NZ620913B2 - Method for the preparation of a heat stable oxygen carrier-containing composition facilitating beta-beta cross-linking - Google Patents
Method for the preparation of a heat stable oxygen carrier-containing composition facilitating beta-beta cross-linking Download PDFInfo
- Publication number
- NZ620913B2 NZ620913B2 NZ620913A NZ62091312A NZ620913B2 NZ 620913 B2 NZ620913 B2 NZ 620913B2 NZ 620913 A NZ620913 A NZ 620913A NZ 62091312 A NZ62091312 A NZ 62091312A NZ 620913 B2 NZ620913 B2 NZ 620913B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- haemoglobin
- crosslinked
- hemoglobin
- red blood
- beta
- Prior art date
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Classifications
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- A61K38/41—Porphyrin- or corrin-ring-containing peptides
- A61K38/42—Haemoglobins; Myoglobins
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- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
Abstract
in vitro method for the preparation of a highly purified and heat stable oxygen carrier containing pharmaceutical composition is disclosed. The oxygen carrier-containing pharmaceutical composition including haemoglobin and the haemoglobin consisting essentially of non-polymeric crosslinked tetrameric haemoglobin having a beta-beta cross-linking of greater than 40% preferably greater than 50%. The method comprises a) providing a sample of mammalian whole blood including at least red blood cells and plasma. The red blood cells are then separated from the plasma in the mammalian whole blood and the red blood cells that were separated from the plasma are filtered to obtain a filtered red blood cell fraction. The filtered red blood cell fraction is washed to remove plasma protein impurities, resulting in washed red blood cells. The washed red blood cells is disrupted to create a solution comprising a lysate of disrupted red blood cells and a filtration is performed filtration to remove at least a portion of the waste retentate from the lysate. A first haemoglobin solution is extracted from the lysate and at least one purification process is performed to remove one or more of viruses, waste retentate, or protein impurities. The first haemoglobin solution is cross-linked by bis-3,5-dibromosalicyl fumarate to form crosslinked haemoglobin in an oxygenated environment. The crosslinked haemoglobin is non-polymeric crosslinked tetrameric haemoglobin having at least 40% beta-beta cross-linking. Any residual chemicals are removed from the solution and the crosslinked haemoglobin are heat treated in a deoxygenated environment to denature and precipitate any residual non-stabilized/ non-crosslinked haemoglobin, any dimeric haemoglobin and any other protein impurities such that the resulting heat stable crosslinked tetrameric haemoglobin has an undetectable concentration of dimer and consists essentially of nonpolymeric crosslinked tetrameric haemoglobin with a beta-beta cross-linking of at least 40% and an oxygen affinity greater than the oxygen affinity of native haemoglobin of the same species measured under substantially similar conditions. Optionally the resulting heat stable crosslinked haemoglobin can be cooled and N-acetyl-cysteine can be added to the cooled crosslinked haemoglobin. The precipitate is removed by a centrifugation or a filtration to form a clear solution; and the purified and heat stable crosslinked tetrameric haemoglobin is added to a pharmaceutically acceptable carrier. meric haemoglobin having a beta-beta cross-linking of greater than 40% preferably greater than 50%. The method comprises a) providing a sample of mammalian whole blood including at least red blood cells and plasma. The red blood cells are then separated from the plasma in the mammalian whole blood and the red blood cells that were separated from the plasma are filtered to obtain a filtered red blood cell fraction. The filtered red blood cell fraction is washed to remove plasma protein impurities, resulting in washed red blood cells. The washed red blood cells is disrupted to create a solution comprising a lysate of disrupted red blood cells and a filtration is performed filtration to remove at least a portion of the waste retentate from the lysate. A first haemoglobin solution is extracted from the lysate and at least one purification process is performed to remove one or more of viruses, waste retentate, or protein impurities. The first haemoglobin solution is cross-linked by bis-3,5-dibromosalicyl fumarate to form crosslinked haemoglobin in an oxygenated environment. The crosslinked haemoglobin is non-polymeric crosslinked tetrameric haemoglobin having at least 40% beta-beta cross-linking. Any residual chemicals are removed from the solution and the crosslinked haemoglobin are heat treated in a deoxygenated environment to denature and precipitate any residual non-stabilized/ non-crosslinked haemoglobin, any dimeric haemoglobin and any other protein impurities such that the resulting heat stable crosslinked tetrameric haemoglobin has an undetectable concentration of dimer and consists essentially of nonpolymeric crosslinked tetrameric haemoglobin with a beta-beta cross-linking of at least 40% and an oxygen affinity greater than the oxygen affinity of native haemoglobin of the same species measured under substantially similar conditions. Optionally the resulting heat stable crosslinked haemoglobin can be cooled and N-acetyl-cysteine can be added to the cooled crosslinked haemoglobin. The precipitate is removed by a centrifugation or a filtration to form a clear solution; and the purified and heat stable crosslinked tetrameric haemoglobin is added to a pharmaceutically acceptable carrier.
Description
METHOD FOR THE PREPARATION OF A HEAT STABLE OXYGEN CARRIER-
CONTAINING COMPOSITION FACILITATING BETA-BETA CROSS-LINKING
ght Notice/Permission
A portion of the disclosure of this patent document contains material which is subject to
copyright protection. The copyright owner has no objection to the facsimile uction by
anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark
Office patent file or records, but otherwise reserves all copyright rights whatsoever. The
following notice applies to the processes, experiments, and data as described below and in the
drawings attached hereto: Copyright © 2011, Billion King International Limited, All Rights
Reserved.
Cross-Reference to Related Applications:
The t application is an International Patent Application claims priority from a
provisional U.S. Patent Application No. 61/529,279 filed August 31, 2011, a U.S. Continuation-
in—Part Application No. 13/225,797 filed September 6, 2011 and a U.S. Continuation-in—Pait
Application No. 13/275,366 filed on October 18, 2011, now Patent U88,106,011, the disclosure
of which are hereby inc01porated by reference in their entirety.
cal Field
The present invention relates to methods for the preparation of a heat stable oxygen—
carrier—containing pharmaceutical composition such that eta cross—linking is favored.
Using the methods of the present invention, the oxygen affinity of the resulting molecule can be
controlled so that obin based oxygen rs tailored for specific applications can be
produced. Lower oxygen affinity crosslinked hemoglobin is useful for applications requiring
rapid tissue oxygenation (e. g. hemorrhagic shock) while higher oxygen affinity crosslinlced
hemoglobin is useful for applications requiring a slower rate of oxygenation (e.g. cancer adjunct
therapy).
ound of Invention
Hemoglobin plays an important role in most vertebrates for gaseous exchange between
the vascular system and tissue. It is responsible for carrying oxygen from the respiratory system
to the body cells via blood circulation and also carrying the metabolic waste product carbon
dioxide away from body cells to the atory system, where the carbon dioxide is d.
Since obin has this oxygen transport feature, it can be used as a potent oxygen supplier if
it can be stabilized ex vivo and used in viva.
Naturally-occurring hemoglobin is a er which is generally stable when present
within red blood cells. However, when naturally-occurring hemoglobin is removed from red
blood cells, it becomes unstable in plasma and splits into two oc-B dimers. Each ofthese dimers is
approximately 32 kDa in molecular weight. These dimers may cause substantial renal injury
when filtered through the kidneys and excreted. The breakdown of the tetramer linkage also
negatively s the nability of the functional hemoglobin in circulation.
In order to solve the problem, recent developments in obin processing have
incorporated various cross-linking techniques to create intramolecular bonds within the tetramer
as well as intermolecular bonds between the tetramers to form polymeric hemoglobin. The prior
art teaches that polymeric hemoglobin is the preferred form in order to se circulatory half—
life of the hemoglobin. However, as determined by the present inventors, polymeric hemoglobin
more readily converts to met-hemoglobin in blood circulation. Met-hemoglobin cannot bind
oxygen and therefore cannot oxygenate . Therefore, the cross-linking taught by the prior
art that causes the formation of polymeric hemoglobin is a problem. There is a need in the art for
PCT/U82012/051959
a technique that permits intramolecular cross-linking to create stable tetramers Without the
simultaneous formation of polymeric hemoglobin.
Further problems with the prior art attempts to stabilize hemoglobin include production
of tetrameric hemoglobin that includes an unacceptably high percentage of dimer units (or non-
crosslinked tetrameric obin that quickly dissociates to dimeric hemoglobin if
administered to a patient); the presence of dimers makes the hemoglobin composition
unsatisfactory for administration to mammals. The dimeric form of the hemoglobin can cause
severe renal injury in a mammalian body; this renal injury can be severe enough to cause death.
Therefore, there is a need in the art to create stable tetrameric hemoglobin with ctable
dimeric form in the final product.
Another problem with prior art hemoglobin products is a sudden increase in blood
pressure ing administration. In the past, nstriction events have been recorded from
older generation of hemoglobin based oxygen carriers. Thus there is a need in the art for a
process to prepare obin which will not cause vasoconstriction and high blood pressure
when applied to a mammal.
Further problems with prior art attempts to create stable hemoglobin include the ce
of n impurities such as immunoglobin G that can cause allergic effects in mammals.
Therefore, there is a need in the art for a process which can e stable tetrameric
hemoglobin Without protein impurities.
In addition to the above ms, there is a need in the art for stabilized tetrameric
hemoglobin that is dimer free, phospholipid free and capable of production on an industrial scale.
Hemoglobin—based oxygen carriers can be used in a wide variety of medical applications;
depending upon the l application, different levels of oxygen affinity are desirable. For
example, a hemoglobin le with low oxygen affinity can transfer oxygen more easily to a
2012/051959
‘ target tissue than a hemoglobin molecule with higher oxygen affinity. Therefore it would be
desirable to control the oxygen affinity of the crosslinked tetrameric hemoglobin. Thus, there is
a need in the art to control the type of linking and cross-linking conditions to produce
crosslinked tetrameric hemoglobin with precise levels of oxygen binding.
Summary of Invention
The present invention provides a method for processing a non—polymeric, heat stable
purified crosslinked tetrameric hemoglobin suitable for use in mammals t causing severe
renal injury, vascular detrimental effects and severe adverse events including death. The present
invention removes the dimeric form of hemoglobin, non-crosslinked tetrameric hemoglobin,
phospholipids and protein impurities. The present ion also provides a technique for
controlling the oxygen affinity of the ant inked tetramer by controlling the type of
cross-linking in the tetramer (e.g., the amount of beta-beta cross-linking, alpha-alpha cross-
linking, alpha-beta cross-linking in the tetramer), the quaternary structure of tetramer, and the
cross-linking ions. Lower oxygen affinity crosslinked hemoglobin is useful for
applications ing rapid tissue oxygenation (e.g. hemorrhagic shock) While higher oxygen
affinity crosslinked hemoglobin is useful for ations requiring a slower rate of oxygenation
(e.g. cancer adjunct therapy). Additionally, the present invention uses (1) an instant cytolysis
apparatus for e and controlled hypotonic lysis, (2) a flowthrough column chromatography,
(3) a high temperature short time (HTST) apparatus for heat processing the hemoglobin solution
in the purification process to remove the undesirable non-stabilized dimers of hemoglobin and to
remove the protein impurities, for example immunoglobin-G, so that renal injury, vascular
detrimental effects and other ty reactions can be avoided, and (4) an air-tight infusion bag
ing to avoid oxygen intrusion into the final product.
PCT/U82012/051959
The method includes a starting material of mammalian whole blood including at least red
blood cells and plasma. Red blood cells are separated from the plasma in the mammalian whole
blood followed by filtering to obtain a filtered red blood cell fraction. The filtered red blood cell
fraction is washed to remove plasma protein impurities. The washed red blood cells are disrupted
by a controlled hypotonic lysis for a time sufficient to lyse red blood cells without lysing white
blood cells in an instant cytolysis apparatus. Filtration is med to remove at least a portion
of the waste retentate from the lysate. A first hemoglobin solution is extracted from the .
One or more purification processes are performed on the hemoglobin solution such as
ultrafiltration and/or chromatography.
The purified hemoglobin is crosslinked by bis-3,5-dibromosalicyl fumarate (DBSF) to
form heat stable crosslinked hemoglobin without the formation of polymeric hemoglobin such
that the molecular weight of the resultant non—polymeric crosslinked tetrameric hemoglobin is
60—70 kDa. The expression “non-polymeric” as used herein, refers to tetrameric hemoglobin
that is not intermolecularly crosslinked with other hemoglobin molecules or any other non-
hemoglobin les such as PEG. Depending upon the hemoglobin source, the quaternary
structure of the obin and on the cross-linking conditions, a tetrameric product with a high
percentage of beta-beta cross-linking can be produced. Further, the oxygen y of the
resultant le can be lled so that hemoglobin based oxygen carriers tailored for
c applications can be produced.
Following this procedure, the crosslinked hemoglobin is heat-treated to remove any
residual non-crosslinked tetrameric hemoglobin and any non-stabilized hemoglobin, for e
the dimeric form of hemoglobin, and any other protein impurities. Prior to the heat treatment N-
acetyl cysteine is optionally added at a tration of imately 0.2% to the crosslinked
tetrameric obin to prevent formation of met-hemoglobin. Immediately following heat
PCT/U82012/051959
treatment and cooling, N-acetyl cysteine is optionally added at a concentration of approximately
0.025% to 0.4% to further prevent formation of met-hemoglobin. The heat treatment is
preferably a high temperature short time treatment conducted at approximately 70°C to 95°C for
s to 3 hours With subsequent cooling to 25°C. Any precipitates formed during the heat
treatment are d by centrifugation or filtration.
The free, phospholipid—free, n ties—free, heat stable, non-polymeric
crosslinked eric hemoglobin is then added to a pharmaceutically acceptable carrier.
Thereafter, the heat stable, crosslinked tetrameric hemoglobin is formulated and
packaged in a —made and air-tight polyethylene, ethylene-vinyl—acetate, and ethylene-
Vinyl alcohol (PE, EVA, EVOH) infusion bag. The packaging prevents oxygen contamination
which results in the formation of inactive met—hemoglobin.
Brief Description of the Drawings
is a flow-chart depicting an overview of a process of the present invention.
schematically depicts an instant cytolysis apparatus used in the process of the
present ion.
depicts high performance liquid chromatography analysis for (a) non-heat treated
crosslinked tetrameric hemoglobin, and (b) heat stable inked tetrameric hemoglobin which
has undergone a heat treatment at 90°C for 45 seconds to 2 minutes or 80°C for 30 minutes.
is an elution profile for flowthrough column chromatography; the hemoglobin
solution is in the flowthrough fraction.
schematically depicts a flowthrough CM column chromatography system with
ultrafiltration for an rial scale operation.
is a schematic ion of an apparatus used for HTST heat processing step.
demonstrates the temperature profile in the HTST processing apparatus and the
time taken to remove unstabilized tetramer (dimer) in the system at 85°C and 90°C of the present
invention.
is a schematic depiction of an infusion bag for the heat stable crosslinked
tetrameric hemoglobin of the present invention.
depicts reverse phase HPLC chromatogram of on and [3 globin chains of bovine
hemoglobin before (dashed line) and after (solid line) reaction with DBSF under deoxygenated
environment.
depicts 15% SDS-PAGE analysis of (A) native bovine hemoglobin and (B)
hemoglobin crosslinked with DBSF under deoxygenated condition.
FIG. ll depicts the peptide mass fingerprint of n-digested peptides from the dimeric
protein band (B6) generated by MALDI—TOF is.
Detailed Description of Invention
obin is an iron-containing oxygen—transport n in red blood cells of the blood
of mammals and other animals. Hemoglobin exhibits characteristics of both the tertiary and
quaternary ures of proteins. Most of the amino acids in hemoglobin form alpha helices
connected by short non-helical segments. Hydrogen bonds ize the helical sections inside the
hemoglobin causing attractions Within the le thereto g each polypeptide chain into a
specific shape. A hemoglobin molecule is assembled from four globular protein subunits. Each
subunit is ed of a polypeptide chain ed into a set of on—helix structural segments
connected in a “myoglobin fold” arrangement with an embedded heme group.
The heme group consists of an iron atom held in a heterocyclic ring, known as a
porphyrin. The iron atom binds equally to all four nitrogen atoms in the center of the ring which
lie in one plane. Oxygen is then able to bind to the iron center perpendicular to the plane of the
PCT/U82012/051959
porphyrin ring. Thus a single hemoglobin molecule has the capacity to combine with four
molecules of oxygen.
In mammals, the most common type of hemoglobin is a tetramer; in humans, it is called
hemoglobin A and consists of two on and two [3 non-covalently bound subunits designated as
(12132, each made of 141 and 146 amino acid residues respectively. The size and structure of cc
and [3 subunits are very similar to each other. Each t has a molecular weight of about 16
kDa for a total molecular weight of the tetramer of about 65 kDa. The four polypeptide chains
are bound to each other by salt bridges, en bonds and hydrophobic interaction. The
structure of bovine hemoglobin is similar to human hemoglobin (90.14% identity in or chain;
84.35% identity in [3 chain). The difference is the two sulfhydryl groups in the bovine
hemoglobin positioned at B Cys 93, while the sulfhydryls in human hemoglobin are at positioned
at on Cys 104, [3 Cys 93 and B Cys 112 tively. Human hemoglobin shares high similarity
with bovine, canine, porcine and equine obin when comparing their amino acid sequences.
In naturally-occurring hemoglobin inside the red blood cells, the association of an a chain
with its corresponding [3 chain is very strong and does not disassociate under physiological
conditions. However, the ation of one (113 dimer with another 01B dimer is fairly weak
outside red blood cells. The bond has a tendency to split into two (‘LB dimers each approximately
32 kDa. These undesired dimers are small enough to be filtered by the kidneys and be excreted,
with the result being ial renal injury and substantially sed intravascular retention
time.
Therefore, it is necessary to stabilize any hemoglobin that is used outside of red blood
cells both for efficacy and safety. The process for producing the stabilized hemoglobin is
outlined below; an overview of the s of the present invention is presented in the flow chart
of
PCT/U82012/051959
Initially, a Whole blood source is selected as a source ofhemoglobin from red blood cells.
Mammalian whole blood is selected ing, but not limited to, human, bovine, porcine, equine,
and canine whole blood. The red blood cells are separated from the plasma, d, and washed
to remove plasma protein impurities.
In order to release the hemoglobin from the red blood cells, the cell membrane is lysed.
gh various techniques can be used to lyse red blood cells, the t invention uses lysis
under hypotonic conditions in a manner which can be precisely controlled at volumes suitable for
industrial-scale production. To this end, an instant cytolysis apparatus as seen in is used
to lyse the red blood cells. Hypotonic lysis creates a solution of lysate including hemoglobin and
a waste retentate. To enable industrial-scale production, the lysis is carefully controlled such that
only red blood cells are lysed without lysing white blood cells or other cells. In one embodiment
the size of the instant cytolysis apparatus is selected such that the red blood cells traverse the
tus in 2 to 30 seconds or otherwise a time sufficient to lyse the red blood cells and
ably, 30 seconds. The instant cytolysis apparatus includes a static mixer. Deionized and
distilled water is used as a hypotonic solution. Of course it is understood that the use of other
hypotonic solutions having different saline concentrations would result in different time periods
for red blood cell lysis. Because the controlled lysis ure lyses the red blood cells only, not
white blood cells or cellular matter, it minimizes the release of toxic proteins, phospholipids or
DNA from white blood cells and other cellular matter. A hypertonic solution is added
ately afier 30 seconds, that is, after the red blood—cell containing on has traversed
the static mixer portion of the t cytolysis apparatus. The resultant hemoglobin has a higher
purity and lower levels of contaminants such as undesired DNA and phospholipids than
hemoglobin resulted from using other lysis ques. Undesired nucleic acids from white blood
cells and phospholipids impurities are not detected in the hemoglobin solution by polymerase
PCT/U82012/051959
chain reaction (detection limit = 64 pg) and high performance liquid chromatography (HPLC,
detection limit = l rig/ml) method respectively.
At this stage in the process, the hemoglobin solution is purified to remove various protein
and other ties. This purification can be ultrafiltration based, chromatography based, or a
combination of one or more ultrafiltration and/or chromatography processes. In an exemplary
embodiment, two ltration processes are performed: one which removes impurities having
molecular weights greater than hemoglobin before flowthrough column chromatography, and
another which removes impurities having molecular weights less than hemoglobin after
ough column chromatography. The latter ultrafiltration process concentrates the
obin. In some embodiments, a 100 kDa filter is used for the first ultrafiltration, while a 30
kDa filter is used for the second ultrafiltration.
Flowthrough column chromatography is used to remove protein impurities in the purified
hemoglobin solution such as globin—G, albumin and carbonic anhydrase. In some
embodiments, column chromatography is d out by using one or a combination of
commercially available ion exchange columns such as a DEAE column, CM column,
hydroxyapatite column, etc. The pH for column chromatography is typically from 6 to 8.5. In
one embodiment, a ough CM column chromatography step is used to remove protein
impurities at pH 8.0. Enzyme-linked sorbent assay (ELISA) and HPLC method are
performed to detect the protein impurities and phospholipids remaining in the sample after
elution from the column chromatography. This unique flowthrough column chromatography
separation enables a continuous separation scheme for industrial-scale production. The ELISA
result shows that the amount of these impurities is substantially low in the eluted hemoglobin
(immunoglobin—G: 44.3 ng/ml; albumin: 20.37 ng/ml; ic anhydrase: 81.2 pig/ml). The
protein impurities removal results using different kinds of columns with different pH values are
shown in Table 1 below.
Table 1
Removal percentage (%)
Column (pH condition)
Carbonic anhydrase Albumin Immunoglobin—G
DEAE (at pH 7.5) ~—- 68 29.8
DEAE (at pH 7.8) --— 60 50.9
CM (at pH 6.2) --- 32 21.8
CM (at pH 8.0) 5.6 53.2 66.4
Hydroxyapatite (at pH 7.5) 4.5 23.5 22.8
Following the column chromatographic process, the hemoglobin is subjected to cross-
g by DBSF. The conditions are selected such that cross-linking occurs between the beta—
beta subunits is favored and the resultant product has greater than 50% beta-beta cross—linking.
For cross-linking under enated ion, the resulting hemoglobin has a low oxygen
affinity with a higher p50 value compared with the native hemoglobin of the same species
measured under substantially similar conditions. For example, for bovine hemoglobin, the native
bovine hemoglobin has a p50 value on the order of 23-29 mm Hg. The crosslinked bovine
hemoglobin formed under deoxygenated conditions in the t invention has a p50 value on
the order of 38-50 mm Hg. Lower oxygen affinity means that the er can d” oxygen
to a target more easily than a material with a higher oxygen affinity. For cross-linking of bovine
obin under oxygenated conditions, a material with a higher oxygen affinity is formed
with a lower p50 value, less than approximately 23 mm Hg, compared with native bovine
hemoglobin which has a p50 value on the order of 23-29 mm Hg. Lower oxygen affinity
2012/051959
compositions are used when rapid oxygenation is desired as in cases of tissue hypoxia resulting
from extensive blood loss (e.g., hemorrhagic shock). Higher oxygen affinity compositions are
useful for ation adjunct therapies in cancer treatment where a slower delivery rate of
oxygen is desirable.
For human hemoglobin, cross-linking under deoxygenated condition typically produces a
majority of alpha-alpha crosslinked hemoglobin with lower oxygen affinity, that is, an oxygen
affinity that is decreased on the order of at least 2-fold from native human obin. Cross-
linking under oxygenated conditions tends to favor production of beta-beta crosslinked
hemoglobin with a higher oxygen affinity (that is, a lower p50, less than approximately 23 mm
Hg), compared with the native human hemoglobin under the same condition (a p50 value on the
order of approximately 23-30 mm Hg).
For deoxygenated cross-linking condition preferably less than 0.1 ppm dissolved oxygen
level, it is maintained with a molar ratio of hemoglobin to DBSF from 1:25 to 114.0 for a period
of time from 3 to 16 hours at ambient temperature (1 5—25°C), at a pH of around 8-10, preferably
around 9.2. The resultant crosslinked hemoglobin is tetrameric hemoglobin having a
lar weight of 60—70 kDa, demonstrating that polymeric hemoglobin is not present. The
yield of the DBSF on is high, > 99% and the dimer concentration in the final product is low.
Optionally, the present process does not require dryl treatment reagents such as
iodoacetamide to react with the hemoglobin before cross—linking as used in various prior art
processes. For cross-linking under oxygenated ions, an oxygenated environment (such as
air, p02 is around l49mmHg; or pure 02, p02 is nearly 760mmHg) is used while the remaining
conditions above are substantially the same.
For bovine hemoglobin, the beta-beta cross-linking is greater than 50%, and preferably
greater than 60% for cross-linking under deoxygenated conditions (less than 0.1 ppm dissolved
PCT/U82012/051959
oxygen . For bovine hemoglobin crosslinked under oxygenated condition, eta cross-
linking is also favored, typically at a level greater that 40% beta-beta crosslinking.
For human hemoglobin, cross-linking under oxygenated conditions favors beta-beta
cross-linking.
ing cross—linking, phosphate buffered saline (PBS), a physiological buffer, is
exchanged for the cross-linking solution and any al chemicals are removed by tangential
flow ion.
Following cross-linking, the present invention provides a heat processing step (High
Temperature Short Time, HTST) for the crosslinked eric hemoglobin solution. The heat
treatment takes place in a deoxygenated environment. Prior to heat treatment, yl cysteine
is optionally added to prevent formation of met-hemoglobin (inactive hemoglobin). After the
heat processing step, the solution is cooled and N—acetyl cysteine is optionally added to maintain
a low level of met-hemoglobin. If N—acetyl cysteine is added before and after heat treatment, the
amount added before heat treatment is approximately 0.2%, while the amount added after heat
treatment is approximately 0.025% to 0.4%. However, if N—acetyl cysteine is added only after
heat treatment, then the amount added is 0.025%—0.4%. In one embodiment, the amount of N-
acetyl cysteine added after heat treatment is 0.2%-0.4%. In another embodiment, the amount of
N—acetyl cysteine added after heat treatment is 0.025%-0.2%.
In some ments, the crosslinked tetrameric hemoglobin solution is heated in a
deoxygenated environment (less than 0.1 ppm dissolved oxygen level) under a range of
temperatures from 50°C to 95°C for durations from 0.5 minutes to 10 hours. In some
embodiments, the inked tetrameric hemoglobin solution is heated under a range of
atures from 70°C to 95°C and for durations from 30 seconds to 3 hours. In some preferred
embodiments, the crosslinked tetrameric hemoglobin solution is heated under 80°C for 30
minutes. And yet in other preferred embodiments, the crosslinked hemoglobin solution is heated
to 90°C for 30 seconds to 3 minutes, then rapidly cooled down to approximately 25°C in
approximately 15 to 30 seconds, and the N-acetyl cysteine is optionally added as set forth above.
To analyze the outcome of the HTST heat processing step, a HPLC analytical method is
used to detect the amount of dimer after this heat sing step. The mobile phase for HPLC
analysis contains magnesium chloride (0.75M) which can separate dimer (non-stabilized
tetramer) and heat stable crosslinked tetrameric hemoglobin. For promoting hemoglobin
dissociation into dimers, magnesium chloride is approximately 30 times more effective than
sodium chloride at the same ionic strength. The heat processing step also acts as a denaturation
step to dramatically remove unwanted protein impurities in the crosslinked tetrameric
hemoglobin ectable in immunoglobin—G; 96.15% decrease in albumin; 99.99% decrease in
carbonic anhydrase). Enzyme-linked immunosorbent assay (ELISA) is performed to detect the
protein impurities in the sample. Thus the purified, heat stable crosslinked eric hemoglobin
on has an undetectable level of dimer (below detection limit: 0.043%), and immunoglobin—
G, and a very low amount of albumin (0.02 rig/ml) and carbonic ase (0.014 .
shows that the dimeric form of hemoglobin is undetectable in a HPLC system. Table 2 shows the
experimental s regarding the protein impurities and dimer removal by the HTST heat
processing step. This HTST heat processing step s the selective separation of heat stable
crosslinked tetramer from unstable tetramer (e. g., non crosslinked tetramer) and dimer.
Table 2
I mmunoglobin-G Albumin Carbonic Tetramer Dimer
(Hg/m1) ) anhydrase (Hg/m1) (%) (%)
No heat 0.36
treatment
80°C for 10min Not detectable . 3.4
80°C for 15min Not detectable . . 2.9
80°C for 30min Not detectable . . Not
detectable
No heat 0.29
treatment
90°C for 1.0min Not detectable . . 2.0
90°C for 1.5min Not detectable . . 0.6
90°C for 2.0n1in Not detectable . . Not
detectable
Following the heat processing step for the crosslinked hemoglobin under a enated
condition, the heat stable crosslinked tetrameric hemoglobin is ready for pharmaceutical
formulation and packaging. The present invention describes an air-tight packaging step of the
heat stable crosslinked tetrameric hemoglobin solution in a deoxygenated nment. Heat
stable crosslinked tetrameric hemoglobin in the present ion is stable when maintained in a
deoxygenated condition for more than two years.
In this invention, the oxygen carrier-containing pharmaceutical composition is primarily
intended for enous injection application. Traditionally, prior ts use conventional
PVC blood bag or Stericon blood bag which has high oxygen permeability which will eventually
shorten the life span of the product since it turns into ve met-hemoglobin rapidly (within a
few days) under oxygenated conditions.
The packaging used in the present invention results in the heat stable crosslinked
tetrameric hemoglobin being stable for more than two years. A multi-layer package of
OH material is used to minimize the gas permeability and to avoid the formation of
inactive met-hemoglobin. A 100 ml infusion bag designed for use with the purified and heat
stable crosslinked tetrameric hemoglobin of the t invention is made from a five layers
EVA/EVOH laminated material with a ess of 0.4 mm that has an oxygen permeability of
0.006-0.132 cm3 per 100 square inches per 24 hours per here at room temperature. This
material is a Class VI plastic (as defined in USP<88>), which meets the in-vz'vo Biological
Reactivity Tests and the Physico-Chemical Test and is suitable for fabricating an infusion bag for
intravenous injection purpose. This primary bag is particularly useful to protect the heat stable
crosslinked tetrameric hemoglobin solution from long term oxygen exposure that causes its
instability and eventually affects its therapeutic properties.
For secondary protection ofblood ts, it has been known to use aluminum overwrap
to protect against ial air leakage and to maintain the product in a deoxygenated state.
However, there is a potential of pin holes in the aluminum overwrap that compromises its air
tightness and makes the product unstable. Therefore the t invention uses as secondary
packaging an aluminum overwrap pouch which prevents oxygenation and also prevents light
exposure. The composition of the overwrap pouch includes 0.012mm of polyethylene
thalate (PET), m of aluminum (Al), m of nylon (NY) and 0.1mm of
polyethylene (PE). The overwrap film has a thickness of 0.14mm and an oxygen transmission
rate of 0.006 cm3 per 100 square inches per 24 hours per atmosphere at room temperature. This
2012/051959
secondary packaging lengthens the stability time for the hemoglobin, extending the product
shelf-life.
The process in this ion is applicable to large scale industrial production of the heat
stable crosslinked tetrameric hemoglobin. In addition, the heat stable crosslinked tetrameric
hemoglobin in combination with a pharmaceutical carrier (e.g. water, physiological buffer, in
e form) is suitable for mammalian use.
The oxygen carrier~containing pharmaceutical composition of the present invention is
useful in improving tissue oxygenation, in cancer treatment, in the treatment of oxygen-
deprivation disorders such as hemorrhagic shock, and in heart preservation under a low oxygen
content environment (e.g. heart transplant). In exemplary embodiments, the dosage is selected to
have a tration range of approximately 0.2-1.3 g/kg with an infusion rate of less than 10
ml/hour/kg body weight.
For the use in the treatment of -deprivation disorders and for heart preservation,
the oxygen carrier-containing pharmaceutical composition with a lower oxygen affinity of the
present ion serves as a blood substitute providing oxygen to a target organ. Lower oxygen
affinity crosslinked hemoglobin is useful for applications requiring rapid tissue oxygenation (e.g.
hemorrhagic shock and ex vivo organ preservation).
For applications in cancer ent, the oxygen carrier-containing pharmaceutical
composition with a higher oxygen affinity of the t invention serves as a tissue oxygenation
agent to improve the oxygenation in tumor s, thereby enhancing chemo- and radiation
sensitivity. A higher oxygen affinity hemoglobin is useful for applications requiring a slower
rate of oxygenation (e.g. cancer adjunct therapy).
Examples
The following examples are ed by way of describing specific embodiments of this
invention without intending to limit the scope of this invention in any way.
Example 1
Process Overview
A schematic flow diagram of the process of the present invention is illustrated in
Bovine whole blood is collected into an enclosed sterile container/bag containing 3.8% (w/V) tri-
sodium citrate solution as anti-coagulant. Blood is then immediately mixed well with tri-sodium
citrate solution to inhibit blood clotting. Red blood cells (RBC) are isolated and collected from
plasma and other smaller blood cells by an apheresis mechanism. A “cell washer” is used for this
procedure with gamma sterilized disposable centrifuge bowl. RBC are washed with an equal
volume of 0.9% (w/v sodium chloride) saline.
Washed RBC are lysed to release hemoglobin content by manipulating hypotonic shock
to the RBC cell membrane. A lized t cytolysis apparatus for RBC lysis device
depicted in is used for this purpose. Following RBC lysis, hemoglobin molecules are
isolated from other proteins by tangential—flow ultrafiltration using a 100 kDa membrane.
Hemoglobin in the filtrate is ted for flowthrough column chromatography and further
concentrated to 12-14 g/dL by a 30 kDa ne. Column tography is carried out to
remove the protein impurities.
The concentrated hemoglobin solution is first reacted with DBSF to form heat stable
inked tetrameric hemoglobin les. A heat processing step is then performed under
deoxygenated conditions at 90°C for 30 seconds to three minutes before final ation and
packaging.
PCT/U82012/051959
Example 2
Time & Controlled Hypotonic lysis and filtration
Bovine whole blood is freshly collected and transported under a cool condition (2 to
°C). The red blood cells are separated from the plasma via a cell washer and subsequently with
a 0.65 pm filtration. After washing the red blood cells (RBC) filtrate with 0.9% saline, the
filtrate is disrupted by hypotonic lysis. The hypotonic lysis is performed by using the instant
cytolysis apparatus depicted in The instant cytolysis apparatus includes a static mixer to
assist in cell lysis. A RBC suspension with controlled hemoglobin tration (12—l4g/dL) is
mixed with 4 volumes of purified water to generate a hypotonic shock to RBC cell membranes.
The period of hypotonic shock is lled to avoid unwanted lysis of white blood cells and
platelets. The nic solution passes h the static mixer portion of the instant sis
apparatus for 2 to 30 seconds or otherwise a time sufficient to lyse the red blood cells and
preferably, 30 s. The shock is terminated after 30 seconds by mixing the lysate with 1/10
volume of hypertonic buffer as it exits the static mixer. The hypertonic solution used is 0.1M
phosphate buffer, 7.4% NaCl, pH 7.4. The instant cytolysis apparatus of can process at 50
to 1000 liters of lysate per hour and, ably at least 300 liters per hour in a continuous
manner.
Following the RBC lysis, the lysate of red blood cells is d by a 0.22 um filter to
obtain a hemoglobin solution. Nucleic acids from white blood cells and phospholipids impurities
= 64
are not detected in the hemoglobin solution by polymerase chain reaction (detection limit
pg) and HPLC (detection limit = 1 ug/ml) method respectively. A first 100 kDa ultrafiltration is
performed to remove impurities having a higher molecular weight than hemoglobin. A
ough column chromatography is followed to further purify the hemoglobin solution‘ A
PCT/U82012/051959
second 30 kDa ultrafiltration is then performed to remove impurities having a lower molecular
weight than hemoglobin and for concentration.
Viral clearance study on stroma-free hemoglobin solution
In order to demonstrate the safety of the product from this invention, the Virus removal
abilities of (1) 0.65 pm diafiltration step and (2) 100 kDa ultrafiltration step are demonstrated by
Virus tion study. This is done by the deliberate spiking of a down-scaled version of these
two processes with different model Viruses (encephalomyocarditis Virus, pseudorabies virus,
bovine viral ea virus and bovine parvovirus). In this study, four types of s (see Table
3) are used. These viruses vary in their sical and structural features and they display a
variation in resistance to physical and al agents or treatments.
Table 3
Size
Model Virus Taxonomy Genome Structure Stability?"
[11ml
Hepatitis Bovine viral diarrhea
Flaviviridae enveloped 40—60
C virus virus (BVDV)
Encephalomyocarditis non-
. _
P1cornav1rus ssRNA 25-30 medium
Virus (EMCV) enveloped
Parvovirus Bovine parvovirus non-
Parvoviridae_ ssDNA 18-26 very hlgh
B19 (BPV) enveloped
Hepatitis Pseudorabies Virus 120-: Low to
Herpesv1r1dae. dsDNA enveloped .
B virus (PRV) 200 medium
The validation scheme is briefly shown in the following Table 4.
PCT/U82012/051959
Table 4
___—_—___—_————————-—_-
Diafiltration Ultrafiltration
________________________.___..____
Cell Wlashing Virus g
Virus ipiking U|trafi|tration
Diafiltration l
1 Virus tests
Virus tests
_.——_—._——
The summary of the log reduction results of the 4 viruses in (1) 0.65 pm diafiltration and
(2) 100 kDa ultrafiltration is shown in the following Table 5. All four viruses, BVDV, BPV,
EMCV and PRV, are effectively removed by 0.65 um diafiltration and 100 kDa ultrafiltration.
Table 5
Viruses
—fiéRun0.65pm Diafiltration
Annotation:
2 no residual infectivity determined
Example 4
Flowthrough column tography
A CM column (commercially ble from GE healthcare) is used to further remove
any protein impurities. The starting buffer is 20mM sodium acetate (pH 8.0), and the elution
buffer is 20mM sodium acetate, 2M NaCl (pH 8.0), After the equilibration of the CM column
with ng buffer, the protein sample is loaded into the column. The unbound protein
impurities are washed with at least 5 column volume of starting buffer. The n is performed
using 25% elution buffer (0-0.5M NaCl) in 8 column volume. The n profile is shown in
the hemoglobin solution is in the flowthrough fraction. The purity of the flowthrough
fraction is analyzed by ELISA. The results are indicated in the following Table 6.
Table 6
Protein ties
Immunoglobin—G Carbonic ase
Efore CM column 1320 ng/ml 860.3 rig/ml
Flowthrough
ining hemoglobin)
As the hemoglobin solution is in the flowthrough from the CM column chromatography
at pH 8 (not in the eluate), it is a good approach for continuous industrial scale operation. The
first ultrafiltration set~up is ted directly to the flowthrough CM column chromatography
system, and the flowthrough tubing can be connected to the second ultrafiltration set-up for
rial scale ion. The schematic industrial process configuration is shown in
Example 5
Preparation of heat stable crosslinked tetrameric hemoglobin
(5a) Cross—linking reaction with DBSF under a deoxygenated condition
The cross-linking reaction is carried out in a deoxygenated condition, that is, less than 0.1
ppm dissolved oxygen level. DBSF is added to the hemoglobin solution to form crosslinked
tetrameric hemoglobin without formation of polymeric hemoglobin. DBSF stabilization
procedure stabilizes the tetrameric form of hemoglobin (65 kDa) and prevents dissociation into
dimers (32 kDa) which are excreted through the kidneys. In this embodiment, a molar ratio of
hemoglobin to DBSF of 1:2.5 is used and the pH is 8.6. This process is carried out for a period
of 3—16 hours at ambient temperature °C) in an inert atmosphere of nitrogen to prevent
oxidation of the hemoglobin to form ferric moglobin which is physiologically ve
(dissolved oxygen level maintained at less than 0.1 ppm). The completeness of DBSF reaction is
monitored by measuring the residual DBSF using HPLC. The yield of the DBSF reaction is high,
> 99%. The production of beta—beta crosslinks is on the order of at least about 40%.
(5b) HTST heat processing step
A High ature Short Time (HTST) processing apparatus is shown in FIG 6. A heat
processing step using the HTST processing apparatus is performed on the crosslinked tetrameric
hemoglobin. In this example, the condition for heat treatment is 90°C for 30 seconds to 3 minutes,
and preferably 45 to 60 seconds although other conditions can be selected as discussed above
and the apparatus modified accordingly. A solution containing crosslinked hemoglobin
optionally with 0.2% of N—acetyl cysteine added thereto is pumped into a HTST processing
apparatus (first n of the HTST heat exchanger is pre-heated and maintained at 90°C) at a
flow rate of 1.0 liter per minute, the residence time of the first section ofthe apparatus is n
45 to 60 seconds, then the solution is passed through at the same flow rate into another section of
the heat exchanger that is maintained at 25°C. The time required for cooling is between 15 to 30
s. After cooling down to 25°C, N—acetyl cysteine is ately added at a tration
of 0.2% to 0.4%. The set-up of the heat processing apparatus is easily controlled for industrial
operation. A temperature profile with dimer content is shown in If the obin is not
crosslinked, it is not heat stable and forms a precipitate after the heat processing step. The
precipitate is then removed by a centrifugation or a filtration to form a clear solution thereafter.
The following Table 7 shows that protein impurities such as globin-G, albumin,
carbonic anhydrase and undesirable non-stabilized tetramer or dimers are removed after the heat
PCT/U82012/051959
processing step. The amount of immunoglobin—G, albumin and carbonic anhydrase are measured
using an ELISA method, while the amount of dimer is ined by an HPLC method. The
purity of heat stable crosslinked tetrameric hemoglobin is extremely high after the HTST heat
processing step, in the range of 98.0 to 100%.
Table 7
Protein impurities (by ELISA)
Sample mmunoglobin—G n Carbonic Tetramer
condition (pg/ml) (pg/m1) anhydrase (rig/ml) (%) (%)
No heat 0.29 0.52 261.80 5.3
treatment
90°C for 2min Not detectable 0.02 0.016 96.1 Not
detectable
Removal (%) 100.0 96.15 -----
(Sc) Prevention of Met-hemoglobin ion by 0.025-0.2% NAC on
Following the heat treatment and cooling, N—acetyl Cysteine (NAC) is immediately added
into the crosslinked tetrameric hemoglobin at a tration of approximately 0.2% to
prevent the formation of met-hemoglobin. At different time interval, the percentage of met-
hemoglobin is measured by metry method. Table 8 shows the percentage of met-
hemoglobin in the crosslinked tetrameric hemoglobin after NAC addition over 5 months. As
shown in Table 8, the met-hemoglobin level in the heat-treated crosslinked tetrameric
hemoglobin is kept steady and very low after NAC addition, in a range of 1.8-5.1%.
WO 32828 PCTfU82012/051959
Table 8
Met-hemoglobin0 o in Heat-treated Crosslinked Tetrameric Hemoglobin
Total NAC After Addin; NAC
level
0.1% NAC
0.025% NAC
Example 6
Packaging
Because the product of the present invention is stable under deoxygenated conditions, the
packaging for the product is important to minimize gas permeability. For intravenous
application, a custom designed, 100 ml infusion bag is made from a yer EVA/EVOH
laminated material with a thickness of 0.4 m that has an oxygen permeability of 0.006 to 0.132
cm3 per 100 square inches per 24 hours per atmosphere at room temperature. This c
material is a Class VI plastic (as defined in USP<88>), which meets the in-vz‘vo biological
reactivity tests and the o-chemical tests and are suitable for fabricating containers for
intravenous injection purpose (note that other forms of packaging can be made from this material
as well depending upon the d application). A secondary packaging aluminum overwrap
pouch is also d to the primary ing infusion bag that provides an additional barrier,
minimizing light exposure and oxygen diffusion. The layers of the pouch comprise: m of
Polyethylene terephthalate (PET), 0.007mm of Aluminum (Al), 0.015mm of Nylon (NY) and
0.1mm of Polyethylene (PB). The overwrap film has a thickness of 0.14mm and oxygen
transmission rate of 0.006 cm3 per 100 square inches per 24 hours per atmosphere at room
temperature. A schematic depiction of the infusion bag is depicted in The overall oxygen
PCT/U82012/051959
permeability for each infusion bag according to the present invention is 0.0025 cm3 per 24 hours
per atmosphere at room temperature.
Example 7
Characterization of crosslinked bovine Hb (Deoxygenated linking ion)
(7a) Separation of globin chains by Reverse Phase High Performance Liquid
Chromatography (HPLC)
The globin chains of native bovine hemoglobin and crosslinked globin chains of DBSF
crosslinked bovine hemoglobin are resolved on a VYDAC C4 column using the nts
developed by Shelton et al.,1984 with minor modification.
(7b) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of
DBSF crosslinked bovine hemoglobin
Native bovine hemoglobin and DBSF crosslinked bovine hemoglobin solution are
prepared by mixing with reducing sample buffer (62 mM Tris—HCl (pH 6.8), 10% (v/v) glycerol,
% (WV) mercaptoethanol and 2.3% (w/v) SDS), and heated at 95°C for 10 min. The sample
mixture is resolved using a 15% acrylamide slab gel with a 4% ng gel. The electrophoresis
is run with a constant current of 60 mA. After electrophoresis, the SDS—PAGE gel is stained with
0.l% (w/v) Coomassie Blue R350, 20% (v/v) ol and 10% (v/V) acetic acid. To te
the percentage of different types of cross-linking in DBSF crosslinked bovine hemoglobin, the
intensities of the resolved protein bands expressed in Black Light Unit (BLU) are quantified
using Lumi-Analyst 3.1 Software.
(7c) Trypsin digestion of reduced globin chain
The protein band corresponding to the major crosslinked globin chain is excised from the
SDS—PAGE gel, cut into cubes (1 X 1 mm), and de-stained with 10% methanol/10% acetic acid.
The de-stained gel cubes are reduced with 10 mM DTT in 25 mM NH4CO3 and alkylated with
PCT/U82012/051959
55 mM idoacetamide in 25 mM NH4C03 for 45 min in dark, and then in-gel digested with 20
ng/ul d trypsin in 25 mM NH4C03 at 37°C overnight. After trypsin digestion, the tiypsin-
digested peptides are extracted by diffusion into 50% (V/v) acetonitrile (ACN) and 1% (v/v)
trifluoroacetic acid (TFA).
(7d) Matrix Assisted Laser Desorption/Ionization Time—of-Flight (MALDI-TOF) mass
ometry (MS) analysis
The trypsin digested peptides extracted from the protein band are spotted onto an
Anchorchip plate, which is otted with 1 pl of matrix solution (2 mg/ml cyano—4—
hydroxycinnamic acid, saturated in 50% ACN/ 0.1% TFA, and allowed to air—dry. After ,
the sample spot is washed with 10 mM monophosphate buffer and tallized using a solution
of ethanol: acetone: 01% TFA (623:1 ratio). MALDI-TOF MS analysis is performed with a
Bruker Autoflex III (Bruker Daltonic GmbH, Bremen, Germany) operated in the reflectron mode
over the m/z range of 00 Da and the parameters are set as follows: ion source 25 kV for
peptide mass fingerprint (PMF), and reflector 26.3 kV for PMF. External calibration is
performed using a Bruker Peptide Mix Calibration Standard. The peaks with a S/N ratio over 4
are automatically labeled by Flex-Analysis (Bruker Daltonic GmbH, Bremen, Germany).
MS data is further analyzed through MASCOT 2.2.04 and Biotools 2.1 software (Bruker
Daltonic GmbH, Bremen, Germany), and these data were searched against Mammalian proteins
in NCBI nonreduntant (NCBInr) database. The following ters are used for database
searches: monoisotopic mass accuracy <250 ppm, parent charge +1, missed cleavages 1,
carbamidomethylation of ne as fixed modification, oxidation of methionine as variable
ation.
(7e) Liquid chromatography ~ electrospray ionization T) tandem mass
spectrometry (MS/MS) analysis
Nano-LC MS/MS analysis of the trypsin digested peptides from the n band is
performed using ary HPLC d directly to HCT Ultra ESI— ion trap mass
in 0.1%
ometer (Bruker Daltonic GmbH, Bremen, Germany). Peptide digests are dissolved
formic acid/2% ACN prior to column injection. A gradient from 4-90% (0.001% formic acid and
0.001% formic acid in 80% ACN) is used for peptide separation using a C18 column (15 cm X
75 nm, LC PACKINGS). The flow rate is 250 ng/min at 25°C. Eluates from a C18 column are
entered into the HCT Ultra ESI— ion trap mass spectrometer, ed in linear mode for online
analysis. The ion trap mass spectrometer is optimized with the nanosource with a spray voltage
of 137V and a heated capillary temperature of 160°C. The accumulation time for peptide ions in
the ion trap is set to be 200 ms, and the mass to charge ratio selected for MS/MS analysis is from
100 to 1800 Da with a charge state l—3.
The reverse phase HPLC on a VYDAC C4 column, monitored at a wavelength of
220nm, is employed to separate different types of cross-linking occurring between on and [3
chains in the DBSF crosslinked bovine hemoglobin. The tographic patterns obtained
using bovine hemoglobin before and after cross—linking with DBSF are shown in In the 0!, chains are more mobile than the [3 chains of native bovine hemoglobin (as shown with
dashed line). Their identities are confirmed by TOF analysis. After the reaction with
DBSF, the [3 chains are crosslinked while a large majority of on chains are left alone (as shown
with solid line). As a consequence of cross—linking with DBSF, 6 major globin peaks with
in the
greater hydrophobicity than the native B chains are . The crosslinked globin chains
DBSF crosslinked bovine hemoglobin are also resolved by 15% SDS-PAGE, as shown in . The major crosslinked globin chain (B6 in ) is subjected to trypsin digestion and
PCT/U82012/051959
subsequent MALDI—TOF analysis, and it is identified as beta globin chain only, based on its
peptide mass fingerprint, as shown in .
While the foregoing invention has been described with respect to various
embodiments, such embodiments are not limiting. us variations and modifications
would be tood by those of ordinary skill in the art. Such variations and modifications are
considered to be included within the scope of the following claims.
Claims (1)
- What is Claimed is:
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161529279P | 2011-08-31 | 2011-08-31 | |
US61/529,279 | 2011-08-31 | ||
US13/225,797 US20130052232A1 (en) | 2011-08-31 | 2011-09-06 | Method for the preparation of a heat stable oxygen carrier-containing composition facilating beta-beta cross-linking |
US13/225,797 | 2011-09-06 | ||
US13/275,366 US8106011B1 (en) | 2011-08-31 | 2011-10-18 | Method for the preparation of a heat stable oxygen carrier-containing composition facilating beta-beta cross-linking |
US13/275,366 | 2011-10-18 | ||
PCT/US2012/051959 WO2013032828A2 (en) | 2011-08-31 | 2012-08-23 | Method for the preparation of a heat stable oxygen carrier-containing composition facilating beta-beta cross-linking |
Publications (2)
Publication Number | Publication Date |
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NZ620913A NZ620913A (en) | 2016-03-31 |
NZ620913B2 true NZ620913B2 (en) | 2016-07-01 |
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