liquid culture. Other culture parameters, such as the pH of the
culture media, the identity and concentration of trace elements,
and other media constituents can also be more readily manipulated
using a bioreactor. As used herein, the term "bioreactor",
"fermentor" and "fermenter" may be used interchangeably.
[0153] Functional: As used herein, a "functional" biological
molecule is a biological molecule in a form in which it exhibits a
property and/or activity by which it is characterized.
[0154] Half-life: As used herein, the term "half-life" is the time
required for a quantity such as nucleic acid or protein
concentration or activity to fall to half of its value as measured
at the beginning of a time period.
[0155] Impurities: As used herein, the term "impurities" refers to
substances inside a confined amount of liquid, gas, or solid, which
differ from the chemical composition of the target material or
compound. Impurities are also referred to as contaminants.
[0156] Improve, increase, or reduce: As used herein, the terms
"improve," "increase" or "reduce," or grammatical equivalents,
indicate values that are relative to a baseline measurement, such
as a measurement in the same individual prior to initiation of the
treatment described herein, or a measurement in a control subject
(or multiple control subject) in the absence of the treatment
described herein. A "control subject" is a subject afflicted with
the same form of disease as the subject being treated, who is about
the same age as the subject being treated.
[0157] In Vitro: As used herein, the term "in vitro" refers to
events that occur in an artificial environment, e.g., in a test
tube or reaction vessel, in cell culture, etc., rather than within
a multi-cellular organism.
[0158] In Vivo: As used herein, the term "in vivo" refers to events
that occur within a multi-cellular organism, such as a human and a
non-human animal. In the context of cell-based systems, the term
may be used to refer to events that occur within a living cell (as
opposed to, for example, in vitro systems).
[0159] Isolated: As used herein, the term "isolated" refers to a
substance and/or entity that has been (1) separated from at least
some of the components with which it was associated when initially
produced (whether in nature and/or in an experimental setting),
and/or (2) produced, prepared, and/or manufactured by the hand of
man. Isolated substances and/or entities may be separated from
about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,
about 70%, about 80%, about 90%, about 91%, about 92%, about 93%,
about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,
or more than about 99% of the other components with which they were
initially associated. In some embodiments, isolated agents are
about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,
about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,
or more than about 99% pure. As used herein, a substance is "pure"
if it is substantially free of other components. As used herein,
calculation of percent purity of isolated substances and/or
entities should not include excipients (e.g., buffer, solvent,
water, etc.). In some embodiments, percentage of purity is
calculated using an assay such as SDS-PAGE (e.g., with Coomassie
Blue staining or silver staining), SE-HPLC, RP-HPLC, IE-HPLC, to
name but a few.
[0160] Load: As used herein, the term "load" refers to, in
chromatography, adding a sample-containing liquid or solid to a
column. In some embodiments, particular components of the sample
loaded onto the column are then captured as the loaded sample
passes through the column. In some embodiments, particular
components of the sample loaded onto the column are not captured
by, or "flow through", the column as the loaded sample passes
through the column.
[0161] Local distribution or delivery: As used herein, the terms
"local distribution," "local delivery," or grammatical equivalent,
refer to tissue specific delivery or distribution. Typically, local
distribution or delivery requires a protein (e.g., enzyme) encoded
by mRNAs be translated and expressed intracellularly or with
limited secretion that avoids entering the patient's circulation
system.
[0162] Medium: The terms as used herein refer to a solution
containing nutrients which nourish growing cells. Typically, these
solutions may provide essential and non-essential amino acids,
vitamins, energy sources, lipids, and trace elements required by
the cell for minimal growth and/or survival. The solution may also
contain components that enhance growth and/or survival above the
minimal rate, including hormones and growth factors. In some
embodiments, the medium may also comprise one or more antibiotics,
which serve as selectable markers to ensure that virtually all
cells retain the plasmid which encodes the target protein. In some
embodiments, medium is formulated to a pH and salt concentration
optimal for cell survival and proliferation. In some embodiments,
medium may be a "chemically defined medium". In some embodiments,
chemically defined medium is a medium in which all components
within the medium have a known chemical structure.
[0163] Optical density: The term "optical density" refers to a
reading of a bacterial culture which is a measure of the light
scattering, which varies depending on the distance between the
sample and the detector. Optical density (OD) is a common method to
quantify the concentration of substances (Beer-Lambert law), since
the absorbance is proportional to the concentration of the
absorbing species in the sample Calibration of each individual
spectrophotometer is required to facilitate accurate conversion of
OD measurements into the number of cells per ml.
[0164] Patient. As used herein, the term "patient" or "subject"
refers to any organism to which a provided composition may be
administered, e.g., for experimental, diagnostic, prophylactic,
cosmetic, and/or therapeutic purposes. Typical patients include
animals (e.g., mammals such as mice, rats, rabbits, non-human
primates, and/or humans). In some embodiments, a patient is a
human. A human includes pre- and post-natal forms.
[0165] Pharmaceutically acceptable: The term "pharmaceutically
acceptable" as used herein, refers to substances that, within the
scope of sound medical judgment, are suitable for use in contact
with the tissues of human beings and animals without excessive
toxicity, irritation, allergic response, or other problem or
complication, commensurate with a reasonable benefit/risk
ratio.
[0166] Polypeptide: As used herein, a "polypeptide", generally
speaking, is a string of at least two amino acids attached to one
another by a peptide bond. In some embodiments, a polypeptide may
include at least 3-5 amino acids, each of which is attached to
others by way of at least one peptide bond. Those of ordinary skill
in the art will appreciate that polypeptides sometimes include
"non-natural" amino acids or other entities that nonetheless are
capable of integrating into a polypeptide chain, optionally.
[0167] Pool: As used herein, the term "pool" in relation to
chromatography refers to combining one or more fractions of fluid
that has passed through a column together. For example, in some
embodiments, one or more fractions which contain a desired
component of a sample that has been separated by chromatography
(e.g., "peak fractions") can be "pooled" together generate a single
"pooled" fraction.
[0168] Protein: As used herein, the term "protein" refers to a
polypeptide (i.e., a string of at least two amino acids linked to
one another by peptide bonds). Proteins may include moieties other
than amino acids (e.g., may be glycoproteins, proteoglycans, etc.)
and/or may be otherwise processed or modified. Those of ordinary
skill in the art will appreciate that a "protein" can be a complete
polypeptide chain as produced by a cell (with or without a signal
sequence), or can be a characteristic portion thereof. In some
embodiments, a protein can sometimes include more than one
polypeptide chain, for example linked by one or more disulfide
bonds or associated by other means. In some embodiments,
polypeptides may contain L-amino acids, D-amino acids, or both and
may contain any of a variety of amino acid modifications or analogs
known in the art. Useful modifications include, e.g., terminal
acetylation, amidation, methylation, etc. In some embodiments,
proteins may comprise natural amino acids, non-natural amino acids,
synthetic amino acids, and combinations thereof. The term "peptide"
is generally used to refer to a polypeptide having a length of less
than about 100 amino acids, less than about 50 amino acids, less
than 20 amino acids, or less than 10 amino acids. In some
embodiments, proteins are antibodies, antibody fragments,
biologically active portions thereof, and/or characteristic
portions thereof.
[0169] Recombinant protein and Recombinant polypeptide: These terms
as used herein refer to a polypeptide expressed from a host cell
that has been genetically engineered to express that polypeptide.
In some embodiments, a recombinant protein may be expressed in a
host cell derived from an animal. In some embodiments, a
recombinant protein may be expressed in a host cell derived from an
insect. In some embodiments, a recombinant protein may be expressed
in a host cell derived from a yeast. In some embodiments, a
recombinant protein may be expressed in a host cell derived from a
prokaryote. In some embodiments, a recombinant protein may be
expressed in a host cell derived from Escherichia coli (e.g.,
BL21). In some embodiments, a recombinant protein may be expressed
in a host cell derived from a mammal. In some embodiments, a
recombinant protein may be expressed in a host cell derived from a
human. In some embodiments, the recombinant expressed polypeptide
may be identical or similar to a polypeptide that is normally
expressed in the host cell. In some embodiments, the recombinantly
expressed polypeptide may be foreign to the host cell, i.e.
heterologous to peptides normally expressed in the host cell.
Alternatively, in some embodiments the recombinantly expressed
polypeptide can be a chimeric, in that portions of the polypeptide
contain amino acid sequences that are identical or similar to
polypeptides normally expressed in the host cell, while other
portions are foreign to the host cell.
[0170] Seeding: The term "seeding" as used herein refers to the
process of providing a cell culture to a bioreactor or another
vessel for large scale cell culture production. In some embodiments
a "seed culture" is used, in which the cells have been propagated
in a smaller cell culture vessel, i.e. culture flask, culture
plate, culture roller bottle, test tube, etc., prior to seeding.
Alternatively, in some embodiments, the cells may have been frozen
and thawed immediately prior to providing them to the bioreactor or
vessel. The term refers to any number of cells, including a single
cell.
[0171] Soluble: As used herein, the term "soluble" refers to the
ability of a therapeutic agent to form a homogenous solution. In
some embodiments, the solubility of the therapeutic agent in the
solution into which it is administered and by which it is
transported to the target site of action is sufficient to permit
the delivery of a therapeutically effective amount of the
therapeutic agent to the targeted site of action. Several factors
can impact the solubility of the therapeutic agents. For example,
relevant factors which may impact protein solubility include ionic
strength, amino acid sequence and the presence of other
co-solubilizing agents or salts (e.g., calcium salts). In some
embodiments, therapeutic agents in accordance with the present
invention are soluble in its corresponding pharmaceutical
composition.
[0172] Stability: As used herein, the term "stable" refers to the
ability of the therapeutic agent (e.g., a recombinant enzyme) to
maintain its therapeutic efficacy (e.g., all or the majority of its
intended biological activity and/or physiochemical integrity) over
extended periods of time. The stability of a therapeutic agent, and
the capability of the pharmaceutical composition to maintain
stability of such therapeutic agent, may be assessed over extended
periods of time (e.g., for at least 1, 3, 6, 12, 18, 24, 30, 36
months or more). In the context of a formulation a stable
formulation is one in which the therapeutic agent therein
essentially retains its physical and/or chemical integrity and
biological activity upon storage and during processes (such as
freeze/thaw, mechanical mixing and lyophilization). For protein
stability, it can be measure by formation of high molecular weight
(HMW) aggregates, loss of enzyme activity, generation of peptide
fragments and shift of charge profiles.
[0173] Systemic distribution or delivery: As used herein, the terms
"systemic distribution," "systemic delivery," or grammatical
equivalent, refer to a delivery or distribution mechanism or
approach that affect the entire body or an entire organism.
Typically, systemic distribution or delivery is accomplished via
body's circulation system, e.g., blood stream compared to the
definition of "local distribution or delivery."
[0174] Subject: As used herein, the term "subject" refers to a
human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat,
cattle, swine, sheep, horse or primate). A human includes pre- and
post-natal forms. In many embodiments, a subject is a human being.
A subject can be a patient, which refers to a human presenting to a
medical provider for diagnosis or treatment of a disease. The term
"subject" is used herein interchangeably with "individual" or
"patient." A subject can be afflicted with or is susceptible to a
disease or disorder but may or may not display symptoms of the
disease or disorder.
[0175] Substantially: As used herein, the term "substantially"
refers to the qualitative condition of exhibiting total or
near-total extent or degree of a characteristic or property of
interest. One of ordinary skill in the biological arts will
understand that biological and chemical phenomena rarely, if ever,
go to completion and/or proceed to completeness or achieve or avoid
an absolute result. The term "substantially" is therefore used
herein to capture the potential lack of completeness inherent in
many biological and chemical phenomena.
[0176] Target tissues: As used herein, the term "target tissues"
refers to any tissue that is affected by a disease to be treated.
In some embodiments, target tissues include those tissues that
display disease-associated pathology, symptom, or feature.
[0177] Therapeutically effective amount: As used herein, the term
"therapeutically effective amount" of a therapeutic agent means an
amount that is sufficient, when administered to a subject suffering
from or susceptible to a disease, disorder, and/or condition, to
treat, diagnose, prevent, and/or delay the onset of the symptom(s)
of the disease, disorder, and/or condition. It will be appreciated
by those of ordinary skill in the art that a therapeutically
effective amount is typically administered via a dosing regimen
comprising at least one unit dose.
[0178] Therapeutic protein: As used herein, the term "therapeutic
protein" refers to a protein with pharmacological activity.
Therapeutic proteins can act by replacing a protein that is
deficient or abnormal; augmenting an existing pathway; providing a
novel function or activity; interfering with a molecule or
organism; and/or delivering other compounds or proteins, such as a
radionuclide, cytotoxic drug, or effector proteins. In some
embodiments, therapeutic proteins can also be grouped based on
their molecular types that include antibody-based drugs, Fc fusion
proteins, anticoagulants, blood factors, bone morphogenetic
proteins, engineered protein scaffolds, enzymes, growth factors,
hormones, interferons, interleukins, and thrombolytics. In some
embodiments, therapeutic proteins can be classified based on their
molecular mechanism of activity as binding non-covalently to
target, e.g., mAbs; affecting covalent bonds, e.g., enzymes; and
exerting activity without specific interactions, e.g., serum
albumin.
[0179] Treating: As used herein, the term "treat," "treatment," or
"treating" refers to any method used to partially or completely
alleviate, ameliorate, relieve, inhibit, prevent, repair, delay
onset of, reduce severity of and/or reduce incidence of one or more
symptoms or features of a particular disease, disorder, and/or
condition. Treatment may be administered to a subject who does not
exhibit signs of a disease and/or exhibits only early signs of the
disease for the purpose of decreasing the risk of developing
pathology associated with the disease.
DETAILED DESCRIPTION
[0180] The present invention provides, among other things, methods
of producing and purifying therapeutic C3 transferase fusion
proteins. In some embodiments, the present invention provides a
method for producing C3 fusion proteins by cultivating host cells
under conditions that promote expression at a specific productivity
rate. In some embodiments, the present invention provides methods
of producing C3 fusion proteins in a large scale vessel under
conditions that involve fermentation. In some embodiments, the
present invention provides a method of purifying a recombinant C3
fusion protein from an impure preparation (e.g., unprocessed
biological materials, such as C3 fusion protein-containing cells
and/or culture medium) using a series of chromatography steps
(e.g., ion exchange, hydrophobic interaction chromatography).
[0181] In some embodiments, the present invention provides a
pharmaceutical composition comprising a therapeutic C3 fusion
protein. In some embodiments, the present invention provides a
pharmaceutical composition comprising a mixture of therapeutic C3
fusion proteins. In various embodiments, pharmaceutical
compositions provided by the present invention are used to
effectively treat spinal cord injury or other CNS trauma,
facilitate axon growth or aid in other tissue repair.
[0182] In some embodiments, the present invention provides a method
of preparing a therapeutic composition by mixing a therapeutically
effective amount of a therapeutic protein (e.g., an ADP-ribosyl
transferase C3 protein) with a fibrinogen composition that does not
contain a thrombin to generate a therapeutic protein-fibrinogen
composition; and combining the therapeutic protein-fibrinogen
composition with a thrombin composition to generate a therapeutic
composition. In some embodiments, a therapeutic composition
prepared according to the present invention is a tissue adhesive
that may be administered to a spinal cord injury site to facilitate
axon growth.
[0183] Various aspects of the invention are described in further
detail in the following subsections. The use of subsections is not
meant to limit the invention. Each subsection may apply to any
aspect of the invention. In this application, the use of "or" means
"and/or" unless stated otherwise.
C3 Fusion Protein
[0184] Among other things, the present invention is used to produce
a C3 fusion protein containing an ADP-ribosyl transferase C3 domain
and a transport domain. In some embodiments, a C3 fusion protein
comprises an amino acid sequence of a transport domain covalently
linked to an amino acid sequence of an ADP-ribosyl transferase C3
domain, wherein the amino acid sequence of said transport domain is
selected from a Tat peptide or antennapedia peptide, a fragment or
subdomain of Tat peptide or antennapedia peptide, a polypeptide
derived from a nucleotide sequence encoding a Tat peptide or
antennapedia peptide, or a polypeptide having an amino acid
sequence having at least 80% (e.g., at least 85%, 90%, 92%, 94%,
95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto and
wherein the amino acid sequence of the active domain of the C3
fusion protein is selected from an ADP-ribosyl transferase C3, a
fragment thereof retaining ADP-ribosyl transferase activity, or an
amino acid sequence having at least 80% (e.g., at least 85%, 90%,
92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity
thereto.
[0185] In some embodiments, a C3 fusion protein amino acid sequence
is as follows:
TABLE-US-00001 (SEQ ID NO: 1)
SAYSNTYQEFTNIDQAKAWGNAQYKKYGLSKSEKEAIVSYTKSASEINGK
LRQNKGVINGFPSNLIKQVELLDKSFNKMKTPENIMLFRGDDPAYLGTEF
QNTLLNSNGTINKTAFEKAKAKFLNKDRLEYGYISTSLMNVSQFAGRPII
TKFKVAKGSKAGYIDPISAFAGQLEMLLPRHSTYHIDDMRLSSDGKQIII
TATMMGTAINPKEFVMNPANAQGRHTPGTRL
[0186] In some embodiments, a C3 fusion protein amino acid sequence
is as follows:
TABLE-US-00002 (SEQ ID NO: 2)
MSAYSNTYQEFTNIDQAKAWGNAQYKKYGLSKSEKEAIVSYTKSASEING
KLRQNKGVINGFPSNLIKQVELLDKSFNKMKTPENIMLFRGDDPAYLGTE
FQNTLLNSNGTINKTAFEKAKAKFLNKDRLEYGYISTSLMNVSQFAGRPI
ITKFKVAKGSKAGYIDPISAFAGQLEMLLPRHSTYHIDDMRLSSDGKQII
ITATMMGTAINPKEFVMNPANAQGRHTPGTRL
[0187] In some embodiments, a C3 fusion protein comprises an amino
acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
100% sequence identity with SEQ ID NO: 1. In some embodiments, a C3
fusion protein comprises an amino acid sequence having at least
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with
SEQ ID NO: 2.
[0188] As used herein, the term "recombinant C3 fusion protein"
refers to any molecule or a portion of a molecule that can
substitute for at least partial activity of naturally-occurring
ADP-ribosyl transferase C3 protein. As used herein, the terms
"recombinant C3 enzyme" and "recombinant C3 protein", and
grammatical equivalents, are used inter-changeably. In some
embodiments, the present invention is used to purify a recombinant
C3 fusion protein that is a polypeptide, wherein the therapeutic
protein comprises an amino acid sequence of a transport domain
covalently linked to an amino acid sequence of an active domain,
said amino acid sequence of said active domain is selected from an
ADP-ribosyl transferase C3, a fragment thereof retaining
ADP-ribosyl transferase activity, or an amino acid sequence having
at least 80% sequence identity thereto, and wherein said amino acid
sequence of said transport domain is selected from a subdomain of
Tat peptide or antennapedia peptide, a fragment of Tat peptide or
antennapedia peptide, a polypeptide derived from a nucleotide
sequence encoding a Tat peptide or antennapedia peptide, or an
amino acid sequence having at least 80% sequence identity
thereto.
[0189] In some embodiments, the DNA sequence encoding a C3 fusion
protein is as follows:
TABLE-US-00003 (SEQ ID NO: 3)
ATGTCGGCTTATTCAAATACTTACCAGGAGTTTACTAATATTGATCAAGC
AAAAGCTTGGGGTAATGCTCAGTATAAAAAGTATGGACTAAGCAAATCAG
AAAAAGAAGCTATAGTATCATATACTAAAAGCGCTAGTGAAATAAATGGA
AAGCTAAGACAAAATAAGGGAGTTATCAATGGATTTCCTTCAAATTTAAT
AAAACAAGTTGAACTTTTAGATAAATCTTTTAATAAAATGAAGACCCCTG
AAAATATTATGTTATTTAGAGGCGACGACCCTGCTTATTTAGGAACAGAA
TTTCAAAACACTCTTCTTAATTCAAATGGTACAATTAATAAAACGGCTTT
TGAAAAGGCTAAAGCTAAGTTTTTAAATAAAGATAGACTTGAATATGGAT
ATATTAGTACTTCATTAATGAATGTTTCTCAATTTGCAGGAAGACCAATT
ATTACAAAATTTAAAGTAGCAAAAGGCTCAAAGGCAGGATATATTGACCC
TATTAGTGCTTTTGCAGGACAACTTGAAATGTTGCTTCCTAGACATAGTA
CTTATCATATAGACGATATGAGATTGTCTTCTGATGGTAAACAAATAATA
ATTACAGCAACAATGATGGGCACAGCTATCAATCCTAAAGAATTCGTGAT
GAATCCCGCAAACGCGCAAGGCAGACATACACCCGGTACCAGACTCTAG
[0190] In some embodiments, a DNA sequence encoding a C3 fusion
protein comprises a nucleic acid sequence having at least 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:
3.
Production of Recombinant C3 Fusion Proteins
[0191] The present invention may be used to purify a recombinant C3
fusion protein produced by various means. For example, a C3 fusion
protein may be recombinantly produced by utilizing a host cell
system engineered to express a C3 fusion protein-encoding nucleic
acid. Typically, cells that are engineered to express recombinant
C3 fusion protein may comprise a transgene that encodes a
recombinant C3 fusion protein described herein. It should be
appreciated that the nucleic acids encoding recombinant C3 fusion
protein may contain regulatory sequences, gene control sequences,
promoters, non-coding sequences and/or other appropriate sequences
for expressing the recombinant C3 fusion protein. Typically, the
coding region is operably linked with one or more of these nucleic
acid components. In some embodiments, SEQ ID NO: 3 is cloned into a
pET9a vector with a Kanamycin resistance gene for antibiotic
selection.
[0192] "Regulatory sequences" typically refer to nucleotide
sequences located upstream (5' non-coding sequences), within, or
downstream (3' non-coding sequences) of a coding sequence, and
which influence the transcription, RNA processing or stability, or
translation of the associated coding sequence. Regulatory sequences
may include promoters, translation leader sequences, introns, and
polyadenylation recognition sequences. Sometimes, "regulatory
sequences" are also referred to as "gene control sequences".
[0193] "Promoter" typically refers to a nucleotide sequence capable
of controlling the expression of a coding sequence or functional
RNA. In general, a coding sequence is located 3' to a promoter
sequence. The promoter sequence consists of proximal and more
distal upstream elements, the latter elements often referred to as
enhancers. Accordingly, an "enhancer" is a nucleotide sequence that
can stimulate promoter activity and may be an innate element of the
promoter or a heterologous element inserted to enhance the level or
tissue-specificity of a promoter. Promoters may be derived in their
entirety from a native gene, or be composed of different elements
derived from different promoters found in nature, or even comprise
synthetic nucleotide segments. It is understood by those skilled in
the art that different promoters may direct the expression of a
gene in different tissues or cell types, or at different stages of
development, or in response to different environmental
conditions.
[0194] The "3' non-coding sequences" typically refer to nucleotide
sequences located downstream of a coding sequence and include
polyadenylation recognition sequences and other sequences encoding
regulatory signals capable of affecting mRNA processing or gene
expression. The polyadenylation signal is usually characterized by
affecting the addition of polyadenylic acid tracts to the 3' end of
the mRNA precursor.
[0195] The "translation leader sequence" or "5' non-coding
sequences" typically refers to a nucleotide sequence located
between the promoter sequence of a gene and the coding sequence.
The translation leader sequence is present in the fully processed
mRNA upstream of the translation start sequence. The translation
leader sequence may affect processing of the primary transcript to
mRNA, mRNA stability or translation efficiency.
[0196] Typically, the term "operatively linked" or "operably
linked" refers to the association of two or more nucleic acid
fragments on a single nucleic acid fragment so that the function of
one is affected by the other. For example, a promoter is
operatively linked with a coding sequence when it is capable of
affecting the expression of that coding sequence (i.e., that the
coding sequence is under the transcriptional control of the
promoter). Coding sequences can be operatively linked to regulatory
sequences in sense or antisense orientation.
[0197] The coding region of a transgene may include one or more
silent mutations to optimize codon usage for a particular cell
type. For example, the codons of a C3 fusion protein transgene may
be optimized for expression in a vertebrate cell. In some
embodiments, the codons of a C3 fusion protein transgene may be
optimized for expression in a mammalian cell. In some embodiments,
the codons of a C3 fusion protein transgene may be optimized for
expression in a human cell.
[0198] Optionally, a construct may contain additional components
such as one or more of the following: a splice site, an enhancer
sequence, a selectable marker gene under the control of an
appropriate promoter, an amplifiable marker gene under the control
of an appropriate promoter, and a matrix attachment region (MAR) or
other element known in the art that enhances expression of the
region where it is inserted.
[0199] Once transfected or transduced into host cells, a suitable
vector can express extrachromosomally (episomally) or integrate
into the host cell's genome.
Host Cells
[0200] As used herein, the term "host cells" refers to cells that
can be used to produce recombinant C3 fusion protein. In
particular, host cells are suitable for producing recombinant C3
fusion protein at a large scale. In some embodiments, host cells
are able to produce C3 fusion protein in an amount ranging from
about or greater than 0.7 g/L of culture, 0.8 g/L of culture, 0.9
g/L of culture, 1.0 g/L of culture, 1.1 g/L of culture, 1.2 g/L of
culture, 1.3 g/L of culture, 1.4 g/L of culture, 1.5 g/L of
culture, 1.6 g/L of culture, 1.7 g/L of culture, 1.8 g/L of
culture, 1.9 g/L of culture, 2.0 g/L of culture, 2.1 g/L of
culture, 2.2 g/L of culture, 2.3 g/L of culture, 2.4 g/L of
culture, 2.5 g/L of culture, 3 g/L of culture, 3.5 g/L of culture,
4 g/L of culture, 4.5 g/L of culture, or 5 g/L of culture according
to the culturing conditions described herein.
[0201] Suitable host cells can be derived from a variety of
organisms, including, but not limited to, mammals, plants, birds
(e.g., avian systems), insects, yeast, and bacteria. In some
embodiments, host cells are mammalian cells. Any mammalian cell
susceptible to cell culture, and to expression of polypeptides, may
be utilized in accordance with the present invention as a host
cell. Non-limiting examples of mammalian cells that may be used in
accordance with the present invention include human embryonic
kidney 293 cells (HEK293), HeLa cells; BALB/c mouse myeloma line
(NSO/l, ECACC No: 85110503); human retinoblasts (PER.C6 (CruCell,
Leiden, The Netherlands)); monkey kidney CV1 line transformed by
SV40 (COS-7, ATCC CRL 1651); human fibrosarcomacell line (e.g.,
HT-1080); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture, Graham et al., J. Gen Virol.,
36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells +/-DHFR (CHO, Urlaub and Chasin, Proc.
Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4,
Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1
ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC
CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2);
canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells
(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75);
human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT
060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.
Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; a human hepatoma
line (Hep G2), human cell line CAP and AGE1.HN, and Glycotope's
panel.
[0202] Non-limiting examples of host cells suitable for the present
invention include cells and cell lines derived from Pichia
pastoris, Pichia methanolica, Pichia angusta, Schizosacccharomyces
pombe, Saccharomyces cerevisiae, and Yarrowia lipolytica for yeast;
Sodoptera frugiperda, Trichoplusis ni, Drosophila melangoster and
Manduca sexta for insects; and Escherichia coli, Salmonella
typhimurium, Bacillus subtilis, Bacillus lichenifonnis, Bacteroides
fragilis, Clostridia perfringens, Clostridia difficile for
bacteria; and Xenopus laevis from amphibian.
[0203] Additionally, any number of available hybridoma cell lines
may be utilized in accordance with the present invention. One
skilled in the art will appreciate that hybridoma cell lines might
have different nutrition requirements and/or might require
different culture conditions for optimal growth and polypeptide or
protein expression, and will be able to modify conditions as
needed.
Culture Medium
[0204] The term "medium" and "culture medium" (and grammatical
equivalents) as used herein refers to a general class of solution
containing nutrients suitable for maintaining and/or growing cells
in vitro. Typically, media solutions provide, without limitation,
essential and nonessential amino acids, vitamins, energy sources,
lipids, and trace elements required by the cell for at least
minimal growth and/or survival. In other embodiments, the medium
may contain an amino acid(s) derived from any source or method
known in the art, including, but not limited to, an amino acid(s)
derived either from single amino acid addition(s) or from a peptone
or protein hydrolysate addition(s) (including animal or plant
source(s)). Vitamins such as, but not limited to, Biotin,
Pantothenate, Choline Chloride, Folic Acid, Myo-Inositol,
Niacinamide, Pyridoxine, Riboflavin, Vitamin B12, Thiamine,
Putrescine and/or combinations thereof. Salts such as, but not
limited to, CaCl.sub.2, KCl, MgCl2, NaCl, Sodium Phosphate
Monobasic, Sodium Phosphate Dibasic, Sodium Selenite, CuSO.sub.4,
ZnCl.sub.2 and/or combinations thereof. Fatty acids such as, but
not limited to, Arachidonic Acid, Linoleic Acid, Oleic Acid, Lauric
Acid, Myristic Acid, as well as Methyl-beta-Cyclodextrin and/or
combinations thereof). In some embodiments, media comprises
additional components such as glucose, glutamine, Na-pyruvate,
insulin or ethanolamine, a protective agent such as Pluronic F68.
In some embodiments, the media may also contain components that
enhance growth and/or survival above the minimal rate, including
hormones and growth factors. Medium may also comprise one or more
buffering agents. The buffering agents may be designed and/or
selected to maintain the culture at a particular pH (e.g., a
physiological pH, (e.g., pH 6.8 to pH 7.4)). A variety of buffers
suitable for culturing cells are known in the art and may be used
in the methods. Suitable buffers (e.g., bicarbonate buffers, HEPES
buffer, Good's buffers, etc.) are those that have the capacity and
efficiency for maintaining physiological pH despite changes in
carbon dioxide concentration associated with cellular respiration.
The solution is preferably formulated to a pH and salt
concentration optimal for cell survival and proliferation.
[0205] In some embodiments, a medium may be a chemically defined
medium. As used herein, the term "chemically-defined nutrient
medium" refers to a medium of which substantially all of the
chemical components are known. Defined media are media composed of
pure ingredients in carefully measured concentrations dissolved in
double distilled water i.e., the exact chemical composition of the
medium is known. In some embodiments, they contain a simple sugar
as the carbon and energy source, an inorganic nitrogen source,
various mineral salts and, if necessary, growth factors (purified
amino acids, vitamins, purines and pyrimidines). In some
embodiments, a chemically defined medium can be prepared by
combining various individual components such as, for example,
essential and nonessential amino acids, vitamins, energy sources,
lipids, salts, buffering agents, and trace elements, at
predetermined weight or molar percentages or ratios.
[0206] In some embodiments, a medium may be a complex medium. As
used herein, the term "complex medium" refers to a medium that may
be rich in nutrients; they may contain water soluble extracts of
plant or animal tissue (e.g., enzymatically digested animal
proteins such as peptone and tryptone). In some embodiments, a
sugar, often glucose, is added to serve as the main carbon and
energy source. The combination of extracts and sugar creates a
medium that is rich in minerals and organic nutrients, but since
the exact composition is unknown, the medium is complex.
[0207] In some embodiments, a medium may be a
selective/differential medium. As used herein, the term
"selective/differential medium" are media based on either of the
two categories above supplemented with growth-promoting or
growth-inhibiting additives. The additives may be species- or
organism-selective (e.g., an antibiotic such as kanamycin, a
specific substrate, or an inhibitor such as cyclohexamide
(artidione), which inhibits all eukaryotic growth and is typically
used to prevent fungal growth in mixed cultures).
[0208] In some embodiments, a medium may be an enrichment medium.
As used herein, the term "enrichment medium" may be a medium
similar to selective media but designed to increase the numbers of
desired microorganisms to a detectable level without stimulating
the rest of the bacterial population.
[0209] In some embodiments, a medium may be a reducing medium. As
used herein, the term "reducing medium" may be used to facilitate
the growth of obligate anaerobes.
[0210] In some embodiments, an exemplary culture medium may be LB,
Terrific, SuperBroth, YT, or 2.times.YT. In some embodiments, a
medium suitable for the present invention is a mixture of one or
more commercially available chemically-defined, complex, selective,
or enrichment media. In various embodiments, a suitable medium is a
mixture of two, three, four, five, six, seven, eight, nine, ten, or
more commercially available chemically-defined media. In some
embodiments, each individual commercially available
chemically-defined medium (e.g., such as those described herein)
constitutes, by weight, 1%, 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, or more, of the mixture. Ratios between each individual
component media may be determined by relative weight percentage
present in the mixture.
Bioreactors
[0211] The invention also provides bioreactors that are useful for
producing recombinant C3 fusion protein. Bioreactors may be, for
example, perfusion, batch, fed-batch, repeated batch, or continuous
(e.g., a continuous stirred-tank reactor model). Typically, the
bioreactors comprise at least one vessel designed and configured to
house medium (e.g., a chemically-defined nutrient medium). The
vessel also typically comprises at least one inlet designed and
configured to flow fresh nutrient media into the vessel. The vessel
also typically comprises at least one outlet designed and
configured to flow waste media out of the vessel. In some
embodiments, the vessel may further comprise at least one filter
designed and configured to minimize the extent to which isolated
cells in the vessel are passed out through the at least one outlet
with waste media. The bioreactor may also be fitted with one or
more other components designed to maintain conditions suitable for
cell growth. For example, the bioreactor may be fitted with one or
more circulation or mixing devices designed and configured to
circulate or mix the nutrient media within the vessel. Typically,
the isolated cells that are engineered to express recombinant C3
fusion protein are suspended in the nutrient medium. Therefore, in
some cases, the circulation device ensures that the isolated cells
remain in suspension in the nutrient medium. In some embodiments,
the cells are attached to a substrate. In some embodiments, the
cells are attached to one or more substrates (e.g., microbeads)
that are suspended in the nutrient medium. The bioreactor may
comprise one or more ports for obtaining a sample of the cell
suspension from the vessel. The bioreactor may be configured with
one or more components for monitoring and/or controlling conditions
of the culture, including conditions such as gas content (e.g.,
air, oxygen, nitrogen, carbon dioxide), flow rates, temperature, pH
and dissolved oxygen levels, and agitation speed/circulation
rate.
Culture Conditions
[0212] Seeding
[0213] The present invention provides a method of producing
recombinant C3 fusion protein at a large scale. Typical large-scale
procedures for producing a recombinant polypeptide of interest
include batch cultures and fed-batch cultures. Batch culture
processes traditionally comprise inoculating a large-scale
production culture with a seed culture of a particular cell
density, growing the cells under conditions (e.g., suitable culture
medium, pH, and temperature) conducive to cell growth, viability,
and/or productivity, harvesting the culture when the cells reach a
specified cell density, and purifying the expressed polypeptide.
Fed-batch culture procedures include an additional step or steps of
supplementing the batch culture with nutrients and other components
that are consumed during the growth of the cells. In some
embodiments, a large-scale production method according to the
present invention uses a fed-batch culture system.
[0214] Typically, a desired cell expressing C3 fusion protein is
first propagated in an initial culture by any of a variety of
methods well-known to one of ordinary skill in the art. The cell is
typically propagated by growing it at a temperature and in a medium
that is conducive to the survival, growth and viability of the
cell. The initial culture volume can be of any size, but is often
smaller than the culture volume of the production bioreactor used
in the final production. The cell culture can be agitated or shaken
to increase oxygenation of the medium and dispersion of nutrients
to the cells. Alternatively or additionally, special sparging
devices that are well known in the art can be used to increase and
control oxygenation of the culture.
[0215] The cell density of the inoculum used to start a seed
culture can be chosen by one of ordinary skill in the art. In some
embodiments, a Master Cell Bank (MCB) vial or a Working Cell Bank
(WCB) vial is used as the inoculum for the seed culture. In some
embodiments, the MCB or WCB vial can be as low as a single cell per
culture volume. In some embodiments, the MCB or WCB inoculum used
to start the seed culture is greater than 1.times.10.sup.6 colony
forming units (CFU) per mL (e.g., greater than about
1.4.times.10.sup.7, 1.4.times.10.sup.8, 1.4.times.10.sup.9,
1.4.times.10.sup.10 CFU/mL and higher).
[0216] Initial cultures may be grown to any desired density before
seeding a final production bioreactor. In some embodiments, once
the optical density (OD) at 600 nm (OD600) reaches an optimal
level, (e.g., 2-10, 2-8, 2-6, 2-4) the culture can be scaled up to
the production bioreactor (e.g., a 30 L bioreactor). In some
embodiments, the initial starting volume of the fermentor can be,
for example, 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, 11
L, 12 L, 13 L, 14 L, 15 L, 16 L, 17 L, 18 L, 19 L, or 20 L. In some
embodiments, once the optical density (OD) at 600 nm (OD600)
reaches an optimal level, (e.g., 2-10, 2-8, 2-6, 2-4) the culture
can be scaled up to the production bioreactor (e.g., a 300 L
bioreactor). In some embodiments, the initial starting volume of
the fermentor can be, for example, 10 L, 20 L, 30 L, 40 L, 50 L, 60
L, 70 L, 80 L, 90 L, 100 L, 110 L, 120 L, 130 L, 140 L, 150 L, 160
L, 170 L, 180 L, 190 L, or 200 L.
[0217] In some embodiments, it may be desirable to wash the removed
seed culture cells with a medium before seeding the next bioreactor
to remove any unwanted metabolic waste products or medium
components. The medium may be the medium in which the cells were
previously grown or it may be a different medium or a washing
solution selected by the practitioner of the present invention.
[0218] The cells may then be diluted to an appropriate density for
seeding the production bioreactor. In some embodiments, the cells
are diluted into the same medium that will be used in the
production bioreactor. Alternatively, the cells can be diluted into
another medium or solution, depending on the needs and desires of
the practitioner of the present invention or to accommodate
particular requirements of the cells themselves, for example, if
they are to be stored for a short period of time prior to seeding
the production bioreactor.
[0219] Fermentation
[0220] Process development focused on two main challenges: low
yields and lack of robustness at the fermentation step. These
challenges were addressed by developing a high-cell density (HCD)
fermentation process, along with optimization of the medium
composition and implementation of an exponential glycerol feeding
strategy. An exemplary fermentation process is shown in FIG. 1.
[0221] In some embodiments, when the cells are ready for the
fermentation process, the culture conditions may be changed to
maximize the production of the recombinant protein of interest. In
some embodiments, such change may be a shift in one or more of a
number of culture conditions including, but not limited to,
temperature, pH, osmolarity, carbon source and medium. In one
embodiment, the carbon source of the culture is shifted. For
example, the carbon source may be shifted from glucose in the batch
phase to glycerol in the feeding phase. In some embodiments, this
change in carbon source is rapid. In some embodiments, this change
in carbon source occurs slowly over a prolonged period of time. In
some embodiments, dissolved oxygen and pH is maintained at 15-50%
(e.g., 20-40%, 20-30%) and 6.5-7.5 respectively throughout the
production process.
[0222] In some embodiments, the temperature is shifted up or down
from the batch phase to feed phase. For example, the temperature
may be shifted up or down from batch phase to the feed phase by
about 1.0.degree. C., 2.0.degree. C., 3.0.degree. C., 4.0.degree.
C., 5.0.degree. C., 6.0.degree. C., 7.0.degree. C., 8.0.degree. C.,
9.0.degree. C., 10.0.degree. C. or more.
[0223] In some embodiments, to maximize yield and robustness, a
high-cell density (HCD) fermentation process may be used. In some
embodiments, an HCD fermentation comprises one or more phases
(e.g., batch-mode phase, fed-batch mode phase). In some
embodiments, an HCD fermentation comprises one or more stages of
feeding (e.g., exponential feeding, constant feeding). In some
embodiments, the feeding stages are the same. In some embodiments,
the feeding stages are not the same.
[0224] In certain embodiments, fermentation is started in batch
mode, in which a defined amount of initial carbon source (glucose)
is provided in the medium. After consumption of the glucose in the
medium, (carbon source limitation), a fed-batch mode is started. In
the fed-batch mode, a glycerol carbon source feed solution was
introduced to the fermentor at an exponential rate (e.g.,
exponential feeding), followed by a second phase of constant
feeding. Thus, in certain embodiments, the feeding strategy
consists of two stages: a first stage of exponential feeding, and a
second stage of constant feeding. The feed parameters are
summarized below:
[0225] 1.sup.st stage: Exponential feeding was controlled
using:
Exp . Feed [ g h ] = 1 .times. 0 .times. 0 .times. 0 * [ ( .mu. Y x
s + m ) V 0 X 0 1 S F . e ( .mu. t ) ] ##EQU00001##
[0226] Where;
[0227] Specific growth rate, .mu., (l/h)
[0228] Biomass yield on glucose, Yx/s, (g/g)
[0229] Maintenance factor, m, (g/g/hr)
[0230] Initial volume of reactor before the start of feed, V.sub.0,
(L)
[0231] Biomass concentration before the start of feed, X.sub.0,
(g/L)
[0232] Feed concentration, S.sub.F, (g/kg)
[0233] Time, t, (hrs)
[0234] 2.sup.nd stage: Constant feed rate=Feed rate at the end of
the exponential feeding stage.
[0235] In some embodiments, the specific growth rate, .mu., is in a
range between 0.12-0.18/hr, the biomass yield on glucose, Yx/s,
ranges from 0.32-0.48 g/g, the maintenance factor, m, ranges from
0.03-0.04 g/g/hr, the initial volume of the reactor before the
start of feed, V.sub.0, ranges from 12 L, the biomass concentration
before the start of feed, X.sub.0, ranges from 8-12 g/L, the feed
concentration, S.sub.F, ranges from 480-720 g/kg, and the time, t,
ranges from 7-8 hrs.
[0236] In certain other embodiments, fermentation is started in
batch mode, in which a defined amount of initial carbon source
(glucose) is provided in the medium. After consumption of the
glucose in the medium, (carbon source limitation), a fed-batch mode
is started. In some embodiments, the fed-batch mode occurs in three
stages wherein a glycerol carbon source feed solution is introduced
to the fermentor at an exponential rate (e.g., exponential
feeding), followed by a second phase of constant feeding. Following
the second stage of constant feeding, a third stage of constant
feeding wherein the feed rate may be the same as the feed rate in
the second stage. In some embodiments, the feed rate in the third
stage is reduced constant feeding at a feed rate about 0.7 times
that of the second feeding stage. Thus, in certain embodiments, the
feeding strategy involves three stages: a first stage of
exponential feeding, a second stage of constant feeding, and a
third stage of constant or reduced constant feeding. The feed
parameters are summarized below:
[0237] 1.sup.st stage: Exponential feeding was controlled
using:
Exp . Feed [ g h ] = 1 .times. 0 .times. 0 .times. 0 * [ ( .mu. Y x
s + m ) V 0 X 0 1 S F . e ( .mu. t ) ] ##EQU00002##
[0238] Where;
[0239] Specific growth rate, .mu., (l/h)
[0240] Biomass yield on glucose, Yx/s, (g/g)
[0241] Maintenance factor, m, (g/g/hr)
[0242] Initial volume of reactor before the start of feed, V.sub.0,
(L)
[0243] Biomass concentration before the start of feed, X.sub.0,
(g/L)
[0244] Feed concentration, S.sub.F, (g/kg)
[0245] Time, t, (hrs)
[0246] 2.sup.nd stage: Constant feed rate=Feed rate at the end of
the exponential feeding stage.
[0247] 3rd stage: Constant feed rate=rate at the end of 2.sup.nd
state, or Reduced constant feed rate=0.7.times.Feed rate of the 2nd
stage (constant feeding).
[0248] Induction
[0249] In some embodiments, cells are induced with an inducing
agent, wherein the inducing agent is Isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG). In some embodiments, the
IPTG is added at a concentration range of 0.1 mM-10 mM. In certain
embodiments, the induction phase begins when the OD is between
145-175. In certain embodiments, the induction phase begins after
8-9 hours of constant feeding. In some embodiments, the third stage
of constant feeding is the induction phase. In some embodiments,
constant feeding is maintained during the induction phase. In other
embodiments, induction triggers a reduced feed rate. In some
embodiments, the temperature is shifted (e.g., reduced) during the
induction phase. In some embodiments, the induction time is between
3-10 hours (e.g., 3-8, 3-6, 3-5, 4-5, 4-6, 4-7, 4-8, or 4-10
hours).
Purification of C3 Fusion Protein
[0250] Embodiments of the invention include purification processes
for the production of C3 fusion protein drug substances. A variety
of techniques, in whole or in part, optionally with modifications
as described herein, may be used to produce purified C3 fusion
protein drug substance. For example, FIGS. 1 and 2 show exemplary
flow diagrams of embodiments of the invention.
[0251] In some embodiments, the present invention provides a method
of purifying recombinant C3 fusion protein from an impure
preparation using a process based on one or more (e.g., two or
more) of cation-exchange and hydrophobic interaction
chromatography. In some embodiments, an inventive method according
to the present invention involves fewer than 4 (e.g., less than 3,
less than 2, or less than 1) chromatography steps. In some
embodiments, an inventive method according to the present invention
involves 2, 3, 4 or 5 chromatography steps. In some embodiments, an
inventive method according to the present invention involves 3
chromatography steps. In some embodiments, an inventive method
according to the present invention conducts one or more (e.g.,
two-or more) cation-exchange chromatography and hydrophobic
interaction chromatography in that order. In some embodiments, an
inventive method disclosed herein further includes one or more
steps (e.g., two or more steps, three or more steps) of
ultrafiltration/diafiltration. In some embodiments, an inventive
method disclosed herein further includes one or more steps of
membrane adsorption.
[0252] Cation Exchange Chromatography
[0253] In some embodiments, provided methods for purifying
recombinant C3 fusion protein include one or more steps of
cation-exchange chromatography. In some embodiments, provided
methods for purifying recombinant C3 fusion protein include two or
more steps of cation-exchange chromatography. In brief, cation
exchange chromatography is a chromatographic technique which relies
on charge-charge interactions between a positively charged compound
and a negatively charged resin. In some embodiments, the
cation-exchange chromatography is strong cation-exchange
chromatography.
[0254] Cation exchange chromatography is generally practiced with
either a strong or weak cation exchange column, containing a
sulfonium ion, or with a weak cation exchanger, having usually a
carboxymethyl (CM) or carboxylate (CX) functional group. Many
suitable cation exchange resins are known in the art and are
commercially available and include, but are not limited to
SP-Sepharose.RTM. (e.g., SP-Sepharose XL resin), CM Sepharose.RTM.;
Amberjet.RTM. resins; Amberlyst.RTM. resins; Amberlite.RTM. resins
(e.g., Amberlite.RTM. IRA120); Capto resins (e.g, Capto SP ImpRes);
Fractogel.RTM. resins (e.g., Fractogel EMD SO3); YMC-BioPro resins
(e.g., YMC-BioPro S30); POROS.RTM. resins (e.g., POROS XS);
Nuvia.TM. resins (e.g., Nuvia H-RS); ProPac.RTM. resins (e.g.,
ProPac.RTM. SCX-10, ProPac.RTM. WCX-10, ProPac.RTM. WCX-10);
Praesto.TM. resins (e.g., Praesto SP45); TSK-GEL.RTM. resins (e.g.,
TSKgel BioAssist S; TSKgel SP-2SW, TSKgel SP-SPW; TSKgel SP-NPR;
TSKgel SCX; TSKgel SP-STAT; TSKgel CM-SPW; TSKgel OApak-A; TSKgel
CM-2SW, TSKgel CM-3SW, and TSKgel CM-STAT); and Acclaim.RTM.
resins. In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5)
cation exchange columns are used. In certain embodiments, the
cation exchange resin is a SP-Sepharose resin. In certain
embodiments, the cation exchange resin is a SP-Sepharose resin,
such as SP-Sepharose XL resin. In certain embodiments, the cation
exchange resin is a YMC BioPro resin, such as YMC BioPro S30 resin.
In further embodiments one or more cation exchange chromatography
steps include a SP Sepharose resin (e.g., SP-Sepharose XL resin)
and a YMC BioPro resin (e.g., YMC BioPro S30 resin).
[0255] Various bed height for the cation exchange chromatography
column may be used. In some embodiments, a suitable cation exchange
chromatography column has a bed height of 5 cm-30 cm. In some
embodiments, a suitable cation exchange chromatography column has a
bed height of 5 cm, 10 cm, 15 cm, 20 cm, 25 cm, or 30 cm. In
particular embodiments, a suitable cation exchange chromatography
column has a bed height of 10 cm.
[0256] Typical mobile phases for cationic exchange chromatography
include relatively polar solutions, such as water, or solutions
containing a buffer, such as 2-(N-morpholino)-ethanesulfonic acid
(MES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
Tris (tris(hydroxymethyl)aminomethane) buffer, or phosphate buffer
(e.g., sodium phosphate buffer). Thus, in certain embodiments, the
mobile phase includes about 0%, 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%,
16%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, or about 100% polar solution. In certain
embodiments, the mobile phase includes between about 1% to about
100%, about 5% to about 95%, about 10% to about 90%, about 20% to
about 80%, about 30% to about 70%, or about 40% to about 60% polar
solution at any given time during the course of the separation.
[0257] Generally, a mobile phase includes a salt. For example, a
salt (e.g., sodium chloride, etc.) can elute a bound protein from a
cation exchange column (e.g., the counter ion is sodium and it is
exchanged for the target protein, which is then released). In some
embodiments, the mobile phase includes a salt concentration between
about 0 to about 1.0M, e.g., between about 0 to about 0.8M, between
about 0 to about 0.6M, between about 0 to about 0.5M, between about
0 to about 0.4M, between about 0.05M to about 0.50M, between about
0.10M to about 0.45M, between about 0.10M to about 0.40M, between
about 0.250 to about 0.5M, between about 0 to about 0.075M, between
about 0.250 to about 0.5M, between about 0.075 to about 0.5M, or
between about 0.15M to about 0.40M. In some embodiments, the mobile
phase includes a salt concentration of approximately 0.01M, 0.02M,
0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.2M, 0.3M,
0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, or 1.0M. In some embodiments,
the salt concentration in the mobile phase is a gradient (e.g.,
linear or non-linear gradient). In some embodiments, the salt
concentration in the mobile phase is constant. In some embodiments,
the salt concentration in the mobile phase may increase or decrease
stepwise.
[0258] Typically, the mobile phase is buffered. In certain
embodiments, the mobile phase is not buffered. In certain
embodiments, the mobile phase is buffered to a pH between about 5
to about 14. In certain embodiments, the mobile phase is buffered
to a pH between about 5 to about 10. In certain embodiments, the
mobile phase is buffered to a pH between about 6 to about 8. In
certain embodiments, the mobile phase is buffered to a pH of about
7.5. In certain embodiments, the mobile phase is buffered to a pH
of about 7.0. In certain embodiments, the mobile phase is buffered
to a pH of about 6.0, 6.5, 7.0, 7.5, or 8.0.
[0259] In some embodiments, an impure preparation or an
intermediate eluate or flow-through is adjusted to conductivity
ranging between about 1 mS/cm and 20 mS/cm (e.g., between about 1
mS/cm and 15 mS/cm, between about 1 mS/cm and 10 mS/cm, between
about 1 mS/cm and 8 mS/cm, between about 1 mS/cm and 6 mS/cm,
between about 1 mS/cm and 4 mS/cm, between about 2 mS/cm and 4
mS/cm) prior to loading in the cation-exchange chromatography
column (e.g., SP column or YMC-BioPro S30 column). In particular
embodiments, an impure preparation or an intermediate eluate or
flow-through is adjusted to conductivity ranging between about 2
mS/cm and 4 mS/cm (e.g., 2, 2.5, 3, 3.5, or 4 mS/cm) prior to
loading in the cation-exchange chromatography column (e.g., SP
column or YMC-BioPro S30 column). The conductivity may be adjusted
by diluting an impure preparation or an intermediate eluate or
flow-through with H.sub.2O at, e.g., 1:1, 1.1:1, 1.2:1, 1.3:1,
1.4:1, 1.5:1, 2.0:1, 2.5:1, 3.0:1, 4.0:1, 5.0:1, or 10:1 ratio. The
conductivity may also be adjusted by diafiltration into a desired
buffer. In some embodiments, a cation-exchange chromatography
column is run at a pH of about 6.5-8.0 (e.g., about 6.5, 7.0, 7.5
or 8.0). In some embodiments, a suitable pH is about 7.0-7.5 (e.g.,
about 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5).
[0260] In some embodiments, prior to loading, the column may be
equilibrated. In some embodiments, the equilibration occurs in one
or more steps (e.g., 1, 2 or 3 steps). In some embodiments, each
equilibration step uses a different buffer. In some embodiments,
the equilibration is performed with one or more column volumes per
step (e.g., 1, 2, 3, 4 or 5 CV).
[0261] In some embodiments, prior to loading a bulk sample
comprising C3 fusion protein onto a chromatography column, the cell
lysate is clarified. In some embodiments, the cell lysate is
clarified and loaded onto a cation exchange column. In certain
embodiments, the clarified cell lysate is generated by TFF, which
results in TFF permeate. In some embodiments, the TFF permeate
containing C3 fusion protein may be conditioned to an optimal
conductivity between 7 and 9 mS/cm (e.g., 7.5.+-.0.5 mS/cm) with a
dilution buffer prior to loading on the cation exchange column. In
some embodiments, the dilution buffer is a HEPES buffer. In certain
embodiments, HEPES is present in the dilution buffer at a
concentration between 10-100 mM. In certain embodiments, HEPES is
present in the dilution buffer at a concentration of 20 mM. In some
embodiments, the dilution buffer further comprises EDTA. In certain
embodiments, EDTA is present in the dilution buffer at a
concentration ranging from 0.1-10 mM, 0.1-5 mM, 1-5 mM (e.g., 1, 2,
3, 4, 5), 0.1-0.60 mM (e.g., 0.1. 0.2, 0.3, 0.4, 0.5, 0.6).
[0262] In further embodiments, to capture the C3 fusion proteins
from most host-cell derived impurities, a cation exchange resin
(e.g., SP Sepharose XL) column may be equilibrated with at least
two column volumes (CV) of equilibration buffer. In some
embodiments, the equilibration buffer comprises 40-50 mM HEPES,
50-250 mM NaCl, 0.3-0.5 mM EDTA, pH 7.0-8.0.
[0263] In some embodiments, the conditioned permeate containing the
C3 fusion is loaded onto the capture column comprising the cation
exchange resin (e.g., SP Sepharose XL), washed and then eluted in a
single step with one or more CV of elution buffer (e.g., 1 CV, 2
CV, 3 CV, 4 CV, 5 CV, 6 CV). In some embodiments, the elution
buffer comprises 40-50 mM HEPES, 75-250 mM NaCl, 0.3-0.5 mM EDTA,
at pH 7-8.0. In some embodiments the peak eluate may be collected.
In particular embodiments, the collection of the C3 fusion protein
product begins when the ascending elution peak reaches an
absorbance of 500 mAU and continues until the absorbance drops on
the tailing edge of the peak to 500 mAU (peak eluate).
[0264] In some embodiments, column buffers may be a sodium
phosphate buffer (e.g., 20-50 mM NaH.sub.2PO.sub.4). In some
embodiments the buffer further comprises 0-500 mM NaCl, 20-30 mM
citric acid, 0.3-0.5 mM EDTA, pH 7.0. After loading, the column may
be washed with one or more column volumes of buffer. In some
embodiments, the wash buffer may be the same as the equilibration
buffer. In some embodiments, the recombinant C3 fusion protein is
eluted using an elution buffer. Protein elution may be performed be
performed using a linear gradient method, a stepwise gradient
method, a combination of gradient and stepwise elution method, or
an isocratic solution method. The gradient can be increasing or
decreasing salt (e.g., NaCl) or pH. In certain embodiments, the
elution is performed with a linear gradient method. In particular
embodiments, the gradient may be started with 5% elution buffer and
continued for 7 CV, at 5% elution buffer per CV, until 40% elution
buffer is reached.
[0265] Hydrophobic Interaction Chromatography
[0266] Hydrophobic Interaction Chromatography (HIC) is a separation
technique that uses the properties of hydrophobicity to separate
proteins from one another. In this type of chromatography,
hydrophobic groups such as phenyl, octyl, or butyl, are attached to
the stationary column. Proteins that pass through the column that
have hydrophobic amino acid side chains on their surfaces are able
to interact with and bind to the hydrophobic groups on the column.
HIC columns are known, and include for example, Butyl Sepharose.
Various bed height for a HIC column may be used. In some
embodiments, a suitable HIC column has a bed height of 5 cm-30 cm.
In particular embodiments, a suitable HIC column has a bed height
of 5 cm, 10 cm, 15 cm, 20 cm, 25 cm, or 30 cm.
[0267] HIC separations are often designed using the opposite
conditions of those used in ion exchange chromatography. In
general, a buffer with a high ionic strength, usually ammonium
sulfate, is initially applied to the column. The salt in the buffer
reduces the solvation of sample solutes thus as solvation
decreases, hydrophobic regions that become exposed are adsorbed by
the medium. The stationary phase is generally designed to form
hydrophobic interactions with other molecules. These interactions
are generally too weak in water, however, addition of salts (e.g.,
Na.sub.2SO.sub.4, K.sub.2SO.sub.4, (NH.sub.4).sub.2SO.sub.4, NaCl,
NH.sub.4Cl, NaBr, and NaSCN) to the buffer results in hydrophobic
interactions. In some embodiments, the mobile phase includes a salt
concentration between about 0.1M to about 3.0M, e.g., between about
0.1M to about 1.6M, between about 0.2M to about 1M, or between
about 0.3M to about 0.75M.
[0268] In certain embodiments, the mobile phase is buffered. In
certain embodiments, the mobile phase is not buffered. In certain
embodiments, the mobile phase is buffered to a pH between about 6
to about 8. In certain embodiments, the mobile phase is buffered to
a pH between about 6.5 to about 7.5. In certain embodiments, the
mobile phase is buffered to a pH of about 7.5. In certain
embodiments, the mobile phase is buffered to a pH of about 7.5.
[0269] In some embodiments, an impure preparation or an
intermediate eluate or flow-through is adjusted to salt (e.g.,
(NH.sub.4).sub.2SO.sub.4) concentration ranging from about 0.5 M to
about 2.0 M (e.g., about 0.5 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M,
1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, or 2.0 M) at pH of about 6.0-8.0
(e.g., about 6.0, 6.5, 7.0, 7.3, or 7.5), prior to loading onto the
hydrophobic interaction chromatography column (e.g., butyl column).
Once loaded, a hydrophobic interaction chromatography column may be
washed using a wash buffer comprising salt (e.g.,
(NH.sub.4).sub.2SO.sub.4) concentration ranging from about 0.5 M to
about 2.0 M (e.g., about 0.5 M, 0.75M, 1.0 M, 1.1 M, 1.2 M, 1.3 M,
1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, or 2.0 M) at pH of about
6.0-8.0 (e.g., about 6.0, 6.5, 7.0, 7.3, or 7.5). In some
embodiments, the hydrophobic interaction chromatography column is
eluted using a elution buffer comprising salt (e.g.,
(NH.sub.4).sub.2SO.sub.4) concentration ranging from about 0.1 M to
about 0.75 M (e.g., about 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M or
0.75M) at pH of about 6.0-8.0 (e.g., about 6.0, 6.5, 7.0, 7.3, or
7.5).
[0270] Ultrafiltration/Diafiltration
[0271] In some embodiments, the purification methods disclosed
herein include one or more steps of ultrafiltration/diafiltration.
Ultrafiltration/diafiltration, as used herein, refers to membrane
filtration with filter pore sizes on the magnitude of 0.001 and 0.1
.mu.m, which may be used for concentrating and desalting dissolved
molecules (proteins, peptides, nucleic acids, carbohydrates, and
other biomolecules), exchanging buffers, and gross fractionation.
Methods of ultrafiltration for use in embodiments of the invention
include tangential flow ultrafiltration or crossflow
filtration.
[0272] Tangential flow filtration and ultrafiltration, as used
herein, refers to arrangements where the feed stream passes
parallel to the membrane face as one portion passes through the
membrane (permeate) while the remainder (retentate) is recirculated
back to the feed reservoir. In some embodiments, pore size of
tangential flow ultrafiltration filters is chosen to allow
recombinant C3 fusion protein to permeate through the filter. In
other embodiments, pore size is chosen so as to retain
substantially all C3 fusion protein in the feed passing across the
filter. As noted elsewhere, C3 fusion protein is approximately 25.7
kD. Using SDS-PAGE analysis, exemplary C3 fusion protein major band
migrates between 21 and 31 kDa. The pH of the feed may be adjusted
in combination with selection of appropriate pore size to either
retain C3 fusion protein on the filter membrane or allow it to pass
through as a permeate.
[0273] In some embodiments, supernatant may be clarified by a
Tangential Flow Filtration (TFF) system. In some embodiments, the
TFF system is equipped flat sheet membrane or a hollow fiber
membrane (e.g., modified Polyethersulfone (mPES), Mixed Cellulose
Ester (ME), Polysulfone (PS) and Polyethersulphone (PES)). In
certain embodiments, the TFF system is equipped with a Sartorius
Sartocon slice membrane (1000 kDa, 0.5 m.sup.2). The filtration may
be performed in two steps: a concentration phase and a
diafiltration phase. Within the diafiltration phase, the retentate
may be continuously washed. In some embodiments, the retentate is
continuously washed with up to 5.0 column volumes, (CV) (e.g., 1
CV, 2CV, 3, CV, 4, CV, 5 CV) volumes of diafiltration buffer. In
some embodiments the diafiltration buffer is a HEPES buffer. In
certain embodiments, HEPES is present in the diafiltration buffer
at a concentration between 10-100 mM. In some embodiments, the
diafiltration buffer further comprises EDTA. In certain
embodiments, EDTA is present in the diafiltration buffer at a
concentration between 50-150 mM.
[0274] Pore size may be selected with molecular weight cutoffs of
at least 5 kDa, at least 10 kDa, at least 20 kDa, at least 30 kDa,
at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa,
at least 80 kDa, at least 90 kDa, at least 100 kDa, at least 200
kDa, at least 300 kDa, at least 400 kDa, at least 500 kDa, 600 kDa,
700 kDa, 800 kDa, 900 kDa, 1000 kDa, or at least 1500 kDa. For
example, a filter with a pore size of at least 5 kDa will retain in
the feed a majority of proteins with molecular weights higher than
approximately 5 kDa. As another example, a filter of a pore size of
at least 400 kDa will retain a majority of proteins with molecular
weights higher than 400 kDa. In some embodiments, the feed
retention rate is at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, or higher. Likewise, pore size may be
selected for isolation of permeates of particular size. For
example, a filter with a pore size of at least 500 kDa will allow a
majority of proteins with molecular weights less than 500 kDa to
permeate through. In some embodiments, the permeation rate is at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%
or higher.
[0275] Filtration area or capacity may also be optimized for use in
the processes disclosed herein. Considerations impacting selection
of filtration area include robustness, cost, feed flow rate (i.e.,
crossflow velocity), transmembrane pressure, permeate flux rate,
plant fit and throughput. In some embodiments, the permeate flux is
about 50-100 liter per meter per hour ("LMH"). In some embodiments,
the feed flow is about 250-600 LMH, inclusive. In some embodiments,
the feed flow is about 250-350 LMH, inclusive. In some embodiments,
the feed flow is about 175-245 LMH, inclusive. In some embodiments,
the feed flow is about 170-230 LMH, inclusive. In some embodiments,
the feed flow is about 120-160 LMH, inclusive. In some embodiments,
the feed flow is about 15-30 LMH, inclusive. In some embodiments,
the fee flow is about 11-21 LMH, inclusive. In some embodiments,
the filtration area is about 0.02 m.sup.2, about 0.14 m.sup.2,
about 0.7 m.sup.2, or about 3.5 m.sup.2. In particular embodiments,
the transmembrane pressure is about 55-60 psi, inclusive. In some
embodiments, the transmembrane pressure is about 15-25 psi,
inclusive. In some embodiments, the transmembrane pressure is about
10-20 psi, inclusive. In some embodiments, the transmembrane
pressure is about 5-15 psi, inclusive.
[0276] Ultrafiltration filters for use in embodiments of the
invention may comprise membrane materials known to those of skill
in the art, including but not limited to polyethersulfone and
stabilized cellulose. One exemplary filter cassette for use in
embodiments of the invention is the Pellicon 3 Ultracell 5 kDa
MWCO. In some embodiments, the filter is preconditioned with buffer
before concentration and/or diafiltration. In some embodiments, the
conditioning or diafiltration buffer comprises NaH.sub.2PO.sub.4.
In some embodiments, the NaH.sub.2PO.sub.4 is present at a
concentration ranging from 5 mM to 50 mM.
[0277] Membrane Adsorption
[0278] Generally, membrane adsorbers are thin, synthetic,
microporous or macroporous membranes that are derivatized with
functional groups akin to those on the equivalent resins. The
membranes are stacked 10-15 layers deep in a comparatively small
cartridge, allowing for increased flow rate and speed in
purification in processing. Adsorption is efficient because the
transport of solutes to their binding sites in a membrane adsorber
occurs mainly by convection, while pore diffusion (the predominant
mechanism in resins) is minimal. Membrane adsorbers are capable of
linear scale-up for parameters such as frontal surface area, bed
volume, flow rate, and static binding capacity.
[0279] Exemplary membrane adsorbers include the Sartobind.RTM.
membrane adsorbers (MA) for ion exchange chromatography. The
Sartobind.RTM. membrane displays a microporous structure with pore
size of >3 Ligands for ion exchange are bound covalently to the
complete internal surface of the membrane, resulting in separation
media of high binding capacity combined with very high flow rates.
Binding of ion exchange ligands is very stable and allows many
cycles of re-use without loss of binding capacity. Sartobind.RTM.
MA units for ion exchange are available as strong or weak anion (Q
and D) and strong cation (S) type. Sartobind.RTM. MA units are
equipped with standard Luer Lock connectors and can be run with
high-performance liquid chromatography (HPLC) or fast protein
liquid chromatography (FPLC) systems or by hand with a syringe.
[0280] In some embodiments, Sartobind.RTM. Q membrane adsorbers
display a macro-porous structure with pore size of >3 .mu.m.
Quaternary ammonium ligands are bound covalently to the complete
internal surface of the membrane, resulting in separation media of
high binding capacity combined with exceptionally high flow rates.
In some embodiments, Sartobind Q membrane may be used for the
flow-through polishing of purified proteins (e.g., C3 fusion
protein). The bed volumes can be kept sufficiently small during the
removal of viruses, DNA, host cell proteins, leached Protein A, and
endotoxins from such pharmaceutical proteins.
Purified C3 Fusion Protein Composition
[0281] Composition Comprising C3 Fusion Protein
[0282] Purified recombinant C3 fusion protein may be characterized
using various methods. In some embodiments, stability indicating
methods (e.g., SDS-PAGE, SE-HPLC, SEC-UV, RP-HPLC, IEX-HPLC, and
LC-MS) may be used to assess the protein purity. According to the
present invention, circular dichroism (CD) can be used for
secondary structure, and differential scanning calorimetry (DSC)
for tertiary structure analysis. The bioactivity may be assessed by
a functional assays such as GH-Assay and/or ADP-ribosylation
activity.
[0283] In some embodiments, a C3 fusion protein comprising SEQ ID
NO: 1 is a recombinant protein composed of 231 amino acids with a
theoretical molecular weight of 25,726 daltons (in some
embodiments, a nominal molecular weight of 26-28 kDa is used for
analytical purposes) and theoretical isoelectric point (pI) of 9.6.
The extinction coefficient has been determined by amino acid
analysis to be 0.72 units of absorbance at A.sub.280 nm per mg of
protein.
[0284] In certain embodiments, upon expression and purification of
the coding region comprising SEQ ID NO: 3, according to the present
invention, the N-terminal methionine is absent as determined by
mass spectrometry in peptides or peptides derived or related to C3
fusion polypeptides. Peptide mapping providing 97%-100% coverage
can be used to confirm the theoretical protein sequence without an
N-terminal methionine, and the amino acid sequence results
correspond with those expected the theoretical sequence.
[0285] In some embodiments, a C3 fusion protein composition
comprises a population of C3 fusion polypeptides. In some
embodiments, a purified C3 fusion protein composition comprises a
population of C3 fusion polypeptide, each having an amino acid
sequence described herein, wherein the first amino acid of each
polypeptide is not a methionine and wherein the population of the
polypeptides constitutes greater than 70%, 75%, 80%, 85%, 90%, 92%,
94%, 95%, 96%, 97%, 98%, 99% of the total amount of polypeptides in
the composition. In some embodiments, a purified C3 fusion protein
composition comprises a C3 fusion protein that does not comprise a
methionine as the first amino acid.
[0286] In some embodiments, a purified C3 fusion protein
composition comprises a population of C3 fusion polypeptide that
comprising a first polypeptide and a second polypeptide, each
having an identical amino acid sequence described herein except
wherein the first polypeptide does not contain a methionine at the
N-terminus. In some embodiments, the weight ratio of the first
polypeptide to the second polypeptide is at least 6:1, 7:1, 8:1,
9:1, 10:1, 12:1, 15:1, 20:1, 50:1, or 100:1.
[0287] The protein concentration of a purified C3 fusion protein
composition may be determined by any suitable method known in the
art for determining protein concentrations. In some embodiments,
the protein concentration is determined by an ultraviolet light
absorbance assay. In some embodiments, such absorbance assays are
typically conducted at about a 280 nm wavelength (A.sub.280). In
some embodiments, a C3 fusion protein composition may comprise C3
fusion protein at a high concentration (e.g., greater than about 8
mg/ml, 9 mg/ml, 10 mg/ml, 13 mg/ml, 15 mg/ml, 19 mg/ml, 20 mg/ml,
25 mg/ml, 30 mg/ml, 31 mg/ml, 32 mg/ml, 33 mg/ml, 34 mg/ml, 35
mg/ml, 36 mg/ml, 37 mg/ml, 38 mg/ml, 39 mg/ml, 40 mg/ml or more).
In some embodiments, a C3 fusion protein composition comprises a
purified C3 fusion protein at a concentration ranging from about
0.1 mg/ml to about 50 mg/ml, about 1 mg/ml to about 40 mg/ml, about
5 mg/ml to about 40 mg/ml, about 8 mg/ml to about 40 mg/ml, about
10 mg/ml to about 40 mg/ml, about 13 mg/ml to about 40 mg/ml, about
15 mg/ml to about 40 mg/ml, about 19 mg/ml to about 40 mg/ml, about
25 mg/ml to about 40 mg/ml, about 25 mg/ml to about 35 mg/ml, about
26 mg/ml to about 34 mg/ml, about 27 mg/ml to about 33 mg/ml, about
30 mg/ml to about 40 mg/ml, about 31 mg/ml to about 38 mg/ml, about
31 mg/ml to about 37 mg/ml, about 33 mg/ml to about 40 mg/ml, about
37 mg/ml to about 40 mg/ml, about 10 mg/ml to about 38 mg/ml, about
10 mg/ml to about 35 mg/ml, about 10 mg/ml to about 30 mg/ml, about
10 mg/ml to about 25 mg/ml, about 10 mg/ml to about 20 mg/ml, about
10 mg/ml to about 15 mg/ml. In some embodiments, a purified C3
fusion protein composition comprises a C3 fusion protein at a
concentration of about 1 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11
mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml,
18 mg/ml, 19 mg/ml, 20 mg/ml, 25 mg/ml, 26 mg/ml, 27 mg/ml, 28
mg/ml, 29 mg/ml, 30 mg/ml, 31 mg/ml, 32 mg/ml, 33 mg/ml, 34 mg/ml,
35 mg/ml, 36 mg/ml or 37 mg/ml.
[0288] Purity
[0289] The purity of a purified C3 fusion protein composition is
typically measured by the percentage of the target C3 fusion
protein in the total amount of purified composition using various
assays. In some embodiments, the purity of a purified recombinant
C3 fusion protein composition is measured by the level of various
impurities (e.g., host cell protein or host cell DNA) present in
the purified composition including final product. In some
embodiments, the level of host cell protein (HCP) is measured by
Host Cell Proteins Immunoassay (such as ELISA), SDS-PAGE, or
size-exclusion chromatography. In some embodiments, ELISA assays
are used to quantify the level of HCP in a purified C3 fusion
protein composition. In some embodiments, various antibodies
against host cell proteins may be used including those commercially
available antibodies against host cell proteins from various cell
types (e.g., bacterial cells such as E. coli, mammalian cells such
as human cells, CHO cells, or others). In some embodiments, the
purified recombinant C3 fusion protein composition contains less
than 150 ng HCP/mg C3 fusion protein (e.g., less than 140, 130,
120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 30, 20, 10 ng HCP/mg C3
fusion protein). Various assays may be used to measure the level of
host cell DNA in a purified C3 fusion protein composition. In some
embodiments, the purified recombinant C3 fusion protein composition
contains less than about 150 pg/mg, 140 pg/mg, 130 pg/mg, 120
pg/mg, 110 pg/mg, 100 pg/mg, 90 pg/mg, 80 pg/mg, 70 pg/mg, 60
pg/mg, 50 pg/mg, 40 pg/mg, 30 pg/mg, 20 pg/mg, or 10 pg/mg Host
Cell DNA.
[0290] In some embodiments, the purified recombinant C3 fusion
protein composition, when subject to SDS-PAGE with Coomassie
Brilliant Blue staining, has no new bands with intensity greater
than the 0.05%, 0.01%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%,
0.45%, or 0.5% assay control. In some embodiments, the purified
recombinant C3 fusion protein composition, when subject to SDS-PAGE
with Western blotting against HCP, has no bands with intensity
greater than the 15 kDa HCP band assay control, and no new bands
with intensity greater than the 0.05%, 0.01%, 0.15%, 0.2%, 0.25%,
0.3%, 0.35%, 0.4%, 0.45%, 0.5%, or 1.0% assay control. In some
embodiments, the purified recombinant C3 fusion protein
composition, when subject to SDS-PAGE with silver staining, has no
new bands with intensity greater than the 0.05%, 0.01%, 0.15%,
0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5% assay control. In
some embodiments, the host cell protein (HCP) log reduction value
(LRV) is between about 0.3 and about 0.6, e.g., between about 0.4
and 0.5. Various assay controls may be used, in particular, those
acceptable to regulatory agencies such as FDA.
[0291] The purity of a purified recombinant C3 fusion protein
composition may also be determined by one or more of size exclusion
chromatography-high performance liquid chromatography (SEC-HPLC),
capillary electrophoresis-SDS PAGE (CE-SDS PAGE), ion-exchange high
performance liquid chromatography (IE-HPLC) and/or reverse
phase-high performance liquid chromatography (RP-HPLC). In some
embodiments, the purity of a purified C3 fusion protein composition
is represented by the percentage of the major peak in the
chromatogram that corresponds to the major species of C3 fusion
protein. In some embodiments, the purity of a purified C3 fusion
protein composition is characterized by percentages of different
peaks indicative of different species of C3 fusion protein present
in the purified composition. In some embodiments, the purity of a
purified C3 fusion protein composition is measured by the
percentage of total impurities present in the composition.
Parameters that may be altered or optimized to increase resolution
include gradient conditions, organic modifier, counter ion,
temperature, column pore size and particle size, solvent
composition and flow rate. Purity levels may be discerned by peak
percentages, as known to those of skill in the art. For example,
purity may be determined by integrating the main and side peaks
observed and calculating the main peak's percentage of the total
area.
[0292] In some embodiments, the purity of a purified C3 fusion
protein composition is determined by SEC-HPLC. In some embodiments,
the purity of a C3 fusion protein composition purified by the
methods disclosed herein is determined by SEC-HPLC. In some
embodiments, the purity of C3 fusion protein composition is
measured by the main peak percentage of SEC-HPLC and is greater
than or equal to about 90%, about 95%, about 96%, about 97%, about
98%, about 99% or higher. The main peak of SEC-HPLC represents the
monomers of C3 fusion protein. In some embodiments, the purity of a
C3 fusion protein composition is indicated by the percentage of
total impurities measured by SEC-HPLC. In some embodiments, the
total impurities measured by SEC-HPLC is less than or equal to
about 10%, about 8%, about 6%, about 5%, about 4%, about 3%, about
2%, about 1%, about 0.5%, about 0.1% or less.
[0293] In some embodiments, the purity of a C3 fusion protein
composition purified by the methods disclosed herein is determined
by RP-HPLC. In some embodiments, the purity of C3 fusion protein
composition is measured by the main peak percentage of RP-HPLC and
is greater than or equal to about 80%, about 85%, about 90%, about
95%, about 96%, about 97%, about 98%, about 99% or higher. In some
embodiments, the purity of a C3 fusion protein composition is
indicated by the percentage of total impurities measured by
RP-HPLC. In some embodiments, the total impurities measured by
RP-HPLC is less than or equal to about 20%, about 18%, about 15%,
about 10%, about 8%, about 6%, about 5%, about 4%, about 3%, about
2%, about 1%, about 0.5%, about 0.1% or less.
[0294] In some embodiments, the purity of a C3 fusion protein
composition purified by the methods disclosed herein is determined
by IE-HPLC (also known as IEX-HPLC). In some embodiments of the
invention, the purity of C3 fusion protein is determined by main
peak percentage of IE-HPLC and is greater than or equal to about
80%, about 82%, about 83%, about 84%, about 85%, about 86%, about
87%, about 88%, about 89%, about 90%, about 91%, about 92%, about
93%, about 94%, about 95%, about 96%, about 97%, about 98%, about
99% or higher. The major peak of IE-HPLC is indicative of the main
species of C3 fusion protein which represents the main product of
C3 fusion protein with a specific charge profile. In some
embodiments, the purity of a C3 fusion protein composition can also
be determined by percentages of side peaks of IE-HPLC. Typical side
peaks of IE-HPLC include, but not limited to, acidic peaks and
basic peaks. In some embodiments, the percentage of total acidic
peaks is less than or equal to about 15%, about 12%, about 11%,
about 10%, about 8%, about 6%, about 5%, about 4%, about 3%, about
2%, about 1%, about 0.5%, about 0.1% or less. In some embodiments,
the percentage of total basic peaks is less than or equal to about
10%, about 8%, about 6%, about 5%, about 4%, about 3%, about 2%,
about 1%, about 0.5%, about 0.1% or less. In some embodiments, the
purified C3 fusion protein composition comprises greater than 83%
main peak of IE-HPLC. In some embodiments, the purified C3 fusion
protein composition comprises greater than 85% main peak of
IE-HPLC, less than 10% total acidic peaks and less than 5% total
basic peaks.
[0295] Glycohydrolase (GH) Activity Assay
[0296] The intracellular action of C3 exoenzymes and C3 fusion
proteins results from transfer of an ADP-ribose moiety to an
asparagine residue in Rho GTPase, trapping this GTPase in its
inactive conformation. An exemplary C3 fusion protein is an
ADP-ribosyltransferase that catalyzes hydrolysis of NAD.sup.+ in
the absence of the specific protein substrate, Rho. C3 fusion
protein catalyzes transfer of an ADP-ribose moiety to the RhoA,
RhoB and RhoC members of the Rho family of small GTPases. In
neurons, the predominant species is RhoA, so the numbering system
for RhoA and the abbreviation "Rho" is used. A spectrofluorometric
glycohydrolase (GH) activity assay was used to measure
glycohydrolase (GH) as the formation of a fluorescent product,
which gives a sensitive and reliable method to serve as a test of
identity and potency.
[0297] The GH assay measures the formation of .epsilon.-ADP-ribose
produced as a result of hydrolysis of .epsilon.-NAD by C3-variants.
Glycohydrolase activity of C3-variants converts .epsilon.-NAD.sup.+
into .epsilon.-ADP-ribose, a molecule with 10 times higher
fluorescence intensity at the selected wavelengths. The
fluorescence intensity of .epsilon.-ADP-ribose is used to measure
the amount of .epsilon.-ADP ribose formed by using a standard curve
of fluorescence of known concentrations of .epsilon.-AMP. The
fluorescence intensities of .epsilon.-AMP and .epsilon.-ADP-ribose
are measured by exciting the reaction at 305 nm and recording the
emission at 410 nm. A unit of activity is defined as nmoles
ADP-ribose formed in 30 minutes at 37.degree. C. In some
embodiments, the purified C3 fusion protein has the activity to
form about 5-60 nmol, about 10-50 nmol, or about 20-40 nmol of
ADP-ribose per mg protein in 30 minutes at 37.degree. C. The assay
is linear with up to at least 12 .mu.g of C3-variant protein and up
to least 180 minutes of incubation time. This assay has been found
to be precise, accurate and reproducible. This assay has also been
found to be useful in measuring decreases in enzymatic activity
after incubation at 70.degree. C., and can be considered to be
stability-indicating when used in a well-designed stability
study.
[0298] Other Attributes
[0299] Other attributes of the purified C3 fusion protein
composition according to the present invention are characterized
including, but not limited to, appearance, pH, molecular weight,
the endotoxin level, osmolality, sterility, subvisible particulate
matter, sub 10 .mu.m particulate matter, and/or container closure
integrity (or dye immersion test). In some embodiments, a purified
C3 fusion protein according to the present invention has an
appearance of a clear, colorless liquid, substantially free of
visible particulates. In some embodiments, a purified C3 fusion
protein according to the present invention has a pH of about
5.5-8.0, about 5.8-7.8, about 6.0-7.5, about 6.0-7.2, or about
6.0-7.0. In some embodiments, a purified C3 fusion protein
according to the present invention has a major band with a
molecular weight of 21-31 kDa when measured by SDS-PAGE with
Coomassie blue staining. In some embodiments, a purified C3 fusion
protein according to the present invention contains an endotoxin
(LAL) level of less than or equal to about 1 EU/mg, about 0.8
EU/mg, about 0.6 EU/mg, about 0.5 EU/mg, about 0.1 EU/mg, or less.
In some embodiments, a purified C3 fusion protein according to the
present invention contains subvisible particulate matter less than
or equal to about 6000 (e.g., less than or equal to about 5000,
about 4000, about 3000, about 2000, about 1000, about 800, about
700, about 600, about 500, or less) NMT with .gtoreq.10 .mu.m per
container, and/or subvisible particulate matter less than or equal
to about 800 (e.g., less than or equal to about 700, about 600,
about 500, about 400, about 300, about 200, about 100, or less) NMT
with .gtoreq.25 .mu.m per container. In some embodiments, no dye
detected in the purified C3 fusion protein according to the present
invention when tested in dye immersion test for container closure
integrity.
Pharmaceutical Compositions
[0300] A recombinant C3 fusion protein or a pharmaceutical
composition containing the same can be formulated with a
physiologically acceptable carrier or excipient to prepare a
pharmaceutical composition. The carrier and therapeutic agent can
be sterile. The formulation should suit the mode of
administration.
[0301] Suitable pharmaceutically acceptable carriers include but
are not limited to water, salt solutions (e.g., NaCl), saline,
buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable
oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates
such as lactose, amylose or starch, sugars such as mannitol,
sucrose, or others, dextrose, magnesium stearate, talc, silicic
acid, viscous paraffin, perfume oil, fatty acid esters,
hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as
combinations thereof. The pharmaceutical preparations can, if
desired, be mixed with auxiliary agents (e.g., lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for
influencing osmotic pressure, buffers, coloring, flavoring and/or
aromatic substances and the like) which do not deleteriously react
with the active compounds or interfere with their activity.
[0302] The composition or medicament, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. The composition can be a liquid solution, suspension,
emulsion, sustained release formulation, or powder. The composition
or medicament can be formulated in accordance with routine
procedures as a pharmaceutical composition adapted for
administration to human beings. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic to
ease pain at the site of the injection. Generally, the ingredients
are supplied either separately or mixed together in unit dosage
form, for example, as a dry lyophilized powder or water free
concentrate in a hermetically sealed container such as an ampule or
sachette indicating the quantity of active agent.
[0303] In some embodiments, the C3 fusion protein pharmaceutical
composition further comprises fibrinogen and does not contain
thrombin. In some embodiments, the C3 fusion protein pharmaceutical
composition further comprises albumin, one or more blood
coagulation factors, globulin, and/or one or more
plasminogen-activator inhibitors or plasmin inhibitors. In some
embodiments, the C3 fusion protein the pharmaceutical composition
comprises aprotinin. In some embodiments, the C3 fusion protein
pharmaceutical composition further comprises thrombin. In some
embodiments, the C3 fusion protein pharmaceutical composition is a
tissue adhesive.
[0304] In some embodiments, the C3 fusion protein pharmaceutical
composition may promote neuroregeneration and neuroprotection. In
some embodiments, the C3 fusion protein pharmaceutical composition
may be used in a method to treat CNS trauma. In some embodiments,
CNS trauma occurs in the form of spinal cord injury, for example,
resulting in the loss of axons and the subsequent inability of
axons to regrow and find appropriate targets. In some embodiments,
spinal cord injury may result in immediate tear of axons. In some
embodiments, spinal cord trauma may cause axons to deteriorate due
to the disruption of vital transport of molecules and cell
components to and from the ends of axons. In some embodiments, CNS
trauma comprises traumatic brain injury. In some embodiments, CNS
trauma comprises stroke.
[0305] In some embodiments, the C3 fusion protein pharmaceutical
composition may be used to facilitating axon growth or tissue
repair (e.g., repair of a central nervous system (CNS) lesion site
or a peripheral nervous system (PNS) lesion site). Treatment with
pharmaceutical compositions according to the present invention can
be used to enhance the rate of axon growth of peripheral nerves and
thereby be effective for repair of peripheral nerves after surgery,
for example after reattaching severed limbs. Also, treatment with
pharmaceutical compositions according to the present invention may
be effective for the treatment of various peripheral neuropathies
because of their axon growth promoting effects.
Therapeutic Uses
[0306] A pharmaceutical composition based on a C3 fusion protein
described herein may be used for various therapeutic purposes. In
particular, the present invention pertains to the field of
mammalian nervous system repair (e.g., repair of a central nervous
system (CNS) lesion site or a peripheral nervous system (PNS)
lesion site). The methods described herein may be extended to use
in many other diseases including, but not limited to, cancer,
metastasis, hypertension, cardiac disease, stroke, diabetic
neuropathy, and neurodegenerative disorders such as stroke,
Alzheimer's disease, Parkinson's disease, and amyotrophic lateral
sclerosis (ALS). Treatment with therapeutic compositions according
to the present invention can be used to enhance the rate of axon
growth of peripheral nerves and thereby be effective for repair of
peripheral nerves after surgery, for example after reattaching
severed limbs. Also, treatment with therapeutic compositions
according to the present invention is expected to be effective for
the treatment of various peripheral neuropathies because of their
axon growth promoting effects.
Spinal Cord Injury
[0307] As used herein, the term "spinal cord injury site" refers to
a site of traumatic nerve injury or nerve injury caused by disease.
The spinal cord injury site may be a single nerve (e.g., sciatic
nerve) or a nerve tract comprised of many nerves (e.g., damaged
region of the spinal cord). The spinal cord injury site may be in
the central nervous system or peripheral nervous system or in any
region needing repair. The spinal injury site may form as a result
of damage caused by stroke.
[0308] Spinal cord injuries result from damage to the vertebrae,
ligaments or disks of the spinal column or to damage to the spinal
cord itself. In some embodiments, a spinal cord injury is a
traumatic injury. In some embodiments, a spinal cord injury is a
nontraumatic injury. A traumatic spinal cord injury may stem from a
sudden, traumatic blow to the spine that fractures, dislocates,
crushes, or compresses one or more vertebrae. It also may result
from a gunshot or knife wound that penetrates and cuts the spinal
cord. Common causes of traumatic spinal cord injury include motor
vehicle accidents, falls, acts of violence and sports and
recreation injuries. Additional damage usually occurs over days or
weeks because of bleeding, swelling, inflammation and fluid
accumulation in and around the spinal cord. A nontraumatic spinal
cord injury may be caused by arthritis, cancer, inflammation,
infections or disk degeneration of the spine.
[0309] Symptoms of spinal cord injury depend on two factors: the
location of the spinal cord injury along the spinal cord and the
severity of the injury. The lowest part of the spinal cord that
functions normally after spinal cord injury is referred to as the
neurological level of the injury. The severity of the injury is
often called the "completeness" is often classified as either
complete or incomplete. A spinal cord injury is complete if almost
all feeling (sensation) and all ability to control movement (motor
function) are lost below the spinal cord injury. A spinal cord
injury is incomplete if some motor or sensory function exists below
the level of the spinal cord injury. There are varying degrees of
incomplete injury. Paralysis from a spinal cord injury may be
referred to as tetraplegia (quadriplegia) or paraplegia.
Tetraplegia means that the arms, hands, trunk, legs, and pelvic
organs are all affected by the spinal cord injury. Paraplegia means
that all or part of the trunk, legs and pelvic organs are affected
by the spinal cord injury.
[0310] Spinal cord injuries may result in one or more of the
following signs and symptoms: loss of movement, loss of sensation,
including the ability to feel heat, cold and touch, loss of bowel
or bladder control, exaggerated reflex activities or spasms,
changes in sexual function, sexual sensitivity and fertility, pain
or an intense stinging sensation caused by damage to the nerve
fibers in the spinal cord, difficulty breathing, coughing or
clearing secretions from the lungs.
[0311] In some embodiments, the present invention provides methods
to promote neuroregeneration and neuroprotection. In some
embodiments, the present invention provides methods for treating
CNS trauma. In some embodiments, CNS trauma occurs in the form of
spinal cord injury, for example, resulting in the loss of axons and
the subsequent inability of axons to regrow and find appropriate
targets. In some embodiments, spinal cord injury may result in
immediate tear of axons. In some embodiments, spinal cord trauma
may cause axons to deteriorate due to the disruption of vital
transport of molecules and cell components to and from the ends of
axons. In some embodiments, CNS trauma comprises traumatic brain
injury. In some embodiments, CNS trauma comprises stroke.
[0312] The Rho family of GTPases regulates axon growth and
regeneration. Inactivation of Rho with a therapeutic protein as
described above (e.g., a C3 transferase) can stimulate regeneration
and sprouting of injured axons. See, for example, Saito et al.,
1995, FEES Lett 371:105-109; Wilde et al 2000. J. Biol. Chem.
275:16478.
[0313] In one embodiment, in the present invention provides methods
of treating spinal cord injury in a subject in need thereof. In
some embodiments, spinal cord injury results from injury or
disease. In some embodiments, spinal cord injury comprises injury
to the cervical spinal cord. In some embodiments, spinal cord
injury comprises injury to the thoracic spinal cord. In some
embodiments, spinal cord injury comprises injury to the lumbar
spinal cord. In some embodiments, spinal cord injury comprises
injury to the sacral spinal cord. In some embodiments, spinal cord
injury comprises incomplete spinal cord injury wherein the spinal
cord is partially severed. In some embodiments, spinal cord injury
comprises complete spinal cord injury, wherein the spinal cord is
completely severed.
[0314] In one embodiment, the present invention provides methods of
treating acute spinal cord injury in a subject in need thereof.
[0315] In one embodiment, the present invention provides methods of
treating nerve injury or compression of a nerve in the peripheral
nervous system, such as a nerve root that extends from the spinal
cord such as a dorsal root (sensory nerve) or a motor root (motor
nerve).
[0316] In other embodiments, the present invention provides method
of treating peripheral nerve injury, such as may occur during
brachial plexus injury where facilitating nerve growth is
needed.
[0317] In some embodiments the present invention facilitates
peripheral nerve injury. Peripheral nerve injury includes injury to
peripheral nerves located in arms, hand, legs, feet or injury to a
hand. In some instances peripheral nerve injury may occur after
biopsy for diagnosis of neurological disease, such as removal of a
segment of sural nerve.
[0318] In one embodiment, the present invention provides methods of
facilitating axon growth in a subject in need thereof. In some
embodiments facilitating axon growth comprises promoting axon
growth. In some embodiments, facilitating axon growth comprises
facilitating axon regeneration and/or regrowth. In some
embodiments, axon growth according to the present invention occurs
in the CNS. In some embodiments, axon growth according to the
present invention occurs in the PNS.
[0319] In one embodiment, the present invention provides methods of
repairing tissue in a subject in need thereof. In some embodiments,
the tissue is neuronal tissue. In some embodiments, the tissue is
in the CNS. In some embodiments, the tissue is in the PNS. In some
embodiments, the tissue is non-neuronal tissue.
[0320] Therapeutic Proteins
[0321] An ADP-ribosyl transferase C3 protein described herein may
be used to treat spinal cord injury or other CNS trauma. Various
methods known in the art may be used to prepare and administer a
therapeutic composition containing an ADP-ribosyl transferase C3
protein described herein.
[0322] In some embodiments, the present invention provides an
improved method of preparing a therapeutic composition by mixing a
therapeutically effective amount of an ADP-ribosyl transferase C3
protein with a fibrinogen composition that does not contain a
thrombin to generate a therapeutic protein-fibrinogen composition;
and combining the therapeutic protein-fibrinogen composition with a
thrombin composition to generate a therapeutic composition. In some
embodiments, a therapeutic composition prepared according to the
present invention is a tissue adhesive that may be administered to
a spinal cord injury site to facilitate axon growth.
[0323] In addition to the various C3 fusion protein described
herein, other therapeutic protein may be prepared and administered
using the improved methods described herein. For example, the
improved methods described herein may be used to prepare and
administer other inhibitors of Rho hyperactivation that can cause
growth-inhibiting proteins to stop axons from regenerating. The
methods described herein are particularly useful for preparing a
therapeutic composition comprising a therapeutic protein containing
a transport domain covalently linked to an amino acid sequence of
an active domain (e.g., a purified C3 fusion protein).
[0324] Transport Domain
[0325] A transport domain comprises a peptide domain that can
freely cross cell membranes. Several transport domains have been
identified that allow a fused protein to efficiently cross cell
membranes in a process known as protein transduction. Studies have
demonstrated that a Tat (twin-arginine translocation) peptide
derived from the HIV Tat protein has the ability to transduce
peptides or proteins into various cells.
[0326] In some embodiments, the transport domain comprises a
wild-type Tat peptide or antennapedia peptide, or a fragment or
subdomain thereof, or a polypeptide derived therefrom. In some
embodiments, the transport domain comprises a wild Tat peptide or
antennapedia peptide. In some embodiments, the transport domain
comprises a polypeptide having an amino acid sequence having at
least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity
with a wild-type Tat peptide or antennapedia peptide. In some
embodiments, the transport domain comprises a fragment or subdomain
of Tat peptide or antennapedia peptide. In some embodiments, the
transport domain comprises a polypeptide having an amino acid
sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity with a fragment or subdomain of wild-type Tat
peptide or antennapedia peptide. In some embodiments, the transport
domain comprises a polypeptide derived from a nucleotide sequence
encoding a wild-type Tat peptide or antennapedia peptide. In some
embodiments, the transport domain comprises a polypeptide having an
amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or 100% sequence identity with a polypeptide derived from a
nucleotide sequence encoding a wild-type Tat peptide or
antennapedia peptide.
[0327] In some embodiments, a Tat peptide or antennapedia peptide
comprises an amino acid sequence having at least 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% sequence identity with a polypeptide
selected from Table 1. In some embodiments, a Tat peptide or
antennapedia peptide comprises an amino acid sequence selected from
Table 1.
TABLE-US-00004 TABLE 1 Transport Domain Amino Acid Sequence SEQ ID
NO: Antennapedia Leader Peptide (CT) KKWKMRRNQFWVKVQRG 15
Antennapedia Peptide RQIKIWFQNRRMKWKK 16 HIV - 1 Tat (48-60)
GRKKRRQRRRPPQ 17 TAT (47-57) YGRKKRRQRRR 18 Tat (48-57) GRKKRRQRRR
19 Tat - Beclin - 1 YGRKKRRQRRRGGTNVFNATFEIW 20 HDGEFGT Tat - C
(48-57) CGRKKRRQRRR 21 TAT - G1uR23A Fusion Peptide
YGRKKRRQRRRAKEGANVAG 22 Tat - G1uR23Y YGRKKRRQRRRYKEGYNVYG 23 TAT -
HA2 Fusion Peptide RRRQRRKKRGGDIMGEWGNEIFGAI 24 AGFLG Tat - NR2Bct
YGRKKRRQRRRKLSSIESDV 25 TAT - NSF222 Fusion Peptide YGRKKRRQRRR -
GGG - 26 LDKEFNSIFRRAFASRVFPPE TAT - NSF700 Fusion Peptide
YGRKKRRQRRR - GGG - 27 LLDYVPIGPRFSNLVLQALLVL TAT - NSF700scr
YGRKKRRQRRRGGGIPPVYFSRLDL 28 NLVVLLLAQL
[0328] In some embodiments, a transport domain amino acid sequence
is as follows: EFVMNPANAQGRHTPGTRL (SEQ ID NO: 10)
[0329] In some embodiments, a transport domain amino acid sequence
is as follows: EFVMNPANAQGR (SEQ ID NO: 11).
[0330] In some embodiments, a transport domain comprises an amino
acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
100% sequence identity with SEQ ID NO: 10. In some embodiments, a
transport domain comprises an amino acid sequence having at least
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with
SEQ ID NO: 11.
[0331] Among other things, the present invention may be used to
prepare and administer a C3 fusion protein containing an
ADP-ribosyl transferase C3 domain and a transport domain described
herein. In some embodiments, a C3 fusion protein comprises an amino
acid sequence of a transport domain covalently linked to an amino
acid sequence of an ADP-ribosyl transferase C3 domain, wherein the
amino acid sequence of said transport domain is selected from a Tat
peptide or antennapedia peptide, a fragment or subdomain of Tat
peptide or antennapedia peptide, a polypeptide derived from a
nucleotide sequence encoding a Tat peptide or antennapedia peptide,
or a polypeptide having an amino acid sequence having at least 80%
sequence identity thereto and wherein the amino acid sequence of
the active domain of the C3 fusion protein is selected from an
ADP-ribosyl transferase C3, a fragment thereof retaining
ADP-ribosyl transferase activity, or an amino acid sequence having
at least 80% sequence identity thereto. In some embodiments, the
transport domain is a cell penetrating peptide or a fusogenic
peptide. In certain embodiments, the transport domain is a
proline-rich fusogenic peptide. In some embodiments, the transport
domain is a highly basic, arginine-rich sequence corresponding to a
reverse Tat sequence. Exemplary transport domains are described in
Winton et al., 2002 J. Biol. Chem. 277:32820-32829, which is hereby
incorporated by reference.
[0332] Active Domain
[0333] In accordance with the present invention, the active domain
of a therapeutic protein may comprise a C3 transferase. C3
transferases cause the addition of one or more ADP-ribose moieties
to Rho-like proteins. In some embodiments, the C3 transferase is
from Clostridium botulinum (C3bot1 and 2). In some embodiments, the
C3 transferase is from Clostridium limosum (C3lim). In some
embodiments, the C3 transferase is from Bacillus cereus (C3cer). In
some embodiments, the C3 transferase is from Staphylococcus aureus
(C3stau1, 2 and 3).
[0334] In some embodiments, the active domain comprises an
ADP-ribosyl transferase C3. In some embodiments, the active domain
comprises a polypeptide having an amino acid sequence having at
least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity
with an ADP-ribosyl transferase C3. In some embodiments, the active
domain comprises a fragment of an ADP-ribosyl transferase C3 that
retains ADP-ribosyl transferase activity. In some embodiments, the
active domain comprises a polypeptide having an amino acid sequence
having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity with a fragment of an ADP-ribosyl transferase C3 that
retains ADP-ribosyl transferase activity. In some embodiments, the
active domain comprises the amino acid sequence of ADP-ribosyl
transferases that act on Rho (Wilde et al. 2001. Biol. Chem.
275-16478-16483; Wilde et al 2001. J. Biol. Chem.
276:9537-9542).
[0335] In some embodiments, an active domain amino acid sequence is
as follows:
TABLE-US-00005 Wild-type C3 transferase of C. botulinum (Swiss-Prot
ent P15879) (SEQ ID NO: 12)
MKGLRKSILCLVLSAGVIAPVTSGMIQSPQKCYAYSINQKAYSNTYQEFT
NIDQAKAWGNAQYKKYGLSKSEKEAIVSYTKSASEINGKLRQNKGVINGF
PSNLIKQVELLDKSFNKMKTPENIMLFRGDDPAYLGTEFQNTLLNSNGTI
NKTAFEKAKAKFLNKDRLEYGYISTSLMNVSQFAGRPIITKFKVAKGSKA
GYIDPISAFAGQLEMLLPRHSTYHIDDMRLSSDGKQIIITATMMGTAINP K
[0336] In some embodiments, an active domain amino acid sequence is
as follows:
TABLE-US-00006 (SEQ ID NO: 13)
SAYSNTYQEFTNIDQAKAWGNAQYKKYGLSKSEKEAIVSYTKSASEINGK
LRQNKGVINGFPSNLIKQVELLDKSFNKMKTPENIMLFRGDDPAYLGTEF
QNTLLNSNGTINKTAFEKAKAKFLNKDRLEYGYISTSLMNVSQFAGRPII
TKFKVAKGSKAGYIDPISAFAGQLEMLLPRHSTYHIDDMRLSSDGKQIII TATMMGTAINPK
[0337] In some embodiments, an active domain amino acid sequence is
as follows:
TABLE-US-00007 (SEQ ID NO: 14)
MSAYSNTYQEFTNIDQAKAWGNAQYKKYGLSKSEKEAIVSYTKSASEING
KLRQNKGVINGFPSNLIKQVELLDKSFNKMKTPENIMLFRGDDPAYLGTE
FQNTLLNSNGTINKTAFEKAKAKFLNKDRLEYGYISTSLMNVSQFAGRPI
ITKFKVAKGSKAGYIDPISAFAGQLEMLLPRHSTYHIDDMRLSSDGKQII
ITATMMGTAINPK
[0338] In some embodiments, an active domain comprises an amino
acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
100% sequence identity with SEQ ID NO: 12. In some embodiments, an
active domain comprises an amino acid sequence having at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID
NO: 13. In some embodiments, an active domain comprises an amino
acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
100% sequence identity with SEQ ID NO: 14.
[0339] Fibrinogen Composition
[0340] In accordance with the present invention, therapeutic
compositions for use as tissue adhesives, sealants or hemostatic
agents can be made using the proteins fibrinogen and thrombin,
Cronkite, E. P. et al., J.A.M.A., 124, 976 (1944), Tidrick, R. T.
and Warner, E. D., Surgery, 15, 90 (1944). Fibrinogen is a soluble
protein found in the blood plasma of all vertebrates that when
contacted by thrombin (another plasma protein) becomes polymerized
to an insoluble gel-like network. In polymerized form, the
fibrinogen is referred to as fibrin. The conversion of fibrinogen
to fibrin is crucial to normal hemostasis in vertebrates.
[0341] There are numerous potential advantages, relative to the use
of synthetic materials, associated with the use of fibrinogen as an
adhesive, sealant or hemostatic agent. For example, when applied to
a wound, polymerized fibrinogen (fibrin) forms a network or
scaffolding through which it is more likely that immunologically
active cells (to defend against invading pathogens) and also
epithelial cells (for tissue regeneration and repair) can migrate.
Additionally, fibrin materials may be dissolved gradually by the
body (a process termed fibrinolysis) after treatment.
[0342] In some embodiments, a pharmaceutically effective amount of
a therapeutic protein as described above is combined with a
fibrinogen composition to generate a therapeutic protein-fibrinogen
composition (see, for example, FIG. 10). The notation "therapeutic
protein-fibrinogen" or "therapeutic polypeptide-fibrinogen" simply
indicates that the composition comprises both components (the
therapeutic protein/polypeptide and fibrinogen), rather than
indicating some form of bond (e.g., covalent bond) between the
components. In some embodiments, a pharmaceutically effective
amount of a therapeutic protein as described above is first added
to a solution comprising one or more plasminogen-activator
inhibitors or plasmin inhibitors before mixing with the fibrinogen
composition. In some embodiments, the one or more
plasminogen-activator inhibitors or plasmin inhibitors comprises
aprotinin. In some embodiments, the fibrinogen composition
comprises a protease inhibitor before it is combined with a
pharmaceutically effective amount of a therapeutic protein as
described above. In some embodiments, the fibrinogen composition
comprises aprotinin. In some embodiments, a suitable fibrinogen
composition further contains albumin, one or more blood coagulation
factors, and/or globulin. In some embodiments, a pharmaceutically
effective amount of a therapeutic protein as described above is
combined with a thrombin composition before it is combined with a
fibrinogen composition (see, for example, FIG. 11).
[0343] In some embodiments, a pharmaceutically effective amount of
a therapeutic protein as described above is combined with a
commercially available fibrin sealant. In some embodiments, the
commercially available fibrin sealant is TISSEEL. In some
embodiments, the commercially available fibrin sealant comprises
fibrinogen, thrombin, Factor XIII, plasmafibronectin, plasminogen,
aprotinin solution and calcium chloride solution.
[0344] Compositions provided by the invention, or used in the
invention, can be defined as "does not contain a thrombin". This
statement indicates that the composition is free from thrombin, or
that any thrombin that is present cannot be detected because it is
below the limit of quantification when the composition is analyzed
by one or more of size exclusion chromatography-high performance
liquid chromatography (SEC-HPLC), SDS-PAGE, capillary
electrophoresis-SDS PAGE (CE-SDS PAGE), and/or reverse phase-high
performance liquid chromatography (RP-HPLC).
[0345] Thrombin Composition
[0346] In accordance with the present invention, a therapeutic
composition comprises a pharmaceutically effective amount of a
therapeutic protein as described above and a fibrinogen composition
that comprises a protease inhibitor, such as aprotinin, and may
further comprise a serine protease, such as, for example, a
thrombin composition.
[0347] Thrombin converts soluble fibrinogen into insoluble strands
of fibrin, as well as catalyzing many other coagulation-related
reactions. In some embodiments, a thrombin composition comprising
thrombin is combined with a therapeutic protein-fibrinogen
composition to generate a therapeutic protein-fibrinogen-thrombin
composition. In some embodiments, a therapeutically effective
amount of a therapeutic protein is combined with each of a
fibrinogen composition comprising a fibrinogen and a thrombin
composition to generate a therapeutic protein-fibrinogen-thrombin
composition, wherein the therapeutic protein comprises an amino
acid sequence of a transport domain covalently linked to an amino
acid sequence of an active domain, said amino acid sequence of said
active domain is selected from an ADP-ribosyl transferase C3, a
fragment thereof retaining ADP-ribosyl transferase activity, or an
amino acid sequence having at least 80% sequence identity thereto,
and wherein said amino acid sequence of said transport domain is
selected from a subdomain of Tat peptide or antennapedia peptide, a
fragment of Tat peptide or antennapedia peptide, a polypeptide
derived from a nucleotide sequence encoding a Tat peptide or
antennapedia peptide, or an amino acid sequence having at least 80%
sequence identity thereto. In some embodiments, the thrombin
composition comprises calcium chloride.
[0348] Administration
[0349] The present invention provides methods for preparing and
administering therapeutic compositions comprising a therapeutic
protein-fibrinogen-thrombin composition to subjects in need
thereof. In some embodiments, administration comprises contacting a
tissue, nerve injury site, open wound, etc. with a therapeutic
composition of the present invention in a pharmaceutically
effective amount sufficient to promote the therapeutic effect of
the therapeutic protein (e.g., facilitating axon growth, tissue
repair, etc.). Application of the therapeutic compositions can be
done by any suitable method known in the art. Specific examples
include dripping or spraying using a cannula or spray set, etc.
See, for example, Tisseel product monograph (2013) and Evicel
product monograph (2010). In some embodiments, the therapeutic
compositions are applied as a layer(s) onto a subject's tissue. In
some embodiments, the therapeutic compositions are applied (e.g.,
sprayed or dripped) onto the tissue in short bursts (for example,
0.1 to 0.2 mL). In some embodiments, the therapeutic compositions
are applied as a layer onto a patient undergoing surgery. In some
embodiments, each of the pharmaceutical compositions described
above is independently employed as the therapeutic composition in
such methods for preparing and administering therapeutic
compositions.
[0350] Kits
[0351] One aspect of the invention relates to kits for the use of
treating spinal cord injury, comprising a therapeutic protein as
described above, for use in facilitating axon growth, treating
spinal cord injury (e.g., nerve injury arising from traumatic nerve
injury or nerve injury caused by disease), preventing (e.g.,
diminishing, inhibiting) cell apoptosis, suppressing the inhibition
of neuronal axon growth, treating ischemic damage related to
stroke, suppressing Rho activity, regenerating injured axons (e.g.,
helping injured axon to recover, partially or totally, their
function), and/or facilitating the formation of new neuronal
connections (e.g., developing axons, dendrites, neurites) with
other (surrounding) cells (e.g., neuronal cells), in a mammal,
(e.g., human).
[0352] In some embodiments, a kit to promote neuroregeneration and
neuroprotection comprises a first container containing a
pharmaceutical composition comprising a therapeutic protein
described herein, a second container containing a fibrinogen
composition, and a third container containing a thrombin
composition. In some embodiments, a fibrinogen composition
comprises fibrinogen, albumin, one or more blood coagulation
factors, and/or globulin. In some embodiments, a kit further
comprises an additional container containing a solution comprising
one or more plasminogen-activator inhibitors or plasmin inhibitors.
In some embodiments, one or more plasminogen-activator inhibitors
or plasmin inhibitors comprise aprotinin. In some embodiments, a
kit further comprises a solution comprising calcium chloride.
[0353] In some embodiments, a kit further comprises a syringe. In
some embodiments, a syringe is a Duploject Syringe (Baxter Product
Code 1501252). As used herein, the term "Duploject Syringe" may be
replaced by a syringe which is defined as one which: comprises a
clip for two disposable syringes for simultaneous application of
two-component products, e.g., a fibrin sealant; is a device that
enables the mixing of the contents of two syringes and then the
mixed contents exit through one outlet; is a device that discharges
the contents of two syringes as if the contents of both syringes
were already mixed in one syringe; and/or enables the mixing of the
contents of two syringes and then the expulsion through one outlet,
when a force is applied to the syringes.
[0354] In more detail, the syringe defined in the previous
paragraph is a device comprising two syringes, wherein the plunger
for each syringe is joined together at one end, wherein in the
nozzle of each syringe is attached to a Y-shaped connector; so when
a force a applied via the plungers, it is applied equally to each
syringe, causing the contents of each syringe to be forced out via
the nozzle into the Y-shaped connector, where the contents mix, and
then the mixed contents exit through the single outlet of the
Y-shaped connector. The single outlet of the Y-shaped connector can
be fitted with a cannula.
[0355] In some embodiments, the syringe defined in the previous two
paragraphs comprises a syringe of therapeutic
polypeptide-fibrinogen composition and a syringe of thrombin,
wherein the plunger of each syringe is joined together at one end,
wherein in the nozzle of each syringe is attached to a Y-shaped
connector; so when a force is applied via the plungers, the force
is applied equally to each syringe, causing the contents of each
syringe to be forced out via the nozzle into the Y-shaped
connector, where the contents mix, initiating fibrin production,
and then the mixed contents exit through the single outlet of the
Y-shaped connector. The single outlet of the Y-shaped connector can
be fitted with a cannula.
[0356] In some embodiments, a kit to promote neuroregeneration and
neuroprotection comprises a first chamber containing a
pharmaceutical composition comprising a therapeutic protein
described herein and a fibrinogen composition, and a second chamber
containing a thrombin composition. In some embodiments, a first
chamber further contains one or more plasminogen-activator
inhibitors or plasmin inhibitors. In some embodiments, one or more
plasminogen-activator inhibitors or plasmin inhibitors comprises
aprotinin. In some embodiments, a thrombin composition comprises
thrombin and calcium chloride. In some embodiments, each of the
pharmaceutical compositions described above is independently
employed as the therapeutic composition in the first chamber of the
kits.
EXAMPLES
[0357] While certain compounds, compositions and methods of the
present invention have been described with specificity in
accordance with certain embodiments, the following examples serve
only to illustrate the compounds of the invention and are not
intended to limit the same.
Example 1. Bacterial Culture Process of C3 Fusion Proteins
[0358] In one exemplary process, C3 fusion protein is expressed
from a T7 promoter-driven vector and E. coli BL21 (DE3) microbial
host expression system yielding a high level of intracellular
expression upon the induction of IPTG. Following expression, cells
are harvested and homogenized, releasing C3 fusion protein and
other host-cell-derived impurities into the culture broth. The
homogenate is cleared of cell debris by centrifugation and
subsequent tangential flow filtration (TFF). C3 fusion protein is
purified by various chromatography steps, followed by a
buffer-exchange step to condition the material for filtration by a
Q-membrane adsorber. Finally, purified C3 fusion protein is
concentrated and exchanged into drug substance (DS) formulation
buffer.
Overview of the Host Vector System
[0359] An exemplary C3 fusion protein was expressed from a T7
promoter driven vector with a Kanamycin selectable marker from an
E. coli BL21 (DE3) microbial host expression system. BL21 has been
widely used as an E. coli host background for expressing
recombinant proteins. It does not contain the Lon protease and also
is deficient in the outer membrane protease, OmpT. The lack of the
two key proteases reduces degradation of heterologous proteins
expressed in the strains.
[0360] BL21 (DE3) is a DE3 derivative of BL21. The DE3 designation
indicates that the strain contains the .lamda.DE3 lysogen that
carries the gene T7 polymerase under the control of the lac UV5
promoter. cDNA comprising SEQ ID NO: 3 encoding an exemplary C3
fusion protein was cloned into a protein expression vector. The
combination of a T7 promoter-driven vector and a BL21 (DE3) host
strain yielded a high level of expression of mRNA encoding the
recombinant protein upon induction of IPTG. Clone selection was
based on SDS-PAGE analysis (Coomassie staining and scanning
densitometry) of the crude extracts from the inductions. The clone
expressing the highest level of C3 fusion protein was selected and
used to establish a Working Cell Bank.
[0361] This example demonstrates that a bacterial culture system
may be used to successfully express C3 fusion proteins.
Seed Culture
[0362] A pre-culture shake flask containing growth medium was
inoculated with a Working Cell Bank vial. The Working Cell Bank
vial may refer to any number of cells, including a single cell. The
cells were cultivated at 37.degree. C. to reach an optical density
(OD, measured at 600 nm) optimal level (e.g., 2-10 OD). This
culture was used for inoculation of the production bioreactor to a
starting OD of 0.004.
Fermentation
[0363] To maximize yield and robustness, a high-cell density (HCD)
fermentation process was used. Cell growth profiles using the HCD
fermentation method provided three times higher OD values (FIG. 4A)
compared to the standard fermentation method (FIG. 4B). The
fermentation process producing exemplary C3 fusion protein was
performed in a Sartorius bioreactor set up. Dissolved oxygen and pH
was controlled throughout the production process. Fermentation
started in batch mode, in which a defined amount of initial carbon
source (glucose) was provided in the medium. After consumption of
the glucose in the medium, (carbon source limitation, typically
within 7.5 hours), a fed-batch mode was started. In the fed-batch
mode, a glycerol carbon source feed solution was introduced to the
fermentor at an exponential rate, followed by a second phase of
constant feeding. Thus, the feeding strategy consisted of two
phases: a first stage of exponential feeding, which lasted 7-8
hours and a second stage of constant feeding for 8.5 hours (8-9
hours). An exemplary fermentation process is shown in FIG. 1.
[0364] 1.sup.st stage: Exponential feeding was controlled
using:
Exp . Feed [ g h ] = 1 .times. 0 .times. 0 .times. 0 * [ ( .mu. Y x
s + m ) V 0 X 0 1 S F . e ( .mu. t ) ] ##EQU00003##
[0365] Where;
[0366] Specific growth rate, .mu., (l/h)=0.12-0.18/hr,
[0367] Biomass yield on glucose, Yx/s, (g/g)=0.32-0.48 g/g,
[0368] Maintenance factor, m, (g/g/hr)=0.32-0.48 g/g/hr,
[0369] Initial volume of reactor before the start of feed, V.sub.0,
(L)=12 L,
[0370] Biomass concentration before the start of feed, X.sub.0,
(g/L)=8-12 g/L,
[0371] Feed concentration, S.sub.F, (g/kg)=480-720 g/kg
[0372] Time, t, (hrs)=7-8 hrs
[0373] 2.sup.nd stage: Constant feed rate=Feed rate at the end of
the exponential feeding stage.
[0374] The temperature during the initial batch mode and the fed
batch mode was maintained at 37.degree. C. until the start of
induction. C3 fusion protein production was triggered by the
addition of an inducing agent, 5 mM IPTG (Isopropyl
.beta.-D-1-thiogalactopyranoside) after the first 8.5 hours of
constant feeding (2.sup.nd stage). The constant feed rate of the
2.sup.nd stage was maintained during induction. At the start of
induction, the temperature was shifted to 28.degree. C. After 4
hours of induction, the fermentor was further cooled to 8.degree.
C. (within an hour) and the culture was harvested for
homogenization. Exemplary C3 fusion protein production is shown in
FIG. 1 and FIG. 3.
Homogenization
[0375] Harvested cells were homogenized in APV1000 (homogenizer) to
lyse the cells. Pressure was maintained at 690 bar with 3 cycles of
homogenization. During this time, the temperature of the feed and
homogenate was maintained at 8.degree. C.-11.degree. C.
Centrifugation
[0376] Centrifugation was performed in a semi-continuous mode using
a CEPA centrifuge (model Z41). Flow was maintained at 30 L/hr in a
17000 g centrifugal force. The temperature of the supernatant was
maintained at 15.degree. C.
Tangential Flow Filtration
[0377] The supernatant comprising the cell lysate was clarified by
a Tangential Flow Filtration (TFF) system equipped with a Sartorius
Sartocon slice membrane (1000 kDa, 0.5 m.sup.2). The filtration was
performed in two steps: a concentration phase and a diafiltration
phase. Within the concentration phase, the supernatant was reduced
by volume factor of 2. Within the diafiltration phase, the
retentate was continuously washed with 5 volumes of diafiltration
buffer while the product was collected on the permeate side. The
temperature of the retentate was maintained at 15.degree. C.
throughout the TFF operation. The permeate was then
sterile-filtered before loading onto the Cation Exchange Column
I.
Example 2. C3 Fusion Protein Capture and Purification Process
[0378] This example demonstrates that a simplified downstream
purification process may be used to capture and purify C3 fusion
proteins. C3 fusion protein is purified by various chromatography
steps, followed by a buffer-exchange step to condition the material
for filtration by a Q-membrane adsorber. Finally, purified C3
fusion protein is concentrated and exchanged into drug substance
(DS) formulation buffer.
Cation Exchange Column I
[0379] TFF permeate containing C3 fusion protein was conditioned to
an optimal conductivity and then filtered using a Sartoban filter.
To capture the C3 fusion proteins from most host-cell derived
impurities, a SP Sepharose cation exchange resin column was
equilibrated and the conditioned permeate containing the C3 fusion
was loaded onto the capture column comprising the cation exchange
resin, washed and then eluted in a single step by increasing salt
concentration (e.g., NaCl). The peak eluate containing the C3
fusion protein was collected.
Cation Exchange Column II
[0380] The peak eluate pool was then conditioned to a conductivity
of 7.5.+-.0.5 mS/cm by diluting with a 10 mM NaH2PO4, 10 mM citric
acid, 0.5 mM EDTA, pH 7.0 buffer. The pool containing the C3 fusion
was loaded onto an intermediate chromatography column with a strong
cation exchange resin (e.g., YMC Biopro S30) to resolve and isolate
C3 fusion protein product from protein contaminants with highly
similar biophysical properties. Prior to loading, the column was
equilibrated. After loading, the column was washed and then eluted
using a linear salt gradient (e.g., NaCl). The addition of an
intermediate chromatography column with a strong cation exchange
resin (e.g., YMC Biopro S30) increased the purity of C3 fusion
protein and reduced batch to batch variability (FIG. 8).
Hydrophobic Interaction Chromatography
[0381] The product collection pool from the intermediate cation
exchange column (e.g., YMC Biopro S30) was adjusted to a final
(NH.sub.4).sub.2SO.sub.4 concentration of 1.6.+-.0.1 M. The pool
was then filtered using a Sartoban P 0.2 .mu.m filter and loaded
onto a hydrophobic interaction chromatography (HIC) column (e.g.,
Butyl 650M), whereby proteins bind at a high lyotropic salt
concentration and elute at low lyotropic salt concentration. HIC
column was equilibrated using one or more column volumes of
equilibration buffer. After loading, the column was washed and the
C3 fusion protein was eluted from the column in a single step by
altering the salt concentration of the mobile phase. The peak
eluate containing the C3 fusion protein was collected.
Ultrafiltration/Diafiltration I
[0382] The HIC collection eluate was then loaded onto an
ultrafiltration/diafiltration (UF/DF) membrane (Pellicon 3
Ultracell 5 kDa MWCO) pre-conditioned with elution buffer from the
HIC column. Diafiltration was performed with 6 diavolumes of
Q-membrane Adsorber buffer to buffer exchange the peak eluate from
the HIC column containing C3 fusion protein.
Q-Membrane Adsorption
[0383] The diafiltration retentate was loaded onto an ion exchange
membrane to remove negatively charged contaminants such as DNA,
host-cell proteins and endotoxins. The ion exchange membrane (e.g.,
Sartobind Q Single Sep Mini) was equilibrated and the diafiltration
retentate was loaded in flow-through mode. The main fraction
collection began when the ascending elution peak reached absorbance
of 50 mAU and continued until absorbance dropped on the tailing
edge of the peak to 50 mAU. The flow-through containing the C3
fusion protein was then filtered using a Sartoban P 0.2 .mu.m
filter.
[0384] Ultrafiltration/Diafiltration II
[0385] C3 fusion protein was then loaded onto a pre-conditioned
UF/DF membrane (e.g., Pellicon Ultracell 5 kDa MWCO). Concentration
was performed until the target C3 fusion protein concentration was
reached (e.g., 10-15 or 30-37 mg/mL). Subsequently, diafiltration
was performed with 5 mM sodium citrate, pH 6.5, until 8 diavolumes
were completed. The product was then filtered using a Sartoban P
0.2 .mu.m filter. The filtered UF/DF II retentate was the C3 fusion
protein Drug Substance.
Example 3. Exemplary High Cell Density Process for Producing
Recombinant C3 Fusion Protein
[0386] Table 2 shows fermentation parameters for an exemplary
process.
TABLE-US-00008 TABLE 2 Parameter Feeding C-source Glycerol Feed
trigger pH spike (~7.5 h) (OD 40-50) Feed profile 7.5 h exponential
8.5 h constant Induction OD.sub.600 145-175 Time (h) 4 End of
OD.sub.600 145-195 Fermentation Time (h) 27.5 Titer 2
[0387] Table 3 shows process steps for an exemplary process.
TABLE-US-00009 TABLE 3 Manufacturing Process Step Drug Substance
Fermentation Scale 300 L Fermentation HCD process Cell Harvest
Fermentation broth taken directly to homogenization Clarification
Homogenate is centrifuged (Centrifugation) to clear cell debris
Clarification (Tangential Sartocon Slice 0.1 .mu.m Flow Filtration)
membrane, high-salt diafiltration buffer, 15.degree. C.
Cation-Exchange 10 .+-. 2 cm bed height Chromatography I
Cation-Exchange Intermediate purification Chromatography II step
Target Concentration of 31-37 mg/mL Bulk Drug Substance (BDS)
Packaging Polycarbonate Drug Product Concentration step Dilute as
necessary for each dose (27-33 mg/mL)
[0388] C3 fusion protein was produced by high cell density
fermentation with an exponential feeding strategy as described
above. After IPTG induction, cells were harvested and homogenized
such that C3 fusion protein was released directly into the
fermentation broth. The resulting mixture was centrifuged and
clarified using TFF as described in Example 1. Following TFF, the
resulting permeate was sterile filtered and purified according to
Example 2. C3 fusion protein purified as described above resulted
in a composition of C3 fusion protein at a concentration of 31-37
mg/mL with an improved purity profile as shown in Table 6.
Example 4. Analytical Methods of Recombinant C3 Fusion Protein
Glycohydrolase (GH) Enzyme Assay
[0389] The intracellular action of C3 exoenzymes and C3 fusion
proteins results from transfer of an ADP-ribose moiety to an
asparagine residue in Rho GTPase, trapping this GTPase in its
inactive conformation. An exemplary C3 fusion protein is an
ADP-ribosyltransferase that catalyzes hydrolysis of NAD+ in the
absence of the specific protein substrate, Rho. C3 fusion protein
catalyzes transfer of an ADP-ribose moiety to the RhoA, RhoB and
RhoC members of the Rho family of small GTPases. In neurons the
predominant species is RhoA, so the numbering system for RhoA and
the abbreviation "Rho" is used. A spectrofluorometric
glycohydrolase (GH) activity assay was used to measure
glycohydrolase (GH) as the formation of a fluorescent product,
which gives a sensitive and reliable method to serve as a test of
identity and potency.
[0390] The GH assay measures the formation of .epsilon.-ADP-ribose
produced as a result of hydrolysis of .epsilon.-NAD by C3-variants.
Glycohydrolase activity of C3-variants converts .epsilon.-NAD.sup.+
into .epsilon.-ADP-ribose, a molecule with 10 times higher
fluorescence intensity at the selected wavelengths. The
fluorescence intensity of .epsilon.-ADP-ribose is used to measure
the amount of .epsilon.-ADP ribose formed by using a standard curve
of fluorescence of known concentrations of .epsilon.-AMP. The
fluorescence intensities of .epsilon.-AMP and .epsilon.-ADP-ribose
are measured by exciting the reaction at 305 nm and recording the
emission at 410 nm. A unit of activity is defined as nmoles
ADP-ribose formed in 30 min at 37.degree. C. The assay is linear
with up to at least 12 .mu.g of C3-variant protein and up to least
180 min of incubation time. This assay has been found to be
precise, accurate and reproducible. This assay has also been found
to be useful in measuring decreases in enzymatic activity after
incubation at 70.degree. C., and can be considered to be
stability-indicating when used in a well-designed stability
study.
[0391] Using the spectrofluorometric GH assay, experiments were
performed with exemplary C3 fusion protein with increasing
concentrations of .epsilon.-NAD (FIG. 2). At 600 .mu.M to 1000
.mu.M .epsilon.-NAD, the plateau in the amount of
.epsilon.-ADP-ribose formed indicates a saturating concentration of
the substrate. The concentration of substrate chosen for the assay
was 0.4 mM .epsilon.-NAD in order to allow more sensitivity in
detecting GH activity.
Glycohydrolase (GH) Enzyme Assay as a Stability-Indicating
Assay
[0392] Hydrolysis, oxidation, deamidation, aggregation,
denaturation or other breakdown pathways during fermentation,
purification, transport or storage of C3-variants could decrease
the specific activity of a preparation. The stability-indicating
potential of the GH enzyme assay using an accelerated temperature
stability experiment was carried out at 70.degree. C.
Purity Profile of Exemplary C3 Fusion Protein
[0393] The purity of C3 fusion protein was evaluated using RP-HPLC,
SEC-HPLC, and SDS-PAGE analysis. Purity levels of product materials
from development batches routinely exceeded 95%. In an E.
coli-derived recombinant protein product, pyrogenicity, bioburden
and residual DNA levels are critical for safety. Another impurity
to be minimized for safety reasons is host cell protein. This is
addressed directly by a quantitative ELISA and indirectly by purity
estimate on SDS-PAGE. Additionally, a significant host cell
contaminant may give a signal upon N-terminal or C-terminal
sequencing or mass spectrometry. Taken together, the
purity/impurity profile for general safety issues associated with
E. coli as a host cell is tolerable. Accelerated stability studies
and release testing indicated a very small amount of multimers,
detected by SE-HPLC. These are known not to be mediated by
cysteine-linked disulfide bond formation, because the exemplary C3
fusion protein lacks a cysteine residue. The impurities detected by
RP-HPLC are in general <1%.
[0394] Exemplary C3 fusion protein drug substance test results are
indicated in Table 4. Test results shown in Table 4 correspond to
drug substance produced using Process 1. Process 1 includes
production using standard fermentation that does not employ high
cell density fermentation with an exponential feeding strategy.
After IPTG induction, cells were harvested by centrifugation and
homogenized. The resulting lysate was clarified using TFF. The TFF
permeate was subjected to cation exchange, followed by HIC, UF/DF,
Q-membrane adsorption and a second UF/DF.
TABLE-US-00010 TABLE 4 Exemplary test results Test Type Test Method
Specification Test Results Strength Protein UV Analysis .sup.1
13-19 mg/mL 15.7 mg/mL Concentration Potency Biological
Glycohydrolase 20-40 Units 33 Units Activity Enzyme Assay .sup.2
(Units/mg C3 fusion protein) Identity Molecular Size Reduced SDS-
Compares to Meets criteria PAGE reference standard; major band
migrates between 21 and 31 kDa Pl cIEF 10.0-10.2 10.1 Purity
RP-HPLC >95% 99% Quality SEC-HPLC >95% 100% monomer Reduced
SDS- >95% as 96.6% PAGE determined by scanning densitometry or
image analysis Appearance Visual Clear, colorless Clear, colorless
Inspection liquid, liquid, essentially free essentially free of
visible of visible particles particles pH @ 25.degree. C. PH
6.8-7.8 7.2 Measurement E. coli. Residual Hybridization <1 ng/mg
.sup.4 <11 pg/mg DNA Method .sup.3 Host Cell Host Cell <10
pg/mg 0.86 pg/mg Proteins Proteins Immunoassay Safety Endotoxin LAL
<1 EU/mg 0.74 EU/mg Bioburden Membrane <1 CFU/10 mL 0 CFU/10
mL Filtration N.T. = not tested .sup.1 Extinction coefficient is
0.72 as determined by amino acid analysis .sup.2 Unit definition: 1
unit = 1 nmol of ADP-ribose/(mg protein .times. 30 min) @
37.degree. C. .sup.3 Selected as a replacement for PicoGreen
method. .sup.4 Current WHO guidelines specify < 10 ng/dose. If a
drug product of exemplary C3 fusion protein dose containing 10 mg
of C3 fusion protein is assumed, then the limit becomes < 1
ng/mg.
[0395] Further characterization attributes may be measured using
LC-MS based methods to determine deamidation, methionine oxidation
and peptide mapping. Additional characterization can include
immunochemical homogeneity via Western blot; residual levels of
PPG-200 defoamer, IPTG, and edetate disodium dihydrate, molecular
ion measurements with MALDI-TOF-MS or high resolution mass
spectrometry; N-terminal and C-terminal sequencing with digestion
and/or Edman degradation; amino acid analysis with HCl hydrolysis;
osmolality; Far-UV Circular Dichroism; Near-UV Circular Dichroism;
Fourier transform infrared spectroscopy (FTIR); Intrinsic
fluorescence; differential scanning calorimetry (DSC); and
sedimentation velocity analytical ultracentrifugation (SV-AUC).
Example 5. Analytical Methods of Recombinant C3 Fusion Protein
[0396] This example demonstrates characterization of an exemplary
purified C3 fusion protein. Specifically, a C3 fusion protein
comprising SEQ ID NO: 1 is a recombinant protein composed of 231
amino acids with a theoretical molecular weight of 25,726 daltons
(a nominal molecular weight of 26-28 kDa is used for analytical
purposes) and theoretical isoelectric point (pI) of 9.6. The
extinction coefficient has been determined by amino acid analysis
to be 0.72 units of absorbance at A.sub.280 nm per mg of
protein.
[0397] Upon expression and purification of the coding region
comprising SEQ ID NO: 3, according to the present invention, the
N-terminal methionine is present in fewer than 15% of the
molecules, as determined by mass spectrometry. Confirmation of this
sequence and the primary structure of C3 fusion protein have been
completed using amino acid analysis, peptide mapping, N-terminal
sequencing, C-terminal sequencing, and mass spectrometry. The
C-terminal sequence was determined to be FVMNPANAQGRHTPGTRL (SEQ ID
NO: 4), in agreement with the theoretical sequence. Molecular ion
by MALDI-TOF was reproducibly found to be approximately 25,726 m/z.
This corresponds to a polypeptide of 231 amino acids matching the
theoretical peptide sequence, at least 85% of the molecules missing
the N-terminal methionine residue. Peptide mapping providing
97%-100% coverage has confirmed the theoretical protein sequence,
and the amino acid sequence results correspond with those expected
the theoretical sequence.
[0398] Exemplary characterization results are shown in Table 5.
TABLE-US-00011 TABLE 5 Exemplary characterization results Target
acceptance Test type Method criteria Result N-terminal Edman
(M)SAYSNTYQEF (M)SAYSNTYQEFTNIDQA sequencing degradation (SEQ ID
NO: 5) (SEQ ID NO: 6) C-terminal MS/MS QGRHTPGTRL
FVMNPANAQGRHTPGTRL sequencing (SEQ ID NO: 7) (SEQ ID NO: 4)
Immunochemical Immunoblot with Immunoreactive Immunoreactive
homogeneity RAb-01 26 kDa band 26 kDa band antiserum Deamidation
TBD To be determined N.T. Methionine Tryptic RP- <15% overall
~10% overall average oxidation HPLC average Amino Acid HCl
hydrolysis 90-110% of 100% of expected mass analysis expected mass
Peptide mapping Tryptic RP- >95% coverage 100% coverage HPLC
Molecular ion MALDI-TOF 25,726 m/z 25,726 m/z MS Biological
Bioassay at two Neurite outgrowth; 2.8-fold (low) Activity
concentrations Increased >2 fold at 6.2-fold (high) low conc.
>3 fold increase at high conc.
[0399] Ion exchange HPLC (IE-HPLC) was used to measure purity
and/or identify product related substances (PRS). Characterization
of exemplary C3 fusion protein drug substance produced by Process 1
for the final drug substance after the downstream processing using
IE-HPLC is shown in FIG. 6. FIG. 7A shows an intermediate stage
(after the fermentation but prior to the downstream processing) of
Process 2. Characterization of exemplary C3 fusion protein drug
substance produced by Process 2 for the final drug substance after
the downstream processing using IE-HPLC is shown in FIG. 7B which
is superimposed with the IE-HPLC of FIG. 6. As shown in FIGS. 6, 7A
and 7B, in addition to the main peak corresponding to C3 fusion
protein (compound X), IE-HPLC identified peaks A, B, C, D, and 1.05
as product related peaks. Process 1 corresponds to C3 fusion
protein production with standard fermentation that does not employ
high cell density fermentation with an exponential feeding strategy
and without an intermediate cation-exchange column (cation exchange
column II described in Example 2). Process 2 corresponds to C3
fusion protein produced with high cell density fermentation with an
exponential feeding strategy and an intermediate cation exchange
column (cation exchange column II described in Example 2) during
the downstream processing. In FIG. 7B, the main peaks for Processes
1 and 2 were about 77% and 95%, respectively. Exemplary samples
from Process 2 after the downstream processing showed .gtoreq.85.0%
main product; .ltoreq.10.0% total acidic peaks; and .ltoreq.5.0%
total basic peaks, as shown in Table 7 below.
[0400] Stability of C3 fusion protein was tested using acid stress
by HCl, base stress by NaOH, oxidation stress by 1% tBHP and by 1%
H.sub.2O.sub.2. RP-HPLC and IE-HPLC (IEP) were used to determine
the drug substance profile of C3 fusion protein after the stability
stress tests (FIG. 5).
[0401] Exemplary analytics shown in Table 6 from development runs
show the exemplary Process 2 is robust and reproducible. As shown
in FIG. 9, purified C3 fusion protein from various batches of the
exemplary Process 2 described herein, exhibits comparable
biological activity by a neurite outgrowth assay.
TABLE-US-00012 TABLE 6 Exemplary analytics Process 2 Process 2
Method Process 1 (30 L) (300 L) UV (g/L) 12 35.8 35.0 SDS-PAGE
(band %) 99.6 100.0 100.0 IEP (area %) 77.0 93.5 94.2 SEC (area %)
96.5 100.0 100.0 RPLC (area %) 93.6 92.5 93.3 GH Assay (units) 24
25 33 Endotoxin (EU/mg) <1 5.1 .times. 10.sup.-4 2.9 .times.
10.sup.- .sup.4 Bioburden (cfu/10 mL) <1 <1 <1 DNA qPCR
(ppb) <1 <8 <9 HCP (ppm) <1 0.2 <0.1
[0402] C3 fusion protein produced and purified according to Example
1 and 2 (Process 2), resulted in a protein composition that is a
clear colorless liquid, with a pH between 6.0-7.0. The protein
concentration was between 31-37 mg/ml. The identity was confirmed
by isoelectric point (pI) using capillary isoelectric focusing and
SDS-PAGE with Coomassie Blue staining. The pI compared to a
reference standard with a pI.+-.0.1. The SDS-PAGE confirmed C3
fusion protein migrates between 21 and 31 kDa. Purity and impurity
analysis was performed with SDS-PAGE and Coomassie Blue staining
.gtoreq.95.0% band purity and .ltoreq.5.0% band total impurities.
Purity and impurity analysis of the C3 fusion protein composition
was performed by SE-HPLC and confirmed .gtoreq.95.0% monomer and
.ltoreq.5.0% total impurities. Purity and impurity analysis of the
C3 fusion protein composition was performed by RP-HPLC and
confirmed .gtoreq.85.0% purity and .ltoreq.15.0% total impurities.
Purity and impurity analysis of the C3 fusion protein composition
was performed by IE-HPLC and confirmed .gtoreq.85.0% major product,
.ltoreq.10% total acidic peaks and .ltoreq.5.0% total basic peaks.
The Glycohydrolase (GH) enzyme assay confirmed enzyme activity was
between 20-40 Units (nmol of ADP-ribose/(mg protein.times.30 min)
at 37.degree. C. Residual DNA was measured by Real-Time PCR and
confirmed to be .ltoreq.1000 pg/mg protein. Host cell proteins were
measured by ELISA using standard off the shelf anti-HCP antibodies
and confirmed to be .ltoreq.1000 ng/mg protein. Endotoxin (LAL) was
confirmed .ltoreq.1 EU/mg protein. Bioburden (TAMC and TYMC) was
confirmed .ltoreq.1 cfu/10 mL.
[0403] Additional attributes measured include Osmolality,
Deamidation (RPLC-MS), Methionine oxidation (RPLC-MS), Peptide Map
confirming .gtoreq.95.0% coverage (RPLC-MS), Molecular ion
confirming molecular weight of 25,726.+-.2 Da (RPLC-MS), and
immunochemical homogeneity confirming a major band migrates between
21 and 31 kDa (Western blot). N-terminal sequencing by Edman
Degradation confirmed (M)SAYSNTYQEF (SEQ ID NO: 5) and C-terminal
sequencing by MS/MS and Edman degradation confirmed QGRHTPGTRL (SEQ
ID NO: 7). Amino acid analysis by HCl hydrolysis confirmed 90-110%
of expected mass. Additional attributes measured include Residual
PPG-2000 Defoamer (LC-MS), Residual Isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG) (LC-MS), Residual Edetate
Disodium Dihydrate (CE), Far-UV Circular Dichroism, Near-UV
Circular Dichroism, FTIR, Intrinsic Fluorescence, DSC, and
Sedimentation velocity analytical ultracentrifugation (SV-AUC).
Exemplary attributes of C3 fusion protein purified using Process 2
are shown in Table 7.
TABLE-US-00013 TABLE 7 Exemplary Attributes of Purified C3 Fusion
Protein ATTRIBUTE ACCEPTANCE CRITERIA Appearance Clear, colorless
liquid pH (at 25.degree. C.) 6.0-7.0 Protein Concentration 31-37
mg/mL (UV at 280 nm) Identity Compares to reference standard
Isoelectric Point (pl) (sample band is within reference (Capillary
Isoelectric Focusing) .sup.a material pl .+-. 0.1) Identity
Compares to reference standard (SDS-PAGE, Coomassie (major band
migrates between 21 Blue Staining) .sup.a and 31 kDa) Purity and
Impurity .gtoreq.95.0 band-% purity; (SDS-PAGE, Coosmassie
.ltoreq.5.0% band-% total impurities Blue Staining) Purity and
Impurity .gtoreq.95.0% monomer; (SE-HPLC) .ltoreq.5.0% total
impurities Purity and Impurity .gtoreq.85.0% purity; (RP-HPLC)
.ltoreq.15.0% total impurities Purity and Impurity .gtoreq.85.0%
major product; (IE-HPLC) .ltoreq.10.0% total acidic peaks
.ltoreq.5.0% total basic peaks Glycohydrolase 20-40 nmol of
ADP-ribose/ Enzyme Assay (mg protein .times. 30 min) at 37.degree.
C. Residual DNA .sup.a (Real-Time PCR) .ltoreq.1000 pg/mg protein
Host Cell Proteins .sup.a (ELISA) .ltoreq.1000 ng/mg protein
Endotoxin (LAL) .ltoreq.1 EU/mg Bioburden TAMC .ltoreq.1 cfu/10 mL
TYMC .ltoreq.1 cfu/10 mL Osmolality .sup.a Report Results
Deamidation (RPLC-MS) Report Results Methionine oxidation (RPLC-MS)
Report Results Peptide Map .sup.a (RPLC-MS) Report Results (Target
coverage: .gtoreq.95%) Molecular ion .sup.a (RPLC-MS) Report
Results (Target value: molecular weight 25,726 .+-. 2 Da)
Immunochemical homogeneity .sup.a Report Results (Western blot)
(Target value: major band migrates between 21 and 31 kDa)
N-terminal sequencing .sup.a Report Results (Edman degradation)
(Target value: (M)SAYSNTYQEF (SEQ ID NO: 5)) C-terminal sequencing
.sup.a Report Results (MS/MS and Edman degradation) (Target value:
QGRHTPGTRL (SEQ ID NO: 7)) Amino acid analysis .sup.a Report
Results (HCI Hydrolysis) (Target value: 90-110% of expected mass)
Residual PPG-2000 Defoamer .sup.a Report Results [LC-MS] Residual
Isopropyl-.beta.-D- Report Results thiogalatopyranoside (IPTG)
.sup.a (LC-MS) Residual Edetate Disodium, Report Results Dihydrate
.sup.a (CE) Far-UV Circular Dichroism .sup.a Report Results Near-UV
Circular Dichroism .sup.a Report Results FTIR .sup.a Report Results
Intrinsic Fluorescence .sup.a Report Results DSC .sup.a Report
Results Sedimentation velocity analytical Report Results
ultracentrifugation (SV-AUC) .sup.a
[0404] Table 8 below summarizes exemplary attributes of a purified
C3 fusion protein composition suitable as drug product.
Specifically, a protein drug product containing a purified C3
fusion protein is a clear, colorless liquid, essentially free of
visible particulates. It has a pH of 6.0-7.0, with a protein
concentration between 27-33 mg/mL. In addition to the quality
attributes described above (see Table 7), the protein product was
further evaluated for osmolality and sterility. Subvisible
particulate matter was .gtoreq.10 .mu.m: NMT 6000 per container or
.gtoreq.25 .mu.m: NMT 600 per container. Sub 10 .mu.m particulate
matter was measured and categorized as 2-5 .mu.m particulate and
5-10 .mu.m particulate. Container closure integrity was measured by
the Dye immersion test and confirmed no dye detected.
TABLE-US-00014 TABLE 8 Exemplary attributes of a final product
ATTRIBUTE ACCEPTANCE CRITERIA Appearance (Visual) Clear, colorless
liquid, essentially free of visible particulates pH (at 25.degree.
C.) 6.0-7.0 Protein Concentration 27-33 mg/mL (UV at 280 nm)
Identity Compares to reference standard; (SDS-PAGE, Coomassie major
band migrates between 21 Blue Staining) .sup.a and 31 KDa Purity
and Impurity .gtoreq.95.0 band-% purity; (SDS-PAGE, Coomassie
.ltoreq.5.0 % band-% total impurities Blue Staining) Purity and
Impurity (SE-HPLC) .gtoreq.95.0% monomer; .ltoreq.5.0% total
impurities Purity and Impurity (RP-HPLC) .gtoreq.85.0% purity;
.ltoreq.15.0% total impurities Purity and Impurity (IE-HPLC)
.gtoreq.85.0% major product; .ltoreq.10.0% total acidic peaks
.ltoreq.5.0% total basic peaks Glycohydrolase Enzyme 20-40 nmol of
ADP-ribose/ Assay (mg protein .times. 30 min) at 37.degree. C.
Endotoxin (LAL) .ltoreq.1 EU/mg Osmolality .sup.a Report Results
(mOsm/kg) Sterility .sup.a Sterile Subvisible Particulate Matter
.gtoreq.10 .mu.m: NMT 6000 per container .gtoreq.25 .mu.m: NMT 600
per container Sub 10 urn Particulate Matter 2-5 .mu.m: Report
Results 5-10 .mu.m: Report Results Container Closure Integrity No
dye detected (Dye immersion test)
Example 6. Compatibility Evaluation of C3 Fusion Protein and Fibrin
Sealant Components
[0405] This example illustrates exemplary methods, compositions and
results for determining the compatibility and in-use stability of a
C3 fusion protein when mixed with separate fibrin sealant
components.
[0406] The exemplary C3 fusion protein used in these experiments
was SEQ ID NO:1.
[0407] Initial methods comprised pre-mixing a C3 fusion protein at
37.degree. C. with a calcium chloride-thrombin solution.
Specifically, after premixing with thrombin, the product was to be
administered within one hour. The fibrinogen was prepared
separately by reconstituting with a solution containing aprotinin.
Thrombin with a C3 fusion protein was administered simultaneously
with fibrinogen/aprotinin using a Duploject Syringe to create the
fibrin clot at the site of spinal cord injury. Several studies have
been conducted to evaluate the compatibility and in-use stability
of a C3 fusion protein when mixed with the separate fibrin sealant
components using three methods: HPLC-MS, a neurite outgrowth
bioassay, and glycohydrolase enzymatic activity (GH assay).
Assessment of C3 Fusion Protein+Thrombin Compatibility
[0408] When the SDS-PAGE method was used to assess the stability of
a C3 fusion protein in a thrombin solution, a slightly faster
migrating form was observed and, at the time, it was ascribed to
limited digestion by proteolysis. Re-evaluation of this finding
using high-performance liquid chromatography-mass spectrometry
(HPLC-MS) confirmed that in the presence of thrombin there was a
time-dependent proteolysis of the C3 fusion protein. The identified
proteolytic site was located 7 amino acids from the C-terminal end
of the protein and truncated the transport sequence as shown below.
SEQ ID NO: 10 is an exemplary full length transport sequence, while
SEQ ID NO: 11 is a truncated version of the exemplary transport
sequence. The proteolytic site was not within the enzymatic region
of the protein and it did not affect enzymatic activity as
confirmed by the GH assay. The specific activity of the C3 fusion
protein with a truncated transport sequence prepared in-house
retained enzymatic activity similar to that of the full-length C3
fusion protein.
TABLE-US-00015 (SEQ ID NO: 10) EFVMNPANAQGRHTPGTRL (SEQ ID NO: 11)
EFVMNPANAQGR
[0409] An in vitro neurite outgrowth model was used to confirm the
biological activity of the C3 fusion protein and the C3 fusion
protein with truncated transport sequence. A comparison of neurite
outgrowth induced by the full length C3 fusion protein or by the C3
fusion protein with truncated transport sequence was measured as a
function of C3 fusion protein concentration. Dissociated primary
human neurons were treated with either C3 fusion protein or C3
fusion protein with a truncated transport sequence (treated with
thrombin and purified) at the indicated concentrations, fixed and
the average neurite length per cell was measured using automated
software. As shown in FIG. 12, when neurite outgrowth was observed
at 24 and 48 hours after treatment, both the C3 fusion protein and
C3 fusion protein with truncated transport sequence led to an
increase in neurite outgrowth over the negative control. A modest
difference in neurite outgrowth was observed between samples
treated with the lower concentrations of the full length or
truncated C3 fusion protein, while higher concentrations of both
versions of the C3 fusion protein led to similar neurite outgrowth.
Comparable results in neurite outgrowth were confirmed using cells
from the rodent neuronal cell line, NG108 (not shown).
Assessment of C3 Fusion Protein+Fibrin Compatibility
[0410] To limit the effect of proteolysis, while still maintaining
the order of addition used for the Ph1/2a studies, the amount of
time for which the C3 fusion protein and thrombin were combined in
the Duploject Syringe prior to administration could be limited to 1
hour. However, changing the order of addition such that the C3
fusion protein is added to the fibrinogen component in the
Duploject Syringe was also considered. In either scenario, all
components are mixed upon administration in order to form the clot.
However, a change to the order of addition would provide for
greater flexibility in preparation time and control variability
upon application; though some truncation of the transport sequence
would still occur once the fibrin clot is formed. Provided below,
compatibility was established using HPLC-MS, a neurite outgrowth
bioassay and the glycohydrolase enzymatic activity assay.
[0411] In the new preparation schema, the volume of C3 fusion
protein solution (0.3 mL) was accommodated by removing 0.3 mL of
aprotinin solution (from an initial volume of 1 mL) and the full
amount of CaCl.sub.2) was used, approximately 1 mL. This allowed
for the same total volume to be delivered with the Duploject
Syringe at the site of injury. Removal of some aprotinin to
introduce the C3 fusion protein was not expected to have an impact
on the rate of clot formation as this is dependent upon the
concentration of thrombin. Although aprotinin was used to stabilize
the fibrin clot, the removal of 0.3 mL was not expected to
significantly decrease the duration of the fibrin clot, which is
approximately 10-14 days. Studies performed in mice with fibrin
sealant with and without antifibrinolytic agents and reported in
Evicel.RTM. Product Monograph US-2010/08/168 (see, for example,
Summary of Preclinical Findings) showed no appreciable change in
clot longevity.
[0412] The compatibility of the C3 fusion protein with components
of the fibrin sealant was studied using HPLC-MS. Samples were
prepared using representative Ph2b/3 GMP material. As shown in FIG.
13, only when the C3 fusion protein was prepared with thrombin
(diamonds) was truncation of the transport sequence observed.
Truncation was not observed when the C3 fusion protein was prepared
with fibrinogen (triangles).
[0413] The transport sequence attached to the C-terminal region of
the C3 protein enhances penetration of the drug into neurons. In
order to evaluate the cellular activity of the C3 fusion protein
and determine whether or not a difference exists when the C3 fusion
protein is prepared with fibrinogen or thrombin, an in vitro
neurite outgrowth model was used.
[0414] Human cell neurite outgrowth was evaluated using a C3 fusion
protein incubated with thrombin, or a C3 fusion protein incubated
with fibrinogen at 37.degree. C. for 4 hours before being applied
to the cells. Dissociated primary human neurons were grown in the
presence of the various samples for 24 hours, after which they were
fixed and the average neurite length per cell was measured using
automated software. Samples consisting of citrate buffer without C3
fusion protein, thrombin or fibrinogen were prepared as negative
controls. Clinical GMP C3 fusion protein drug product (10 mg/mL)
was combined with either thrombin or fibrinogen to create samples
that were then diluted to a test concentration of 1 .mu.g/mL. The
same lot of C3 fusion drug product was also used as the C3 fusion
protein positive control and was diluted to a concentration of 1
.mu.g/mL.
[0415] As shown in FIG. 14, after 24 hours of incubation, the C3
fusion protein combined with fibrinogen for 4 hours at 37.degree.
C. led to similar levels of neurite outgrowth as treatment with the
C3 fusion protein alone. In comparison, C3 fusion protein combined
with thrombin for 4 hours at 37.degree. C. led to less neurite
outgrowth compared to treatment with the C3 fusion protein
alone.
[0416] In addition to HPLC-MS and a neurite outgrowth bioassay, a
modified GH assay was used to determine and confirm the
compatibility of the C3 fusion protein GMP drug product with
components of the fibrin sealant. The GH assay was modified to use
HPLC-fluorescence, instead of a plate reader, to separate
interfering protein fluorescence. The results shown in Table 9
confirm that C3 fusion protein specific activity was constant over
time in the presence of either the fibrinogen or thrombin sealant
components. In enzymatic assays, the measured absolute rate of
reaction is affected by the conditions of the test. In this case,
the addition of fibrinogen affected the measured activity in the in
vitro enzymatic assay but neither thrombin nor fibrinogen affected
the potency of the drug (enzymatic activity). The reduced specific
activity of the C3 fusion protein measured in the presence of
fibrinogen was attributed to the increased viscosity of the
fibrinogen solutions. The test concentration for the GH assay was
0.3 mg/mL C3 fusion protein; hence, the 10 mg/mL dose contained a
greater concentration of fibrinogen than the 30 mg/mL strength
after dilutions. Therefore, a higher concentration of fibrinogen
interfered more with the assay in the 10 mg/mL strength as depicted
in Table 9. In summary, whether the C3 fusion protein was premixed
with thrombin or fibrinogen, the rate of enzymatic activity
remained constant, demonstrating compatibility of the C3 fusion
protein in the presence of the fibrin components.
TABLE-US-00016 TABLE 9 Effect of Thrombin and Fibrinogen Components
of Fibrin Sealant on C3 Fusion Protein Glycohydrolase Enzyme
Activity Time Point Sample Description and Conditions 0 hours 3
hours 6 hours 24 hours C3 Fusion Protein in thrombin 10 mg/mL C3
fusion protein, 37.degree. C. 28 NT.sup.a 24 28 30 mg/mL C3 fusion
protein, 37.degree. C. 25 NT.sup.a 26 27 C3 Fusion Protein in
fibrinogen 10 mg/mL C3 fusion protein, 37.degree. C. 3.3 2.9.sup.b
4.8 4.7 30 mg/mL C3 fusion protein, 37.degree. C. 16 16 19 19 Units
are nmol eADPr/protein weight/37.degree. C. at 30 minutes .sup.aNot
tested per study design .sup.bTested at 1 hour
Example 7. Formulation of C3-Thrombin-Fibrinogen Composition
[0417] This example provides exemplary methods of preparing a
pharmaceutically acceptable composition comprising a C3 fusion
protein, a fibrinogen composition and a thrombin composition.
C3 Fusion Protein
[0418] Exemplary C3 fusion proteins comprise an amino acid sequence
of a transport domain covalently linked to an amino acid sequence
of an active domain, as described above. The amino acid sequence of
the active domain can be selected from an ADP-ribosyl transferase
C3, a fragment thereof retaining ADP-ribosyl transferase activity,
or an amino acid sequence having at least 80% sequence identity
thereto. The amino acid sequence of the transport domain can be
selected from a subdomain of HIV Tat peptide or antennapedia
peptide, a fragment of Tat peptide or antennapedia peptide, a
Histidine tag, or an amino acid sequence having at least 80%
sequence identity thereto.
[0419] Exemplary C3 fusion protein amino acid sequences are as
follows:
TABLE-US-00017 (SEQ ID NO: 1)
SAYSNTYQEFTNIDQAKAWGNAQYKKYGLSKSEKEAIVSYTKSASEINGK
LRQNKGVINGFPSNLIKQVELLDKSFNKMKTPENIMLFRGDDPAYLGTEF
QNTLLNSNGTINKTAFEKAKAKFLNKDRLEYGYISTSLMNVSQFAGRPII
TKFKVAKGSKAGYIDPISAFAGQLEMLLPRHSTYHIDDMRLSSDGKQIII
TATMMGTAINPKEFVMNPANAQGRHTPGTRL (SEQ ID NO: 2)
MSAYSNTYQEFTNIDQAKAWGNAQYKKYGLSKSEKEAIVSYTKSASEING
KLRQNKGVINGFPSNLIKQVELLDKSFNKMKTPENIMLFRGDDPAYLGTE
FQNTLLNSNGTINKTAFEKAKAKFLNKDRLEYGYISTSLMNVSQFAGRPI
ITKFKVAKGSKAGYIDPISAFAGQLEMLLPRHSTYHIDDMRLSSDGKQII
ITATMMGTAINPKEFVMNPANAQGRHTPGTRL (SEQ ID NO: 8)
SAYSNTYQEFTNIDQAKAWGNAQYKKYGLSKSEKEAIVSYTKSASEINGK
LRQNKGVINGFPSNLIKQVELLDKSFNKMKTPENIMLFRGDDPAYLGTEF
QNTLLNSNGTINKTAFEKAKAKFLNKDRLEYGYISTSLMNVSQFAGRPII
TKFKVAKGSKAGYIDPISAFAGQLEMLLPRHSTYHIDDMRLSSDGKQIII
TATMMGTAINPKEFVMNPANAQGR (SEQ ID NO: 9)
MSAYSNTYQEFTNIDQAKAWGNAQYKKYGLSKSEKEAIVSYTKSASEING
KLRQNKGVINGFPSNLIKQVELLDKSFNKMKTPENIMLFRGDDPAYLGTE
FQNTLLNSNGTINKTAFEKAKAKFLNKDRLEYGYISTSLMNVSQFAGRPI
ITKFKVAKGSKAGYIDPISAFAGQLEMLLPRHSTYHIDDMRLSSDGKQII
ITATMMGTAINPKEFVMNPANAQGR
Fibrinogen Composition
[0420] An exemplary fibrinogen composition comprising a fibrinogen
is a component of a TISSEEL kit. In a TISSEEL kit, the fibrinogen
composition further comprises aprotinin, a fibrinolysis inhibitor.
In these examples, a fibrinogen composition may also be referred to
as "Sealer Protein Concentrate" or "Sealer Protein".
Thrombin Composition
[0421] An exemplary thrombin composition comprising thrombin is a
component of a TISSEEL kit. In a TISSEEL kit, the thrombin
composition further comprises calcium chloride.
Exemplary Formulation Protocol
[0422] The following is an exemplary formulation protocol for
generating a therapeutic C3 fusion protein-thrombin-fibrinogen
composition.
[0423] Exemplary preparation of a C3 fusion
protein-thrombin-fibrinogen composition comprises 4 steps: (1)
pre-warming of TISSEEL kit vials and thawing C3 fusion protein drug
product; (2) thrombin preparation; (3) C3 fusion protein
reconstitution in Sealer Protein Concentrate; and (4) loading of
Thrombin and Sealer Protein+C3 fusion protein in DUPLOJECT syringe
holder.
Pre-Warming of TISSEEL Kit Vials and Thawing of C3 Fusion
Protein/Placebo Drug Product
[0424] FIBRINOTHERM device is turned on and all vials from TISSEEL
kit (Sealer Protein Concentrate, Fibrinolysis Inhibitor Solution,
Thrombin and Calcium Chloride Solution) are placed into the wells
of the FIBRINOTHERM, using the appropriately sized adapter rings.
The vials are allowed to warm up for up to 5 minutes and magnetic
stirring is not turned on at this time. C3 fusion protein drug
product vials are thawed by being held in the palm of the hand for
1 to 2 minutes.
Thrombin Preparation
[0425] Flip-off caps are removed from Thrombin and Calcium Chloride
Solution vials and wiped with a non-iodine based disinfectant.
Packet A of the DUPLOJECT 2 mL/4 mL Fibrin Sealant Preparation and
Application system kit within the TISSEEL kit is opened. One needle
from Packet A is attached to the Black-Scaled sterile syringe also
provided in Packet A. Using this Black-Scaled syringe, the entire
volume from the Black-Capped Calcium Chloride Solution vial is
withdrawn and transferred into the Black-Capped Thrombin vial. The
empty syringe and needle are discarded in a sharps container. Care
is taken not to inject air into the vial.
[0426] Contents of the Thrombin vial are gently swirled (not
inverted) to ensure that the product is completely soaked and then
the vial is returned to an appropriately sized heating well in the
FIBRINOTHERM device. The Thrombin vial is left in the FIBRINOTHERM
device at 37.degree. C. until the solution is ready to be passed
into the sterile field.
C3 Fusion Protein Reconstitution in Sealer Protein Concentrate
[0427] Flip-off caps are removed from the Blue-Capped Sealer
Protein Concentrate and Fibrinolysis Inhibitor Solution vials and
wiped with a non-iodine based disinfectant. C3 fusion protein drug
product (or placebo) vial is wiped with a non-iodine based
disinfectant. One needle from Packet A of the DUPLOJECT 2 mL/4 mL
Fibrin Sealant Preparation and Application system kit within the
TISSEEL kit is attached to the Blue-Scaled sterile syringe also
provided in Packet A. Using this Blue-Scaled syringe, 0.3 mL is
withdrawn from the Blue-Capped Fibrinolysis Inhibitor Solution vial
and discarded in a waste container.
[0428] Packet A of the additional DUPLOJECT 2 mL/4 mL Fibrin
Sealant Preparation and Application system kit is opened and one
needle from this Packet A is attached to the Blue-Scaled syringe
from this Packet A. Using this Blue-Scaled syringe, 0.3 mL is drawn
from aluminum-capped C3 fusion protein drug product (or placebo)
and injected into Blue-Capped Fibrinolysis Inhibitor Solution vial.
C3 fusion protein drug product (or placebo) and Fibrinolysis
Inhibitor Solution are mixed by swirling the vial gently.
[0429] Packet B of the additional DUPLOJECT 2 mL/4 mL Fibrin
Sealant Preparation and Application system kit is opened and one
needle from this Packet B is attached to the Blue-Scaled syringe
from the Packet. Using this Blue-Scaled syringe, the entire volume
is withdrawn from the Blue-Capped Fibrinolysis Inhibitor Solution
vial (C3 fusion protein drug product (or placebo)+Fibrinolysis
Inhibitor Solution), with the vial tilted slightly to facilitate
removal of the entire solution. The C3 fusion protein drug product
(or placebo)+Fibrinolysis Inhibitor Solution is injected into the
Blue-Capped Sealer Protein Concentrate vial. The vial is then
swirled (not inverted) to ensure that the product is completely
soaked. The empty syringe and needle are discarded in a sharps
container. Care is taken not to inject air into the vial.
[0430] The Blue-Capped Sealer Protein+C3 fusion protein (or
placebo) vial is placed into the largest opening of the
FIBRINOTHERM device with the appropriate adaptor. Magnetic stirring
by the FIBRINOTHERM device is then turned on. The contents of the
vial are allowed to stir until all the Sealer Protein is dissolved.
Sealer Protein is effectively reconstituted once there were no
visible clumps of the powder. At this time, the magnetic stirring
by the FIBRINOTHERM device is turned off. If Sealer Protein does
not dissolve within 20 minutes, the vial is discarded and a new
TISSEEL kit and a new vial of C3 fusion protein (or placebo) are
used. The Sealer protein solution is kept in the FIBRINOTHERM
device (at 37.degree. C.) without stirring until the solution is
ready to be passed onto the sterile field. The solution is stirred
shortly before being drawn up to ensure homogeneity.
Loading of Thrombin and Sealer Protein+C3 Fusion Protein (or
Placebo) in DUPLOJECT Syringe Holder
[0431] Solutions are loaded into DUPLOJECT according to the
manufacturer's instructions by a Circulating Nurse and Scrub Nurse
or designees. A brief description of the preparation procedure is
included below.
[0432] Packet B of the DUPLOJECT 2 mL/4 mL Fibrin Sealant
Preparation and Application system kit within the TISSEEL kit is
opened into the sterile field. Within Packet B, Packets 1, 2, and 3
are opened. The Blue-Scaled and Black-Scaled syringes from Packet 1
of Packet B are assembled with the needles provided in Packet 1 of
Packet B. While the Circulating Nurse or designee holds the
Blue-Capped Sealer Protein+C3 fusion protein (or placebo) vial
slightly tilted, Scrub Nurse or designee inserts the needle
attached to the Blue-Scaled syringe from Packet 1 of Packet B into
the Blue-Capped Sealer Protein+C3 fusion protein (or placebo) vial
(bevel side down) and withdraws all of the Sealer Protein+C3 fusion
protein (or placebo) Solution using firm, constant aspiration.
Needle is discarded in sharps container. While the Circulating
Nurse or designee holds the Black-Capped Thrombin Solution vial
slightly tilted, Scrub Nurse inserts needle attached to
Black-Scaled syringe from Packet 1 of Packet B into vial (bevel
side down) and withdraws all of the Thrombin Solution using firm,
constant aspiration. Needle is discarded in sharps container. Air
bubbles are removed by striking the side of each syringe 1 or 2
times while keeping the tip in an upright position. It is then
ensured that both syringes contain the same volume. The filled
syringes (Blue-Scaled syringe and Black-Scaled Syringe) are snapped
into the 2-syringe clip from Packet 2 of Packet B with the flanges
in an up/down position (referring to the illustration in the
instructions provided in TISSEEL kit). The joining piece from
Packet 3 of Packet B is attached to the syringe nozzles, ensuring
that both were firmly seated. The joining piece is secured by
fastening the retaining strap to the double syringe clip. The
syringe nozzles are aligned toward the middle for proper alignment.
One of the application needles from Packet 3 of Packet B is fitted
onto the joining piece. The needles are not primed because
premature mixing will lead to blockage. The DUPLOJECT applicator is
then ready for use.
[0433] Preparation of the C3 fusion protein (or placebo)+TISSEEL 2
mL kit is done within 4 hours prior to application. The preparation
time begins when the C3 fusion protein (or placebo) is withdrawn
from the freezer.
EQUIVALENTS
[0434] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. The scope of the present invention is not intended to be
limited to the above Description, but rather is as set forth in the
following claims.
[0435] Use of ordinal terms such as "first," "second," "third,"
etc., in the claims to modify a claim element does not by itself
connote any priority, precedence, or order of one claim element
over another or the temporal order in which acts of a method are
performed, but are used merely as labels to distinguish one claim
element having a certain name from another element having a same
name (but for use of the ordinal term) to distinguish the claim
elements.
[0436] The articles "a" and "an" as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to include the plural referents.
Claims or descriptions that include "or" between one or more
members of a group are considered satisfied if one, more than one,
or all of the group members are present in, employed in, or
otherwise relevant to a given product or process unless indicated
to the contrary or otherwise evident from the context. The
invention includes embodiments in which exactly one member of the
group is present in, employed in, or otherwise relevant to a given
product or process. The invention also includes embodiments in
which more than one, or the entire group members are present in,
employed in, or otherwise relevant to a given product or process.
Furthermore, it is to be understood that the invention encompasses
all variations, combinations, and permutations in which one or more
limitations, elements, clauses, descriptive terms, etc., from one
or more of the listed claims is introduced into another claim
dependent on the same base claim (or, as relevant, any other claim)
unless otherwise indicated or unless it would be evident to one of
ordinary skill in the art that a contradiction or inconsistency
would arise. Where elements are presented as lists, (e.g., in
Markush group or similar format) it is to be understood that each
subgroup of the elements is also disclosed, and any element(s) can
be removed from the group. It should be understood that, in
general, where the invention, or aspects of the invention, is/are
referred to as comprising particular elements, features, etc.,
certain embodiments of the invention or aspects of the invention
consist, or consist essentially of, such elements, features, etc.
For purposes of simplicity those embodiments have not in every case
been specifically set forth in so many words herein. It should also
be understood that any embodiment or aspect of the invention can be
explicitly excluded from the claims, regardless of whether the
specific exclusion is recited in the specification. The
publications, websites and other reference materials referenced
herein to describe the background of the invention and to provide
additional detail regarding its practice are hereby incorporated by
reference.
Sequence CWU 1
1
281231PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 1Ser Ala Tyr Ser Asn Thr Tyr Gln
Glu Phe Thr Asn Ile Asp Gln Ala1 5 10 15Lys Ala Trp Gly Asn Ala Gln
Tyr Lys Lys Tyr Gly Leu Ser Lys Ser 20 25 30Glu Lys Glu Ala Ile Val
Ser Tyr Thr Lys Ser Ala Ser Glu Ile Asn 35 40 45Gly Lys Leu Arg Gln
Asn Lys Gly Val Ile Asn Gly Phe Pro Ser Asn 50 55 60Leu Ile Lys Gln
Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met Lys65 70 75 80Thr Pro
Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr Leu 85 90 95Gly
Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile Asn 100 105
110Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp Arg
115 120 125Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser
Gln Phe 130 135 140Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val Ala
Lys Gly Ser Lys145 150 155 160Ala Gly Tyr Ile Asp Pro Ile Ser Ala
Phe Ala Gly Gln Leu Glu Met 165 170 175Leu Leu Pro Arg His Ser Thr
Tyr His Ile Asp Asp Met Arg Leu Ser 180 185 190Ser Asp Gly Lys Gln
Ile Ile Ile Thr Ala Thr Met Met Gly Thr Ala 195 200 205Ile Asn Pro
Lys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly Arg 210 215 220His
Thr Pro Gly Thr Arg Leu225 2302232PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 2Met Ser Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn
Ile Asp Gln1 5 10 15Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr
Gly Leu Ser Lys 20 25 30Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys
Ser Ala Ser Glu Ile 35 40 45Asn Gly Lys Leu Arg Gln Asn Lys Gly Val
Ile Asn Gly Phe Pro Ser 50 55 60Asn Leu Ile Lys Gln Val Glu Leu Leu
Asp Lys Ser Phe Asn Lys Met65 70 75 80Lys Thr Pro Glu Asn Ile Met
Leu Phe Arg Gly Asp Asp Pro Ala Tyr 85 90 95Leu Gly Thr Glu Phe Gln
Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 100 105 110Asn Lys Thr Ala
Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 115 120 125Arg Leu
Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln 130 135
140Phe Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val Ala Lys Gly
Ser145 150 155 160Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Ala
Gly Gln Leu Glu 165 170 175Met Leu Leu Pro Arg His Ser Thr Tyr His
Ile Asp Asp Met Arg Leu 180 185 190Ser Ser Asp Gly Lys Gln Ile Ile
Ile Thr Ala Thr Met Met Gly Thr 195 200 205Ala Ile Asn Pro Lys Glu
Phe Val Met Asn Pro Ala Asn Ala Gln Gly 210 215 220Arg His Thr Pro
Gly Thr Arg Leu225 2303699DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 3atgtcggctt attcaaatac ttaccaggag tttactaata
ttgatcaagc aaaagcttgg 60ggtaatgctc agtataaaaa gtatggacta agcaaatcag
aaaaagaagc tatagtatca 120tatactaaaa gcgctagtga aataaatgga
aagctaagac aaaataaggg agttatcaat 180ggatttcctt caaatttaat
aaaacaagtt gaacttttag ataaatcttt taataaaatg 240aagacccctg
aaaatattat gttatttaga ggcgacgacc ctgcttattt aggaacagaa
300tttcaaaaca ctcttcttaa ttcaaatggt acaattaata aaacggcttt
tgaaaaggct 360aaagctaagt ttttaaataa agatagactt gaatatggat
atattagtac ttcattaatg 420aatgtttctc aatttgcagg aagaccaatt
attacaaaat ttaaagtagc aaaaggctca 480aaggcaggat atattgaccc
tattagtgct tttgcaggac aacttgaaat gttgcttcct 540agacatagta
cttatcatat agacgatatg agattgtctt ctgatggtaa acaaataata
600attacagcaa caatgatggg cacagctatc aatcctaaag aattcgtgat
gaatcccgca 660aacgcgcaag gcagacatac acccggtacc agactctag
699418PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 4Phe Val Met Asn Pro Ala Asn Ala Gln
Gly Arg His Thr Pro Gly Thr1 5 10 15Arg Leu511PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"VARIANT(1)..(1)/replace="
"MISC_FEATURE(1)..(11)/note="Variant residues given in the sequence
have no preference with respect to those in the annotations for
variant positions" 5Met Ser Ala Tyr Ser Asn Thr Tyr Gln Glu Phe1 5
10617PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide"VARIANT(1)..(1)/replace="
"MISC_FEATURE(1)..(17)/note="Variant residues given in the sequence
have no preference with respect to those in the annotations for
variant positions" 6Met Ser Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr
Asn Ile Asp Gln1 5 10 15Ala710PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 7Gln Gly Arg His Thr Pro Gly Thr Arg Leu1 5
108224PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 8Ser Ala Tyr Ser Asn Thr Tyr Gln
Glu Phe Thr Asn Ile Asp Gln Ala1 5 10 15Lys Ala Trp Gly Asn Ala Gln
Tyr Lys Lys Tyr Gly Leu Ser Lys Ser 20 25 30Glu Lys Glu Ala Ile Val
Ser Tyr Thr Lys Ser Ala Ser Glu Ile Asn 35 40 45Gly Lys Leu Arg Gln
Asn Lys Gly Val Ile Asn Gly Phe Pro Ser Asn 50 55 60Leu Ile Lys Gln
Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met Lys65 70 75 80Thr Pro
Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr Leu 85 90 95Gly
Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile Asn 100 105
110Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp Arg
115 120 125Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser
Gln Phe 130 135 140Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val Ala
Lys Gly Ser Lys145 150 155 160Ala Gly Tyr Ile Asp Pro Ile Ser Ala
Phe Ala Gly Gln Leu Glu Met 165 170 175Leu Leu Pro Arg His Ser Thr
Tyr His Ile Asp Asp Met Arg Leu Ser 180 185 190Ser Asp Gly Lys Gln
Ile Ile Ile Thr Ala Thr Met Met Gly Thr Ala 195 200 205Ile Asn Pro
Lys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly Arg 210 215
2209225PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 9Met Ser Ala Tyr Ser Asn Thr Tyr
Gln Glu Phe Thr Asn Ile Asp Gln1 5 10 15Ala Lys Ala Trp Gly Asn Ala
Gln Tyr Lys Lys Tyr Gly Leu Ser Lys 20 25 30Ser Glu Lys Glu Ala Ile
Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile 35 40 45Asn Gly Lys Leu Arg
Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser 50 55 60Asn Leu Ile Lys
Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met65 70 75 80Lys Thr
Pro Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 85 90 95Leu
Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 100 105
110Asn Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp
115 120 125Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val
Ser Gln 130 135 140Phe Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val
Ala Lys Gly Ser145 150 155 160Lys Ala Gly Tyr Ile Asp Pro Ile Ser
Ala Phe Ala Gly Gln Leu Glu 165 170 175Met Leu Leu Pro Arg His Ser
Thr Tyr His Ile Asp Asp Met Arg Leu 180 185 190Ser Ser Asp Gly Lys
Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr 195 200 205Ala Ile Asn
Pro Lys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly 210 215
220Arg2251019PRTUnknownsource/note="Description of Unknown
Transport domain" 10Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly Arg
His Thr Pro Gly1 5 10 15Thr Arg
Leu1112PRTUnknownsource/note="Description of Unknown Transport
domain" 11Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly Arg1 5
1012251PRTClostridium botulinum 12Met Lys Gly Leu Arg Lys Ser Ile
Leu Cys Leu Val Leu Ser Ala Gly1 5 10 15Val Ile Ala Pro Val Thr Ser
Gly Met Ile Gln Ser Pro Gln Lys Cys 20 25 30Tyr Ala Tyr Ser Ile Asn
Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu 35 40 45Phe Thr Asn Ile Asp
Gln Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys 50 55 60Lys Tyr Gly Leu
Ser Lys Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr65 70 75 80Lys Ser
Ala Ser Glu Ile Asn Gly Lys Leu Arg Gln Asn Lys Gly Val 85 90 95Ile
Asn Gly Phe Pro Ser Asn Leu Ile Lys Gln Val Glu Leu Leu Asp 100 105
110Lys Ser Phe Asn Lys Met Lys Thr Pro Glu Asn Ile Met Leu Phe Arg
115 120 125Gly Asp Asp Pro Ala Tyr Leu Gly Thr Glu Phe Gln Asn Thr
Leu Leu 130 135 140Asn Ser Asn Gly Thr Ile Asn Lys Thr Ala Phe Glu
Lys Ala Lys Ala145 150 155 160Lys Phe Leu Asn Lys Asp Arg Leu Glu
Tyr Gly Tyr Ile Ser Thr Ser 165 170 175Leu Met Asn Val Ser Gln Phe
Ala Gly Arg Pro Ile Ile Thr Lys Phe 180 185 190Lys Val Ala Lys Gly
Ser Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala 195 200 205Phe Ala Gly
Gln Leu Glu Met Leu Leu Pro Arg His Ser Thr Tyr His 210 215 220Ile
Asp Asp Met Arg Leu Ser Ser Asp Gly Lys Gln Ile Ile Ile Thr225 230
235 240Ala Thr Met Met Gly Thr Ala Ile Asn Pro Lys 245
25013212PRTClostridium botulinum 13Ser Ala Tyr Ser Asn Thr Tyr Gln
Glu Phe Thr Asn Ile Asp Gln Ala1 5 10 15Lys Ala Trp Gly Asn Ala Gln
Tyr Lys Lys Tyr Gly Leu Ser Lys Ser 20 25 30Glu Lys Glu Ala Ile Val
Ser Tyr Thr Lys Ser Ala Ser Glu Ile Asn 35 40 45Gly Lys Leu Arg Gln
Asn Lys Gly Val Ile Asn Gly Phe Pro Ser Asn 50 55 60Leu Ile Lys Gln
Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met Lys65 70 75 80Thr Pro
Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr Leu 85 90 95Gly
Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile Asn 100 105
110Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp Arg
115 120 125Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser
Gln Phe 130 135 140Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val Ala
Lys Gly Ser Lys145 150 155 160Ala Gly Tyr Ile Asp Pro Ile Ser Ala
Phe Ala Gly Gln Leu Glu Met 165 170 175Leu Leu Pro Arg His Ser Thr
Tyr His Ile Asp Asp Met Arg Leu Ser 180 185 190Ser Asp Gly Lys Gln
Ile Ile Ile Thr Ala Thr Met Met Gly Thr Ala 195 200 205Ile Asn Pro
Lys 21014213PRTUnknownsource/note="Description of Unknown Active
domain sequence" 14Met Ser Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr
Asn Ile Asp Gln1 5 10 15Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys
Tyr Gly Leu Ser Lys 20 25 30Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr
Lys Ser Ala Ser Glu Ile 35 40 45Asn Gly Lys Leu Arg Gln Asn Lys Gly
Val Ile Asn Gly Phe Pro Ser 50 55 60Asn Leu Ile Lys Gln Val Glu Leu
Leu Asp Lys Ser Phe Asn Lys Met65 70 75 80Lys Thr Pro Glu Asn Ile
Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 85 90 95Leu Gly Thr Glu Phe
Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 100 105 110Asn Lys Thr
Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 115 120 125Arg
Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln 130 135
140Phe Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val Ala Lys Gly
Ser145 150 155 160Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Ala
Gly Gln Leu Glu 165 170 175Met Leu Leu Pro Arg His Ser Thr Tyr His
Ile Asp Asp Met Arg Leu 180 185 190Ser Ser Asp Gly Lys Gln Ile Ile
Ile Thr Ala Thr Met Met Gly Thr 195 200 205Ala Ile Asn Pro Lys
2101517PRTUnknownsource/note="Description of Unknown Antennapedia
Leader Peptide" 15Lys Lys Trp Lys Met Arg Arg Asn Gln Phe Trp Val
Lys Val Gln Arg1 5 10 15Gly1616PRTDrosophila sp. 16Arg Gln Ile Lys
Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys1 5 10
151713PRTHuman immunodeficiency virus 17Gly Arg Lys Lys Arg Arg Gln
Arg Arg Arg Pro Pro Gln1 5 101811PRTHuman immunodeficiency virus
18Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5 101910PRTHuman
immunodeficiency virus 19Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5
102031PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 20Tyr Gly Arg Lys Lys Arg Arg Gln
Arg Arg Arg Gly Gly Thr Asn Val1 5 10 15Phe Asn Ala Thr Phe Glu Ile
Trp His Asp Gly Glu Phe Gly Thr 20 25 302111PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 21Cys Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5
102220PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 22Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Ala Lys Glu Gly Ala1 5 10 15Asn Val Ala Gly
202320PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 23Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Tyr Lys Glu Gly Tyr1 5 10 15Asn Val Tyr Gly
202430PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 24Arg Arg Arg Gln Arg Arg Lys Lys
Arg Gly Gly Asp Ile Met Gly Glu1 5 10 15Trp Gly Asn Glu Ile Phe Gly
Ala Ile Ala Gly Phe Leu Gly 20 25 302520PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 25Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Lys Leu Ser
Ser Ile1 5 10 15Glu Ser Asp Val 202635PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 26Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly
Gly Leu Asp1 5 10 15Lys Glu Phe Asn Ser Ile Phe Arg Arg Ala Phe Ala
Ser Arg Val Phe 20 25 30Pro Pro Glu 352736PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 27Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly
Gly Leu Leu1 5 10 15Asp Tyr Val Pro Ile Gly Pro Arg Phe Ser Asn Leu
Val Leu Gln Ala 20 25 30Leu Leu Val Leu 352835PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 28Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly
Gly Ile Pro1 5
10 15Pro Val Tyr Phe Ser Arg Leu Asp Leu Asn Leu Val Val Leu Leu
Leu 20 25 30Ala Gln Leu 35
* * * * *