U.S. patent application number 12/824618 was filed with the patent office on 2012-02-23 for method of producing hydroxyalkyl starch derivatives.
Invention is credited to Harald Conradt, Wolfram Eichner, Norbert Zander.
Application Number | 20120046240 12/824618 |
Document ID | / |
Family ID | 31995520 |
Filed Date | 2012-02-23 |
United States Patent
Application |
20120046240 |
Kind Code |
A9 |
Zander; Norbert ; et
al. |
February 23, 2012 |
Method of Producing Hydroxyalkyl Starch Derivatives
Abstract
The present invention relates to methods of producing
hydroxyalkyl starch (HAS) derivatives having a structure according
to formula (I) comprising reacting HAS of formula (I) at its
optionally oxidized reducing end or a HAS derivative, obtainable by
reacting HAS of formula (I) at its optionally oxidized reducing end
with a compound (D) comprising at least one functional group
Z.sub.1 capable of being reacted with the optionally oxidized
reducing end of the HAS and at least one functional group W, with a
compound (L) comprising at least one functional group Z.sub.1
capable of being reacted with said HAS, or at least one functional
group Z.sub.2 capable of being reacted with functional group W
comprised in said HAS derivative, and at least one functional group
X capable of being reacted with a functional group Y of a further
compound. The present invention further relates to HAS derivatives
and pharmaceutical compositions comprising them.
Inventors: |
Zander; Norbert; (Meine,
DE) ; Conradt; Harald; (Braunschweig, DE) ;
Eichner; Wolfram; (Butzbach, DE) |
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20100317609 A1 |
December 16, 2010 |
|
|
Family ID: |
31995520 |
Appl. No.: |
12/824618 |
Filed: |
June 28, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11078098 |
Mar 11, 2005 |
|
|
|
12824618 |
|
|
|
|
PCT/EP2003/008829 |
Aug 8, 2003 |
|
|
|
11078098 |
|
|
|
|
60409781 |
Sep 11, 2002 |
|
|
|
Current U.S.
Class: |
514/43 ;
536/50 |
Current CPC
Class: |
C07K 14/505 20130101;
C07K 14/56 20130101; C08B 31/00 20130101; A61K 47/61 20170801; C07K
14/5412 20130101; C08B 31/12 20130101; A61K 38/00 20130101; C08B
31/006 20130101; C07K 17/10 20130101; C07K 14/565 20130101; A61P
43/00 20180101; A61P 7/00 20180101; C08H 1/00 20130101; A61P 7/06
20180101; A61K 9/0021 20130101; C07K 14/55 20130101; C08B 31/185
20130101; C08B 31/08 20130101; C07K 14/53 20130101 |
Class at
Publication: |
514/43 ;
536/50 |
International
Class: |
A61K 31/70 20060101
A61K031/70; C08B 31/08 20060101 C08B031/08 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 11, 2002 |
EP |
02020425.1 |
Claims
1-59. (canceled)
60. A hydroxyalkyl starch (HAS) derivative obtainable by a method
comprising reacting (a) a first HAS derivative obtained by reacting
HAS of formula (I) ##STR00120## at its optionally oxidized reducing
end with a compound (D), said compound (D) comprising at least one
functional group Z.sub.1 capable of being reacted with the
optionally oxidized reducing end of the HAS, and at least one
functional group W, said reacting being carried out via the
reaction of the functional group Z.sub.1 comprised in the compound
(D) with the optionally oxidized reducing end of the HAS wherein
the functional group Z.sub.1 is selected from the group consisting
of ##STR00121## wherein G is O or S and, if present twice,
independently O or S, with (b) a compound (L) comprising at least
one functional group Z.sub.2 capable of being reacted with the at
least one functional group W of said first HAS derivative, and at
least one functional group X capable of being reacted with a
functional group Y of a further compound (M), said reaction of said
first HAS derivative with the compound (L) being carried out via
the reaction of the functional group Z.sub.2 comprised in the
compound (L) with the functional group W comprised in the compound
(D) to give a second HAS derivative, (i) wherein the functional
group W or the functional group Z.sub.2 is --SH and the functional
group Z.sub.2 or the functional group W is selected from the group
consisting of ##STR00122## wherein Hal is Cl, Br, or I, (ii) or
wherein the functional group W or the functional group Z.sub.2 is
selected from the group consisting of an activated ester or a
carboxy group which is optionally transformed into an activated
ester and the functional group Z.sub.2 or the functional group W is
selected from the group consisting of ##STR00123## wherein G is O
or S and, if present twice, independently O or S, and R' is methyl,
(c) said method further comprising reacting said second HAS
derivative with the further compound (M) via the reaction of the
functional group X comprised in the compound (L) with the
functional group Y comprised in the further compound (M), wherein
said functional group Y is a thio group, and the functional group X
is selected from the group consisting of ##STR00124## wherein Hal
is Cl, Br or I.
61. A pharmaceutical composition comprising, in a therapeutically
effective amount, a HAS derivative according to claim 60.
Description
[0001] The present invention relates to hydroxyalkyl starch
derivates, particularly hydroxyalkyl starch derivatives obtainable
by a process in which hydroxyalkyl starch is reacted with a primary
or secondary amino group of a crosslinking compound or with two
crosslinking compounds wherein the resulting hydroxaylkyl starch
derivative has at least one functional group X which is capable of
being reacted with a functional group Y of a further compound and
wherein this group Y is an aldehyd group, a keto group, a
hemiacetal group, an acetal group, or a thio group. According to an
especially preferred embodiment, the present invention relates to
hydroxyalkyl starch derivatives obtainable by a process according
to which hydroxyalkyl starch is reacted with a primary or secondary
amino group of a crosslinking compound, the resulting reaction
product optionally being further reacted with a second crosslinking
compound, wherein the resulting hydroxaylkyl starch derivative has
at least one functional group X which is capable of being reacted
with a functional group Y of a further compound and wherein this
group Y is an aldehyd group, a keto group, a hemiacetal group, an
acetal group, or a thio group, and the resulting reaction product
is reacted with a polypeptide, preferably with a polypeptide such
as AT III, IFN-beta or erythropoietin and especially preferably
with erythropoietin, which comprises at least one of these
functional groups Y. A hydroxyalkyl starch which is especially
preferred is hydroxyethyl starch. According to the present
invention, the hydroxyalkyl starch and preferably the hydroxylethyl
starch is reacted with the linker compound at its reducing end
which is optionally oxidized prior to said reaction.
[0002] Hydroxyethyl starch (HES) is a derivative of naturally
occurring amylopectin and is degraded by alpha-amylase in the body.
HES is a substituted derivative of the carbohydrate polymer
amylopectin, which is present in corn starch at a concentration of
up to 95% by weight. HES exhibits advantageous biological
properties and is used as a blood volume replacement agent and in
hemodilution therapy in the clinics (Sommermeyer et al., 1987,
Krankenhauspharmazie, 8(8), 271-278; and Weidler et al., 1991,
Arzneim.-Forschung/Drug Res., 41, 494-498).
[0003] Amylopectin consists of glucose moieties, wherein in the
main chain alpha-1,4-glycosidic bonds are present and at the
branching sites alpha-1,6-glycosidic bonds are found. The
physical-chemical properties of this molecule are mainly determined
by the type of glycosidic bonds. Due to the nicked
alpha-1,4-glycosidic bond, helical structures with about six
glucose-monomers per turn are produced. The physicochemical as well
as the biochemical properties of the polymer can be modified via
substitution. The introduction of a hydroxyethyl group can be
achieved via alkaline hydroxyethylation. By adapting the reaction
conditions it is possible to exploit the different reactivity of
the respective hydroxy group in the unsubstituted glucose monomer
with respect to a hydroxyethylation. Owing to this fact, the
skilled person is able to influence the substitution pattern to a
limited extent.
[0004] Some ways of producing a hydroxyethyl starch derivative are
described in the art.
[0005] DE 26 16 086 discloses the conjugation of hemoglobin to
hydroxyethyl starch wherein, in a first step, a cross-linking
agent, e.g. bromocyane, is bound to hydroxyethyl starch and
subsequently hemoglobin is linked to the intermediate product.
[0006] One important field in which HES is used is the
stabilisation of polypeptides which are applied, e.g., to the
circulatory system in order to obtain a particular physiological
effect. One specific example of these polypeptides is
erythropoietin, an acid glycoprotein of approximately 34,000 kD
which is essential in regulating the level of red blood cells in
the circulation.
[0007] A well-known problem with the application of polypeptides
and enzymes is that these proteins often exhibit an unsatisfactory
stability. Especially erythropoietin has a relatively short plasma
half live (Spivak and Hogans, 1989, Blood 73, 90; McMahon et al.,
1990, Blood 76, 1718). This means that therapeutic plasma levels
are rapidly lost and repeated intravenous administrations must be
carried out. Furthermore, in certain circumstances an immune
response against the peptides is observed.
[0008] It is generally accepted that the stability of polypeptides
can be improved and the immune response against these polypeptides
is reduced when the polypeptides are coupled to polymeric
molecules. WO 94/28024 discloses that physiologically active
polypeptides modified with polyethyleneglycol (PEG) exhibit reduced
immunogenicity and antigenicity and circulate in the bloodstream
considerably longer than unconjugated proteins, i.e. have a longer
clearance rate. However, PEG-drug conjugates exhibit several
disadvantages, e.g. they do not exhibit a natural structure which
can be recognized by elements of in vivo degradation pathways.
Therefore, apart from PEG-conjugates, other conjugates and protein
polymerates have been produced. A plurality of methods for the
cross-linking of different proteins and macromolecules such as
polymerase have been described in the literature (see e.g. Wong,
Chemistry of protein conjugation and cross-linking, 1993, CRCS,
Inc.).
[0009] In summary, there is still a need for further improved
polypeptides with improved stability and/or bioactivity. This
applies especially to erythropoietin where isoforms with a high
degree of sialic acids and therefore high activity have to be
purified from isoforms with a low degree of sialic acids (see EP 0
428 267 B1). Therefore, it would be highly advantageous if
production methods were available which provide highly active
polypeptides without requiring extensive purification.
Unfortunately, the production of polypeptides in bacteria or insect
cells is often difficult, because the polypeptides are often not
produced in a properly folded, native confirmation and lack proper
glycosylation.
[0010] WO 02/08079 A2 discloses compounds comprising a conjugate of
an active agent and a hydroxyalkyl starch wherein active agent and
hydroxyalykl starch are either linked directly or via a linker
compound. As far as the direct linkage is concerned, the reaction
of active agent and hydroxyalkyl starch is carried out in an
aqueous medium which comprises at least 10 wt.-% of water. No
examples are given which are directed to a hydroxyalkyl starch
derivative which is linked to a carbonyl group comprised in the
active reagent, neither an aldehyd or keto group nor a an acetal or
a hemiacetal group.
[0011] Consequently, it is an object of the present invention to
provide hydroxyalkyl starch derivatives which are capable of
forming a chemical linkage to a further compound, e.g. a
polypeptide, which comprises, as functional group, a thio group or
an aldehyd group, a keto group, a hemiacetal group, or an acetal
group. Preferably, the aldehyd group, the keto group, the
hemiacetal group, or the acetal group are comprised in a
carbohydrate moiety of the further compound.
[0012] Therefore, the present invention relates to a method of
producing a hydroxyalkyl starch derivative, said hydroxyalkyl
starch having a structure according to formula (I)
##STR00001##
comprising reacting [0013] hydroxyalkyl starch of formula (I) at
its optionally oxidized reducing end or [0014] hydroxyalkyl starch
derivative, obtainable by reacting hydroxyalkyl starch of formula
(I) at its optionally oxidized reducing end with a compound (D),
said compound (D) comprising [0015] at least one functional group
Z.sub.1 capable of being reacted with the optionally oxidized
reducing end of the hydroxyalkyl starch, and [0016] at least one
functional group W, with a compound (L) comprising [0017] at least
one functional group Z.sub.1 capable of being reacted with said
hydroxyalkyl starch, or at least one functional group Z.sub.2
capable of being reacted with functional group W comprised in said
hydroxyalkyl starch derivative, and [0018] at least one functional
group X capable of being reacted with a functional group Y of a
further compound (M), wherein said functional group Y is selected
from the group consisting of an aldehyd group, a keto group, a
hemiacetal group, an acetal group, or a thio group.
[0019] In the context of the present invention, the term
"hydroxyalkyl starch" (HAS) refers to a starch derivative which has
been substituted by at least one hydroxyalkyl group. Therefore, the
term hydroxyalkyl starch as used in the present invention is not
limited to compounds where the terminal carbohydrate moiety
comprises hydroxyalkyl groups R.sub.1, R.sub.2, and/or R.sub.3 as
depicted, for the sake of brevity, in formula (I), but also refers
to compounds in which at least one hydroxy group present anywhere,
either in the terminal carbohydrate moiety and/or in the remaining
part of the starch molecule, HAS', is substituted by a hydroxyalkyl
group R.sub.1, R.sub.2, or R.sub.3.
[0020] In this context, the alkyl group may be a linear or branched
alkyl group which may be suitably substituted. Preferably, the
hydroxyalkyl group contains 1 to 10 carbon atoms, more preferably
from 1 to 6 carbon atoms, more preferably from 1 to 4 carbon atoms,
and even more preferably 2-4 carbon atoms. "Hydroxyallyl starch"
therefore preferably comprises hydroxyethyl starch, hydroxypropyl
starch and hydroxybutyl starch, wherein hydroxyethyl starch and
hydroxypropyl starch are particularly preferred.
[0021] Hydroxyalkyl starch comprising two or more different
hydroxyalkyl groups is also comprised in the present invention.
[0022] The at least one hydroxyalkyl group comprised in HAS may
contain two or more hydroxy groups. According to a preferred
embodiment, the at least one hydroxyalkyl group comprised HAS
contains one hydroxy group.
[0023] The expression "hydroxyalkyl starch" also includes
derivatives wherein the alkyl group is mono- or polysubstituted. In
this context, it is preferred that the alkyl group is substituted
with a halogen, especially fluorine, or with an aryl group,
provided that the HAS remains soluble in water. Furthermore, the
terminal hydroxy group a of hydroxyalkyl group may be esterified or
etherified.
[0024] Furthermore, instead of alkyl, also linear or branched
substituted or unsubstituted alkene groups may be used.
[0025] Hydroxyalkyl starch is an ether derivative of starch.
Besides of said ether derivatives, also other starch derivatives
can be used in the context of the present invention. For example,
derivatives are useful which comprise esterified hydroxy groups.
These derivatives may be, e.g., derivatives of unsubstituted mono-
or dicarboxylic acids with 2-12 carbon atoms or of substituted
derivatives thereof. Especially useful are derivatives of
unsubstituted monocarboxylic acids with 2-6 carbon atoms,
especially derivatives of acetic acid. In this context, acetyl
starch, butyl starch and propyl starch are preferred.
[0026] Furthermore, derivatives of unsubstituted dicarboxylic acids
with 2-6 carbon atoms are preferred.
[0027] In the case of derivatives of dicarboxylic acids, it is
useful that the second carboxy group of the dicarboxylic acid is
also esterified. Furthermore, derivatives of monoalkyl esters of
dicarboxylic acids are also suitable in the context of the present
invention.
[0028] For the substituted mono- or dicarboxylic acids, the
substitute groups may be preferably the same as mentioned above for
substituted alkyl residues.
[0029] Techniques for the esterification of starch are known in the
art (see e.g. Klemm D. et al, Comprehensive Cellulose Chemistry
Vol. 2, 1998, Whiley-VCH, Weinheim, N.Y., especially chapter 4.4,
Esterification of Cellulose (ISBN 3-527-29489-9).
[0030] Hydroxyethyl starch (HES) is most preferred for all
embodiments of the present invention.
[0031] Therefore, the present invention also relates to a method as
described above wherein the hydroxyalkyl starch is hydroxyethyl
starch.
[0032] HES is mainly characterized by the molecular weight
distribution and the degree of substitution. There are two
possibilities of describing the substitution degree: [0033] 1. The
substitution degree can be described relatively to the portion of
substituted glucose monomers with respect to all glucose moieties
(DS). [0034] 2. The substitution degree can be described as the
"molar substitution" (MS), wherein the number of hydroxyethyl
groups per glucose moiety are described.
[0035] HES solutions are present as polydisperse compositions,
wherein each molecule differs from the other with respect to the
polymerisation degree, the number and pattern of branching sites,
and the substitution pattern. HES is therefore a mixture of
compounds with different molecular weight. Consequently, a
particular HES solution is determined by average molecular weight
with the help of statistical means. In this context, M.sub.n is
calculated as the arithmetic mean depending on the number of
molecules. Alternatively, M.sub.w, the weight mean, represents a
unit which depends on the mass of the HES.
[0036] In the context of the present invention, hydroxyethyl starch
may have a mean molecular weight (weight mean) of from 1 to 300
kDa, wherein a mean molecular weight of from 5 to 100 kDa is more
preferred. Hydroxyethyl starch can further exhibit a molar degree
of substitution of from 0.1 to 0.8 and a ratio between
C.sub.2:C.sub.6 substitution in the range of from 2 to 20 with
respect to the hydroxyethyl groups.
[0037] As far as the residues R.sub.1, R.sub.2 and R.sub.3
according to formula (I) are concerned there are no specific
limitations given that compound (I) remains capable of being
reacted with a compound (D) or a compound (L). According to a
preferred embodiment, R.sub.1, R.sub.2 and R.sub.3 are
independently hydrogen or a hydroxyalkyl group, a hydroxyaryl
group, a hydroxyaralkly group or a hydroxyalkarly group having of
from 1 to 10 carbon atoms. Hydrogen and hydroxyalkyl groups having
of from 1 to 6 carbon atoms are preferred. The alkyl, aryl, aralkyl
and/or alkaryl group may be linear or branched and suitably
substituted.
[0038] Therefore, the present invention also related to a method as
described above wherein R.sub.1, R.sub.2 and R.sub.3 are
independently hydrogen or a linear or branched hydroxyalkyl group
with from 1 to 6 carbon atoms.
[0039] Thus, R.sub.1, R.sub.2 and R.sub.3 may be hydroxyhexyl,
hydroxypentyl, hydroxybutyl, hydroxypropyl such as 1-hydroxypropyl,
2-hydroxypropyl, 3-hydroxypropyl, 1-hydroxyisopropyl,
2-hydroxyisopropyl, hydroxyethyl such as 1-hydroxyethyl,
2-hydroxyethyl, or hydroxymethyl. Hydrogen and hydroxyethyl groups
are preferred, hydrogen and the 2-hydroxyethyl group being
especially preferred.
[0040] Therefore, the present invention also relates to a method as
described above wherein R.sub.1, R.sub.2 and R.sub.3 are
independently hydrogen or a 2-hydroxyethyl group.
[0041] According to the present invention either compound (D) or
compound (L) is reacted with the reducing end of the hydroxyalkyl
starch via the reaction of the functional group Z.sub.1 with the
reducing end where group Z.sub.1 is comprised in compound (D) or
compound (L).
[0042] According to a first preferred embodiment of the present
invention, compound (D) or compound (L) is reacted with the
reducing end of the hydroxyalkyl starch and where the reducing end
is oxidized prior to the reaction.
[0043] This oxidation of the reducing end leads to hydroxyalkyl
starch in which the terminal carbohydrate group comprises a lactone
group, or in which the terminal carbohydrate group, depending of
the chemical reaction conditions and/or the oxidizing agents, has a
non-cyclic structure comprising a carboxy group.
[0044] According to one embodiment of the present invention, the
hydroxyalkyl starch which is oxidized at its reducing end is
present as a mixture of a compound comprising the lactone group and
a compound comprising the carboxy group. In the mixture, the
respective compounds may be present at any conceivable ratio.
[0045] Therefore, the present invention also relates to a method as
described above wherein the reducing end of the hydroxyalkyl starch
is oxidized prior to the reaction with compound (D) or compound
(L), said hydroxyalkyl starch thus having a structure according to
formula (IIa)
##STR00002##
and/or according to formula (IIb)
##STR00003##
[0046] The oxidation of the reducing end of the hydroxyalkyl starch
may be carried out according to each method or combination of
methods which result compounds having the above-mentioned
structures (IIa) and/or (IIb).
[0047] Although the oxidation may be carried out according to all
suitable method or methods resulting in the oxidized reducing end
of hydroxyalkyl starch, it is preferably carried out using an
alkaline iodine solution as described, e.g., in 196 28 705 A1.
[0048] Therefore, the present invention also relates to a method as
mentioned above wherein the reducing end is oxidized by an alkaline
iodine solution.
[0049] According to a second preferred embodiment of the present
invention, compound (D) or compound (L) is reacted with the
reducing end of the hydroxyalkyl starch and where the reducing end
is not oxidized prior to the reaction.
[0050] Therefore, the present invention also relates to a method as
mentioned above wherein the reducing end of the hydroxyalkyl starch
is not oxidized prior to the reaction with compound (D) or compound
(L), said hydroxyalkyl starch thus having a structure according to
formula (I)
##STR00004##
[0051] The formation of a chemical linkage between either compound
(L) and hydroxyalkyl starch or compound (D) and hydroxyalkyl starch
is achieved by reaction of the functional group Z.sub.1 with the
optionally oxidized reducing end of the hydroxyalkyl starch.
[0052] As functional group Z.sub.1, each functional group may be
used which is capable of forming a chemical linkage with the
optionally oxidized reducing end of the hydroxyalkyl starch.
[0053] According to a preferred embodiment of the present
invention, the functional group Z.sub.1 comprises the chemical
structure --NH--.
[0054] Therefore, the present invention also relates to a method as
described above wherein the functional group Z.sub.1 comprises the
structure --NH--.
[0055] According to one preferred embodiment of the present
invention, the functional group Z.sub.1 is a group having the
structure R'--NH-- where R' is hydrogen or a alkyl, cycloalkyl,
aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue
where the cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or
cycloalkylaryl residue may be linked directly to the NH group or,
according to another embodiment, may be linked by an oxygen bridge
to the NH group. The alkyl, cycloalkyl, aryl, aralkyl,
arylcycloalkyl, alkaryl, or cycloalkylaryl residues may be suitably
substituted. As preferred substituents, halogens such as F, Cl or
Br may be mentioned. Especially preferred residues R' are hydrogen,
alkyl and alkoxy groups, and even more preferred are hydrogen and
unsubstituted alkyl and alkoxy groups.
[0056] Among the alkyl and alkoxy groups, groups with 1, 2, 3, 4,
5, or 6 C atoms are preferred. More preferred are methyl, ethyl,
propyl, isopropyl, methoxy, ethoxy, propoxy, and isopropoxy groups.
Especially preferred are methyl, ethyl, methoxy, ethoxy, and
particular preference is given to methyl or methoxy.
[0057] Therefore, the present invention also relates to a method as
described above wherein R' is hydrogen or a methyl or a methoxy
group.
[0058] According to another preferred embodiment of the present
invention, the functional group Z.sub.1 has the structure
R'--NH--R''-- where R'''' preferably comprises the structure unit
--NH-- and/or the structure unit --(C=G)- where G is O or S, and/or
the structure unit --SO.sub.2--. According to more preferred
embodiments, the functional group R'' is selected from the group
consisting of
##STR00005##
where, it G is present twice, it is independently O or S.
[0059] Therefore, the present invention also relates to a method as
mentioned above wherein the functional group Z.sub.1 is selected
from the group consisting of
##STR00006##
wherein G is O or S and, if present twice, independently O or S,
and R' is methyl.
[0060] It is an object of the present invention to provide a
hydroxyalkyl starch derivative which comprises a functional group X
which is capable to react with the functional group Y of a further
compound (M) to give as reaction product a hydroxyalkyl starch
derivative which comprises the hydroxyalkyl starch, compound (L),
optionally compound (D), and the further compound.
[0061] As to functional group X, there are no specific restrictions
provided that a chemical linkage can be formed with the functional
group Y which is comprised in the further compound (M).
[0062] If the functional group Y is selected from the group
consisting of an aldehyd group, a keto group, a hemiacetal group,
and an acetal group, the functional group X, the functional group X
preferably comprises the chemical structure --NH--.
[0063] Therefore, the present invention also relates to a method as
described above wherein the functional group Y is selected from the
group consisting of an aldehyd group, a keto group, a hemiacetal
group, and an acetal group, and the functional group X comprises
the structure --NH--.
[0064] According to one preferred embodiment of the present
invention, the functional group X is a group having the structure
R'--NH-- where R' is hydrogen or a allyl, cycloalkyl, aryl,
aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue where
the cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or
cycloalkylaryl residue may be linked directly to the NH group or,
according to another embodiment, may be linked by an oxygen bridge
to the NH group. The alkyl, cycloalkyl, aryl, aralkyl,
arylcycloalkyl, alkaryl, or cycloalkylaryl residues may be suitably
substituted. As preferred substituents, halogenes such as F, Cl or
Br may be mentioned. Especially preferred residues R' are hydrogen,
alkyl and alkoxy groups, and even more preferred are hydrogen and
unsubstituted alkyl and alkoxy groups.
[0065] Among the alkyl and alkoxy groups, groups with 1, 2, 3, 4,
5, or 6 C atoms are preferred. More preferred are methyl, ethyl,
propyl, isopropyl, methoxy, ethoxy, propoxy, and isopropoxy groups.
Especially preferred are methyl, ethyl, methoxy, ethoxy, and
particular preference is given to methyl or methoxy.
[0066] Therefore, the present invention also relates to a method as
described above wherein R' is hydrogen or a methyl or a methoxy
group.
[0067] According to another preferred embodiment of the present
invention, the functional group X has the structure R'--NH--R''--
where R'''' preferably comprises the structure unit --NH-- and/or
the structure unit --(C=G)- where G is O or S, and/or the structure
unit --SO.sub.2--. According to more preferred embodiments, the
functional group R'' is selected from the group consisting of
##STR00007##
where, if G is present twice, it is independently O or S.
[0068] Therefore, the present invention also relates to a method as
mentioned above wherein the functional group X is selected from the
group consisting of
##STR00008##
wherein G is O or S and, if present twice, independently O or S,
and R' is methyl.
[0069] If the functional group Y is a thio group, the functional
group X is preferably selected from the groups consisting of
##STR00009##
wherein Hal is Cl, Br or I, preferably Br or I.
[0070] Therefore, the present invention also relates to a method as
described above wherein the functional group Y is --SH and the
functional group X is selected from the group consisting of
##STR00010##
wherein Hal is Cl, Br or I.
[0071] According to one embodiment of the present invention,
hydroxyalkyl starch is reacted with a compound (D) and the
resulting reaction product is further reacted with compound (L)
where the chemical linkage between compound (L) and the reaction
product is formed by reaction of functional group Z.sub.2 comprised
in compound (L) and functional group W comprised in compound (D)
being part of the reaction product.
[0072] Regarding the functional groups Z.sub.2 and W, there are
generally no restrictions provided that the desired chemical
linkage is formed.
[0073] As possible functional groups W or Z.sub.2, the following
functional groups are to be mentioned, among others: [0074]
C--C-double bonds or C--C-triple bonds or aromatic C--C-bonds;
[0075] the thio group or the hydroxy groups; [0076] alkyl sulfonic
acid hydrazide, aryl sulfonic acid hydrazide; [0077] 1,2-dioles;
[0078] 1,2-aminoalcohols; [0079] the amino group --NH.sub.2 or
derivatives of the amino groups comprising the structure unit
--NH-- such as aminoalkyl groups, aminoaryl group, aminoaralkyl
groups, or alkarlyaminogoups; [0080] the hydroxylamino group
--O--NH.sub.2, or derivatives of the hydroxylamino group comprising
the structure unit --O--NH--, such as hydroxylalkylamino groups,
hydroxylarylamino groups, hydroxylaralkylamino groups, or
hydroxalalkarylamino groups; [0081] alkoxyamino groups,
aryloxyamino groups, aralkyloxyamino groups, or alkaryloxyamino
groups, each comprising the structure unit --NH--O--; [0082]
residues having a carbonyl group, -Q-C(=G)-M, wherein G is O or S,
and M is, for example, [0083] --OH or --SH; [0084] an alkoxy group,
an aryloxy group, an aralkyloxy group, or an alkaryloxy group;
[0085] an alkylthio group, an arylthio group, an aralkylthio group,
or an alkarylthio group; [0086] an alkylcarbonyloxy group, an
arylcarbonyloxy group, an aralkylcarbonyloxy group, an
alkarylcarbonyloxy group; [0087] activated esters such as esters of
hydroxylamines having imid structure such as N-hydroxysuccinimide
or having a structure unit O--N where N is part of a heteroaryl
compound or, with G=O and Q absent, such as aryloxy compounds with
a substituted aryl residue such as pentafluorophenyl,
paranitrophenyl or trochlorophenyl; [0088] wherein Q is absent or
NH or a heteroatom such as S or O; [0089] --NH--NH.sub.2, or
--NH--NH--; [0090] --NO.sub.2; [0091] the nitril group; [0092]
carbonyl groups such as the aldehyde group or the keto group;
[0093] the carboxy group; [0094] the --N.dbd.C.dbd.O group or the
--N.dbd.C.dbd.S group; [0095] vinyl halide groups such as the vinyl
iodide or the vinyl bromide group or triflate; [0096]
--C.ident.C--H; [0097] --(C.dbd.NH.sub.2Cl)--OAlkyl [0098] groups
--(C.dbd.O)--CH.sub.2--Hal wherein Hal is Cl, Br, or I; [0099]
--CH.dbd.CH--SO.sub.2--; [0100] a disulfide group comprising the
structure --S--S--; [0101] the group
[0101] ##STR00011## [0102] the group
##STR00012##
[0102] where Z.sub.2 and W, respectively, is a group capable of
forming a chemical linkage with one of the above-mentioned
groups.
[0103] According to preferred embodiments of the present invention,
both W and Z.sub.2 are groups from the list of groups given
above.
[0104] According to a first especially preferred embodiment of the
present invention, Z.sub.2 or W is a thio group. In this particular
case, the functional group W is preferably selected from the group
consisting of
##STR00013##
wherein Hal is Cl, Br, or I, preferably Br or I.
[0105] Therefore, the present invention also relates to a method as
described above wherein the functional group W or the functional
group Z.sub.2 is --SH and the functional group Z.sub.2 or the
functional group W is selected from the group consisting of
##STR00014##
wherein Hal is Cl, Br, or I.
[0106] According to a second especially preferred embodiment of the
present invention, Z.sub.2 or W is selected from the group
consisting of an activated ester, as described above, or a carboxy
group which is optionally transformed into an activated ester. In
this particular case, the functional group W or Z.sub.2,
respectively, comprises the chemical structure --NH--.
[0107] Therefore, the present invention also relates to a method as
described above wherein Z.sub.2 or W is selected from the group
consisting of an activated ester, as described above, or a carboxy
group which is optionally transformed into an activated ester, and
the functional group W or Z.sub.2, respectively, comprises the
chemical structure --NH--.
[0108] According to one preferred embodiment of the present
invention, the functional group W or Z.sub.2 comprising the
structure --NH-- is a group having the structure R'--NH-- where R'
is hydrogen or a alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl,
alkaryl or cycloalkylaryl residue where the cycloalkyl, aryl,
aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue may be
linked directly to the NH group or, according to another
embodiment, may be linked by an oxygen bridge to the NH group. The
alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl, or
cycloalkylaryl residues may be suitably substituted. As preferred
substituents, halogenes such as F, Cl or Br may be mentioned.
Especially preferred residues R' are hydrogen, alkyl and alkoxy
groups, and even more preferred are hydrogen and unsubstituted
alkyl and alkoxy groups.
[0109] Among the alkyl and alkoxy groups, groups with 1, 2, 3, 4,
5, or 6 C atoms are preferred. More preferred are methyl, ethyl,
propyl, isopropyl, methoxy, ethoxy, propoxy, and isopropoxy groups.
Especially preferred are methyl, ethyl, methoxy, ethoxy, and
particular preference is given to methyl or methoxy.
[0110] Therefore, the present invention also relates to a method as
described above wherein W or Z.sub.2 is selected from the group
consisting of an activated ester, as described above, or a carboxy
group which is optionally transformed into an activated ester, and
the functional group W or Z.sub.2, respectively, is R'--NH--
wherein R' is hydrogen or a methyl or a methoxy group.
[0111] According to another preferred embodiment of the present
invention, the functional group W or Z.sub.2 has the structure
R'--NH--R''-- where R'' preferably comprises the structure unit
--NH-- and/or the structure unit --(C=G)- where G is O or S, and/or
the structure unit --SO.sub.2--. According to more preferred
embodiments, the functional group R'' is selected from the group
consisting of
##STR00015##
where, if G is present twice, it is independently O or S.
[0112] Therefore, the present invention also relates to a method as
mentioned above wherein the functional group W or Z.sub.2 is
selected from the group consisting of
##STR00016##
wherein G is O or S and, if present twice, independently O or S,
and R' is methyl.
[0113] According to yet another aspect of the present invention,
the at least one functional group X, Z.sub.2 and/or W may be a
group which is not capable of reacting directly with a given
further compound, but which may be chemically modified in order to
be capable of reacting in the desired way.
[0114] As an example of a functional group to be modified prior to
the reaction with a further compound, a 1,2-amino alcohol or a
1,2-diol may be mentioned which is modified, e.g., by oxidation to
form an aldehyd or a keto group.
[0115] Another example for a functional group to be modified prior
to the reaction with a further compound is a --NH.sub.2 group which
is modified by the reaction with, e.g., a compound according to the
following formula
##STR00017##
to give a structure of the following formula
##STR00018##
which is, e.g., reactive towards a thio group.
[0116] Another example for a functional group to be modified prior
to the reaction with a further compound is a --NH.sub.2 group which
is modified by the reaction with, e.g., a compound according to the
following formula
##STR00019##
to give a structure of the following formula
##STR00020##
which is, e.g., reactive towards a thio group.
[0117] Yet another example for a functional group to be modified
prior to the reaction with a further compound is an amino group
which is reacted with bromoacetic anhydride or N-succinimidyl iodo
acetate.
[0118] According to a preferred embodiment of the present
invention, a compound (L) has the structure Z.sub.1-L'X or
Z.sub.2-L' X, L' being an organic residue separating the functional
groups and being optionally absent, the structure depending on
whether a compound (D) is reacted with the hydroxyalkyl starch or
not.
[0119] According to a first preferred embodiment, no compound (D)
is involved and Y is selected from the group consisting of an
aldehyd group, a keto group, a hemiacetal group, and an acetal
group.
[0120] In this particular case, the following compounds, among
others, are preferred as compound (L) having the structure
Z.sub.1-L'-X where L' is absent:
##STR00021##
[0121] If, in this particular case, L' is not absent, L' may be a
linear or branched alkyl or cycloalkyl of aryl or aralkyl or
arylcycloalkyl or alkaryl or cycloalkylaryl group, wherein L' may
comprise at least one heteroatom such as N, O, S, and wherein L'
may be suitably substituted. The size of the group L' may be
adapted to the specific needs. Generally, the separating group L'
generally has from 1 to 60, preferably from 1 to 40, more
preferably from 1 to 20, more preferably from 1 to 10, more
preferably from 1 to 6 and especially preferably from 1 to 4 carbon
atoms. If heteroatoms are present, the separating group comprises
generally from 1 to 20, preferably from 1 to 8 and especially
preferably from 1 to 4 heteroatoms. According to particularly
preferred embodiments of the present invention, the separating
group L' comprises 1 to 4 oxygen atoms. The separating group L' may
comprise an optionally branched alkyl chain or an aryl group or a
cycloalkyl group having, e.g., from 5 to 7 carbon atoms, or be a
aralkyl group, an alkaryl group where the alkyl part may be a
linear and/or cyclic alkyl group. According to an even more
preferred embodiment, the separating group is an alkyl chain of
from 1 to 20, preferably from 1 to 8, more preferably from 1 to 6,
more preferably from 1 to 4 and especially preferably from 2 to 4
carbon atoms. In case heteroatoms are present, a chain comprising 1
to 4 oxygen atoms is particularly, preferred.
[0122] As to this particular case where Y is selected from the
group consisting of an aldehyd group, a keto group, a hemiacetal
group, and an acetal group, the following compounds, among others,
are preferred as compound (L) having the structure Z.sub.1-L'-X
where L' is not absent:
##STR00022##
[0123] According to a second preferred embodiment, a compound (D)
is involved.
[0124] According to a further preferred embodiment of the present
invention, a compound (D) has the structure Z.sub.1-D'-W, D' being
an organic residue separating the functional groups and being
optionally absent.
[0125] In this particular case, the following compounds, among
others, are preferred as compound (D) having the structure
Z.sub.1-D'-W where D' is absent:
##STR00023##
[0126] A specific example of a compound D where D' is absent is
NH.sub.3.
[0127] If, in this particular case, D' is not absent, D' may be a
linear or branched alkyl or cycloalkyl or aryl or aralkyl or
arylcycloalkyl or alkaryl or cycloalkylaryl group, wherein D' may
comprise at least one heteroatom such as N, O, S, and wherein D'
may be suitably substituted. The size of the group D' may be
adapted to the specific needs. Generally, the separating group D'
generally has from 1 to 60, preferably from 1 to 40, more
preferably from 1 to 20, more preferably from 1 to 10, more
preferably from 1 to 6 and especially preferably from 1 to 4 carbon
atoms. If heteroatoms are present, the separating group comprises
generally from 1 to 20, preferably from 1 to 8 and especially
preferably from 1 to 4 heteroatoms. According to particularly
preferred embodiments of the present invention, the separating
group D' comprises 1 to 4 oxygen atoms. The separating group D' may
comprise an optionally branched alkyl chain or an aryl group or a
cycloalkyl group having, e.g., from 5 to 7 carbon atoms, or be an
aralkyl group, an alkaryl group where the alkyl part may be a
linear and/or cyclic alkyl group. According to an even more
preferred embodiment, the separating group is an alkyl chain of
from 1 to 20, preferably from 1 to 8, more preferably from 1 to 6,
more preferably from 1 to 4 and especially preferably from 2 to 4
carbon atoms. In case heteroatoms are present, a chain comprising 1
to 4 oxygen atoms is particularly preferred.
[0128] As to this particular case, preferred compounds (D) having
the structure Z.sub.1-D'-W where D' is not absent are:
##STR00024##
[0129] Depending on the chemical nature of the functional group W
comprised in compound (D) and the functional group Y, specific
compounds (L) may be used according to the specific needs.
[0130] If, e.g., the functional group Y is a thio group and the
functional group W comprises the structure --NH--, as described
above in detail, the following types of compounds (L) are, among
others, preferred:
TABLE-US-00001 Type of compound (L) Functional group X Functional
group Z.sub.2 C Iodoalkyl N-succinimide ester D Bromoalkyl
N-succinimide ester E Maleimido N-succinimide ester F
Pydridyldithio N-succinimide ester G Vinylsulfone N-succinimide
ester
[0131] If, e.g., the functional group Y is selected from the group
consisting of an aldehyd group, a keto group, a hemiacetal group,
and an acetal group, and the functional group W is a thio group,
the following types of compounds (L) are, among others,
preferred:
TABLE-US-00002 Type of compound (L) Functional group X Functional
group Z.sub.2 A Hydrazide Maleimido B Hydrazide Pyridyldithio
[0132] In Table 1 at the end of the present description, some
preferred examples of compounds (L) according to the types given
above are listed.
[0133] The separating groups L' and/or D' may be suitably
substituted. Preferred substituents are, e.g, halides such as F,
Cl, Br or I.
[0134] The separating groups L' and/or D' may comprise one or more
cleavage sites such as
##STR00025##
which allow for an easy cleavage of a resulting compound at a
pre-determined site.
[0135] Especially preferred examples of compounds (L) which may be
linked to hydroxyalkyl starch wherein the resulting hydroxyalkyl
starch derivative comprises the functional group X capable of being
reacted with a functional group Y comprised in a further compound
(M) and wherein said functional group Y is selected from the group
consisting of an aldehyd group, a keto group, a hemiacetal group,
an acetal group, are
##STR00026##
the compounds (L)
##STR00027##
being particularly preferred.
[0136] Especially preferred examples of compounds (D) which may be
linked to hydroxyalkyl starch wherein the resulting hydroxyalkyl
starch derivative comprises the functional group W capable of being
reacted with a functional group Z.sub.2 comprised in a compound (L)
wherein the resulting hydroxyallyl starch derivative which
comprises hydroxyalkyl starch, compound (D) and compound (L), is
capable of being reacted with the functional group Y of a further
compound (M) and wherein said functional group Y is a thio group,
are
##STR00028##
the compounds (D)
##STR00029##
being particularly preferred.
[0137] Together with the above-mentioned preferred compounds (D),
the following compounds (L)
##STR00030##
are preferred, the compound (L)
##STR00031##
being especially preferred.
[0138] According to a first preferred embodiment of the present
invention, a compound (D) or a compound (L) is reacted with the
reducing end of the hydroxyalkyl starch which is not oxidised.
[0139] Depending on the reaction conditions such as the solvent or
solvent mixture used, the temperature, pressure or pH of the
reaction mixture, the reaction product of a compound (D) or a
compound (L) is reacted with the reducing end of the hydroxyalkyl
starch which is not oxidised may have different constitutions.
[0140] According to a preferred embodiment of the present
invention, this reaction is carried out in an aqueous system.
[0141] The term "aqueous system" as used in the context of the
present invention refers to a solvent or a mixture of solvents
comprising water in the range of from at least 10% per weight,
preferably at least 50% per weight, more preferably at least 80%
per weight, even more preferably at least 90% per weight or up to
100% per weight, based on the weight of the solvents involved. As
additional solvents, solvents such as DMSO, DMF, ethanol or
methanol may be mentioned.
[0142] If the reaction is carried out in an aqueous system and the
functional group Z.sub.1 is a group R'--NH--, as described above,
the hydroxyalkyl starch derivative may have a constitution
according to formula (IIIa)
##STR00032##
[0143] If the reaction is carried out in an aqueous system and the
functional group Z.sub.1 is a group R'--NH-- with R'.dbd.H, as
described above, the hydroxyalkyl starch derivative may have a
constitution according to formula (IIIa) or formula (IIIb) or be a
mixture of compounds according to formulae (IIIa) and (IIIb)
##STR00033##
[0144] Depending on the reaction conditions and/or the chemical
nature of compounds (L) or compound (D) used for the reaction, the
compounds according to formula (IIIa) may be present with the N
atom in equatorial or axial position where also a mixture of both
forms may be present having a certain equilibrium distribution.
[0145] Depending on the reaction conditions and/or the chemical
nature of compounds (L) or (D) used for the reaction, the compounds
according to formula (IIIb) may be present with the C--N double
bond in E or Z conformation where also a mixture of both forms may
be present having a certain equilibrium distribution.
[0146] Therefore, the present invention also relates to a
hydroxyalkyl starch derivative as described above having a
constitution according to formula (IIIb) or according to formula
(IIIb) or according to formulae (IIIa) and (IIIb).
[0147] In some cases it may be desirable to stabilize the compound
according to formula (IIIa). This is especially the case where the
compound according to formula (IIIa) is produced and/or used in an
aqueous solution. As stabilizing method, acylation of the compound
according to formula (IIIa) is particularly preferred, especially
in the case where R' is hydrogen. As acylation reagent, all
suitable reagents may be used which result in the desired
hydroxyalkyl starch derivative according to formula (IVa)
##STR00034##
[0148] According to especially preferred embodiments of the present
invention, the residue Ra being part of the acylation reagent is
methyl. As acylation reagents, carboxylic acid anhydrides,
carboxylic acid halides, and carboxylic acid active esters are
preferably used.
[0149] The acylation is carried at a temperature in the range of
from 0 to 30.degree. C., preferably in the range of from 2 to
20.degree. C. and especially preferably in the range of from 4 to
10.degree. C.
[0150] Therefore, the present invention also relates to a
hydroxyalkyl starch derivate obtainable by a method as described
above wherein said derivative has a constitution according to
formula (IVa).
[0151] In other cases it may be desirable to stabilize the compound
according to formula (IIIb). This is especially the case where the
compound according to formula (IIIb) is produced and/or used in an
aqueous solution. As stabilizing method, reduction of the compound
according to formula (IIIb) is particularly preferred, especially
in the case where R' is hydrogen. As reduction reagent, all
suitable reagents may be used which result in the desired
hydroxyalkyl starch derivative according to formula (IVb)
##STR00035##
[0152] According to especially preferred embodiments of the present
invention, as reduction reagents boro hydrides such as NaCNBH.sub.3
or NaBH.sub.4 are used.
[0153] The reduction is carried at a temperature in the range of
from 4 to 100.degree. C., preferably in the range of from 10 to
90.degree. C. and especially preferably in the range of from 25 to
80.degree. C.
[0154] Therefore, the present invention also relates to a
hydroxyalkyl starch derivate obtainable by a method as described
above wherein said derivative has a constitution according to
formula (IVb).
[0155] The present invention further relates to mixtures of
compounds having constitutions according to formulae (IIIa) and
(IIIb), (IVa) and (IVb), (IIIa) and (IVa), (IIIa) and (IVb), (IIIb)
and (IVa), (IIIb) and (IVb), (IIIa) and (IIIb) and (IVa), (IIIa)
and (IIIb) and (IVb), (IVa) and (IVb) and (IIIa), and (IVa) and
(IVb) and (IIIb) wherein (IIIa) and/or (IVa) may be independently
present in a conformation where the N atom in equatorial or axial
position and/or wherein (IIIb) may be present with the C--N double
bond in E or Z conformation.
[0156] According to a second preferred embodiment of the present
invention, a compound (D) or a compound (L) is reacted with the
reducing end of the hydroxyalkyl starch which is oxidised.
[0157] In this case, preferably polar aprotic solvents are used
which may also contain a certain amount of water, such as up to 10
wt.-%. Preferred aprotic solvents are, among others, DMSO or DMF.
An example of a preferred reaction temperature range is from room
20 to 65.degree. C., and the reaction times are generally in the
range of 1 minute to several hours and up to several days,
depending on the chemical nature of the functional group which is
reacted with the oxidized reducing end og the hydroxyalkyl starch
and the other reaction conditions.
[0158] If, in this case, the functional group Z.sub.1 is a group
R'--NH--, as described above, the hydroxyalkyl starch derivative
may have a constitution according to formula (Va)
##STR00036##
[0159] Therefore, the present invention also relates to a
hydroxyalkyl starch derivate obtainable by a method as described
above wherein said derivative has a constitution according to
formula (Va).
[0160] As far as the reactions of hydroxyalkyl starch with compound
(D) and/or compound (L) as well as compound (M) are concerned, all
possible sequences are comprised by the present invention.
[0161] A preferred embodiment of the present invention relates to a
method as described above wherein hydroxyalkyl starch is reacted
with a compound (L) via the reaction of functional group Z.sub.1
with the optionally oxidized reducing end of the hydroxyalkyl
starch and the resulting reaction product is reacted with a further
compound (M) via the reaction of the functional group X comprised
in compound (L) with the functional group Y comprised in compound
(M).
[0162] Another embodiment of the present invention relates to a
method as described above wherein hydroxyalkyl starch is reacted
with a compound (L) via the reaction of functional group Z.sub.1
with the optionally oxidized reducing end of the hydroxyalkyl
starch, where compound (L), prior to the reaction with hydroxyalkyl
starch, is reacted with a further compound (M) via the reaction of
functional group X comprised in compound (L) with the functional
group Y comprised in compound (M).
[0163] Still another embodiment of the present invention relates to
a method as described above wherein hydroxyallyl starch is reacted
with a compound (D) via the reaction of the functional group
Z.sub.1 comprised in compound (D), with the optionally oxidized
reducing end of the hydroxyalkyl starch to give a first
hydroxyalkyl starch derivative, and where the first hydroxyalkyl
starch derivative is reacted with a compound (L) via the reaction
of functional group Z.sub.2 comprised in compound (L) with the
functional group W comprised in compound (D) to give a second
hydroxyalkyl starch derivative.
[0164] Yet another embodiment of the present invention relates to
the latter method wherein the second hydroxyalkyl starch derivative
is reacted with a further compound (M) via the reaction of
functional group X comprised in compound (L) with the functional
group Y comprised in compound (M).
[0165] Still yet another embodiment of the present invention
relates to a method as described above wherein hydroxyalkyl starch
is reacted with a compound (D) via the reaction of functional group
Z.sub.1 comprised in compound (D) with the optionally oxidized
reducing end of the hydroxyalkyl starch to give a first
hydroxyalkyl starch derivative, and where the first hydroxyalkyl
starch derivative is reacted, via the reaction of the functional
group W, comprised in compound (D), and the functional group
Z.sub.2, comprised in compound (L), with compound (L), where
compound (L), prior to the reaction with the first hydroxyalkyl
starch derivative, is reacted with a further compound (M) via the
reaction of functional group X comprised in compound (L) with the
functional group Y comprised in compound (M).
[0166] As far as the reaction conditions of each of the
above-described reactions steps are concerned, all parameters such
as temperature, pressure, pH, or solvent or solvent mixture may be
adapted to the specific needs and the chemical nature of compounds
to be reacted.
[0167] According to an especially preferred embodiment of the
present invention, water is used as solvent, either alone or in
combination with at least one other solvent. As at least one other
solvent, DMSO, DMF, methanol and ethanol may be mentioned.
Preferred solvents other than water are DMSO, DMF, methanol and
ethanol. In this embodiment, hydroxylalkyl starch is preferably
reacted via the non-oxidized reducing end.
[0168] If hydroxyalkyl starch is reacted with compound (D) or
compound (L) in an aqueous medium and compound (D) or compound (L)
is a hydroxylamine or a hydrazide, the temperature of the reaction
is preferably in the range of from 5 to 45.degree. C., more
preferably in the range of from 10 to 30.degree. C. and especially
preferably in the range of from 15 to 25.degree. C.
[0169] If hydroxyalkyl starch is reacted with compound (D) or
compound (L) in an aqueous medium and the reaction being a
reductive amination, the temperature is preferably in the range of
up to 100.degree. C., more preferably in the range of from 70 to
90.degree. C. and especially preferably in the range of from 75 to
85.degree. C.
[0170] During the course of the reaction the temperature may be
varied, preferably in the above-given ranges, or held essentially
constant.
[0171] The reaction time for the reaction of hydroxyalkyl starch
with compound (D) or compound (L) may be adapted to the specific
needs and is generally in the range of from 1 h to 7 d.
[0172] In case compound (D) or compound (L) is a hydroxylamine or a
hydrazide, the reaction time is preferably in the range of from 1 h
to 3 d and more preferably of from 2 h to 48 h.
[0173] In case the reaction of hydroxyalkyl starch with compound
(D) or compound (L) is a reductive amination, the reaction time is
preferably in the range of from 2 h to 7 d.
[0174] The pH value for the reaction of hydroxyalkyl starch with
compound (D) or compound (L) may be adapted to the specific needs
such as the chemical nature of the reactants.
[0175] In case compound (D) or compound (L) is a hydroxylamine or a
hydrazide, the pH value is preferably in the range of from 4.5 to
6.5.
[0176] In case the reaction of hydroxyalkyl starch with compound
(D) or compound (L) is a reductive amination, the pH value is
preferably in the range of from 8 to 12.
[0177] The suitable pH value of the reaction mixture may be
adjusted, for each reaction step, by adding at least one suitable
buffer. Among the preferred buffers, sodium acetate buffer,
phosphate or borate buffers may be mentioned.
[0178] If necessary, the at least one functional group X may be
protected with at least one suitable protecting group prior to the
reaction of hydroxyalkyl starch with compound (L) or prior to the
reaction of compound (D) with compound (L) or prior to the reaction
of compound (L) with the reaction product of the reaction of
hydroxyalkyl starch with compound (D). In this respect, all
conceivable protecting groups are possible which prevent the
protected compound (L) from reacting via the at least one
functional group X. Hence, the protecting group may be chosen
depending from the chemical nature of the functional group X to be
protected, from, e.g., the solvent the reaction is carried out in
or the pH of the reaction mixture. Preferred protecting groups are,
among others, the benzyloxycarbonyl group, the tert-butoxycarbonyl
group, the methoxyphenyl group, the 2,4-dimethoxyphenyl group,
triarly methyl groups, trityl, the monomethoxytrityl group, the
dimethoxytrityl group, the monomethyltrityl group, the
dimethyltrityl group, the trifluoracetyl group, phthalimin
compounds, 2-(trialkylsilyl)ethoxy carbonyl compounds, Fmoc, the
tert-butyl group, or trialkyl silyl groups.
[0179] If two or more different functional groups X are present in
compound (L), at least one group may be protected whereas at least
one other group may be left unprotected.
[0180] After the reaction of compound (L), the at least one
protecting group may be left in the reaction product or removed by
suitable methods such as conventional methods known to the person
skilled in the art. If two different functional groups X are
protected by suitable protecting groups, it is possible to remove
at least one protecting group so as to make at least one functional
group X available for further reaction with at least one further
compound (M), and leave at least one other functional group
protected until the reaction product comprising compound (L) is
reacted with the further compound (M). Afterwards, the protecting
group of the functional group still protected may be removed to
make the remaining functional group X available for reaction with
yet a further compound (M).
[0181] The use of at least one protecting group may be important
for preventing the reaction from resulting in a hydroxyalkyl starch
derivative comprising a compound (L) or compound (D) which has been
reacted with two or more hydroxyalkyl starch molecules, i.e. a
multiple HAS substituted compound (L) or (D). The same result,
however, may be achieved by reacting hydroxyalkyl starch with an
excess of compound (L) or (D). If an excess amount of compound (L)
or (D) is used in the process of the present invention, the molar
ratio of compound (L) or (D) to hydroxyalkyl starch is preferably
in the range of from 2 to 100.
[0182] Once the reaction product of the respective reaction step,
as described above, is formed, it may be isolated from the reaction
mixture by at least one suitable method. If necessary, the reaction
product may be precipitated prior to the isolation by at least one
suitable method.
[0183] If the reaction product is precipitated first, it is
possible, e.g., to contact the reaction mixture with at least one
solvent or solvent mixture other than the solvent or solvent
mixture present in the reaction mixture at suitable temperatures.
According to a particularly preferred embodiment of the present
invention where an aqueous system is used as solvent, the reaction
mixture is contacted with a mixture of ethanol and acetone,
preferably a 1:1 mixture, indicating equal volumes of said
compounds, at a temperature, preferably in the range of from -20 to
+50.degree. C. and especially preferably in the range of from 0 to
25.degree. C.
[0184] Isolation of the reaction product may be carried out by a
suitable process which may comprise one or more steps. According to
a preferred embodiment of the present invention, the reaction
product is first separated off the reaction mixture or the mixture
of the reaction mixture with, e.g., the ethanol-acetone mixture, by
a suitable method such as centrifugation or filtration. In a second
step, the separated reaction product may be subjected to a further
treatment such as an after-treatment like dialysis, centrifugal
filtration or pressure filtration, ion exchange chromatography,
HPLC, MPLC, gel filtration and/or lyophilisation. According to an
even more preferred embodiment, the separated reaction product is
first dialysed, preferably against water, and then lyophilized
until the solvent content of the reaction product is sufficiently
low according to the desired specifications of the product.
Lyophilisation may be carried out at temperature of from 20 to
35.degree. C., preferably of from 25 to 30.degree. C.
[0185] According to preferred embodiments of the present invention,
the hydroxyalkyl starch derivative comprising hydroxyalkyl starch
and compound (L) or comprising hydroxyalkyl starch, compound (D)
and compound (L) is further reacted with the further compound (M)
which comprises at least one functional group Y.
[0186] Generally, there are no limitations regarding compound (M).
Preferably, a polypeptide is used as compound (M) in the context of
the present invention. However, other compounds (M) are also
possible, either polymers or oligomers or monomolecular compounds
or mixtures of two or more thereof.
[0187] The term "polypeptide" as used in the context of the present
invention refers to a compound which comprises at least 2 amino
acids which are linked via a peptide bond, i.e. a bond with
structure --(C.dbd.O)--NH--. The polypeptide may be a naturally
occurring compound or a polypeptide which does not occur naturally,
the latter comprising naturally occurring amino acids and/or at
least one amino acid which does not naturally occur. The backbone
of the polypeptide, the polypeptide chain, may be further
substituted with at least one suitable substituent thus having at
least one side-chain. The at least one functional group Y may be
part of the polypeptide backbone or of at least one substituent of
the backbone wherein embodiments are possible comprising at least
one functional group being part of the polypeptide backbone and at
least one functional group being part of at least one substituent
of the polypeptide backbone.
[0188] As far as the polypeptide is concerned, there exist no
restrictions, given that the polypeptide comprises at least one
functional group Y. Said functional group Y may be linked directly
to the polypeptide backbone or be part of a side-chain of the
backbone. Either side-chain or functional group Y or both may be
part of a naturally occurring polypeptide or may be introduced into
a naturally occurring polypeptide or into a polypeptide which, at
least partially, does not occur naturally, prior to the reaction
with the functional group X.
[0189] Moreover, the polypeptide can be, at least partly, of any
human or animal source. In a preferred embodiment, the polypeptide
is of human source.
[0190] The polypeptide may be a cytokine, especially
erythropoietin, an antithrombin (AT) such as AT III, an
interleukin, especially interleukin-2, IFN-beta, IFN-alpha, G-CSF,
CSF, interleukin-6 and therapeutic antibodies.
[0191] According to a preferred embodiment, the polypeptide is an
antithrombin (AT), preferably AT III (Levy J H, Weisinger A, Ziomek
C A, Echelard Y, Recombinant Antithrombin: Production and Role in
Cardiovascular Disorder, Seminars in Thrombosis and Hemostasis 27,
4 (2001) 405-416; Edmunds T, Van Patten S M, Pollock J, Hanson E,
Bernasconi R, Higgins E, Manavalan P, Ziomek C, Meade H, McPherson
J, Cole E S, Transgenically Produced Human Antithrombin: Structural
and Functional Comparison to Human Plasma-Derived Antithrombin,
Blood 91, 12 (1998) 4661-4671; Minnema M C, Chang A C K, Jansen P
M, Lubbers Y T P, Pratt B M, Whittaker B G, Taylor F B, Hack C E,
Friedman B, Recombinant human antithrombin III improves survival
and attenuates inflammatory responses in baboons lethally
challenged with Escherichia coli, Blood 95, 4 (2000) 1117-1123; Van
Patten S M, Hanson E H, Bernasconi R., Zhang K, Manavaln P, Cole E
S, McPherson J M, Edmunds T, Oxidation of Methionine Residues in
Antithrombin, J. Biol. Chemistry 274, 15 (1999) 10268-10276).
[0192] According to another preferred embodiment, the polypeptide
is human IFN-beta, in particular IFN-beta 1a (cf. Avonex.RTM.,
REBIF.RTM.) and IFN-beta 1b (cf. BETASERON.RTM.).
[0193] A further preferred polypeptide is human G-CSF (granulocyte
colony stimulating factor). See, e.g., Nagata et al., The
chromosomal gene structure and two mRNAs for human granulocyte
colony-stimulating factor, EMBO J. 5: 575-581, 1986; Souza et al.,
Recombinant human granulocyte colony-stimulating factor: effects on
normal and leukemic myeloid cells, Science 232 (1986) 61-65; and
Herman et al., Characterization, formulation, and stability of
Neupogen.RTM. (Filgrastim), a recombinant human granulocyte-colony
stimulating factor, in: Formulation, characterization, and
stability of protein drugs, Rodney Pearlman and Y. John. Wang,
eds., Plenum Press, New York, 1996, 303-328.
[0194] If a mixture of at least two different polypeptides is used,
the at least two polypeptides may differ, e.g., in the molecular
mass, the number and/or sequence of amino acids, the number and/or
chemical nature of the substituents or the number of polypeptide
chains linked by suitable chemical bonds such as disulfide
bridges.
[0195] According to a preferred embodiment of the present
invention, the reaction product of hydroxyalkyl starch and compound
(L) or the reaction product of hydroxyalkyl starch and compound (D)
which is further reacted with compound (L) is isolated, preferably
according to at least one of the above-mentioned processes, and
then reacted with a polypeptide having at least one functional
group Y. According to a preferred embodiment of the present
invention, the functional group Y is comprised in a carbohydrate
moiety of the polypeptide.
[0196] In the context of the present invention, the term
"carbohydrate moiety" refers to hydroxyaldehydes or hydroxyketones
as well as to chemical modifications thereof (see Rompp
Chemielexikon, Thieme Verlag Stuttgart, Germany, 9.sup.th edition
1990, Volume 9, pages 2281-2285 and the literature cited therein).
Furthermore, it also refers to derivatives of naturally occurring
carbohydrate moieties like glucose, galactose, mannose, sialic acid
and the like. The term also includes chemically oxidized, naturally
occurring carbohydrate moieties. The structure of the oxidized
carbohydrate moiety may be cyclic or linear.
[0197] The carbohydrate moiety may be linked directly to the
polypeptide backbone. Preferably, the carbohydrate moiety is part
of a carbohydrate side chain. More preferably, the carbohydrate
moiety is the terminal moiety of the carbohydrate side chain.
[0198] In an even more preferred embodiment, the carbohydrate
moiety is a galactose residue of the carbohydrate side chain,
preferably the terminal galactose residue of the carbohydrate side
chain. This galactose residue can be made available for reaction
with the functional group X comprised in the reaction product of
hydroxyalkyl starch and compound (L) or the reaction product of
hydroxyalkyl starch and compound (D) which is further reacted with
compound (L), by removal of terminal sialic acids, followed by
oxidation, as described hereinunder.
[0199] In a still further preferred embodiment, the reaction
product of hydroxyalkyl starch and compound (L) or the reaction
product of hydroxyalkyl starch and compound (D) which is further
reacted with compound (L) is linked to a sialic acid residue of the
carbohydrate side chains, preferably the terminal sialic acid
residue of the carbohydrate side chain.
[0200] Oxidation of terminal carbohydrate moieties can be performed
either chemically or enzymatically.
[0201] Methods for the chemical oxidation of carbohydrate moieties
of polypeptides are known in the art and include the treatment with
perjodate (Chamow et al., 1992, J. Biol. Chem., 267,
15916-15922).
[0202] By chemically oxidizing, it is in principle possible to
oxidize any carbohydrate moiety, being terminally positioned or
not. However, by choosing mild conditions (1 mM periodate,
0.degree. C. in contrast to harsh conditions: 10 mM periodate 1 h
at room temperature), it is possible to preferably oxidize the
terminal sialic acid of a carbohydrate side chain.
[0203] Alternatively, the carbohydrate moiety may be oxidized
enzymatically. Enzymes for the oxidation of the individual
carbohydrate moieties are known in the art, e.g. in the case of
galactose the enzyme is galactose oxidase. If it is intended to
oxidize terminal galactose moieties, it will be eventually
necessary to remove terminal sialic acids (partially or completely)
if the polypeptide has been produced in cells capable of attaching
sialic acids to carbohydrate chains, e.g. in mammalian cells or in
cells which have been genetically modified to be capable of
attaching sialic acids to carbohydrate chains. Chemical or
enzymatic methods for the removal of sialic acids are known in the
art (Chaplin and Kennedy (eds.), 1996, Carbohydrate Analysis: a
practical approach, especially Chapter 5 Montreuill, Glycoproteins,
pages 175-177; IRL Press Practical approach series (ISBN
0-947946-44-3)).
[0204] According to another preferred embodiment of the present
invention, the functional group of the polypeptide is the thio
group. Therefore, the reaction product of hydroxyalkyl starch and
compound (L) or the reaction product of hydroxyalkyl starch and
compound (D) which is further reacted with compound (L) may be
linked to the polypeptide via a thioether group wherein the S atom
can be derived from any thio group comprised in the
polypeptide.
[0205] In the context of this embodiment, it is particularly
preferred to react the polypeptide with a reaction product of
hydroxyalkyl starch and compound (D) which is further reacted with
compound (L).
[0206] Therefore, the present invention also relates to a method as
described above wherein the reaction product of hydroxyalkyl starch
and compound (D) is further reacted with compound (L) is reacted
with the polypeptide via a thio group comprised in the
polypeptide.
[0207] Therefore, the present invention also relates to a method as
described above wherein the reaction product of hydroxyalkyl starch
and compound (D) which is further reacted with compound (L) is
reacted with the polypeptide via an oxidized carbohydrate moiety
and a thio group comprised in the polypeptide.
[0208] The thio group may be present in the polypeptide as such.
Moreover, it is possible to introduce a thio group into the
polyeptide according to a suitbale method. Among others, chemical
methods may be mentioned. If a disulfide bridge is present in the
polypeptide, it is possible to reduce the --S--S-- structure to get
a thio group. It is also possible to transform an amino group
present in the polypeptide into a SH group by reaction the
polypeptide via the amino group with a compound which has at least
two different functional groups, one of which is capable of being
reacted with the amino group and the other is an SH group or a
precursor of an SH group. This modification of an amino group may
be regarded as an example where the protein is first reacted with a
compound (L) which has at least two different functional groups,
one of which is capable of being reacted with the amino group and
the other is an SH group, and the resulting reaction product is
then reacted with, e.g., a HAS derivative comprising HAS and a
compound (D), said derivative comprising a functional group being
capable of reacting with the SH group. It is also possible to
introduce an SH group by mutation of the polypeptide such as by
introducing a cystein or a suitable SH functional amino acid into
the polypeptide or such as removing a cystein from the polypeptide
so as to disable another cystein in the polypeptide to form a
disulfide bridge.
[0209] As an especially preferred polypeptide, erythropoietin (EPO)
is used.
[0210] Therefore, the present invention also relates to a method as
described above wherein the polypeptide is erythropoietin.
[0211] The EPO can be of any human (see e.g. Inoue, Wada, Takeuchi,
1994, An improved method for the purification of human
erythropoietin with high in vivo activity from the urine of anemic
patients, Biol. Pharm. Bull. 17(2), 180-4; Miyake, Kung,
Gold-wasser, 1977, Purification of human erythropoietin., J. Biol.
Chem., 252(15), 5558-64) or another mammalian source and can be
obtained by purification from naturally occurring sources like
human kidney, embryonic human liver or animal, preferably monkey
kidney. Furthermore, the expression "erythropoietin" or "EPO"
encompasses also an EPO variant wherein one or more amino acids
(e.g. 1 to 25, preferably 1 to 10, more preferred 1 to 5, most
preferred 1 or 2) have been exchanged by another amino acid and
which exhibits erythropoietic activity (see e.g. EP 640 619 B1).
The measurement of erythropoietic activity is described in the art
(for measurement of activity in vitro see e.g. Fibi et al., 1991,
Blood, 77, 1203 ff; Kitamura et al, 1989, J. Cell Phys., 140,
323-334; for measurement of EPO activity in vivo see Ph. Eur. 2001,
911-917; Ph. Eur. 2000, 1316 Erythropoietini solutio concentrata,
780-785; European Pharmacopoeia (1996/2000); European
Pharmacopoeia, 1996, Erythropoietin concentrated solution,
Pharmaeuropa., 8, 371-377; Fibi, Hermentin, Pauly, Lauffer,
Zettlmeissl., 1995, N- and O-glycosylation muteins of recombinant
human erythropoietin secreted from BHK-21 cells, Blood, 85(5),
1229-36; (EPO and modified EPO forms were injected into female NMRI
mice (equal amounts of protein 50 ng/mouse) at day 1, 2 and 3 blood
samples were taken at day 4 and reticulocytes were determined)).
Further publications where tests for the measurement of the
activity of EPO are Barbone, Aparicio, Anderson, Natarajan,
Ritchie, 1994, Reticulocytes measurements as a bioassay for
erythropoietin, J. Pharm. Biomed. Anal., 12(4), 515-22; Bowen,
Culligan, Beguin, Kendall, Villis, 1994, Estimation of effective
and total erythropoiesis in myelodysplasia using serum transferrin
receptor and erythropoietin concentrations, with automated
reticulocyte parameters, Leukemi, 8(1), 151-5; Delorme, Lorenzini,
Giffin, Martin, Jacobsen, Boone, Elliott, 1992, Role of
glycosylation on the secretion and biological activity of
erythropoietin, Biochemistry, 31(41), 9871-6; Higuchi, Oheda,
Kuboniwa, Tomonoh, Shimonaka, Ochi, 1992; Role of sugar chains in
the expression of the biological activity of human erythropoietin,
J. Biol. Chem., 267(11), 7703-9; Yamaguchi, Akai, Kawanishi, Ueda,
Masuda, Sasaki, 1991, Effects of site-directed removal of
N-glycosylation sites in human erythropoietin on its production and
biological properties, J. Biol. Chem., 266(30), 20434-9; Takeuchi,
Inoue, Strickland, Kubota, Wada, Shimizu, Hoshi, Kozutsumi,
Takasaki, Kobata, 1989, Relationship between sugar chain structure
and biological activity of recombinant human erythropoietin
produced in Chinese hamster ovary cells, Proc. Natl. Acad. Sci.
USA, 85(20), 7819-22; Kurtz, Eckardt, 1989, Assay methods for
erythropoietin, Nephron., 51(1), 11-4 (German); Zucali, Sulkowski,
1985, Purification of human urinary erythropoietin on
controlled-pore glass and silicic acid, Exp. Hematol., 13(3),
833-7; Krystal, 1983, Physical and biological characterization of
erythroblast enhancing factor (EEF), a late acting erythropoietic
stimulator in serum distinct from erythropoietin, Exp. Hematol.,
11(1), 18-31.
[0212] Preferably, the EPO is recombinantly produced. This includes
the production in eukaryotic or prokaryotic cells, preferably
mammalian, insect, yeast, bacterial cells or in any other cell type
which is convenient for the recombinant production of EPO.
Furthermore, the EPO may be expressed in transgenic animals (e.g.
in body fluids like milk, blood, etc.), in eggs of transgenic
birds, especially poultry, preferred chicken, or in transgenic
plants.
[0213] The recombinant production of a polypeptide is known in the
art. In general, this includes the transfection of host cells with
an appropriate expression vector, the cultivation of the host cells
under conditions which enable the production of the polypeptide and
the purification of the polypeptide from the host cells. For
detailed information see e.g. Krystal, Pankratz, Farber, Smart,
1986, Purification of human erythropoietin to homogeneity by a
rapid five-step procedure, Blood, 67(1), 71-9; Quell; Caslake,
Burkert, Wojchowski, 1989, High-level expression and purification
of a recombinant human erythropoietin produced using a baculovirus
vector, Blood, 74(2), 652-7; EP 640 619 B1 and EP 668 351 B1.
[0214] In a preferred embodiment, the EPO has the amino acid
sequence of human EPO (see EP 148 605 B2).
[0215] The EPO may comprise one or more carbohydrate side chains,
preferably 1 to 12, more preferably 1 to 9, even more preferably 1
to 6 and particularly 1 to 4, especially preferably 4 carbohydrate
side chains, attached to the EPO via N- and/or O-linked
glycosylation, i.e. the EPO is glycosylated. Usually, when EPO is
produced in eukaryotic cells, the polypeptide is
posttranslationally glycosylated. Consequently, the carbohydrate
side chains may have been attached to the EPO during biosynthesis
in mammalian, especially human, insect or yeast cells. The
structure and properties of glycosylated EPO have, been extensively
studied in the art (see EP 428 267 B1; EP 640 619 B1; Rush, Derby,
Smith, Merry, Rogers, Rohde, Katta, 1995, Microheterogeneity of
erythropoietin carbohydrate structure, Anal Chem., 67(8), 1442-52;
Takeuchi, Kobata, 1991, Structures and functional roles of the
sugar chains of human erythropoietins, Glycobiology, 1(4), 337-46
(Review).
[0216] Therefore, the hydroxyalkyl starch derivative according to
the present invention may comprise at least one, preferably 1 to
12, more preferably 1 to 9, even more preferably 1 to 6 and
particularly preferably 1 to 4 HAS molecules per EPO molecule. The
number of HAS-molecules per EPO molecule can be determined by
quanatitative carbohydrate compositional analysis using GC-MS after
hydrolysis of the product and derivatisation of the resulting
monosaccharides (see Chaplin and Kennedy (eds.), 1986, Carbohydrate
Analysis: a practical approach, IRL Press Practical approach series
(ISBN 0-947946-44-3), especially Chapter 1, Monosaccharides, page
1-36; Chapter 2, Oligosaccharides, page 37-53, Chapter 3, Neutral
Polysaccharides, page 55-96).
[0217] According to an especially preferred embodiment of the
present invention, the carbohydrate moiety linked to EPO, is part
of a carbohydrate side chain. More preferably, the carbohydrate
moiety is the terminal moiety of the carbohydrate side chain. In an
even more preferred embodiment, the carbohydrate moiety is a
galactose residue of the carbohydrate side chain, preferably the
terminal galactose residue of the carbohydrate side chain. This
galactose residue can be made available for reaction with the
reaction product of compound (I) and compound (II) by removal of
terminal sialic acids, followed by oxidation, as described
hereinunder. In a further preferred embodiment, the reaction
product of compound (I) and (II) is linked to a sialic acid residue
of the carbohydrate side chains, preferably the terminal sialic
acid residue of the carbohydrate side chain. The sialic acid is
oxidized as described hereinunder.
[0218] Particularly preferably this galactose residue is made
available for reaction with the reaction product of hydroxyalkyl
starch and compound (L) or the reaction product of hydroxyalkyl
starch and compound (D) which is further reacted with compound (L)
via the functional group X by removal of terminal sialic acid
followed by oxidation.
[0219] More preferably, this galactose residue is made available
for reaction with the reaction product of hydroxyalkyl starch and
compound (L) or the reaction product of hydroxyalkyl starch and
compound (D) which is further reacted with compound (L) via the
functional group X by oxidation wherein terminal sialic acid is not
removed.
[0220] As mentioned above, the reaction product of hydroxyalkyl
starch and compound (L) or the reaction product of hydroxyalkyl
starch and compound (D) which is further reacted with compound (L)
be reacted with a thio group comprised in EPO.
[0221] It is also possible to react the reaction product of
hydroxyalkyl starch and compound (L) or the reaction product of
hydroxyalkyl starch and compound (D) which is further reacted with
compound (L) with a thio group as well as with a carbohydrate
moiety, each of them comprised in the at least one further
compound, preferably a polypeptide, more preferably
erythropoietin.
[0222] According to a preferred embodiment, this SH group may be
linked to a preferably oxidized carbohydrate moiety, e.g. by using
a hydroxylamine derivative, e.g. 2-(aminooxy)ethylmercaptan
hydrochloride (Bauer L. et al., 1965, J. Org. Chem., 30, 949) or by
using a hydrazide derivative, e.g. thioglycolic acid hydrazide
(Whitesides et al., 1977, J. Org. Chem., 42, 332.)
[0223] According to a further preferred embodiment, the thio group
is preferably introduced in an oxidized carbohydrate moiety of EPO,
more preferably an oxidized carbohydrate moiety which is part of a
carbohydrate side chain of EPO.
[0224] Preferably, the thio group is derived from a naturally
occurring cysteine or from an added cysteine. More preferably, the
EPO has the amino acid sequence of human EPO and the naturally
occurring cysteines are cysteine 29 and/or 33. In a more preferred
embodiment, t the reaction product of hydroxyalkyl starch and
compound (L) or the reaction product of hydroxyalkyl starch and
compound (D) which is further reacted with compound (L) is reacted
with cysteine 29 whereas cysteine 33 is replaced by another amino
acid. Alternatively, the reaction product of hydroxyalkyl starch
and compound (L) or the reaction product of hydroxyalkyl starch and
compound (D) which is further reacted with compound (L) is reacted
with cysteine 33 whereas cysteine 29 is replaced by another amino
acid.
[0225] In the context of the present invention, the term "added
cysteines" indicates that the polypeptides, preferably EPO,
comprise a cysteine residue which is not present in the wild-type
polypeptide.
[0226] In the context of this aspect of the invention, the cysteine
may be an additional amino acid added at the N- or C-terminal end
of EPO.
[0227] Furthermore, the added cysteine may have been added by
replacing a naturally occurring amino acid by cysteine or a
suitably substituted cysteine. Preferably, in the context of this
aspect of the invention, the EPO is human EPO and the replaced
amino acid residue is serine 126.
[0228] As to the reaction conditions regarding the reaction of the
reaction product of hydroxyalkyl starch and compound (L),
optionally with compound (D), with the further compound (M), no
specific limitations exist, and the reaction conditions may be
adjusted to the specific needs. According to an especially
preferred embodiment of the present invention, water is used as
solvent, either alone or in combination with at least one other
solvent. As at least one other solvent, DMSO, DMF, methanol or
ethanol may be mentioned. Preferred solvents other than water are
methanol and ethanol. According to other preferred embodiments,
DMSO or DMF or methanol or ethanol or a mixture of two or more
thereof is used as solvent.
[0229] If, e.g., hydroxyallyl starch is reacted with compound (L)
in an aqueous system, as it is the case, e.g., when hydroxyethyl
starch is reacted with a hydroxyamine such as
O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine, via reaction of the
non-oxidised reducing end of the starch, and the reaction product
is further reacted with a polypeptide, preferably erythropoietin,
via an aldehyde, keto, acetal or hemiacetale group, the reaction
temperature is preferably in the range of from 4 to 37.degree. C.,
more preferably of from 10 to 30.degree. C. and especially
preferably of from 15 to 25.degree. C.
[0230] Isolation of the reaction product comprising the further
compound (M), preferably the polypeptide and especially preferably
erythropoietin, can be performed by using known procedures for the
purification of natural and recombinant EPO (e.g. size exclusion
chromatography, ion-exchange chromatography, RP-HPLC,
hydroxyapatite chromatography, hydrophobic interaction
chromatography or combinations thereof). Isolation of the reaction
product may be carried out by a suitable process which may comprise
one or more steps. According to a preferred embodiment of the
present invention, the reaction product is first separated off the
reaction mixture or the mixture of the reaction mixture with, e.g.,
the ethanol-acetone mixture, by a suitable method such as
centrifugation or filtration. In a second step, the separated
reaction product may be subjected to a further treatment such as an
after-treatment like dialysis, centrifugal filtration or pressure
filtration, ion exchange chromatography such as, e.g., by a column
containing Q-sepharose, HPLC, MPLC, gel filtration and/or
lyophilisation. According to one preferred embodiment, the
separated reaction product is first dialysed, preferably against
water, and then lyophilized until the solvent content of the
reaction product is sufficiently low according to the desired
specifications of the product. Lyophilisation may be carried out at
temperature of from 20 to 35.degree. C., preferably of from 25 to
30.degree. C. According to another preferred embodiment, the
reaction mixture comprising the reaction product is applied to a
column containing Q-Sepharose to give an eluate which is
concentrated, e.g. by centrifugal filtration.
[0231] It is another object of the present invention to provide
hydroxyalkyl starch derivatives which are produced by one or more
of the aforesaid methods.
[0232] Therefore, the present invention relates to a hydroxyalkyl
starch derivative obtainable by a method of producing a
hydroxyalkyl starch derivative, said hydroxyalkyl starch having a
structure according to formula (I)
##STR00037##
comprising reacting [0233] hydroxyalkyl starch of formula (I) at
its optionally oxidized reducing end or [0234] a hydroxyalkyl
starch derivative, obtainable by reacting hydroxyalkyl starch of
formula (I) at its optionally oxidized reducing end with a compound
(D), said compound (D) comprising [0235] at least one functional
group Z.sub.1 capable of being reacted with the optionally oxidized
reducing end of the hydroxyalkyl starch, and [0236] at least one
functional group W, with a compound (L) comprising [0237] at least
one functional group Z.sub.1 capable of being reacted with said
hydroxyalkyl starch, or at least one functional group Z.sub.2
capable of being reacted with functional group W comprised in said
hydroxyallyl starch derivative, and [0238] at least one functional
group X capable of being reacted with a functional group Y of a
further compound (M), wherein said functional group Y is selected
from the group consisting of an aldehyd group, a keto group, a
hemiacetal group, an acetal group, or a thio group.
[0239] According to a preferred embodiment, the present invention
relates to a hydroxyalkyl starch derivative as described above
wherein R.sub.1, R.sub.2 and R.sub.3 are independently hydrogen or
a linear or branched hydroxyalkyl group.
[0240] According to an even more preferred embodiment, the present
invention relates to a hydroxyalkyl starch derivative as described
above wherein R.sub.1, R.sub.2 and R.sub.3 are independently
hydrogen or a 2-hydroxyethyl group.
[0241] According to a further preferred embodiment, the present
invention relates to a hydroxyalkyl starch derivative as described
wherein the hydroxyalkyl starch is hydroxyethyl starch.
[0242] According to a further preferred embodiment, the present
invention relates to a hydroxyalkyl starch derivative as described
above wherein the functional group Z.sub.1 comprises the structure
--NH--.
[0243] According to an especially preferred embodiment, the present
invention relates to a hydroxyalkyl starch derivative as described
above wherein Z.sub.1 is selected from the group consisting of
##STR00038##
wherein G is O or S and, if present twice, independently O or S,
and R' is methyl.
[0244] According to a further preferred embodiment, the present
invention relates to a hydroxyalkyl starch derivative as described
above wherein the functional group Y is selected from the group
consisting of an aldehyd group, a keto group, a hemiacetal group,
and an acetal group, and the functional group X comprises the
structure --NH--.
[0245] According to an especially preferred embodiment, the present
invention relates to a hydroxyalkyl starch derivative as described
above X is selected from the group consisting of
##STR00039##
wherein G is O or S and, if present twice, independently O or S,
and R is methyl.
[0246] According to a further preferred embodiment, the present
invention relates to a hydroxyallyl starch derivative as described
above wherein the functional group Y is --SH and the functional
group X is selected from the group consisting of
##STR00040##
wherein Hal is Cl, Br or I.
[0247] According to a further preferred embodiment, the present
invention relates to a hydroxyalkyl starch derivative as described
above wherein the functional group W or the functional group
Z.sub.2 is --SH and the functional group Z.sub.2 or the functional
group W is selected from the group consisting of
##STR00041##
wherein Hal is Cl, Br, or I.
[0248] According to a further preferred embodiment, the present
invention relates to a hydroxyalkyl starch derivative as described
above wherein the functional group W or the functional group
Z.sub.2 is selected from the group consisting of an activated
ester, as described above, or a carboxy group which is optionally
transformed into an activated ester and the functional group
Z.sub.2 or the functional group W is selected from the group
consisting of
##STR00042##
wherein G is O or S and, if present twice, independently O or S,
and R is methyl.
[0249] According to an especially preferred embodiment, the present
invention relates to a hydroxyalkyl starch derivative as described
above wherein the reducing end of the hydroxyalkyl starch is not
oxidized prior to the reaction with compound (D) or compound (L),
said hydroxyalkyl starch thus having a structure according to
formula (I)
##STR00043##
[0250] According to another especially preferred embodiment, the
present invention relates to a hydroxyalkyl starch derivative as
described above wherein the reducing end of the hydroxyalkyl starch
is oxidized prior to the reaction with compound (D) or compound
(L), said hydroxyalkyl starch thus having a structure according to
formula (IIa)
##STR00044##
and/or according to formula (IIb)
##STR00045##
[0251] According to a further preferred embodiment, the present
invention relates to a hydroxyalkyl starch derivative as described
above wherein the reducing end is oxidized by an alkaline iodine
solution.
[0252] According to a further preferred embodiment, the present
invention relates to a hydroxyalkyl starch derivative as described
above wherein hydroxyalkyl starch is reacted with a compound (L)
via the reaction of functional group Z.sub.1 with the optionally
oxidized reducing end of the hydroxyalkyl starch and the resulting
reaction product is reacted with a further compound (M) via the
reaction of the functional group X comprised in compound (L) with
the functional group Y comprised in compound (M).
[0253] According to yet a further preferred embodiment, the present
invention relates to a hydroxyalkyl starch derivative as described
above hydroxyalkyl starch is reacted with a compound (L) via the
reaction of functional group Z.sub.1 with the optionally oxidized
reducing end of the hydroxyalkyl starch, where compound (L), prior
to the reaction with hydroxyalkyl starch, is reacted with a further
compound (M) via the reaction of functional group X comprised in
compound (L) with the functional group Y comprised in compound
(M).
[0254] According to still a further preferred embodiment, the
present invention relates to a hydroxyalkyl starch derivative as
described above wherein hydroxyalkyl starch is reacted with a
compound (D) via the reaction of the functional group Z.sub.1
comprised in compound (D), with the optionally oxidized reducing
end of the hydroxyalkyl starch to give a first hydroxyalkyl starch
derivative, and where the first hydroxyalkyl starch derivative is
reacted with a compound (L) via the reaction of functional group
Z.sub.2 comprised in compound (L) with the functional group W
comprised in compound (D) to give a second hydroxyalkyl starch
derivative.
[0255] According to an especially preferred embodiment, the present
invention relates to the aforesaid hydroxyalkyl starch derivative
wherein the second hydroxyalkyl starch derivative is reacted with a
further compound (M) via the reaction of functional group X
comprised in compound (L) with the functional group Y comprised in
compound (M).
[0256] According to a further preferred embodiment, the present
invention relates to a hydroxyalkyl starch derivative as described
above wherein hydroxyalkyl starch is reacted with a compound (D)
via the reaction of functional group Z.sub.1 comprised in compound
(D) with the optionally oxidized reducing end of the hydroxyalkyl
starch to give a first hydroxyalkyl starch derivative, and where
the first hydroxyalkyl starch derivative is reacted, via the
reaction of the functional group W, comprised in compound (D), and
the functional group Z.sub.2, comprised in compound (L), with
compound (L), where compound (L), prior to the reaction with the
first hydroxyalkyl starch derivative, is reacted with a further
compound (M) via the reaction of functional group X comprised in
compound (L) with the functional group Y comprised in compound
(M).
[0257] According to an especially preferred embodiment, the present
invention relates to a hydroxyalkyl starch derivative as described
above wherein the at least one further compound (M) is a
polypeptide.
[0258] According to a particularly preferred embodiment, the
present invention relates to a hydroxyalkyl starch derivative as
described above wherein the polypeptide is erythropoietin.
[0259] The hydroxyalkyl starch derivative which in the following is
referred to as HAS-EPO conjugate and which is formed by reaction of
hydroxyalkyl starch with compound (L) and optionally-compound (D)
and erythrpoietin, has the advantage that it exhibits an improved
biological stability when compared to the erythropoietin before
conjugation. Furthermore, it exhibits a higher biological activity
than standard BRP EPO. This is mainly due to the fact that this
hydroxyalkyl starch derivative is less or even not recognized by
the removal systems of the liver, and kidney and therefore persists
in the circulatory system for a longer period of time. Furthermore,
since the HAS is attached site-specifically, the risk of destroying
the in-vivo biological activity of EPO by conjugation of HAS to EPO
is minimized.
[0260] The HAS-EPO conjugate of the invention may exhibit
essentially the same in-vitro biological activity as recombinant
native EPO, since the in-vitro biological activity only measures
binding affinity to the EPO receptor. Methods for determining the
in-vitro biological activity are known in the art.
[0261] Furthermore, the HAS-EPO exhibits a greater in-vivo activity
than the EPO used as a starting material for conjugation
(unconjugated EPO). Methods for determining the in vivo biological
activity are known in the art.
[0262] The HAS-EPO conjugate may exhibit an in vivo activity of
from 110% to 500%, preferably of from 300 to 400%, or preferably of
from 110 to 300%, more preferably from 110% to 200%, more
preferably from 110% to 180% or from 110 to 150% most preferably
from 110% to 140%, if the in-vivo activity of the unconjugated EPO
is set as 100%.
[0263] Compared to the highly sialylated EPO of Amgen (see EP 428
267 B1), the HAS-EPO exhibits preferably at least 50%, more
preferably at least 70%, even more preferably at least 85% or at
least 95%, at least 150%, at least 200% or at least 300% of the in
vivo activity of the highly sialylated EPO if the in-vivo activity
of highly sialylated EPO is set as 100%. Most preferably, it
exhibits at least 95% of the in vivo activity of the highly
sialylated EPO.
[0264] The high in-vivo biological activity of the HAS-EPO
conjugate of the invention mainly results from the fact that the
HAS-EPO conjugate remains longer in the circulation than the
unconjugated EPO because it is less recognized by the removal
systems of the liver and because renal clearance is reduced due to
the higher molecular weight. Methods for the determination of the
in-vivo half life time of EPO in the circulation are known in the
art (Sytkowski, Lunn, Davis, Feldman, Siekman, 1998, Human
erythropoietin dimers with markedly enhanced in vivo activity,
Proc. Natl. Acad. Sci. USA, 95(3), 1184-8).
[0265] Consequently, it is a great advantage of the present
invention that a HAS-EPO conjugate is provided which may be
administered less frequently than the EPO preparations commercially
available at present. While standard EPO preparations have to be
administered at least every 3 days, the HAS-EPO conjugate of the
invention is preferable administered twice a week, more preferably
once a week.
[0266] Furthermore, the method of the invention has the advantage
that an effective EPO derivative can be produced at reduced costs
since the method does not comprise extensive and time consuming
purification steps resulting in low final yield, e.g. it is not
necessary to purify away under-sialylated EPO forms which are known
to exhibit low or no in-vivo biological activity. Especially
Example 8.11(d) demonstrates that a HES-EPO produced with few
modifications steps exhibits a 3-fold activity over standard BRP
EPO.
[0267] It is yet another object of the present invention to provide
a pharmaceutical composition which comprises, in a therapeutically
effective amount, the HAS-EPO conjugate of the present
invention.
[0268] Furthermore, the present invention relates to a
pharmaceutical composition comprising, in a therapeutically
effective amount, the HAS-polypeptide conjugate, preferably the
HAS-EPO conjugate, more preferably the HES-EPO conjugate of the
present invention. In a preferred embodiment, the pharmaceutical
composition comprises further at least one pharmaceutically
acceptable diluent, adjuvant and/or carrier useful in
erythropoietin therapy.
[0269] Therefore, the present invention also relates to a
pharmaceutical composition comprising, in a therapeutically
effective amount, a hydroxyalkyl starch derivative obtainable by a
method of producing a hydroxyalkyl starch derivative, said
hydroxyalkyl starch having a structure according to formula (I)
##STR00046##
comprising reacting, [0270] hydroxyalkyl starch of formula (I) at
its optionally oxidized reducing end or [0271] a hydroxyalkyl
starch derivative, obtainable by reacting hydroxyalkyl starch of
formula (I) at its optionally oxidized reducing end with a compound
(D), said compound (D) comprising [0272] at least one functional
group Z.sub.1 capable of being reacted with the optionally oxidized
reducing end of the hydroxyalkyl starch, and [0273] at least one
functional group W, with a compound (L) comprising [0274] at least
one functional group Z.sub.1 capable of being reacted with said
hydroxyalkyl starch, or at least one functional group Z.sub.2
capable of being reacted with functional group W comprised in said
hydroxyalkyl starch derivative, and [0275] at least one functional
group X capable of being reacted with a functional group Y of a
further compound (M), wherein said functional group Y is selected
from the group consisting of an aldehyd group, a keto group, a
hemiacetal group, an acetal group, or a thio group, said method of
producing a hydroxyalkyl starch derivative further comprising
reacting the reaction product comprising hydroxyalkyl starch,
compound (L) and optionally compound (D) with a further compound
(M) wherein the at least one further compound is a polypeptide.
[0276] Moreover, the present invention relates to the use of a
hydroxyalkyl starch derivative as described for the preparation of
a medicament for the treatment of anemic disorders or hematopoietic
dysfunction disorders or diseases related thereto.
[0277] According to a preferred embodiment, the present invention
relates to a pharmaceutical composition as described above wherein
the polypeptide is an antithrombin (AT), preferably AT III (Levy J
H, Weisinger A, Ziomek C A, Echelard Y, Recombinant Antithrombin:
Production and Role in Cardiovascular Disorder, Seminars in
Thrombosis and Hemostasis 27, 4 (2001) 405-416; Edmunds T, Van
Patten S M, Pollock J, Hanson E, Bernasconi R, Higgins E, Manavalan
P, Ziomek C, Meade H, McPherson J, Cole E S, Transgenically
Produced. Human Antithrombin: Structural and Functional Comparison
to Human Plasma-Derived Antithrombin, Blood 91, 12 (1998)
4661-4671; Minnema M C, Chang A C K, Jansen P M, Lubbers Y T P,
Pratt B M, Whittaker B G, Taylor F B, Hack C E, Friedman B,
Recombinant human antithrombin III improves survival and attenuates
inflammatory responses in baboons lethally challenged with
Escherichia coli, Blood 95, 4 (2000) 1.117-1123; Van Patten S M,
Hanson E H, Bernasconi R, Zhang K, Manavaln P, Cole E S, McPherson
J M, Edmunds T, Oxidation of Methionine Residues in Antithrombin,
J. Biol. Chemistry 274, 15 (1999) 10268-10276).
[0278] According to other preferred embodiments, the present
invention relates to pharmaceutical compositions wherein the
polypeptide is G-CSF or IFN-beta.
[0279] According to an especially preferred embodiment, the present
invention relates to a pharmaceutical composition as described
above wherein the polypeptide is erythropoietin.
[0280] According to a further embodiment, the present invention
relates to a pharmaceutical composition as described above wherein
the functional group Y is --SH and compound (L) is a compound of
general formula Z.sub.1-L'-X where the functional group Z.sub.1 is
selected from the group consisting of
##STR00047##
wherein G is O or S and, if present twice, independently O or S,
and R' is methyl, and where the functional group X is selected from
the group consisting of
##STR00048##
wherein Hal is Cl, Br or I, and where L' is an organic chain
bridging Z.sub.1 and X or where L' is absent.
[0281] According to a preferred embodiment, the present invention
relates to a pharmaceutical composition as described above wherein
the functional group Y is selected from the group consisting of an
aldehyd group, a keto group, a hemiacetal group, and an acetal
group, and compound (L) is a compound of general formula
Z.sub.1-L'-X where the functional group Z.sub.1 is selected from
the group consisting of
##STR00049##
wherein G is O or S and, if present twice, independently O or S,
and R' is methyl, and where the functional group X is selected from
the group consisting of
##STR00050##
wherein G is O or S and, if present twice, independently O or S,
and R' is methyl, and where L' is an organic chain bridging Z.sub.1
and X or where L' is absent.
[0282] According to another embodiment, the present invention
relates to a pharmaceutical composition as described above wherein
the functional group Y is --SH, compound (D) is a compound of
general formula Z.sub.1-D'-W, and compound (L) is a compound of
general formula Z.sub.2-L'-X, where the functional group Z.sub.1 is
selected from the group consisting of
##STR00051##
wherein G is O or S and, if present twice, independently O or S,
and R' is methyl, where the functional group X is selected from the
group consisting of
##STR00052##
wherein Hal is Cl, Br or I, where the functional group W or the
functional group Z.sub.2 is --SH and the functional group Z.sub.2
or the functional group W is selected from the group consisting
of
##STR00053##
wherein Hal is Cl, Br, or I, or where the functional group W or the
functional group Z.sub.2 is selected from the group consisting of
an activated ester, as described above, or a carboxy group which is
optionally transformed into an activated ester and the functional
group Z.sub.2 or the functional group W is selected from the group
consisting of
##STR00054##
wherein G is O or S and, if present twice, independently O or S,
and R' is methyl, and where D' is an organic chain bridging Z.sub.1
and W or where D' is absent and where L' is an organic chain
bridging Z.sub.2 and X or where L' is absent.
[0283] According to yet another embodiment, the present invention
relates to a pharmaceutical composition as described above wherein
the functional group Y is selected from the group consisting of an
aldehyd group, a keto group, a hemiacetal group, and an acetal
group, compound (D) is a compound of general formula Z.sub.1-D'-W,
and compound (L) is a compound of general formula Z.sub.2-L'-X,
where the functional group Z.sub.1 is selected from the group
consisting of
##STR00055##
wherein G is O or S and, if present twice, independently O or S,
and R' is methyl, where the functional group X is selected from the
group consisting of
##STR00056##
wherein G is O or S and, if present twice, independently O or S,
and R' is methyl, the functional group W or the functional group
Z.sub.2 is --SH and the functional group Z.sub.2 or the functional
group W is selected from the group consisting of
##STR00057##
wherein Hal is Cl, Br, or I, or where the functional group W or the
functional group Z.sub.2 is selected from the group consisting of
an activated ester, as described above, or a carboxy group which is
optionally transformed into an activated ester and the functional
group Z.sub.2 or the functional group W is selected from the group
consisting of
##STR00058##
wherein G is O or S and, if present twice, independently O or S,
and R' is methyl, and where D' is an organic chain bridging Z.sub.1
and W or where D' is absent and where L' is an organic chain
bridging Z.sub.2 and X or where L' is absent.
[0284] According to a particularly preferred embodiment, the
present invention relates to a pharmaceutical composition as
described above wherein hydroxyethyl starch is reacted in an
aqueous medium with a compound according to the following
formula
##STR00059##
and the reaction product is reacted with erythropoietin.
[0285] According to an even more preferred embodiment, the present
invention relates to the aforementioned pharmaceutical composition
wherein the erythropoietin is oxidised with sodium periodate prior
to the reaction.
[0286] According to a further preferred embodiment, the present
invention relates to pharmaceutical composition as described above
wherein the erythropoietin is partially desialylated and
subsequently oxidised with sodium periodate prior to the
reaction.
[0287] According to a further preferred embodiment of the present
invention, pharmaceutical compositions comprising a hydroxyallyl
starch derivative which are produced on the basis of a completely
reduced Thio-EPO according to Example 6 are excluded.
[0288] The above-mentioned pharmaceutical composition is especially
suitable for the treatment of anemic disorders or hematopoietic
dysfunction disorders or diseases related thereto.
[0289] A "therapeutically effective amount" as used herein refers
to that amount which provides therapeutic effect for a given
condition and administration regimen. The administration of
erythropoietin isoforms is preferably by parenteral routes. The
specific route chosen will depend upon the condition being treated.
The administration of erythropoietin isoforms is preferably done as
part of a formulation containing a suitable carrier, such as human
serum albumin, a suitable diluent, such as a buffered saline
solution, and/or a suitable adjuvant. The required dosage will be
in amounts sufficient to raise the hematocrit of patients and will
vary depending upon the severity of the condition being treated,
the method of administration used and the like.
[0290] The object of the treatment with the pharmaceutical
composition of the invention is preferably an increase of the
hemoglobin value of more than 6.8 mmol/l in the blood. For this,
the pharmaceutical composition may be administered in a way that
the hemoglobin value increases between from 0.6 mmol/l and 1.6
mmol/l per week. If the hemoglobin value exceeds 8.7 mmol/l, the
therapy should be preferably interrupted until the hemoglobin value
is below 8.1 mmol/l.
[0291] The composition of the invention is preferably used in a
formulation suitable for subcutaneous or intravenous or parenteral
injection. For this, suitable excipients and carriers are e.g.
sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium
chlorate, polysorbate 80, HSA and water for injection. The
composition may be administered three times a week, preferably two
times a week, more preferably once a week, and most preferably
every two weeks.
[0292] Preferably, the pharmaceutical composition is administered
in an amount of 0.01-10 .mu.g/kg body weight of the patient, more
preferably 0.1 to 5 .mu.g/kg, 0.1 to 1 .mu.g/kg, or 0.2-0.9
.mu.g/kg, most preferably 0.3-0.7 .mu.g/kg, and most preferred
0.4-0.6 .mu.g/kg body weight.
[0293] In general, preferably between 10 .mu.g and 200 .mu.g,
preferably between 15 .mu.g and 100 .mu.g are administered per
dosis.
[0294] The invention further relates to a HAS-polypeptide according
to the present invention for use in method for treatment of the
human or animal body.
[0295] The invention further relates to the use of a HAS-EPO
conjugate of the present invention for the preparation of a
medicament for the treatment of anemic disorders or hematopoietic
dysfunction disorders or diseases related hereto.
[0296] In case compound (L) used in according to the present
invention comprises one or more chiral centers, compound (II) may
be present in R conformation or in S conformation or as racemic
compound with respect to each chiral center.
[0297] In case compound (D) optionally used in the present
invention comprises one or more chiral centers, compound (D) may be
present in R conformation or in S conformation or as racemic
compound with respect to each chiral center.
[0298] The invention is further illustrated by the following
examples, tables an figures which are in no way intended to
restrict the scope of the present invention.
SHORT DESCRIPTION OF THE FIGURES
[0299] FIG. 1
[0300] FIG. 1 shows an SDS page analysis of the HES-EPO conjugate,
produced according to example 5.1. [0301] Lane A: Protein marker
Roti.RTM.-Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D);
molecular weights (in kD) of the protein marker from top to bottom:
245, 123, 77, 42, 30, 25.4, and 17. [0302] Lane B: Crude product
after conjugation according to example 5.1. [0303] Lane C: EPO
starting material.
[0304] FIG. 2
[0305] FIG. 2 shows an SDS page analysis of the HES-EPO conjugate,
produced according to example 5.3. [0306] Lane A: Crude product
after conjugation according to example 5.3. [0307] Lane B: EPO
starting material. [0308] Lane C: Protein marker Roti.RTM.-Mark
PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D); molecular weights (in
kD) of the protein marker from top to bottom: 245, 123, 77, 42, 30,
25.4, and 17.
[0309] FIG. 3
[0310] FIG. 3 shows an SDS page analysis of the HES-EPO conjugate,
produced according to example 5.4 and 5.5. [0311] Lane A: Protein
marker Roti.RTM.-Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D);
molecular weights (in kD) of the protein marker from top to bottom:
245, 123, 77, 42, 30, 25.4, and 17. [0312] Lane B: Crude product
after conjugation according to example 5.4. [0313] Lane C: Crude
product after conjugation according to example 5.5. [0314] Lane D:
EPO starting material.
[0315] FIG. 4
[0316] FIG. 4 shows an SDS page analysis of HES-EPO conjugates,
produced according to examples 7.1 and 7.4. [0317] Lane A: Protein
marker Roti.RTM.-Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D);
molecular weights (in kD) of the protein marker from top to bottom:
245, 123, 77, 42, 30, 25.4, and 17. [0318] Lane B: Crude product
after conjugation according to example 7.4. [0319] Lane C: Crude
product after conjugation according to example 7.1. [0320] Lane D:
EPO starting material.
[0321] FIG. 5
[0322] FIG. 5 shows an SDS page analysis of HES-EPO conjugates,
produced according to examples 7.2, 7.3, 7.5, and 7.6. [0323] Lane
A: Protein marker Roti.RTM.-Mark PRESTAINED (Carl Roth GmbH+Co,
Karlsruhe, D); molecular weights (in kD) of the protein marker from
top to bottom: 245, 123, 77, 42, 30, 25.4, and 17. [0324] Lane B:
Crude product after conjugation according to example 7.6, based on
Example 1.3 b). [0325] Lane C: Crude product after conjugation
according to example 7.5, based on Example 1.1 b). [0326] Lane D:
Crude product after conjugation according to example 7.6, based on
Example 1.3 a). [0327] Lane E: Crude product after conjugation
according to example 7.5, based on Example 1.1 a). [0328] Lane F:
Crude product after conjugation according to example 7.2. [0329]
Lane G: Crude product after conjugation according to example 7.3.
[0330] Lane K: EPO starting material.
[0331] FIG. 6
[0332] FIG. 6 shows an SDS page analysis of HES-EPO conjugates,
produced according to examples 7.7, 7.8, 7.9, 7.10, 7.11, and 7.12.
[0333] Lane A; Protein marker Roti.RTM.-Mark PRESTAINED (Carl Roth
GmbH+Co, Karlsruhe, D); molecular weights (in kD) of the protein
marker from top to bottom: 245, 123, 77, 42, 30, 25.4, and 17.
[0334] Lane B: Crude product after conjugation according to example
7.11. [0335] Lane C: Crude product after conjugation according to
example 7.10. [0336] Lane D: Crude product after conjugation
according to example 7.7. [0337] Lane E: Crude product after
conjugation according to example 7.8. [0338] Lane F: Crude product
after conjugation according to example 7.12. [0339] Lane G: EPO
starting material. [0340] Lane K: Crude product after conjugation
according to example 7.9.
[0341] FIG. 7
[0342] SDS-PAGE analyses of EPO-GT-1 subjected to mild acid
treatment for 5 min.=lane 2; 10 min.=lane 3; 60 min.=lane 4 and
untreated EPO=lane 1; the mobility shift of EPO after removal of
N-glycans is shown (+PNGASE).
[0343] FIG. 8
[0344] HPAEC-PAD pattern of oligosaccharides isolated from
untreated EPO and from EPO incubated for 5 min., 10 min. and 60
min. under mild acid hydrolysis conditions. Roman numbers I-V
indicate the elution position of I=desialylated diantennary
structure, II=trisialylated triantennary structures (two isomers);
III=tetrasialylated tetraantennary structure+2 N-acetyllactosamine
repeats, IV=tetrasialylated tetraantennary structure+1
N-acetyllactosamine repeat; V=tetrasialylated tetraantennary
structure+without N-acetyllactosamine repeat. The elution area of
oligosaccharides structures without, with 1-4 sialic acid is
indicated by brackets.
[0345] FIG. 9
[0346] HPAEC-PAD of N-linked oligosaccharides after desialylation;
the elution position of N-acetylneuraminic acid is shown; numbers
1-9 indicate the elution position of standard oligosaccharides:
1=diantennary; 2=triantennary (2-4 isomer), 3=triantennary (2-6
isomer); 4=tetraantennary; 5=triantennary plus 1 repeat;
6=tetraantennary plus 1 repeat; 7=triantennary plus 2 repeats;
8=tetraantennary plus 2 repeats and 9=tetraantennary plus 3
repeats.
[0347] FIG. 10
[0348] SDS-PAGE analysis of mild treated and untreated EPO which
were subjected to periodate oxidation of sialic acid residues.
1=periodate oxidized without acid treatment; 2=periodate oxidized 5
min. acid treatment; 3=periodate oxidized and acid treatment 10
min.; 4=periodate oxidized without acid treatment; 5=BRP EPO
standard without periodate and without acid treatment.
[0349] FIG. 11
[0350] HPAEC-PAD pattern of native oligosaccharides isolated from
untreated EPO and from EPO incubated for 5 min and 10 min under
mild acid hydrolysis conditions and subsequent periodate treatment.
The elution area of oligosaccharides structures without and with
1-4 sialic acid is indicated by brackets 1-5.
[0351] FIG. 12
[0352] SDS-PAGE analysis of the time course of HES-modification of
EPO-GT-1-A: 20 .mu.g aliquots of EPO-GT-1-A were reacted with
hydroxylamine-modified HES derivative X for 30 min, 2, 4 and 17
hours. Lane 1=30 min reaction time; land 2=2 hour reaction time;
land 3=4 hours reaction time; lane 4=17 hours reaction time; lane
5=EPO-GT-1-A without HES-modification. Left figure shows the shift
in mobility of EPO-GT-1-A with increasing incubation time in the
presence of the with hydroxylamine-modified HES derivative (flow
rate: 1 mlmin.sup.-1) X: Lane 1=30 min reaction time; lane 2=2
hours reaction time; lane 3=4 hours reaction time, land 4=17 hours
reaction time; lane 5=EPO-GT-1-A with HES modification. The figure
on the right shows analysis of the same samples after their
treatment with N-glycosidase.
[0353] FIG. 13
[0354] SDS-PAGE analysis of Q-Sepharose fractions of HES-EPO
conjugates. Each 1% of the flow-through and 1% of the fraction
eluting at high salt concentrations were concentrated in a Speed
Vac concentrator and were loaded onto the gels in sample buffer.
EPO protein was stained by Coomassie Blue. A=sample I; B=sample II;
C=sample III; K=control EPO-GT-1; A1, B1, C1 and K1 indicated the
flow-through fraction; A2, B2, C2 and K2 indicates the fraction
eluted with high salt concentration.
[0355] FIG. 14a
[0356] SDS-PAGE analysis of HES-modified EPO sample A2 (see FIG.
13), control EPO sample K2 and EPO-GT-1-A EPO preparation were
digested in the presence of N-glycosidase in order to remove
N-linked oligosaccharides. All EPO samples showed the mobility
shift towards low molecular weight forms lacking or containing
O-glycan. A lower ratio of the O-glycosylated and nonglycosylated
protein band was observed for the HES-modified EPO sample A2 after
de-N-glycosylation and a diffuse protein band was detected around
30 KDa, presumably representing HES-modification at the sialic acid
of O-glycan residue (see arrow marked by an asterisk).
[0357] FIG. 14b
[0358] SDS-PAGE analysis after mild hydrolysis of HES-modified EPO
sample A2 (see FIG. 13), control EPO sample K2 and EPO-GT-1A which
were untreated or digested in the presence of N-glycosidase in
order to remove N-linked oligosaccharides (see FIG. 14a). Both high
molecular weight form of A2 before and A after N.glycosidase
treatment (see brackets with and without arrow) disappeared upon
acid treatment of the samples. The BRP EPO standard which was run
for comparison was not subjected to mild acid treatment.
[0359] FIG. 15
[0360] HPAEC-PAD analysis of N-linked oligosaccharide material
liberated from HES-modified sample A, from EPO-GT-1-A and from a
control EPO sample incubated with unmodified HES (K). Roman numbers
I-V indicate the elution position of I=disialylated diantennary
structure, II=trisialylated triantennary structures (two isomers),
III=tetrasialylated tetraantennary structure+2 N-acetyllactosamine
repeats, IV=tetrasialylated tetraantennary structure+1
N-acetyllactosamine repeat, V=tetrasialylated tetraantennary
structure+without N-acetyllactosamine repeat; brackets indicate the
elution area of di-, tri- and tetrasialylated N-glycans as reported
in the legends of FIGS. 8 and 11.
[0361] FIG. 16
[0362] HPAEC-PAD analysis of N-linked oligosaccharide material
liberated from HES-modified sample A, from EPO-GT-1A and from a
control EPO sample (K) incubated with unmodified HES. The retention
times of a mixture of standard oligosaccharides is shown: numbers
1-9 indicate the elution position of standard oligosaccharides:
1=diantennary; 2=triantennary (2-4 isomer); 3=triantennary (2-6
isomer); 4=tetraantennary; 5=triantennary plus 1 repeat
6=tetraantennary plus 1 repeat 7=triantennary plus 2 repeats;
8=tetraantennary plus 2 repeats and 9=tetraantennary plus 3
repeats.
[0363] FIGS. 17 to 23
[0364] FIGS. 17 to 23 represent MALDI/TOF mass spectra of the
enzymatically liberated and chemically desialylated N-glycans
isolated from HES-modified EPO and control EPO preparations. Major
signals at m/z 1809.7, 2174.8, 2539.9, 2905.0 and 3270.1
([M+Na].sup.+) correspond to di- to tetraantennary complex-type
N-glycan structures with no, one or two N-acetyllactosamine repeats
accompanied by weak signals due to loss of fucose or galactose
which are due to acid hydrolysis conditions employed for the
desialylation of samples for MS analysis.
[0365] FIG. 17
[0366] MALDI/TOF spectrum: desialylated oligosaccharides of
HES-modified EPO A2.
[0367] FIG. 18
[0368] MALDI/TOF spectrum: desialylated oligosaccharides of EPO
GT-1-A.
[0369] FIG. 19
[0370] MALDI/TOF spectrum: desialylated oligosaccharides of EPO
K2.
[0371] FIG. 20
[0372] MALDI/TOF spectrum: desialylated oligosaccharides of
EPO-GT-1.
[0373] FIG. 21
[0374] MALDI/TOF spectrum: desialylated oligosaccharides of
EPO-GT-1 subjected to acid hydrolysis for 5 min.
[0375] FIG. 22
[0376] MALDI/TOF spectrum: desialylated oligosaccharides of
EPO-GT-1 subjected to acid hydrolysis for 10 min.
[0377] FIG. 23
[0378] MALDI/TOF spectrum: desialylated oligosaccharides of
EPO-GT-1 subjected to acid hydrolysis for 60 min.
[0379] FIG. 24
[0380] FIG. 24 shows an SDS page analysis of two HES-EPO conjugates
[0381] mw: marker [0382] Lane 1: HES-EPO produced according to
example protocol 8B: EPO is conjugated to hydrazido-HES 12KD L
[0383] Lane 2: HES-EPO produced according to example protocol 9B:
EPO is conjugated to hydroxylamino HES 12 KD K [0384] C: control
(unconjugated EPO); the upper band represents EPO dimer
[0385] FIG. 25
[0386] FIG. 25 demonstrates that the HES is conjugated to a
carbohydrate moiety of a carbohydrate side chain by showing a
digestion of HAS modified EPO forms with polypeptide N-glycosidase
[0387] Lane 1: HES-EPO produced according to example protocol 8B
after digestion with N-glycosidase [0388] Lane 2: HES-EPO produced
according to example protocol 9B after digestion with N-glycosidase
[0389] Lane 3: BRP EPO standard [0390] Lane 4: BRP EPO standard
after digestion with N-glycosidase [0391] mw: marker (Bio-Rad
SDS-PAGE Standards Low range Catalog No 161-0305, Bio-Rad
Laboratories, Hercules, Calif., USA)
[0392] In the context of the present invention, the degree of
substitution, denoted as DS, relates to the molar substitution, as
described above (see also Sommermeyer et al., 1987,
Krankenhauspharmazie, 8(8), 271-278, in particular p. 273).
Throughout the invention, the DS of the HES18/04 when measured
according to Sommermeyer et al., 1987, Krankenhauspharmazie, 8(8),
271-278 was 0.5.
EXAMPLES
Example 1
Formation of Hydroxyethyl Starch Derivatives by Reductive Amination
of the Non-Oxidised Reducing End
Example 1.1
Reaction of Hydroxyethyl Starch with 1,3-diamino-2-hydroxy
Propane
[0393] ##STR00060## [0394] a) To a solution of 200 mg hydroxyethyl
starch (HES18/0.4 (MW=18,000 D, DS=0.4)) in 5 ml water, 0.83 mmol
1,3-diamino-2-hydroxy propane (Sigma Aldrich, Taufkirchen, D) and
50 mg sodium cyanoborohydrate NaCNBH.sub.3 were added. The
resulting mixture was incubated at 80.degree. C. for 17 h. The
reaction mixture was added to 160 ml of a cold 1:1 mixture of
acetone and ethanol (v/v). The precipitate was collected by
centrifugation and dialysed for 4 d against water (SnakeSkin
dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH,
Bonn, D), and lyophilized. [0395] b) Incubation of the mixture
resulting from adding 0.83 mmol 1,3-diamino-2-hydroxy propane and
50 mg sodium cyanoborohydrate NaCNBH.sub.3 to the solution of 200
mg hydroxyethyl starch was also possible and carried out at
25.degree. C. for 3 d.
Example 1.2
Reaction of Hydroxyethyl Starch with 1,2-dihydroxy-3-amino
Propane
[0396] ##STR00061## [0397] a) To a solution of 200 mg hydroxyethyl
starch (HES18/0.4 (MW=18,000 D, DS=0.4)) in 5 ml water, 0.83 mmol
1,2-dihydroxy-3-amino propane (Sigma Aldrich, Taufkirchen, D) and
50 mg sodium cyanoborohydrate NaCNBH.sub.3 were added. The
resulting mixture was incubated at 80.degree. C. for 17 h. The
reaction mixture was added to 160 ml of a cold 1:1 mixture of
acetone and ethanol (v/v). The precipitate was collected by
centrifugation and dialysed for 4 d against water (SnakeSkin
dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH,
Bonn, D), and lyophilized. [0398] b) Incubation of the mixture
resulting from adding 0.83 mmol 1,2-dihydroxy-3-amino propane and
50 mg sodium cyanoborohydrate NaCNBH.sub.3 to the solution of 200
mg hydroxyethyl starch was also possible and carried out at
25.degree. C. for 3 d.
[0399] The reaction of 1,2-dihydroxy-3-amino propane with HES was
confirmed indirectly by quantification of formaldehyde, resulting
from the oxidative cleavage of the 1,2-diole in the reaction
product by periodate as described by G. Avigad, Anal. Biochem. 134
(1983) 449-504.
Example 1.3
Reaction of Hydroxyethyl Starch with 1,4-diamino Butane
[0400] ##STR00062## [0401] a) To a solution of 200 mg hydroxyethyl
starch (HES18/0.4 (MW=18,000 D, DS=0.4)) in 5 ml water, 0.83 mmol
1,4-diamino butane (Sigma Aldrich, Taufkirchen, D) and 50 mg sodium
cyanoborohydrate NaCNBH.sub.3 were added. The resulting mixture was
incubated at 80.degree. C. for 17 h. The reaction mixture was added
to 160 ml of a cold 1:1 mixture of acetone and ethanol (v/v). The
precipitate was collected by centrifugation and dialysed for 4 d
against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio
Science Deutschland GmbH, Bonn, D), and lyophilized. [0402] b)
Incubation of the mixture resulting from adding 0.83 mmol
1,4-diamino butane and 50 mg sodium cyanoborohydrate NaCNBH.sub.3
to the solution of 200 mg hydroxyethyl starch was also possible and
carried out at 25.degree. C. for 3 d.
Example 1.4
Reaction of Hydroxyethyl Starch with 1-mercapto-2-amino Ethane
[0403] ##STR00063## [0404] a) To a solution of 200 mg hydroxyethyl
starch (HES18/0.4 (MW=18,000 D, DS=0.4)) in 5 ml water, 0.83 mmol
1-mercapto-2-amino ethane (Sigma Aldrich, Taufkirchen, D) and 50 mg
sodium cyanoborohydrate NaCNBH.sub.3 were added. The resulting
mixture was incubated at 80.degree. C. for 17 h. The reaction
mixture was added to 160 ml of a cold 1:1 mixture of acetone and
ethanol (v/v). The precipitate was collected by centrifugation and
dialysed for 4 d against water (SnakeSkin dialysis tubing, 3.5 KD
cut off, Perbio Science Deutschland GmbH, Bonn, D), and
lyophilized. [0405] b) Incubation of the mixture resulting from
adding 0.83 mmol 1-mercapto-2-amino ethane and 50 mg sodium
cyanoborohydrate NaCNBH.sub.3 to the solution of 200 mg
hydroxyethyl starch was also possible and carried out at 25.degree.
C. for 3 d.
Example 2
Formation of Hydroxyethyl Starch Derivatives by Conjugation with
the Non-Oxidised Reducing End
Example 2.1
Reaction of Hydroxyethyl Starch with Carbohydrazide
##STR00064##
[0407] 0.96 g of HES18/0.4 (MW=18,000 D, DS=0.4) were dissolved in
8 ml aqueous 0.1 M sodium acetate buffer, pH 5.2, and 8 mmol
carbohydrazide (Sigma Aldrich, Taufkirchen, D) were added. After
stirring for 18 h at 25.degree. C., the reaction mixture was added
to 160 ml of a cold 1:1 mixture of acetone and ethanol (v/v). The
precipitated product was collected by centrifugation, re-dissolved
in 40 ml water, and dialysed for 3 d against water (SnakeSkin
dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH,
Bonn, D), and lyophilized.
Example 2.2
Reaction of Hydroxyethyl Starch with Adepic Dihydrazide
##STR00065##
[0409] 0.96 g of HES18/0.4 (MW=18,000 D, DS=0.4) were dissolved in
8 ml aqueous 0.1. M sodium acetate buffer, pH 5.2, and 8 mmol
adepic dihydrazide (Lancaster Synthesis, Frankfurt/Main, D) were
added. After stirring for 18 h at 25.degree. C., the reaction
mixture was added to 160 ml of a cold 1:1 mixture of acetone and
ethanol (v/v). The precipitated product was collected by
centrifugation, re-dissolved in 40 ml water, and dialysed for 3 d
against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio
Science Deutschland GmbH, Bonn, D), and lyophilized.
Example 2.3
Reaction of Hydroxyethyl Starch with
1,4-phenylene-bis-3-thiosemicarbazide
##STR00066##
[0411] 0.96 g of HES18/0.4 (MW=18,000 D, DS=0.4) were dissolved in
8 ml aqueous 0.1 M sodium acetate buffer, pH 5.2, and 8 mmol
1,4-phenylene-bis-3-thiosemicarbazide (Lancaster Synthesis,
Frankfurt/Main, D) were added. After stirring for 18 h at
25.degree. C., 8 ml water was added to the reaction mixture, and
the suspension was centrifugated for 15 min at 4,500 rpm. The clear
supernatant was decanted and subsequently added to 160 ml of a cold
1:1 mixture of acetone and ethanol (v/v). The precipitated product
was collected by centrifugation, re-dissolved in 40 ml water, and
centrifugated for 15 min at 4,500 rpm. The clear supernatant was
dialysed for 3 d against water (SnakeSkin dialysis tubing, 3.5 KD
cut off, Perbio Science Deutschland GmbH, Bonn, D), and
lyophilized.
Example 2.4
Reaction of Hydroxyethyl Starch with
O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl Amine
##STR00067##
[0413] O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine was
synthesized as described in Boturyn et al. Tetrahedron 53 (1997) p.
5485-5492 in 2 steps from commercially available materials.
[0414] 0.96 g of HES18/0.4 (MW=18,000 D, DS=0.4) were dissolved in
8 ml aqueous 0.1 M sodium acetate buffer, pH 5.2, and 8 mmol
O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine were added. After
stirring for 18 h at 25.degree. C., the reaction mixture was added
to 160 ml of a cold 1:1 mixture of acetone and ethanol (v/v). The
precipitated product was collected by centrifugation, re-dissolved
in 40 ml water, and dialysed for 3 d against water (SnakeSkin
dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH,
Bonn, D), and lyophilized.
Example 3
Formation of Hydroxyethyl Starch Derivatives by Reaction with the
Oxidised Reducing End
Example 3.1
Reaction of Hydroxyethyl Starch with Carbohydrazide
##STR00068##
[0416] 0.12 mmol Oxo-HES 10/0.4 (MW=10,000 D, DS=0.4, prepared
according to DE 196 28 705 A1) were dissolved in 3 ml absolute
dimethyl sulfoxide (DMSO) and added dropwise under nitrogen to a
mixture of 15 mmol of carbohydrazide (Sigma Aldrich, Taufkirchen,
D) in 15 ml DMSO. After stirring for 88 h at 65.degree. C., the
reaction mixture was added to 160 ml of a cold 1:1 mixture of
acetone and ethanol (v/v). The precipitate was collected by
centrifugation and was dialysed for 4 d against water (SnakeSkin
dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH,
Bonn, D) and lyophilised.
Example 3.2
Reaction of Hydroxyethyl Starch with
1,4-phenylene-bis-3-thiosemicarbazide
##STR00069##
[0418] 0.12 mmol Oxo-HES 10/0.4 (MW=10,000 D, DS=0.4, prepared
according to DE 196 28 705 A1) were dissolved in 3 ml absolute
dimethyl sulfoxide (DMSO) and added dropwise under nitrogen to a
mixture of 15 mmol of 1,4-phenylene-bis-3-thiosemicarbazide
(Lancaster Synthesis, Frankfurt/Main, D) in 15 ml DMSO. After
stirring for 88 h at 65.degree. C., the reaction mixture was added
to 160 ml of a cold 1:1 mixture of acetone and ethanol (v/v). The
precipitate was collected by centrifugation and was dialysed for 4
d against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio
Science Deutschland GmbH, Bonn, D) and lyophilized.
Example 3.3
Reaction of Hydroxyethyl Starch with Hydrazine
[0419] H.sub.2N--NH.sub.2
[0420] 1.44 g (0.12 mmol) of Oxo-HES 10/0.4 (MW=10,000 D, DS=0.4,
prepared according to DE 196 28 705 A1) were dissolved in 3 ml
absolute dimethyl sulfoxide (DMSO) and were added dropwise under
nitrogen to a mixture of 0.47 ml (15 mmol) hydrazine in 15 ml DMSO.
After stirring for 19 h at 40.degree. C. the reaction mixture was
added to 160 ml of a 1:1 mixture of ethanol and acetone (v/v). The
precipitated product was collected by centrifugation, redissolved
in 40 mL of water and dialysed for 2 days against a 0.5% (v/v)
triethylamine in water solution and for 2 days against water
(SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science
Deutschland GmbH, Bonn, Germany) and lyophilized.
Example 3.4
Reaction of Hydroxyethyl Starch with Hydroxylamine
##STR00070##
[0422] O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine was
synthesized as described by Boturyn et al in 2 steps from
commercially available materials (Boturyn, Boudali, Constant,
Defrancq, Lhomme, 1997, Tetrahedron, 53, 5485).
[0423] 1.44 g (0.12 mmol) of Oxo-HES 10/0.4 (MW=10,000 D, DS=0.4,
prepared according to DE 196 28 705 A1) were dissolved in 3 ml
absolute dimethyl sulfoxide (DMSO) and were added dropwise under
nitrogen to a mixture of 2.04 g (15 mmol)
O-[2-(2-aminooxy-ethoxy)-ethyl]hydroxylamine in 15 ml DMSO. After
stirring for 48 h at 65.degree. C. the reaction mixture was added
to 160 ml of a 1:1 mixture of ethanol and acetone (v/v). The
precipitated product was collected by centrifugation, re-dissolved
in 40 ml of water and dialysed for 4 days against water (SnakeSkin
dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH,
Bonn, Germany) and lyophilized.
Example 3.5
Reaction of Hydroxyethyl Starch with Adepic Dihydrazide
##STR00071##
[0425] 1.74 g (15 mmol) adepic dihydrazide (Lancaster Synthesis,
Frankfurt/Main, D) were dissolved in 20 ml absolute dimethyl
sulfoxide (DMSO) at 65.degree. C. and 1.44 g (0.12 mmol) of Oxo-HES
10/0.4 (MW=10,000 D, DS=0.4, prepared according to DE 196 28 705
A1), dissolved in 3 ml absolute DMSO were added dropwise under
nitrogen. After stirring for 68 h at 60.degree. C. the reaction
mixture was added to 200 ml of water The solution containing the
reaction product was dialysed for 2 days against a 0.5% (v/v)
triethylamine in water solution and for 2 days against water
(SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science
Deutschland GmbH, Bonn, Germany) and lyophilized.
Example 3.6
Reaction of Hydroxyethyl Starch with 1,4-diamino Butane
##STR00072##
[0427] 1.44 g (0.12 mmol) of Oxo-HES 10/0.4 (MW=10,000 D, DS=0.4,
prepared according to DE 196 28 705 A1) were dissolved in 3 ml dry
dimethyl sulfoxide (DMSO) and were added dropwise under nitrogen to
a mixture of 1.51 ml (15 mmol) 1,4-diaminobutane (Sigma Aldrich,
Taufkirchen, D) in 15 ml DMSO. After stirring for 19 h at
40.degree. C. the reaction mixture was added to 160 ml of a 1:1
mixture of ethanol and acetone (v/v). The precipitate
Amino-HES10KD/0.4 was collected by centrifugation, redissolved in
40 ml of water and dialysed for 4 days against water (SnakeSkin
dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH,
Bonn, Germany) and lyophilized.
Example 4
Oxidation of Erythropoietin
[0428] Oxidized erythropoietin was produced as described in Example
8. As oxidised erythropoietin, EPO-GT-1-A as described in Example
8.11(c) was used (EPO-GT-1 without acid hydroylsis, treated with
mild periodate oxidation).
Example 5
Conjugation of Hydroxyethyl Starch Derivatives with Oxidized
Erythropoietin of Example 4
Example 5.1
Reaction of Oxidized Erythropoietin with the Reaction Product of
Example 2.1
[0429] Oxidized EPO (1.055 .mu.g/.mu.l) in 20 mM PBS buffer was
adjusted to pH 5.3 with 5 M sodium acetate buffer, pH 5.2. To 19
.mu.l of the EPO solution, 18 .mu.l of a solution of the HES
derivate as produced according to example 2.1 (MW 18 KD; 18.7
.mu.g/.mu.l in 0.1 M sodium acetate buffer, pH 5.2) was added, and
the mixture was incubated for 16 h at 25.degree. C. After
lyophilisation, the crude product was analyzed by SDS-Page with
NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, Calif.,
USA) as described in the instructions given by Invitrogen. The gel
is stained with Roti-Blue Coomassie staining reagent (Roth,
Karlsruhe, D) overnight.
[0430] The experimental result is shown in FIG. 1. A successful
conjugation is indicated by the migration of the protein band to
higher molecular weights. The increased bandwidth is due to the
molecular weight distribution of the HES derivatives used and the
number of HES derivatives linked to the protein.
Example 5.2
Reaction of Oxidized Erythropoietin with the Reaction Product of
Example 2.3
[0431] Oxidized EPO (1.055 .mu.g/.mu.l) in 20 mM PBS buffer was
adjusted to pH 5.3 with 5 M sodium acetate buffer, pH 5.2. To 19
.mu.l of the EPO solution, 18 .mu.l of a solution of the HES
derivate as produced according to example 2.3 (MW 18 KD; 18.7
.mu.g/.mu.l in 0.1 M sodium acetate buffer, pH 5.2) was added, and
the mixture was incubated for 16 h at 25.degree. C. After
lyophilisation, the crude product was analyzed by SDS-Page with
NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, Calif.,
USA) as described in the instructions given by Invitrogen.
Example 5.3
Reaction of Oxidized Erythropoietin with the Reaction Product of
Example 2.4
[0432] Oxidized EPO (1.055 .mu.g/.mu.l) in 20 mM PBS buffer was
adjusted to pH 5.3 with 5 M sodium acetate buffer, pH 5.2. To 19
.mu.l of the EPO solution, 18 .mu.l of a solution of the HES
derivate as produced according to example 2.4 (MW 18 kD; 18.7
.mu.g/.mu.l in 0.1 M sodium acetate buffer, pH 5.2) was added, and
the mixture was incubated for 16 h at 25.degree. C. After
lyophilisation, the crude product was analyzed by SDS-Page with
NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, Calif.,
USA) as described in the instructions given by Invitrogen. The gel
is stained with Roti-Blue Coomassie staining reagent (Roth,
Karlsruhe, D) overnight.
[0433] The experimental result is shown in FIG. 2. A successful
conjugation is indicated by the migration of the protein band to
higher molecular weights. The increased bandwidth is due to the
molecular weight distribution of the HES derivatives used and the
number of HES derivatives linked to the protein.
Example 5.4
Reaction of Oxidized Erythropoietin with the Reaction Product of
Example 3.1
[0434] Oxidized EPO (1.055 .mu.g/.mu.l) in 20 mM PBS buffer was
adjusted to pH 5.3 with 5 M sodium acetate buffer, pH 5.2. To 19
.mu.l of the EPO solution, 18 .mu.l of a solution of the HES
derivate as produced according to example 3.1 (MW 10 kD; 18.7
.mu.g/.mu.l in 0.1 M sodium acetate buffer, pH 5.2) was added, and
the mixture was incubated for 16 h at 25.degree. C. After
lyophilisation, the crude product was analyzed by SDS-Page with
NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, Calif.,
USA) as described in the instructions given by Invitrogen. The gel
is stained with Roti-Blue Coomassie staining reagent (Roth,
Karlsruhe, D) overnight.
[0435] The experimental result is shown in FIG. 3. A successful
conjugation is indicated by the migration of the protein band to
higher molecular weights. The increased bandwidth is due to the
molecular weight distribution of the HES derivatives used and the
number of HES derivatives linked to the protein.
Example 5.5
Reaction of Oxidized Erythropoietin with the Reaction Product of
Example 3.2
[0436] Oxidized EPO (1.055 .mu.g/.mu.l) in 20 mM PBS buffer was
adjusted to pH 5.3 with 5 M sodium acetate buffer, pH 5.2. To 19
.mu.l of the EPO solution, 18 .mu.l of a solution of the HES
derivate as produced according to example 3.1 (MW 10 kD; 18.7
.mu.g/.mu.l in 0.1 M sodium acetate buffer, pH 5.2) was added, and
the mixture was incubated for 16 h at 25.degree. C. After
lyophilisation, the crude product was analyzed by SDS-Page with
NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, Calif.,
USA) as described in the instructions given by Invitrogen. The gel
is stained with Roti-Blue Coomassie staining reagent (Roth,
Karlsruhe, D) overnight.
[0437] The experimental result is shown in FIG. 3. A successful
conjugation is indicated by the migration of the protein band to
higher molecular weights. The increased bandwidth is due to the
molecular weight distribution of the HES derivatives used and the
number of HES derivatives linked to the protein.
Example 6
Formation of Thio-EPO by Reduction of Erythropoietin
[0438] 241.5 .mu.g erythropoietin (EPO-GT-1, see Example 8) in 500
.mu.l of a 0.1 M sodium borate buffer, 5 mM EDTA, 10 mM DTT
(Lancaster, Morcambe, UK), pH 8.3, were incubated for 1 h at
37.degree. C. The DTT was removed by centrifugal filtration with a
VIVASPIN 0.5 ml concentrator, 10 KD MWCO (VIVASCIENCE, Hannover, D)
at 13,000 rpm, subsequent washing 3 times with the borate buffer
and twice with a phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA,
pH 7.2). The gel is stained with Roti-Blue Coomassie staining
reagent (Roth, Karlsruhe, D) overnight.
Example 7
Conjugation of Hydroxyethyl Starch Derivatives with
Thio-Erythropoietin Using a Crosslinking Compound
[0439] In each of the following examples,
N-(alpha-maleimidoacetoxy) succinimide ester (AMAS)
##STR00073##
was used as crosslinking compound.
Example 7.1
Reaction of Thio-Erythropoietin with the Reaction Product of
Example 2.1 and the Crosslinking Compound
[0440] To 50 nmol HES derivate as produced according to example 2.1
and dissolved in 200 .mu.l of a 0.1 M sodium phosphate buffer (0.1
M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 .mu.l of a solution of 2.5
.mu.mol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO were added.
The clear solution was incubated for 80 min at 25.degree. C. and 20
min at 40.degree. C. Remaining AMAS was removed by centrifugal
filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO
(VIVASCIENCE, Hannover, D) at 13,000 rpm, washing 4 times and 30
min with the phosphate buffer.
[0441] To the residual solution, 15 .mu.g of ThioEPO as produced
according to example 5 (1 .mu.g/.mu.l in phosphate buffer) were
added, and the mixture was incubated for 16 h at 25.degree. C.
After lyophilisation, the crude product was analysed by SDS-Page
with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad,
USA) as described in the instructions given by Invitrogen. The gel
is stained with Roti-Blue Coomassie staining reagent (Roth,
Karlsruhe, D) overnight.
[0442] The experimental result is shown in FIG. 4. A successful
conjugation is indicated by the migration of the protein band to
higher molecular weights. The increased bandwidth is due to the
molecular weight distribution of the HES derivatives used and the
number of HES derivatives linked to the protein.
Example 7.2
Reaction of Thio-Erythropoietin with the Reaction Product of
Example 2.2 and the Crosslinking Compound
[0443] To 50 nmol HES derivate as produced according to example 2.2
and dissolved in 200 .mu.l of a 0.1 M sodium phosphate buffer (0.1
M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 .mu.l of a solution of 2.5
.mu.mol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO were added.
The clear solution was incubated for 80 min at 25.degree. C. and 20
min at 40.degree. C. Remaining AMAS was removed by centrifugal
filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO
(VIVASCIENCE, Hannover, D) at 13,000 rpm, washing 4 times and 30
min with the phosphate buffer.
[0444] To the residual solution, 15 .mu.g of ThioEPO as produced
according to example 5 (1 .mu.g/.mu.l in phosphate buffer) were
added, and the mixture was incubated for 16 h at 25.degree. C.
After lyophilisation, the crude product was analysed by SDS-Page
with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad,
USA) as described in the instructions given by Invitrogen. The gel
is stained with Roti-Blue Coomassie staining reagent (Roth,
Karlsruhe, D) overnight.
[0445] The experimental result is shown in FIG. 5. A successful
conjugation is indicated by the migration of the protein band to
higher molecular weights. The increased bandwidth is due to the
molecular weight distribution of the HES derivatives used and the
number of HES derivatives linked to the protein.
Example 7.3
Reaction of Thio-Erythropoietin with the Reaction Product of
Example 2.3 and the Crosslinking Compound
[0446] To 50 nmol HES derivate as produced according to example 2.3
and dissolved in 200 .mu.l of a 0.1 M sodium phosphate buffer (0.1
M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 .mu.l of a solution of 2.5
.mu.mol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO were added.
The clear solution was incubated for 80 min at 25.degree. C. and 20
min at 40.degree. C. Remaining AMAS was removed by centrifugal
filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO
(VIVASCIENCE, Hannover, D) at 13,000 rpm, washing 4 times and 30
min with the phosphate buffer.
[0447] To the residual solution, 15 .mu.g of ThioEPO as produced
according to example 5 (1 .mu.g/.mu.l in phosphate buffer) were
added, and the mixture was incubated for 16 h at 25.degree. C.
After lyophilisation, the crude product was analysed by SDS-Page
with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad,
USA) as described in the instructions given by Invitrogen. The gel
is stained with Roti-Blue Coomassie staining reagent (Roth,
Karlsruhe, D) overnight.
[0448] The experimental result is shown in FIG. 5. A successful
conjugation is indicated by the migration of the protein band to
higher molecular weights. The increased bandwidth is due to the
molecular weight distribution of the HES derivatives used and the
number of HES derivatives linked to the protein.
Example 7.4
Reaction of Thio-Erythropoietin with the Reaction Product of
Example 2.4 and the Crosslinking Compound
[0449] To 50 nmol HES derivate as produced according to example 2.4
and dissolved in 200 .mu.l of a 0.1 M sodium phosphate buffer (0.1
M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 .mu.l of a solution of 2.5
.mu.mol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO were added.
The clear solution was incubated for 80 min at 25.degree. C. and 20
min at 40.degree. C. Remaining AMAS was removed by centrifugal
filtration with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO
(VIVASCIENCE, Hannover, D) at 13,000 rpm, washing 4 times and 30
min with the phosphate buffer.
[0450] To the residual solution, 15 .mu.g of ThioEPO as produced
according to example 5 (1 .mu.g/.mu.l in phosphate buffer) were
added, and the mixture was incubated for 16 h at 25.degree. C.
After lyophilisation, the crude product was analysed by SDS-Page
with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad,
USA) as described in the instructions given by Invitrogen. The gel
is stained with Roti-Blue Coomassie staining reagent (Roth,
Karlsruhe, D) overnight.
[0451] The experimental result is shown in FIG. 4. A successful
conjugation is indicated by the migration of the protein band to
higher molecular weights. The increased bandwidth is due to the
molecular weight distribution of the HES derivatives used and the
number of HES derivatives linked to the protein.
Example 7.5
Reaction of Thio-Erythropoietin with the Reaction Product of
Example 1.1 and the Crosslinking Compound
[0452] To 50 nmol HES derivate as produced according to example
1.1, at incubation conditions of 80.degree. C. and 17 h as well as
of 25.degree. C. and 3 d, and dissolved in 200 .mu.l of a 0.1 M
sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2),
10 .mu.A of a solution of 2.5 .mu.mol AMAS (Sigma Aldrich,
Taufkirchen, D) in DMSO were added. The clear solution was
incubated for 80 min at 25.degree. C. and 20 min at 40.degree. C.
Remaining AMAS was removed by centrifugal filtration with a
VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, D)
at 13,000 rpm, washing 4 times and 30 min with the phosphate
buffer.
[0453] To the residual solution, 15 .mu.g of ThioEPO as produced
according to example 5 (1 .mu.g/.mu.l in phosphate buffer) were
added, and the mixture was incubated for 16 h at 25.degree. C.
After lyophilisation, the crude product was analysed by SDS-Page
with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad,
USA) as described in the instructions given by Invitrogen. The gel
is stained with Roti-Blue Coomassie staining reagent (Roth,
Karlsruhe, D) overnight.
[0454] The experimental result is shown in FIG. 5. A successful
conjugation is indicated by the migration of the protein band to
higher molecular weights. The increased bandwidth is due to the
molecular weight distribution of the HES derivatives used and the
number of HES derivatives linked to the protein.
Example 7.6
Reaction of Thio-Erythropoietin with the Reaction Product of
Example 1.3 and the Crosslinking Compound
[0455] To 50 nmol HES derivate as produced according to example
1.3, at incubation conditions of 80.degree. C. and 17 h as well as
of 25.degree. C. and 3 d, and dissolved in 200 .mu.l of a 0.1 M
sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2),
10 .mu.l of a solution of 2.5 .mu.mol AMAS (Sigma Aldrich,
Taufkirchen, D) in DMSO were added. The clear solution was
incubated for 80 min at 25.degree. C. and 20 min at 40.degree. C.
Remaining AMAS was removed by centrifugal filtration with a
VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, D)
at 13,000 rpm, washing 4 times and 30 min with the phosphate
buffer.
[0456] To the residual solution, 15 .mu.g of ThioEPO as produced
according to example 5 (1 .mu.g/.mu.l in phosphate buffer) were
added, and the mixture was incubated for 16 h at 25.degree. C.
After lyophilisation, the crude product was analysed by SDS-Page
with NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad,
USA) as described in the instructions given by Invitrogen. The gel
is stained with Roti-Blue Coomassie staining reagent (Roth,
Karlsruhe, D) overnight.
[0457] The experimental result is shown in FIG. 5. A successful
conjugation is indicated by the migration of the protein band to
higher molecular weights. The increased bandwidth is due to the
molecular weight distribution of the HES derivatives used and the
number of HES derivatives linked to the protein.
Example 7.7
Reaction of Thio-Erythropoietin with the Reaction Product of
Example 3.1 and the Crosslinking Compound
[0458] To 50 nmol HES derivate, produced according to Example 3.1
and dissolved in 200 .mu.l phosphate buffer (0.1 M, 9.15 M NaCl, 50
mM EDTA, pH 7.2), 10 .mu.l of a solution of 2.5 .mu.mol AMAS (Sigma
Aldrich, Taufkirchen, D) in DMSO was added, and the clear solution
was incubated for 80 min at 25.degree. C. and 20 min at 40.degree.
C. The AMAS was removed by centrifugal filtration with a VIVASPIN
0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, Germany) at
13,000 rpm and washing 4 times for 30 min with the phosphate
buffer.
[0459] To the residual solution, 15 .mu.g Thio-EPO (1 .mu.g/.mu.l
in phosphate buffer) were added, and the mixture was incubated for
16 h at 25.degree. C. After lyophilisation, the crude product was
analysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer
(Invitrogen, Carlsbad, Calif., USA) as described in the
instructions given by Invitrogen. The gel is stained with Roti-Blue
Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
[0460] The experimental result is shown in FIG. 6. A successful
conjugation is indicated by the migration of the protein band to
higher molecular weights. The increased bandwidth is due to the
molecular weight distribution of the HES derivatives used and the
number of HES derivatives linked to the protein.
Example 7.8
Reaction of Thio-Erythropoietin with the Reaction Product of
Example 3.2 and the Crosslinking Compound
[0461] To 50 nmol HES derivate, produced according to Example 3.2
and dissolved in 200 .mu.l phosphate buffer (0.1 M, 9.15 M NaCl, 50
mM EDTA, pH 7.2), 10 .mu.l of a solution of 2.5 .mu.mol AMAS (Sigma
Aldrich, Taufkirchen, D) in DMSO was added, and the clear solution
was incubated for 80 min at 25.degree. C. and 20 min at 40.degree.
C. The AMAS was removed by centrifugal filtration with a VIVASPIN
0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, Germany) at
13,000 rpm and washing 4 times for 30 min with the phosphate
buffer.
[0462] To the residual solution, 15 .mu.g Thio-EPO (1 .mu.g/.mu.l
in phosphate buffer) were added, and the mixture was incubated for
16 h at 25.degree. C. After lyophilisation, the crude product was
analysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer
(Invitrogen, Carlsbad, Calif., USA) as described in the
instructions given by Invitrogen. The gel is stained with Roti-Blue
Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
[0463] The experimental result is shown in FIG. 6. A successful
conjugation is indicated by the migration of the protein band to
higher molecular weights. The increased bandwidth is due to the
molecular weight distribution of the HES derivatives used and the
number of HES derivatives linked to the protein.
Example 7.9
Reaction of Thio-Erythropoietin with the Reaction Product of
Example 3.3 and the Crosslinking Compound
[0464] To 50 nmol HES derivate, produced according to Example 3.3
and dissolved in 200 .mu.l phosphate buffer (0.1 M, 9.15 M NaCl, 50
mM EDTA, pH 7.2), 10 .mu.l of a solution of 2.5 .mu.mol AMAS (Sigma
Aldrich, Taufkirchen, D) in DMSO was added, and the clear solution
was incubated for 80 min at 25.degree. C. and 20 min at 40.degree.
C. The AMAS was removed by centrifugal filtration with a VIVASPIN
0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, Germany) at
13,000 rpm and washing 4 times for 30 min with the phosphate
buffer.
[0465] To the residual solution, 15 .mu.g Thio-EPO (1 .mu.g/.mu.l
in phosphate buffer) were added, and the mixture was incubated for
16 h at 25.degree. C. After lyophilisation, the crude product was
analysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer
(Invitrogen, Carlsbad, Calif., USA) as described in the
instructions given by Invitrogen. The gel is stained with Roti-Blue
Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
[0466] The experimental result is shown in FIG. 6. A successful
conjugation is indicated by the migration of the protein band to
higher molecular weights. The increased bandwidth is due to the
molecular weight distribution of the HES derivatives used and the
number of HES derivatives linked to the protein.
Example 7.10
Reaction of Thio-Erythropoietin with the Reaction Product of
Example 3.4 and the Crosslinking Compound
[0467] To 50 nmol HES derivate, produced according to Example 3.4
and dissolved in 200 .mu.l phosphate buffer (0.1 M, 9.15 M NaCl, 50
mM EDTA, pH 7.2), 10 .mu.l of a solution of 2.5 .mu.mol AMAS (Sigma
Aldrich, Taufkirchen, D) in DMSO was added, and the clear solution
was incubated for 80 min at 25.degree. C. and 20 min at 40.degree.
C. The AMAS was removed by centrifugal filtration with a VIVASPIN
0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, Germany) at
13,000 rpm and washing 4 times for 30 min with the phosphate
buffer.
[0468] To the residual solution, 15 .mu.g Thio-EPO (1 .mu.g/.mu.l
in phosphate buffer) were added, and the mixture was incubated for
16 h at 25.degree. C. After lyophilisation, the crude product was
analysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer
(Invitrogen, Carlsbad, Calif., USA) as described in the
instructions given by Invitrogen. The gel is stained with Roti-Blue
Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
[0469] The experimental result is shown in FIG. 6. A successful
conjugation is indicated by the migration of the protein band to
higher molecular weights. The increased bandwidth is due to the
molecular weight distribution of the HES derivatives used and the
number of HES derivatives linked to the protein.
Example 7.11
Reaction of Thio-Erythropoietin with the Reaction Product of
Example 3.5 and the Crosslinking Compound
[0470] To 50 nmol HES derivate, produced according to Example 3.5
and dissolved in 200 .mu.l phosphate buffer (0.1 M, 9.15 M NaCl, 50
mM EDTA, pH 7.2), 10 .mu.l of a solution of 2.5 .mu.mol AMAS (Sigma
Aldrich, Taufkirchen, D) in DMSO was added, and the clear solution
was incubated for 80 min at 25.degree. C. and 20 min at 40.degree.
C. The AMAS was removed by centrifugal filtration with a VIVASPIN
0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, Germany) at
13,000 rpm and washing 4 times for 30 min with the phosphate
buffer.
[0471] To the residual solution, 15 .mu.g Thio-EPO (1 .mu.g/.mu.l
in phosphate buffer) were added, and the mixture was incubated for
16 h at 25.degree. C. After lyophilisation, the crude product was
analysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer
(Invitrogen, Carlsbad, Calif., USA) as described in the
instructions given by Invitrogen. The gel is stained with Roti-Blue
Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
[0472] The experimental result is shown in FIG. 6. A successful
conjugation is indicated by the migration of the protein band to
higher molecular weights. The increased bandwidth is due to the
molecular weight distribution of the HES derivatives used and the
number of HES derivatives linked to the protein.
Example 7.12
Reaction of Thio-Erythropoietin with the Reaction Product of
Example 3.6 and the Crosslinking Compound
[0473] To 50 nmol HES derivate, produced according to Example 36
and dissolved in 200 .mu.l phosphate buffer (0.1 M, 9.15 M NaCl, 50
mM EDTA, pH 7.2), 10 .mu.l of a solution of 2.5 .mu.mol AMAS (Sigma
Aldrich, Taufkirchen, D) in DMSO was added, and the clear solution
was incubated for 80 min at 25.degree. C. and 20 min at 40.degree.
C. The AMAS was removed by centrifugal filtration with a VIVASPIN
0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, Germany) at
13,000 rpm and washing 4 times for 30 min with the phosphate
buffer.
[0474] To the residual solution, 15 .mu.g Thio-EPO (1 .mu.g/.mu.l
in phosphate buffer) were added, and the mixture was incubated for
16 h at 25.degree. C. After lyophilisation, the crude product was
analysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer
(Invitrogen, Carlsbad, Calif., USA) as described in the
instructions given by Invitrogen. The gel is stained with Roti-Blue
Coomassie staining reagent (Roth, Karlsruhe, D) overnight.
[0475] The experimental result is shown in FIG. 6. A successful
conjugation is indicated by the migration of the protein band to
higher molecular weights. The increased bandwidth is due to the
molecular weight distribution of the HES derivatives used and the
number of HES derivatives linked to the protein.
Example 8
Preparative Production of HES-EPO Conjugates
Summary
[0476] HES-EPO conjugates were synthesized by coupling of HES
derivatives (average mw of 18,000 Dalton; hydroxyethyl substitution
degree of 0.4) to the partially (mild periodate) oxidized sialic
acid residues on the oligosaccharide chains of recombinant human
EPO. Based on carbohydrate structural analysis the modifications
introduced did not affect the structural integrity of the core
oligosaccharide chains since MALDI/TOF-MS of the mild acid treated
HES-modified glycans revealed intact neutral
N-acetyllactosamine-type chains which were indistinguishable from
those observed in unmodified EPO product. The results obtained
indicate that at least 3 modified HES-residues are attached per EPO
molecule in the case of the EPO preparation which was subjected to
modification without prior partial sialic acid removal. An EPO
variant lacking about 50% of the sialic acid residues of the former
protein showed a similar apparent high molecular weight mobility in
SDS-PAGE (60-110 KDa vs 40 KDa for the BRP EPO standard). The HES
modified EPO is stable under standard ion-exchange chromatography
conditions at room temperature at pH 3-10.
[0477] The EPO-bioassay in the normocythaemic mouse system
indicates that the HES-modified EPO has 2.5-3.0 fold higher
specific activity (IU/mg) in this assay when compared to the
International BRP EPO reference standard based on protein
determination using the UV absorption value from the European
Pharmacopeia and an RP-HPLC EPO protein determination method
calibrated against the BRP EPO standard preparation.
Example 8.1
Materials and Methods
[0478] (a) Liberation of N-Linked Oligosaccharides by Digestion
with N-Glycosidase
[0479] Samples were incubated with 25 units (according to
manufacturer's specification, Roche Diagnostics, Germany) of
recombinant PNGase F over night at 37.degree. C. Complete digestion
was monitored by the specific mobility shift of the protein in
SDS-PAGE. The released N-glycans were separated from the
polypeptide by addition of 3 volumes of cold 100% ethanol and
incubation at -20.degree. C. for at least 2 hours (Schroeter S et
al., 1999). The precipitated protein was removed by centrifugation
for 10 minutes at 4.degree. C. at 13000 rpm. The pellet was then
subjected to two additional washes with 500 .mu.l of ice-cold 75%
ethanol. The oligosaccharides in the pooled supernatants were dried
in a vacuum centrifuge (Speed Vac concentrator, Savant Instruments
Inc., USA). The glycan samples were desalted using Hypercarb
cartridges (25 mg or 100 mg of HyperCarb) as follows prior to use:
the columns were washed with 3.times.500 .mu.l of 80% acetonitrile
(v/v) in 0.1% TFA followed by washes with 3.times.500 .mu.l of
water. The samples were diluted with water to a final volume of 300
.mu.l-600 .mu.l before loading onto the cartridge which then was
rigorously washed with water. Oligosaccharides were eluted with 1.2
ml (25 mg cartridges; 1.8 ml in the case of 100 mg cartridges) 25%
acetonitrile in water containing 0.1% trifluoroacetic acid (v/v).
The eluted oligosaccharides were neutralized with 2 M NH.sub.4OH
and were dried in a Speed Vac concentrator. In some cases desalting
of N-glycosidase released oligosaccharides was performed by
adsorption of the digestion mixture from samples <100 .mu.g of
total (glyco)protein onto 100 mg Hypercarb cartridges.
(b) Analysis of Oligosaccharides by Matrix-Assisted Laser
Desorption/Ionization Time-of-Flight Mass-Spectrometry
(MALDI/TOF/TOF-MS)
[0480] A Bruker ULTRAFLEX time-of-flight (TOF/TOF) instrument was
used: native desialylated oligosaccharides were analyzed using
2,5-dihydroxybenzoic acid as UV-absorbing material in the positive
as well as in the negative ion mode using the reflectron in both
cases. For MS-MS analyses, selected parent ions were subjected to
laser induced dissociation (LID) and the resulting fragment ions
separated by the second TOF stage (LIFT) of the instrument Sample
solutions of 1 .mu.l and an approximate concentration of 1-10
.mu.pmol.mu.l.sup.-1 were mixed with equal amounts of the
respective matrix. This mixture was spotted onto a stainless steel
target and dried at room temperature before analysis.
Example 8.2
Preparation and Characterization of Recombinant Human EPO
(EPO-GT-1)
[0481] EPO was expressed from recombinant CHO cells as described
(Mueller P P et al., 1999, Dorner A J et al., 1984) and the
preparations were characterized according to methods described in
the Eur. Phar. (Ph. Eur. 4, Monography 01/2002:1316: Erythropoietin
concentrated solution). The final product had a sialic acid content
of 12 nMol (+/-1.5 nMol) per nMol of protein. The structures of
N-linked oligosaccharides were determined by HPAEC-PAD and by
MALDI/TOF-MS as described (Nimtz et al., 1999, Grabenhorst, 1999).
The EPO preparations that were obtained contained di-, tri- and
tetrasialylated oligosaccharides (2-12%, 15-28% and 60-80%,
respectively, sulphated and pentasialylated chains were present in
small amounts). The overall glycosylation characteristics of EPO
preparations were similar to that of the international BRP EPO
standard preparation.
[0482] The isoelectric focusing pattern of the recombinant EPO was
comparable to that of the international BRP Reference EPO standard
preparation showing the corresponding isoforms. 25% of the EPO
protein lacked O-glycosylation at Ser.sub.126 of the polypeptide
chain.
Example 8.3
Preparation of Partially Desialylated EPO Forms
[0483] EPO GT-1 protein (2.84 mg/ml) was heated to 80.degree. C. in
2a mM Na-phosphate buffer pH 7.0 and then 100 .mu.l of 1 N
H.sub.2SO.sub.4 was added per 1 ml of the EPO solution; incubation
was continued for 5 min, 10 min and 60 min, respectively, yielding
EPO preparations of different degree of sialylation. Quantitation
of oligosaccharides with 0-4 sialic acids was performed after
liberation of oligosaccharides with polypeptide N-glycosidase and
isolation of N-linked chains was performed by desalting using
Hypercarb cartridges (25 mg HyperSep Hypercarb;
ThermoHypersil-Keystone, UK). EPO preparations were neutralized by
addition of 1 N NaOH and were frozen in liquid N.sub.2 and were
stored at -20.degree. C. until further use.
Example 8.4
Periodate Oxidation of Sialylated EPO Forms
[0484] To 10 mg of untreated or mild acid treated EPO dissolved in
3.5 ml of 20 mM Na-phosphate buffer pH 7.0 was added 1.5 ml of 0.1
M Na-acetate buffer pH 5.5 and the mixture was cooled to 0.degree.
C. in an ice-bath; 500 .mu.l of 10 mM Na-periodate was added and
the reaction mixture was kept in the dark for 60 min at 0.degree.
C. Then 10 .mu.l of glycerol was added and incubation was continued
for further 10 min in the dark. The partially oxidized EPO forms
were separated from reagents by desalting using VIVASPIN
concentrators (10,000 MWCO, PES Vivascience AG, Hannover, Germany)
according to manufacturer's recommendation at 3000 rpm in a
laboratory centrifuge equipped with a fixed angle rotor. After
freezing in liquid nitrogen the EPO preparations were stored in a
final volume of 4 ml at -20.degree. C.
[0485] 100 .mu.g aliquots of the partially oxidized EPO preparation
were subjected to N-glycosidase treatment and oligosaccharides were
isolated using Hypercarb cartridges as described. Oligosaccharides
were desialylated by mild acid treatment and were analyzed by
HPAEC-PAD and their retention times were compared to those of
authentic standard oligosaccharides as described (Nimtz et al.,
1990 and 1993).
Example 8.5
Reduction of EPO Disulfides with Dithioerythreitol
[0486] 5 mg of EPO-GT-1 was incubated in 5 ml of 0.1 M Tris/HCl
buffer pH 8.1 in the presence of 30 mM dithioerythreitol (DTT) at
37.degree. C. for 60 minutes; removal of DTT was achieved by using
a Vivaspin concentrator at 4.degree. C., 4 cycles of buffer
exchange. The final reduced EPO preparation was frozen in liquid
nitrogen and stored at -20.degree. C. in 50 mM Na-acetate buffer pH
5.5.
Example 8.6
EPO Protein Determination
[0487] Quantitative determination of EPO protein was performed by
measuring UV absorption at 280 nm according to the Eur. Phar.
(European Pharmacopeia 4, Monography 01/2002: 1316: erythropoietin
concentrated solution) in a cuvette with 1 cm path length. In
addition, EPO was quantitated by applying a RP-HPLC method using a
RP-C4 column (Vydac Protein C4, Cat. #214TP5410, Grace Vydac, Ca,
US); the HPLC method was calibrated using the erythropoietin BRP 1
reference standard (European Pharmacopeia, Conseil de l'Europe B.P.
907-F67029, Strasbourg Cedex 1).
Example 8.7
Oxidation of Desialylated EPO with Galactose Oxidase
[0488] 4.485 mg of completely desialylated EPO was incubated in 20
mM Na-phosphate buffer pH 6.8 in the presence of 16 .mu.l catalase
(6214 units/200 ml) and 80 .mu.l of galactose oxidase (2250
units/ml from Dactylium dendroides (Sigma-Aldrich, Steinheim,
Germany); incubation at 37.degree. C. was over night; 2 times 20
.mu.l of galactose oxidase was added after 4 hours and after 8
hours after starting of the incubation.
Example 8.8
Preparation of EPO Samples for Bioassays
Purification of EPO from Incubations of Periodate- or
Galactose-Oxidase-Oxidized EPO Protein Preparations with Activated
HES
[0489] Purification of EPO samples (removal of unreacted HES
derivatives) was carried out at room temperature. The EPO
incubation mixtures (approximately 5 mg of EPO protein) were
diluted 1:10 with buffer A (20 mM N-morpholine propane sulfonic
acid [MOPS/NaOH] in H.sub.2O bidest, pH 8.0) and were applied to a
column containing 3 ml Q-Sepharose HP (Pharmacia Code no.
17-1014-03, Lot no. 220211) equilibrated with 10 column volumes
(CV) of buffer A by using a flow rate of 0.5 ml/min. The column was
washed with 6-8 CV of buffer A (flow rate=0.8 ml/min) and elution
was performed by using buffer B (20 mM morpholine ethane sulfonic
acid [MES/NaOH], 0.5 M NaCl in H.sub.2O bidest, pH 6.5) at a flow
rate of 0.5 ml/min. EPO was detected by UV absorption at 280 nm and
eluted in about 6 ml. The column was regenerated by using 3 CV of
buffer C (20 mM MES, 1.5 M NaCl in H.sub.2O adjusted to pH 6.5) and
was re-equilibrated by using 10 CV of buffer A (flow rate=0.7
ml/min).
[0490] Buffer exchange of EPO eluates obtained from the Q-Sepharose
step was performed using Vivaspin concentrators and phosphate
buffered saline (PBS) with each 3 centrifugation cycles per sample;
samples were adjusted to 2 ml with PBS and were stored at
-20.degree. C.
[0491] Only <25% of the partially desialylated and subsequently
mild periodate oxidized EPO forms that were subjected to
HES-modification were obtained from the Q-Sepharose eluate since
under the conditions employed the basic EPO forms did not bind
Q-Sepharose and were found in the flow-through together with
nonreacted HES derivatives.
Example 8.9
High-pH Anion-Exchange Chromatography with Pulsed Amperometric
Detection (HPAEC-PAD)
[0492] Purified native and desialylated oligosaccharides were
analyzed by high-pH anion-exchange (HPAE) chromatography using a
Dionex BioLC system (Dionex, USA) equipped with a CarboPac PA1
column (0.4.times.25 cm) in combination with a pulsed amperometric
detector (PAD) (Schroter et al., 1999; Nimtz et al., 1999).
Detector potentials (E) and pulse durations (T) were: E1: +50 mV,
T1: 480 ms; E2: +500 mV, T2: 120 ms; E3: -500 mV, T3: 60 ms, and
the output range was 500-1500 nA. The oligosaccharides were then
injected onto the CarboPac PA1 column which was equilibrated with
100% solvent A. For desialylated oligosaccharides elution (flow
rate: 1 ml-min.sup.-1) was performed by applying a linear gradient
(0-20%) of solvent B over a period of 40 min followed by a linear
increase from 20-100% solvent B over 5 min. Solvent A was 0.2 M
NaOH in bidistilled H.sub.2O, solvent B consisted of 0.6 M NaOAc in
solvent A. For native oligosaccharides the column was equilibrated
with 100% solvent C (0.1 M NaOH in bidistilled H.sub.2O) and
elution (flow rate: 1 mlmin.sup.-1) was performed by applying a
linear gradient (0-35%) of solvent D over a period of 48 min
followed by a linear increase from 35-100% solvent D over 10 min.
Solvent D consisted of 0.6 M NaAc in solvent C.
Example 8.10
Monosaccharide Compositional Analysis of N-Glycans, HES-Modified
N-Glycans and EPO protein by GC-MS
[0493] Monosaccharides were analyzed as the corresponding methyl
glycosides after methanolysis, N-reacetylation and
trimethylsilylation by GC/MS [Chaplin, M. F. (1982) A rapid and
sensitive method for the analysis of carbohydrate. Anal. Biochem.
123, 336-341]. The analyses were performed on a Finnigan GCQ ion
trap mass spectrometer (Finnigan MAT corp., San Jose, Calif.)
running in the positive ion E1 mode equipped with a 30 m DB5
capillary column. Temperature program: 2 min isotherm at 80.degree.
C., then 10 degrees min.sup.-1 to 300.degree. C.
[0494] Monosaccharides were identified by their retention time and
characteristic fragmentation pattern. The uncorrected results of
electronic peak integration were used for quantification.
Monosaccharides yielding more than one peak due to anomericity
and/or the presence of furanoid and pyranoid forms were quantified
by adding all major peaks. 0.5 .mu.g of myo-inositol was used as an
internal standard compound.
Example 8.11
Results
Example 8.11(a)
Characterization of N-Glycans of Mild Acid Treated (Partially
Desialylated) EPO-GT-1
[0495] EPO-GT-1 preparations subjected to mild acid treatment for
5, 10 or 60 min. were analyzed by SDS-PAGE before and after
liberation of N-linked oligosaccharides by incubation with
N-glycosidase as shown in FIG. 7. N-linked oligosaccharides were
subjected to HPAEC-PAD oligosaccharide mapping (FIG. 8). The
untreated EPO-GT-1 contained >90% of N-linked oligosaccharides
with 3 or 4 sialic acid residues whereas after 5 min of incubation
in the presence of mild acid <40% of carbohydrate chains had 3
or 4 sialic acid residues. HPAEC-PAD of the desialylated N-glycans
revealed that the ratio of neutral oligosaccharides that were
detected for the untreated EPO-GT-1 and remained stable in the
preparations subjected to acid treatment for 5, 10 or 60 min.
MALDI/TOF-MS of the desialylated glycans revealed that <90% of
the proximal fucose was present after mild acid treatment of the
protein.
Example 8.11(b)
Characterization of Periodate Treated EPO-GT-1
[0496] SDS-PAGE mobility of mild periodate treated EPO forms that
were previously subjected to a 5 and 10 minute treatment with acid
or were not treated are compared in FIG. 10. The conditions used
for periodate oxidation of sialic acids did not change the SDS-PAGE
pattern of EPO preparations (compare FIG. 7). Oxidation of sialic
acids resulted in HPAEC-PAD analysis a shift of oligosaccharides to
earlier elution times (compare FIGS. 8 and 11).
Example 8.11(c)
Characterization of HES-Modified EPO Derivatives
[0497] (aa) Time Course of HES Modification of EPO-GT-1-A with
Hydroxylamine-Modified HES Derivative X, Produced According to
Example 2.4
[0498] 400 .mu.g of hydroxylamine-modified HES derivative X was
added to 20 .mu.g of EPO-GT-1-A (mild periodate oxidized EPO, not
acid hydrolyzed prior to mild periodate oxidation) in 20 .mu.L of
0.5 M NaOAc buffer pH 5.5 and the reaction was stopped after 30
min, 2, 4, and 17 hours, respectively, by freezing samples in
liquid nitrogen. Subsequently samples were stored at -20.degree. C.
until further analysis.
[0499] SDS-PAGE sample buffer was added and the samples were heated
to 90.degree. C. and applied onto SDS-gels. As shown in FIG. 12,
increasing incubation times resulted in an increased shift towards
higher molecular weight of the protein. After 17 hours of
incubation in the presence of the hydroxylamine-modified HES
derivative X a diffuse Coomassie stained protein band was detected
migrating in an area between 60 and 11 KDa, based on the position
of molecular weight standards (see left part of FIG. 12). Upon
treatment with N-glycosidase most of the protein was shifted
towards the position of de-N-glycosylated EPO (see FIG. 12, right
gel; arrow A indicates migration position of N-glycosidase, arrow B
indicates migration position of de-N-glycosylated EPO; the diffuse
protein band visible in the region between the 28 KDa and 36 KDa
molecular weight standards presumably represents EPO-forms which
are modified by HES and the O-glycosylation site of the molecule.
In view of the specificity of N-glycosidase we conclude from this
result that in fact HES-modification occurs at the periodate
oxidized sialic acid residues of glycans of the EPO protein.
(bb) Characterization of HES-EPO Conjugates
[0500] HES-EPO conjugates I (originating from EPO-GT-1 after mild
periodate oxidation, i.e. from EPO-GT-1-A), II (resulting from
EPO-GT-1 subjected to 5 min acid hydrolysis and mild periodate
oxidation), III (resulting from EPO-GT-1 subjected to 10 min acid
hydrolysis and mild periodate oxidation) were synthesized as
described before. A control incubation (K) was included containing
unmodified EPO-GT-1 under the same buffer conditions to which an
equivalent amount of unmodified HES was added. The incubation
mixtures were subjected to further purification for subsequent
biochemical analysis of the HES-EPO derivatives.
[0501] Incubations HES-EPO conjugates I, II and III as well as the
control incubation K were subjected to a Q-Sepharose purification
step as described under "Material and Methods" (Example 8.8) in
order to remove the excess of nonreacted HES-reagent which was
expected in flow through of the ion-exchange column. Due to the
high amounts of basic EPO forms contained in previously acid
treated samples II and III we expected considerable amounts of
modified EPO product from these incubations in the flow through. As
is shown in FIG. 13, almost all of the EPO material from samples I
was retained by Q-Sepharose column whereas only approximately
20-30% of the samples III and II was recovered in the fraction
eluting with high salt concentration. All of the protein material
from the incubations with HES derivative X, both in the
flow-through and the fractions eluting with high salt, had apparent
higher molecular weight in SDS-PAGE when compared to the control
EPO.
[0502] In order to characterize in more detail the HES-modified EPO
sample A and K (see FIG. 11) were compared to periodate oxidized
form EPO-GT-1-A. The samples were subjected to N-glycosidase
treatment and as is depicted in FIGS. 14a and 14b the release of
N-glycans resulted in the two low molecular weight bands at the
position of the O-glycosylated and nonglycosylated EPO forms of the
standard EPO preparation. In the case of sample A a further band
migrating at the position of the 28 KDa mw standard was detected
suggesting HES-modification at the O-glycan of this EPO variant
(cf. Example 8.11(c)(aa)). This band (and also the heavily
HES-modified high mw form of N-glycosylated EPO, see FIGS. 14a and
14b) disappeared after subjecting the samples to mild hydrolysis
which is in agreement with the view that HES modification was
achieved at the periodate oxidised sialic acid residues of
erythropoietin.
[0503] Aliquots of the N-glycosidase incubation mixtures were
hydrolyzed using conditions enabling the complete removal of sialic
acids residues (and also the sialic acid linked HES derivative)
from oligosaccharides; after neutralization, the mixtures were then
absorbed onto small Hypercarb columns for their desalting. The
columns were washed rigorously with water followed by elution of
bound neutral oligosaccharides with 40% acetonitrile in H.sub.2O
containing 0.1% of trifuloacetic acid. The resulting
oligosaccharides were subjected to MALDI/TOF-MS. The spectra of the
desialylated oligosaccharide fractions from sample A, EPO-GT-1-A
and sample K showed identical masses for complex type
oligosaccharides at m/z=1810 Da (diantennary), 2175=triantennary,
2540=tetraantennary, 2906=tetraantennary plus 1 N-acetyllactosamine
repeat and 3271=tetraantennary plus 2 N-acetyllactosamine repeats;
small signals corresponding to lack of fucose (-146) and galactose
(minus 162) were detected which are attributable to the acid
hydrolysis conditions applied for sialic acid removal (see
MALDI-FIGS. 17, 18 and 19).
[0504] In a parallel experiment the N-glycosidase digestion mixture
was absorbed onto 1 ml RP-C18 cartridge (without prior acid
hydrolysis of oligosaccharides) and elution was performed with 5%
acetonitrile in water containing 0.1% TFA; under these conditions
the EPO protein was completely retained onto the RP-material and
oligosaccharides were washed off from the column with 5%
acetonitrile in H.sub.2O containing 0.1% TFA. The de-N-glycosylated
EPO protein was eluted with 70% acetonitrile in H.sub.2O containing
0.1% TFA. The oligosaccharide fractions from the RP-C18 step of
N-glycosidase-treated sample A, EPO GT-1-A and sample K were
neutralized and subjected to desalting using Hypercarb cartridges
as described before. The isolated oligosaccharides were subjected
to HPAEC-PAD mapping before (see FIG. 15) and after mild acid
treatment under conditions which enabled quantitative removal of
sialic acids from glycans (see FIG. 16).
[0505] The HPAEC-PAD profile for the native material obtained from
the HES-modified sample A showed only neglectable signals for
oligosaccharides whereas EPO GT-1-A-derived oligosaccharides
exhibited the same glycan profile as the one shown in FIG. 11
(sample named EPO-GT-1 after mild periodate treatment). The elution
profile of oligosaccharides obtained from the control EPO sample
(K) yielded the expected pattern (compare profile in FIG. 8). For
comparison, the native oligosaccharide profile of the international
BRP-EPO standard is included for comparison and as reference
standard.
[0506] After mild acid hydrolysis, all oligosaccharide preparations
showed an identical elution profile of neutral oligosaccharide
structures (see FIG. 16) with the expected qualitative and
quantitative composition of di-, tri- and tetraantennary
complex-type carbohydrate chains as described in the methods
section for the EPO preparation which was used as a starting
material in the present study. This result demonstrates that the
HES-modification of the EPO sample results in a covalent linkage of
the PIES derivative which is detached from the EPO-protein by
N-glycosidase and is acid-labile since it is removed from the
N-glycans using mild acid treatment conditions known to desialylate
carbohydrates (see FIGS. 14a+b).
(cc) Monosaccharide Compositional Analysis of HES-EPO and HES-EPO
N-Glycans by GC-MS
[0507] In order to further confirm HES-modification of EPO at the
N-glycans of the molecule, EPO samples were digested with
N-glycosidase and the EPO protein was adsorbed onto RP-C18
cartridges whereas oligosaccharide material was washed off as
described above. As shown in Table 2, glucose and hydroxyethylated
glucose derivatives were detected only in the EPO protein which was
subjected to HES-modification at cysteine residues and in
oligosaccharide fractions of EPO sample A2.
Example 8.11(d)
In-Vivo Assay of the Biological Activity of HES-Modified EPO
[0508] The EPO-bioassay in the normocythaemic mouse system
indicates was performed according to the procedures described in
the European Pharmacopeia; the laboratory that carried out the EPO
assay was using the International BRP EPO reference standard
preparation. For the HES-modified EPO A2 preparation a mean value
for the specific activity of 294,600 units per mg EPO of protein
was determined indicating an approximately 3-fold higher specific
activity when compared to the International BRP EPO reference
standard preparation that was included in the samples sent for
activity assays.
[0509] The results of the study are summarized in Table 3.
REFERENCES FOR EXAMPLES 1 TO 8
[0510] Nimtz M, Noll G, Paques E P, Conradt H S. [0511]
Carbohydrate structures of a human tissue plasminogen activator
expressed in recombinant Chinese hamster ovary cells. [0512] FEBS
Lett. 1990 Oct. 1; 271(1-2):14-8 [0513] Dorner A J, Wasley L C,
Kaufman R J. [0514] Increased synthesis of secreted proteins
induces expression of glucose-regulated proteins in
butyrate-treated Chinese hamster ovary cells. [0515] J Biol. Chem.
1989 Dec. 5; 264 (34):20602-7 [0516] Mueller P P, Schlenke P, Nimtz
M, Conradt H S, Hauser H [0517] Recombinant glycoprotein quality in
proliferation-controlled BHK-21 cells. [0518] Biotechnol Bioeng.
1999 Dec. 5; 65(5):529-36 [0519] Nimtz M, Martin W, Wray V, Kloppel
K D, Augustin J, Conradt H S. [0520] Structures of sialylated
oligosaccharides of human erythropoietin expressed in recobminant
BHK-21 cells. [0521] Eur J. Biochem. 1993. Apr. 1; 213(1):39-56
[0522] Hermentin P, Witzel R, Vliegenthart J F, Kamerling J P,
Nimtz M, Conradt H S. [0523] A strategy for the mapping of
N-glycans by high-ph anion-exchange chromatography with pulsed
amperometric detection. [0524] Anal Biochem. 1992 June;
203(2):281-9 [0525] Schroter S, Derr P, Conradt H S, Nimtz M, Hale
G, Kirchhoff C. [0526] Male specific modification of human CD52.
[0527] J Biol. Chem. 1999 Oct. 15; 274(42):29862-73
Example 9
Production of Recombinant EPO
A) Production in Mammalian Cells
[0528] Recombinant EPO was produced in CHO cells as follows
[0529] A plasmid harbouring the human EPO cDNA was cloned into the
eukaryotic expression vector (pCR3 and named afterwards pCREPO).
Site directed mutagenesis was performed using standard procedures
as described (Grabenhorst, Nimtz, Costa et al., 1998, In vivo
specificity of human alpha 1,3/4-fucosyltransferases III-VII in the
biosynthesis of Lewis(x) and sialyl Lewis(x) motifs on complex-type
N-glycans-Coexpression studies from BHK-21 cells together with
human beta-trace protein, J. Biol. Chem., 273(47),
30985-30994).
[0530] CHO cells stably expressing human EPO or amino acid variants
(e.g. Cys-29.fwdarw.Ser/Ala, or Cys-33.fwdarw.Ser/Ala,
Ser-126.fwdarw.Ala etc.) thereof were generated with the calcium
phosphate precipitation method and selected with G418-sulfate as
described (Grabenhorst et al.). Three days after transfection, the
cells were subcultivated 1:5 and selected in DMEM containing 10%
FBS and 1.5 g/liter G418 sulfate.
[0531] Using this selection procedure, usually 100-500 clones
survived and where propagated in selection medium for a further
time period of 2-3 weeks. Cell culture supernatants of confluently
growing monolayers were then analyzed for EPO expression levels by
Western blot analysis and by IEF/Western Blot analysis.
[0532] EPO was produced from stable subclones in spinner flasks or
in 21 perfusion reactors. Different glycoforms of EPO with
different amounts of NeuAc (e.g. 2-8, 4-10, 8-12 NeuAc residues)
were isolated according to published protocols using combinations
various chromatographic procedures as described below.
Literature:
[0533] Grabenhorst, Conradt, 1999, The cytoplasmic, transmembrane,
and stem regions of glycosyltransferases specify their in vivo
functional sublocalization and stability in the Golgi., J Biol.
Chem., 274(51), 36107-16; Grabenhorst, Schlenke, Pohl, Nimtz,
Conradt, 1999, Genetic engineering of recombinant glycoproteins and
the glycosylation pathway in mammalian host cells, Glycoconj J.,
16(2), 81-97; Mueller, Schlenke, Nimtz, Conradt, Hauser, 1999,
Recombinant glycoprotein product quality in
proliferation-controlled BHK-21 cells, Biotechnology and
bioengineering, 65(5), 529-536; Schlenke, Grabenhorst, Nimtz,
Conradt, 1999, Construction and characterization of stably
transfected BHK-21 cells with human-type sialylation
characteristic, Cytotechnology, 30(1-3), 17-25.
B) Production in Insect Cells
[0534] Recombinant human EPO is produced from insect cell lines SF9
and SF 21 after infection of cells with recombinant baculovirus
vector containing the human EPO cDNA under control of the
polyhedrin promoter as described in the literature.
[0535] Cells grown in serum-free culture medium are infected at
cell density of 2.times.10.sup.6 or .times.10.sup.7 cells per mL
and EPO titers are determined every day in the cell culture
supernatants. EPO is purified by Blue sepharose chromatography,
ion-exchange chromatography on Q-Sepharose and finally RP-HPLC on
C.sub.4-Phase.
[0536] Purity of the product is checked by SDS-PAGE and N-terminal
sequencing. Detailed carbohydrate structural analysis (N- and
O-glycosylation) may be performed according to published
procedures.
Literature:
[0537] Grabenhorst, Hofer, Nimtz, Jager, Conradt, 1993,
Biosynthesis and secretion of human interleukin 2 glycoprotein
variants from baculovirus-infected Sf21 cells. Characterization of
polypeptides and posttranslational modifications, Eur J. Biochem.,
215(1), 189-97; Quelle, Caslake, Burkert, Wojchowski, 1989,
High-level expression and purification of a recombinant human
erythropoietin produced using a baculovirus vector, Blood, 74(2),
652-7
Example 10A
Formation of Reactive HES Derivatives
1. SH-Reactive HES
[0538] 1.1 Reaction of EMCH with Oxo-HES12KD to Form SH-Reactive
HES12KD B
[0539] 0.144 g (0.012 mmol) of Oxo-HES12KD (Fresenius German Patent
DE 196 28 705 A1)
##STR00074##
are dissolved in 0.3 mL absolute dimethyl sulfoxide (DMSO) and are
added dropwise under nitrogen to a mixture of 34 mg (0.15 mmol)
EMCH (Perbio Science, Deutschland GmbH, Bonn, Germany) in 1.5 mL
DMSO. After stirring for 19 h at 60.degree. C. the reaction mixture
is added to 16 mL of a 1:1 mixture of ethanol and acetone. The
precipitate is collected by centrifugation, redissolved in 3 mL
DMSO and again precipitated as described. The SH-reactiv-HES12KD B
is obtained by centrifugation and drying in vaccuo. The conjugation
reaction with Thio-EPO is described in Example 11, 2.2.
Alternatives:
[0540] In this reaction, all cross-linkers can be used, which
exhibit a hydrazide- and a maleimide function, separated by a
spacer. Further examples for molecules of that group, available
from Perbio Science, Deutschland GmbH, Bonn, Germany, are shown in
table 2; marked with an "A". Furthermore, another group of
cross-linkers exhibiting an activated disulfide function instead of
a maleimide function could also be used.
1.2 Halogenacetamide-Derivatives of HES Glycosylamines
[0541] a) Glycosylamine-Formation 1 1Manger, Wong, Rademacher,
Dwek, 1992, Biochemistry, 31, 10733-10740; Manger, Rademacher,
Dwek, 1992, Biochemistry, 31, 10724-10732
[0542] A 1 mg sample of HES12KD is dissolved in 3 mL of saturated
ammonium bicarbonate. Additional solid ammonium bicarbonate is then
added to maintain saturation of the solution during incubation for
120 h at 30.degree. C. The Amino-HES12KD C is desalted by direct
lyophilization of the reaction mixture.
b) Acylation of the Glycosylamine C with Chloroacetic Acid
Anhydride
[0543] A 1 mg sample of Amino-HES12KD C is dissolved in 1 mL of 1 M
sodium bicarbonate and cooled on ice. To this is added a crystal of
solid chloroacetic acid anhydride (.about.5 mg), and the reaction
mixture is allowed to warm to room temperature. The pH is monitored
and additional base is added if the pH dropped below 7.0. After two
hours at room temperature a second aliquot of base and anhydride is
added. After six hours the product Chloroacetamide-HES D1
(X.dbd.Cl) is desalted by passage over a mixed bed Amberlite
MB-3(H)(OH) ion exchange resins.
c) Acylation of the Glycosylamine with Bromoacetic Anhydride2
2Black, Kiss, Tull, Withers, 1993, Carbohydr. Res., 250, 195
[0544] Bromoacetic anhydride is prepared as described by Thomas.3 A
1 mg sample of amino-HES12KD C is dissolved in 0.1 mL of dry DMF
and cooled on ice and 5 mg bromoacetic anhydride is added. The
reaction mixture is brought slowly to room temperature and the
solution is stirred for 3 h. The reaction mixture is added to 1 mL
of a 1:1 mixture of ethanol and acetone with -20.degree. C. The
precipitate is collected by centrifugation, redissolved in 0.1 mL
DMF and again precipitated as described. The Bromoacetamide-HES D2
(X.dbd.Br) is obtained by centrifugation and drying in vaccuo. The
conjugation reaction with Thio-EPO is described in Example 11, 1.2.
3Thomas, 1977, Methodes Enzymol., 46, 362
d) The corresponding Iodo-derivative D3 (X.dbd.I) is synthesized as
described for D2. Instead bromoacetic anhydride N-succinimidyl
iodoacetate is used and all steps are performed in the dark.
##STR00075##
Alternatives:
[0545] For acylation of amino groups, other activated forms of
halogen acidic acids can be used, e.g. [0546] -bromides or
-chlorides [0547] esters, e.g. N-hydroxysuccinimide ester, esters
with substituted phenoles (p-nitrophenole, pentafluorophenole,
trichlorophenole etc)
[0548] Furthermore, all cross-linkers having an amino reactive
group and a halogen acetyl function, separated by a spacer, could
be used. An example thereof is SBAP. This molecule and others are
available from Perbio Science Deutschland GmbH, Bonn, Germany. They
are marked in table 2 with an "D". For the use as cross-linkers for
the ligation of amino-HES with thio-EPO without isolation of the
halogenacetamide-HES derivatives see remarks in example 11,
1.2.
1.3 Halogenacetamide-Derivatives of Amino-HES E.sup.1
[0549] a) Reaction of 1,4-Diaminobutane with Oxo-HES12KD to
Amino-HES12KD E4
[0550] 1.44 g (0.12 mmol) of Oxo-HES12KD are dissolved in 3 mL dry
dimethyl sulfoxide (DMSO) and are added dropwise under nitrogen to
a mixture of 1.51 mL (15 mmol) 1,4-diaminobutane in 15 mL DMSO.
After stirring for 19 h at 40.degree. C. the reaction mixture is
added to 160 mL of a 1:1 mixture of ethanol and acetone. The
precipitate Amino-HES12KD E is collected by centrifugation,
redissolved in 40 mL of water an dialyzed for 4 days against water
(SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science
Deutschland GmbH, Bonn, Germany) and lyophilized.
##STR00076##
b) Chloroacetamide-HES12KD F1 is prepared as described for
chloroacetamide-HES12KD D1 in 1.3 above. c) Bromoacetamide-HES12KD
F2 (X.dbd.Br) is prepared as described for Bromoacetamide-HES12KD
D2 in 1.3 above. The conjugation reaction with Thio-EPO is
described in Example 11, 1.2. d) The corresponding Iodo-derivative
F3 (X.dbd.I) is not isolated before its reaction with Thio-EPO. The
experiment is described in Example 11, 1.1. 4S. Frie, Diplomarbeit,
Fachhochschule Hamburg, 1998
Alternatives:
[0551] See 1.2 above
2. CHO-Reactive HES
2.1 Hydrazide-HES
[0552] a) Reaction of Hydrazine with Oxo-HES12KD
##STR00077##
[0553] 1.44 g (0.12 mmol) of Oxo-HES12KD are dissolved in 3 mL
absolute dimethyl sulfoxide (DMSO) and are added dropwise under
nitrogen to a mixture of 0.47 mL (15 mmol) hydrazine in 15 mL DMSO.
After stirring for 19 h at 40.degree. C. the reaction mixture is
added to 160 mL of a 1:1 mixture of ethanol and acetone. The
precipitated product J is collected by centrifugation, redissolved
in 40 mL of water and dialyzed for 2 days against a 0.5% (v/v)
triethylamine in water solution and for 2 days against water
(SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science
Deutschland GmbH, Bonn, Germany) and lyophilized. The conjugation
reaction with oxidized Glyco-EPO is described in Example 12,
2.2.
b) Reaction of Adipic Dihydrazide with Oxo-HES12KD
##STR00078##
[0554] 1.74 g (15 mmol) adipic dihydrazide are dissolved in 20 mL
absolute dimethyl sulfoxide (DMSO) at 65.degree. C. and 1.44 g
(0.12 mmol) of Oxo-HES12KD, dissolved in 3 mL absolute DMSO are
added dropwise under nitrogen. After stirring for 68 h at
60.degree. C. the reaction mixture is added to 200 mL of water The
solution containing L is dialyzed for 2 days against a 0.5% (v/v)
triethylamine in water solution and for 2 days against water
(SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science
Deutschland GmbH, Bonn, Germany) and lyophilized. The conjugation
reaction with oxidized Glyco-EPO is described in Example 12,
2.2.
Alternatives:
[0555] Furthermore, derivatives can be used, wherein 2 hydrazide
groups are separated by any spacer.
3. Further Amino-HES12KD Derivatives I and H.sup.1
[0556] Ammonolysis of D or F is performed separately by dissolving
a 1 mg sample of each halogenacetamide in 0.1 mL of saturated
ammonium carbonate. Additional solid ammonium carbonate is then
added to maintain saturation of the solution during incubation of
120 h at 30.degree. C. The reaction mixture is added to 1 mL of a
1:1 mixture of ethanol and acetone with -20.degree. C. The
precipitate is collected by centrifugation, redissolved in 0.05 mL
water and again precipitated as described. The product amino-HES H
or I is obtained by centrifugation and drying in vaccuo. The
conjugation reaction with oxidized Glyco-EPO is described in
Example 12, 4.1.
##STR00079##
4. Hydroxylamine-modified HES12KD K
##STR00080##
[0558] O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine is synthesized
as described by Boturyn et al in 2 steps from commercially
available materials.5 1.44 g (0.12 mmol) of Oxo-HES12KD are
dissolved in 3 mL absolute dimethyl sulfoxide (DMSO) and are added
dropwise under nitrogen to a mixture of 2.04 g (15 mmol)
O-[2-(2-aminooxy-ethoxy)ethyl]-hydroxylamine in 15 mL DMSO. After
stirring for 48 h at 65.degree. C. the reaction mixture is added to
160 mL of a 1:1 mixture of ethanol and acetone. The precipitated
product K is collected by centrifugation, redissolved in 40 mL of
water and dialyzed for 4 days against water (SnakeSkin dialysis
tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH, Bonn,
Germany) and lyophilized. The conjugation reaction with oxidized
Glyco-EPO is described in Example 12, 3.1. 5Boturyn, Boudali,
Constant, Defrancq, Lhomme, 1997, Tetrahedron, 53, 5485
Alternatives:
[0559] Furthermore, derivatives could be used, wherein the two
hydroxylamine groups are separated by any spacer.
5. Thio-HES12KD
5.1 Addition to Oxo-HES12KD
##STR00081##
[0561] 1.44 g (0.12 mmol) of Oxo-HES12KD are dissolved in 3 mL
absolute dimethyl sulfoxide (DMSO) and are added to a mixture of
1.16 g (15 mmol) cysteamine in 15 mL DMSO under nitrogen dropwise.
After stirring for 24 h at 40.degree. C. the reaction mixture is
added to 160 mL of a 1:1 mixture of ethanol and acetone. The
precipitated product M is collected by centrifugation, redissolved
in 40 mL of water and dialyzed for 2 days against a 0.5% (v/v)
triethylamine in water solution and for 2 days against water
(SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science
Deutschland GmbH, Bonn, Germany) and lyophilized. The conjugation
reaction with oxidized Glyco-EPO is described in Example 12,
2.1.
Alternatives:
[0562] Derivatives could be used, wherein the amino group and the
thio-function are separated by any spacer. Furthermore, the amino
group in the derivatives could be replaced by a hydrazine, a
hydrazide or a hydroxylamine. The thio-function could be protected
in the form of e.g. a disulfide or a trityl-derivative. However, in
this case, a further deprotection step must be preformed before the
conjugation, which would release a component being analogous to
M.
5.2 Modification of Amino-HES12KD E, H or I
[0563] a) Modification with SATA/SATP
[0564] 1.44 g (0.12 mmol) of Amino-HES12KD E, H or I are dissolved
in 3 mL absolute dimethyl sulfoxide (DMSO) and are added to a
mixture of 139 mg (0.6 mmol) SATA in 5 mL DMSO under nitrogen
dropwise. After stirring for 24 h at room temperature the reaction
mixture is added to 160 mL of a 1:1 mixture of ethanol and acetone.
The precipitated product N is collected by centrifugation,
redissolved in 40 mL of water and dialyzed for 2 days against water
(SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science
Deutschland GmbH, Bonn, Germany) and lyophilized.
[0565] The deprotection is performed in a 50 mM sodium phosphate
buffer, containing 25 mM EDTA and 0.5M hydroxylamine, pH7.5 for 2
hours at room temperature and the product O is purified by dialysis
against a 0.1 M sodium acetate buffer pH 5.5, containing 1 mM EDTA.
The deprotection reaction is performed immediately before the
conjugation reaction which is described in Example 12, 2.1.
##STR00082##
b) Modification with SPDP
[0566] 1.44 g (0.12 mmol) of Amino-HES12KD E, H or I are dissolved
in 3 mL absolute dimethyl sulfoxide (DMSO) and are dropwise added
to a mixture of 187 mg (0.6 mmol) SPDP in 5 mL DMSO under nitrogen.
After stirring for 24 h at room temperature the reaction mixture is
added to 160 mL of a 1:1 mixture of ethanol and acetone. The
precipitated product P is collected by centrifugation, redissolved
in 40 mL of water and dialyzed for 2 days against water (SnakeSkin
dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH,
Bonn, Germany) and lyophilized.
[0567] The deprotection is performed in a solution of 12 mg
dithiothreitol (DTT) per 0.5 mL 100 mM sodium acetate buffer,
containing 100 mM sodium chloride at pH 4.5 for 30 min at room
temperature and the product Q was purified by dialysis against a
0.1 M sodium acetate buffer pH 5.5, containing 1 mM EDTA. The
deprotection reaction is performed immediately before the
conjugation reaction which is described in Example 12, 2.1.
Alternatives:
[0568] For the conversion of amino- to thiol-groups, either in free
form or protected, several reagants are available. After the
modification, the products could be isolated. Alternatively, as
accepted for the use of cross-linkers, they could be directly used
for the conjugation reaction, preferably after a purification step.
For the isolation and storage of thio-HES derivatives, the
synthesis of thio-HES derivatives in a protected form may be
useful. For this, all derivatives being analogous to SATA could be
used, which have an active ester-function and a thioester-function,
separated by any spacer. SATP, being a further member of this
group, is found in table 2, marked with an "H". The derivatives
being analogous to SPDP could have an active ester-function and a
disulfide-function, separated by any spacer. Further members of
these groups are found in table 2, marked with an "F". Further
analogous derivatives could have an active ester-function and a
thiol-function, protected as a trityl derivative, separated by any
spacer.
Example 10B
Formation of Reactive HES Derivatives
1. Halogenacetamide-derivatives of Amino-HES E.sup.1
[0569] Reaction of 1,4-diaminobutane with Oxo-HES18KD to
amino-HES18KD E6
[0570] 1.44 g (0.12 mmol) of Oxo-HES18KD were dissolved in 3 mL dry
dimethyl sulfoxide (DMSO) and were added dropwise under nitrogen to
a mixture of 1.51 mL (15 mmol) 1,4-diaminobutane in 15 mL DMSO.
After stirring for 19 h at 40.degree. C. the reaction mixture was
added to 160 mL of a 1:1 mixture of ethanol and acetone. The
precipitate Amino-HES18KD E was collected by centrifugation,
redissolved in 40 mL of water an dialyzed for 4 days against water
(SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science
Deutschland GmbH, Bonn, Germany) and lyophilized. 6S. Frie,
Diplomarbeit, Fachhochschule Hamburg, 1998
##STR00083##
2. CHO-Reactive HES
[0571] a. Hydrazide-HES a) Reaction of Hydrazine with
Oxo-HES18KD
##STR00084##
[0572] 1.44 g (0.12 mmol) of Oxo-HES18KD were dissolved in 3 mL
absolute dimethyl sulfoxide (DMSO) and were added dropwise under
nitrogen to a mixture of 0.47 mL (15 mmol) hydrazine in 15 mL DMSO.
After stirring for 19 h at 40.degree. C. the reaction mixture was
added to 160 mL of a 1:1 mixture of ethanol and acetone. The
precipitated product J was collected by centrifugation, redissolved
in 40 mL of water and dialyzed for 2 days against a 0.5% (v/v)
triethylamine in water solution and for 2 days against water
(SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science
Deutschland GmbH, Bonn, Germany) and lyophilized. The conjugation
reaction with oxidized Glyco-EPO is described in Example 12,
2.2.
b) Reaction of Adipic Dihydrazide with Oxo-HES18KD
##STR00085##
[0573] 1.74 g (15 mmol) adipic dihydrazide were dissolved in 20 mL
absolute dimethyl sulfoxide (DMSO) at 65.degree. C. and 1.44 g
(0.12 mmol) of Oxo-HES18KD, dissolved in 3 mL absolute DMSO were
added dropwise under nitrogen. After stirring for 68 h at
60.degree. C. the reaction mixture was added to 200 mL of water The
solution containing L was dialyzed for 2 days against a 0.5% (v/v)
triethylamine in water solution and for 2 days against water
(SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science
Deutschland GmbH, Bonn, Germany) and lyophilized. The conjugation
reaction with oxidized Glyco-EPO is described in Example 12,
2.2.
3. Hydroxylamine-Modified HES18KD K
##STR00086##
[0575] O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine was
synthesized as described by Boturyn et al in 2 steps from
commercially available materials.7 1.44 g (0.12 mmol) of
Oxo-HES18KD were dissolved in 3 mL absolute dimethyl sulfoxide
(DMSO) and were added dropwise under nitrogen to a mixture of 2.04
g (15 mmol) O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine in 15 mL
DMSO. After stirring for 48 h at 65.degree. C. the reaction mixture
was added to 160 mL of a 1:1 mixture of ethanol and acetone. The
precipitated product K was collected by centrifugation, redissolved
in 40 mL of water and dialyzed for 4 days against water (SnakeSkin
dialysis tubing, 3.5 KD cut off, Perbio Science Deutschland GmbH,
Bonn, Germany) and lyophilized. The conjugation reaction with
oxidized Glyco-EPO is described in Example 12, 3.1. 7Boturyn,
Boudali, Constant, Defrancq, Lhomme, 1997, Tetrahedron, 53,
5485
4. Thio-HES18KD
[0576] a. Addition to Oxo-HES18KD
##STR00087##
[0577] 1.44 g (0.12 mmol) of Oxo-HES18KD were dissolved in 3 mL
absolute dimethyl sulfoxide (DMSO) and were added to a mixture of
1.16 g (15 mmol) cysteamine in 15 mL DMSO under nitrogen dropwise.
After stirring for 24 h at 40.degree. C. the reaction mixture was
added to 160 mL of a 1:1 mixture of ethanol and acetone. The
precipitated product M was collected by centrifugation, redissolved
in 40 mL of water and dialyzed for 2 days against a 0.5% (v/v)
triethylamine in water solution and for 2 days against water
(SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio Science
Deutschland GmbH, Bonn, Germany) and lyophilized. The conjugation
reaction with oxidized Glyco-EPO is described in Example 12,
2.1.
Example 11
Conjugation Reactions with Thio-EPO
[0578] 1. Reaction of Thio-EPO with a Halogenacetamide-Modified
SH-Reactive HES
1.1 Example Protocol 1
[0579] Conjugation of ThioEPO to Amino-HES12KD (E, H or I) with a
Cross-linker containing a NHS-active-ester and an iodoacetamide
group, e.g. SIA.8 8Cumber, Forrester, Foxwell, Ross, Thorpe, 1985,
Methods Enzymol., 112, 207
Materials
[0580] A. Borate buffer. Composition is 50 mM sodium borate, pH
8.3, 5 mM EDTA. B. PBS, phosphate buffered saline: 10 mM sodium
phosphate, 150 mM NaCl, pH 7.4. C. AminoHES12KD E, H or I. Prepared
at 1 mg/mL in borate buffer. D. Crosslinker stock solution: 14 mg
SIA were dissolved in 1 mL DMSO E. D-Salt.TM. Dextran Desalting
Columns, 2.times.5 mL bed volume (Perbio Science Deutschland GmbH,
Bonn, Germany)
F. Coomassie.RTM. Protein Assay Reagent (Perbio Science Deutschland
GmbH, Bonn, Germany)
[0581] G. ThioEPO solution: 5 mg/mL of ThioEPO 1 in borate buffer.
H. Microconcentrator: Microcon YM-3 (amicon, Milipore GmbH,
Eschborn, Germany)
Method
[0582] 100 .mu.l, SIA solution is added to 400 .mu.L of the
aminoHES12KD E solution and is allowed to react with agitation for
0.5 hours at room temperature. The excess crosslinker is removed by
centrifuging the sample at 14000.times.g for 60 minutes using a
microconcentrator. After centrifuging the sample is brought up to
its original volume in borate buffer and this process is repeated
two more times. The residual solution is added to 1 mL of ThioEPO
solution and the reaction mixture is incubated for 16 hour at room
temperature. Reactivity of the excess iodoacetamide is quenched at
the end of the incubation period by the addition of cysteine to a
final concentration of 10 mM. The reaction mixture is applied to a
desalting column equilibrated with PBS buffer and the protein
content of the fractions are monitored with a Coomassie protein
assay reagent. All fractions containing the protein conjugate are
pooled and the conjugate was obtained by lyophylization after
dialysis against water over night.
Alternatives:
[0583] In this reaction, all cross-linkers could be used, which
have a succinimide- or a sulfosuccinimide function and a
iodoacetamide function separated by a spacer. Further examples are
found in table 2. They are marked with a "C" and are available from
Perbio Science Deutschland GmbH, Bonn, Germany.
1.2 Example Protocol 2
[0584] Conjugation of ThioEPO 1 to SH reactiveHES12KD
bromoacetamide D2, F2 or iodoacetamide D3. 9 9de Valasco, Merkus,
Anderton, Verheul, Lizzio, Van der Zee, van Eden, Hoffmann,
Verhoef, Snippe, 1995, Infect. Immun., 63, 961
Materials
[0585] A. Phosphate buffer. Composition is 100 mM sodium phosphate,
pH 6.1, 5 mM EDTA. B. PBS, phosphate buffered saline: 10 mM sodium
phosphate, 150 mM NaCl, pH 7.4. C. SH reactiveHES12KD
bromoacetamide D2. Prepared at 10 mg/mL in phosphate buffer. D.
D-Salt.TM. Dextran Desalting Columns, 2.times.5 mL bed volume
(Perbio Science Deutschland GmbH, Bonn, Germany)
E. Coomassie.RTM. Protein Assay Reagent (Perbio Science Deutschland
GmbH, Bonn, Germany)
[0586] F. ThioEPO solution: 5 mg/mL of ThioEPO 1 in phosphate
buffer.
Method
[0587] 1 mL SH reactiveHES12KD bromoacetamide D2 solution and 1 mL
of ThioEPO solution are combined and the reaction mixture is
incubated for 48 hours at room temperature. Reactivity of the
excess bromoacetamide is quenched at the end of the incubation
period by the addition of cysteine to a final concentration of 10
mM. The reaction mixture is applied to a desalting column,
equilibrated with PBS buffer. The protein content of the fractions
are monitored with a Coomassie protein assay reagent, all fractions
containing the protein conjugate are pooled and the conjugate is
obtained by lyophylization after dialysis against water over
night.
Alternatives:
[0588] Instead of the isolation of the SH reactive
HES12KD-bromoacetamid D2, amino HES12KD (E, H, I) could be linked
with a cross-linker via a succinimide- and a bromoacetamide
function (see 1.1 above). SBAP is a member of this group of
cross-linkers and is found in table 2, marked with a "D".
2. Reaction of Thio-EPO with a Maleimide-Modified SH-Reactive
HES
2.1 Example Protocol 3
[0589] Conjugation of ThioEPO to HES12KD with a cross-linker
containing a hydrazide and a maleimide functional group, e.g.
M.sub.2C.sub.2H.
Materials
[0590] A. M.sub.2C.sub.2H stock: 10 mg/mLM2C2H in DMSO, prepared
fresh B. HES12KD: 10 mg/mL in 0.1 M sodium acetate buffer, pH 5.5
C. ThioEPO solution: 5 mg/mL of ThioEPO in phosphate/NaCl-buffer D.
Phosphate/NaCl: 0.1 M sodium phosphate, 50 mM NaCl, pH 7.0 E.
Microconcentrator: Microcon YM-3 (amicon, Milipore GmbH, Eschborn,
Germany) F. Gel filtration column: for example, Sephadex.RTM. G-200
(1.5.times.45 cm)
G. Coomassie.RTM. Protein Assay Reagent (Perbio Science Deutschland
GmbH, Bonn, Germany)
[0591] H. PBS, phosphate buffered saline: 10 mM sodium phosphate,
150 mM NaCl, pH 7.4.
Method
[0592] M.sub.2C.sub.2H solution is added to 400 .mu.L of the
HES12KD solution to a final concentration of 1 mM and is allowed to
react with agitation for 2 hours at room temperature. The excess
cross-linker is removed by centrifuging the sample at 14000.times.g
for 60 minutes using a microconcentrator. After centrifuging the
sample is brought up to its original volume in phosphate/NaCl
buffer and this process is repeated two more times. To the
M.sub.2C.sub.2H-modified HES12KD 0.5 mL of ThioEPO solution is
added and the reaction mixture is incubated for 2 hours at room
temperature. Reactivity of the excess maleimides is quenched at the
end of the incubation period by the addition of cysteine to a final
concentration of 10 mM. The reaction mixture is applied to
Sephadex.RTM. G-200 (1.5.times.45 cm) equilibrated with PBS buffer
and 1 mL fractions are collected. The protein content of the
fractions is monitored with a Coomassie protein assay reagent. All
fractions containing the protein conjugate are pooled and the
conjugate was obtained by lyophylization after dialysis against
water over night.
Procedural Notes
[0593] The hydrazone adduct is slightly less stable at extremes of
pH. For applications that may involve treatment at low pH, we
reduced the hydrazone by treatment with 30 mM sodium
cyanoborohydride in PBS buffer to a hydrazine. For most
applications, this extra step is unnecessary.
2.2 Example Protocol 4
[0594] Conjugation of ThioEPO to Maleimido-HES12KD B.
Materials
[0595] A. Maleimido-HES12KD B: 10 mg/mL in 0.1 M sodium acetate
buffer, pH 5.5 B. ThioEPO solution: 5 mg/mL of ThioEPO in
phosphate/NaCl-buffer C. Phosphate/NaCl: 0.1 M sodium phosphate, 50
mM NaCl, pH 7.0 D. Gel filtration column: for example,
Sephadex.RTM. G-200 (1.5.times.45 cm)
E. Coomassie.RTM. Protein Assay Reagent (Perbio Science Deutschland
GmbH, Bonn, Germany)
[0596] F. PBS, phosphate buffered saline: 10 mM sodium phosphate,
150 mM NaCl, pH 7.4.
Method
[0597] 1 mL SH-reactive-HES12KD B solution and 1 mL of ThioEPO 1
solution are combined and the reaction mixture is incubated for 2
hours at room temperature. Reactivity of the excess maleimides is
quenched at the end of the incubation period by the addition of
cysteine to a final concentration of 10 mM. The reaction mixture is
applied to Sephadex.RTM. G-200 (1.5.times.45 cm) equilibrated with
PBS buffer and 1 mL fractions are collected. The protein content of
the fractions is monitored with a Coomassie protein assay reagent.
All fractions containing the protein conjugate are pooled and the
conjugate is obtained by lyophylization after dialysis against
water over night.
2.3 Example Protocol 12
[0598] Conjugation of ThioEPO to aminoHES12KD (E, H, I) with a
Cross-linker containing a NHS-active-ester and a maleimide group,
e.g. SMCC
Materials
[0599] A: Microconcentrator: Microcon YM-10 (amicon, Milipore GmbH,
Eschborn, Germany). B. PBS, phosphate buffered saline: 10 mM sodium
phosphate, 150 mM NaCl, pH 7.4. C. AminoHES12KD E, H or I. Prepared
at 10 mg/mL in PBS buffer. D. SMCC solution: 1 mg SMCC were
dissolved in 50 .mu.L DMSO E. D-Salt.TM. Dextran Desalting Columns,
2.times.5 mL bed volume (Perbio Science Deutschland GmbH, Bonn,
Germany)
F. Coomassie.RTM. Protein Assay Reagent (Perbio Science Deutschland
GmbH, Bonn, Germany)
[0600] G. ThioEPO 1 solution: 5 mg/mL of ThioEPO 1 in PBS
buffer.
Method
[0601] To 50 .mu.L SMCC solution 400 .mu.L of the aminoHES12KD E
solution is added and the reaction mixture is allowed to react with
agitation for 80 min at room temperature and for 10 min at
46.degree. C. The excess crosslinker is removed by centrifugation
of the reaction mixture through a microconcentrator at
14000.times.g for 60 min. The volume is brought up to 450 .mu.L
with PBS buffer and the process is repeated two more times. After
the last centrifugation, the residual solution is brought up to 450
.mu.L with PBS and is added to 1 mL of ThioEPO solution and the
reaction mixture are incubated for 16 hours at room temperature.
Reactivity of the excess maleimide is quenched at the end of the
incubation period by the addition of cysteine to a final
concentration of 10 mM. The reaction mixture is applied to a
desalting column equilibrated with PBS buffer. The protein content
of the fractions are monitored with a Coomassie protein assay
reagent, all fractions containing the protein conjugate are pooled
and the conjugate is obtained by lyophylization after dialysis
against water over night.
Alternatives:
[0602] In this reaction, all cross-linkers could be used which have
a succinimide- or a sulfosuccinimide function and a
maleimide-function, separated by a spacer. Further examples for
this group of molecules, available from Perbio Science Deutschland
GmbH, Bonn, Germany, are found in table, 2, marked with an "E".
There is a further group of cross-linkers, which have instead of a
maleimide function an activated disulfide function. These
cross-linkers could also be used for the conjugation. However, the
disulfide bond of the conjugate is cleavable under reductive
conditions. Members of this group are marked in table 2 with a "F".
A third group of cross-linkers uses instead of a maleimide function
a vinylsulfone function as a SH-reactive group. A member of this
group "SVSB" is marked in table 2 with a "G".
Example 12
Conjugation Reactions with Oxidized EPO
1. Oxidation of Glyco-EPO
[0603] 1.1 Oxidation of Glyco-EPO with sodium meta-periodate:
Example Protocol 5
Materials
[0604] A. Glyco-EPO solution: 10 mg/mL of Glyco-EPO in acetate
buffer B. Sodium meta-periodate solution: 10 mM or 100 mM sodium
periodate in acetate buffer, prepared fresh. Keep in dark. Using
these solutions, the final concentration of sodium periodate in the
oxidation mixture is 1 mM or 10 mM, respectively. C. acetate
buffer: 0.1 M sodium acetate buffer, pH 5.5
D. Glycerol
E. Microconcentrator: Microcon YM-3 (Amicon, Milipore GmbH,
Eschborn, Germany)
Method
[0605] All steps were performed in the dark.
[0606] To 1 mL of cold Glyco-EPO solution 0.1 mL of cold sodium
meta-periodate solution were added and the oxidation reaction was
allowed to proceed for 1 hour in the dark. If the Glyco-EPO to be
oxidized contained sialic acid residues, then the oxidation
conditions were 1 mM sodium periodate, 0.degree. C. Otherwise, 10
mM sodium periodate at room temperature was used. To stop the
oxidation glycerol was added to a final concentration of 15 mM and
incubated for 5 minutes at 0.degree. C. The excess reagents and
byproducts were remove by centrifuging of the product at
14000.times.g for 60 minutes using a microconcentrator. After
centrifuging, sample was brought up to its original volume in the
buffer used in the next modification step, e.g. in the acetate
buffer. This process was repeated two more times.
1.2 Enzymatic Oxidation of Glyco-EPO: Example Protocol 6
[0607] The enzymatic oxidation of EPO is described elsewhere
(Chamow et al., 1992, J. Biol. Chem., 267, 15916-15922).
2. Conjugation with Hydrazine/Hydrazide-Derivatives
2.1 Example Protocol 7
[0608] Conjugation of oxidized Glyco-EPO to Thio-HES12 KD M, O or Q
with a Cross-linker containing a hydrazide and a maleimide
functional group, e.g. M.sub.2C.sub.2H (Perbio Science, Deutschland
GmbH, Bonn, Germany).
Materials
[0609] A. M.sub.2C.sub.2H stock: 10 mg/mL M.sub.2C.sub.2H in DMSO,
prepared fresh B. Oxidized Glyco-EPO solution from 6.1.1: 5 mg/mL
of Glyco-EPO in acetate buffer C. Thio-HES12KD M, O or Q: 10 mg/mL
in phosphate/NaCl buffer D. Acetate buffer: 0.1 M sodium acetate
buffer, pH 5.5 E. Phosphate/NaCl: 0.1 M sodium phosphate, 50 mM
NaCl, pH 7.0 F. Microconcentrator: Microcon YM-3 (amicon, Milipore
GmbH, Eschborn, Germany) G. Gel filtration column: for example,
Sephadex.RTM. G-200 (1.5.times.45 cm)
H. Coomassie.RTM. Protein Assay Reagent (Perbio Science Deutschland
GmbH, Bonn, Germany)
[0610] I. PBS, phosphate buffered saline: 10 mM sodium phosphate,
150 mM NaCl, pH 7.4
Method
[0611] M.sub.2C.sub.2H stock solution is added to 1 mL of oxidized
Glyco-EPO to a final concentration of 1 mM and is allowed to react
with agitation for 2 hours at room temperature. The excess
crosslinker is removed by centrifuging the sample at 14000.times.g
for 60 minutes using a microconcentrator. After centrifuging the
sample is brought up to its original volume in phosphate/NaCl
buffer and this process was repeated two more times. To the
M.sub.2C.sub.2H-modified Glyco-EPO 1 mL of Thio-HES12KD M, O or Q
solution is added and the reaction mixture is incubated for 16
hours at room temperature. Reactivity of the excess maleimides is
quenched at the end of the incubation period by the addition of
cysteine. The reaction mixture is applied to Sephadex.RTM. G-200
(1.5.times.45 cm) equilibrated with PBS and 1 mL fractions are
collected. The protein content of the fractions is monitored with a
Coomassie protein assay reagent, all fractions containing the
protein conjugate are pooled and the conjugate is obtained by
lyophylization after dialysis against water over night.
Procedural Notes
[0612] The hydrazone adduct is slightly less stable at extremes of
pH. For applications that may involve treatment at low pH, we
reduced the hydrazone by treatment with 30 mM sodium
cyanoborohydride in PBS buffer to a hydrazine. For most
applications, this extra step was unnecessary.
2.2 Example Protocol 8A
[0613] Direct conjugation of oxidized Glyco-EPO to
Hydrazido-HES12KD L or J.
Materials
[0614] A. Oxidized Glyco-EPO solution from 6.1.1: 5 mg/mL of
Glyco-EPO in acetate buffer B. Hydrazido-HES12KD L or J: 10 mg/mL
in acetate buffer C. Acetate buffer: 0.1 M sodium acetate buffer,
pH 5.5 D. Gel filtration column: for example, Sephadex.RTM. G-200
(1.5.times.45 cm)
E. Coomassie.RTM. Protein Assay Reagent (Perbio Science Deutschland
GmbH, Bonn, Germany)
[0615] F. PBS, phosphate buffered saline: 10 mM sodium phosphate,
150 mM NaCl, pH 7.4
Method
[0616] 1 mL of Hydrazido-HES12KD L or J solution and 1 mL of
oxidized Glyco-EPO solution are combined and the reaction mixture
is allowed to react with agitation for 16 hours at room
temperature. The reaction mixture is applied to Sephadex.RTM. G-200
(1.5.times.45 cm) equilibrated with PBS and 1 mL fractions are
collected. The protein content of the fractions are monitored with
a Coomassie protein assay reagent, all fractions containing the
protein conjugate are pooled and the conjugate is obtained by
lyophylization after dialysis against water over night.
Procedural Notes
[0617] The hydrazone adduct is slightly less stable at extremes of
pH. For applications that may involve treatment at low pH, the
hydrazone may be reduced by treatment with 30 mM sodium
cyanoborohydride in PBS buffer to a hydrazine. For most
applications, this extra step is unnecessary.
2.3 Example Protocol 8B
[0618] To 100 .mu.l of a 0.5 mg/ml solution of oxidized EPO in a
buffer containing 0.1 M sodium acetate and 150 mM sodium chloride
at a pH of 5.2, 50 .mu.L of a 20 mg/ml solution of HES18/0.5 L
dissolved in a 0.1 M sodium acetate buffer, pH 5.2 (synthesized
corresponding to Example 102.1b) were added and the mixture was
incubated at 22.degree. C. for 14.5 h. The crude reaction mixture
was analyzed by SDS gel electrophoresis and stained with Coomassie.
The result of the conjugation is shown in FIG. 24. The observed
molecular shift demonstrates that the conjugation was successful.
The smear results from the heterogeneity of HES. FIG. 25
demonstrates that HES is conjugated to a carbohydrate moiety of a
carbohydrate side chain.
3. Conjugation with Hydroxylamine-Derivatives10 10Rose, 1994, Am.
Chem. Soc., 116, 30
3.1 Example Protocol 9A
[0619] Conjugation of oxidized Glyco-EPO to Hydroxylamino-HES12KD
K
Materials
[0620] A. Oxidized Glyco-EPO solution from 6.1.1: 5 mg/mL of
Glyco-EPO in acetate buffer B. Hydroxylamino-HES12KD K: 10 mg/mL in
acetate buffer C. Acetate buffer: 0.1 M sodium acetate buffer, pH
5.5 D. Gel filtration column: for example, Sephadex.RTM. G-200
(1.5.times.45 cm)
E. Coomassie.RTM. Protein Assay Reagent (Perbio Science Deutschland
GmbH, Bonn, Germany)
[0621] F. PBS, phosphate buffered saline: 10 mM sodium phosphate,
150 mM NaCl, pH 7.4
Method
[0622] 1 mL of Hydroxylamino-HES12KD K solution and 1 mL of
oxidized Glyco-EPO solution are combined and the reaction mixture
is allowed to react with agitation for 16 hours at room
temperature. The reaction mixture is applied to Sephadex.RTM. G-200
(1.5.times.45 cm) equilibrated with PBS and 1 mL fractions were
collected. The protein content of the fractions are monitored with
a Coomassie protein assay reagent, all fractions containing the
protein conjugate are pooled and the conjugate is obtained by
lyophylization after dialysis against water over night.
3.2 Example Protocol 9B
[0623] To 100 .mu.l of a 0.5 mg/ml solution of oxidized EPO in a
buffer containing 0.1 M sodium acetate and 150 mM sodium chloride
at a pH of 5.2, 50 .mu.L of a 20 mg/ml solution of HES18/0.5 K
dissolved in a 0.1 M sodium acetate buffer, pH 5.2 (synthesized
corresponding to Example 10. 4) were added and the mixture was
incubated at 22.degree. C. for 14.5 h. The crude reaction mixture
was analyzed by SDS gel electrophoresis and stained with Coomassie.
The result of the conjugation is shown in FIG. 24. The observed
molecular shift demonstrates that the conjugation was successful.
The smear results from the heterogeneity of HES. FIG. 25
demonstrates that HES is conjugated to a carbohydrate moiety of a
carbohydrate side chain.
Example 13
Characterization of Galactose Oxidase Treated EPO N-Glycans
[0624] Recombinant EPO or partially desialylated EPO forms
(generated by limited mild acid hydrolysis) were incubated with
galactose oxidase in the presence of catalase at 37.degree. C. from
30 min-4 hours at 37.degree. C. in 0.05 M Na-phosphate buffer pH
7.0. Progress of the reaction was monitored by removal of 50 .mu.g
aliquots of the EPO and subsequent treatment of the protein with
polypeptide N-glycanase.
[0625] Liberated N-linked oligosaccharides (monitored by SDS-PAGE
detection of the de-N-glycosylated polypeptide) were subjected to
HPAEC-PAD mapping as described (Grabenhorst et al., 1999, Nimtz et
al., 1993/1994; Schlenke et al., 1999) before and after removal of
sialic acids. Quantitation of oxidized galactose residues in
individual EPO oligosaccharides was performed by the typical shift
observed in HPAEC-PAD and was also verified by MALDI/TOF MS of the
oligosaccharide mixtures.
Example 14
Characterization of HAS modified EPO
[0626] Separation of HAS modified EPO forms from nonreacted EPO and
HAS-precursor molecules was achieved by gel filtration using e.g.
Ultrogel AcA 44/54 or similar gel filtration media. Alternatively,
nonreacted HAS was removed by immuno affinity isolation of EPO on a
4 mL column containing a monoclonal antibody coupled to Affigel
(BioRad) and subsequent separation of unmodified EPO by gel
filtration (e.g. using a matrix enabling the separation of globular
proteins of a relative molecular mass between 20 kDa and 200
kDa).
[0627] HAS modified EPOs were identified by SDS-PAGE analysis
(using 12.5 or 10% acrylamide gels) through detection of their
higher molecular weight compared to unmodified EPO upon staining of
gels with Coomassie Brilliant Blue. The higher molecular weight of
HAS modified EPO polypeptides was also identified by Western Blot
analysis of samples using a polyclonal antibody raised against
recombinant human EPO.
[0628] N-glycan modification of EPO forms was demonstrated by their
successful removal from the EPO protein with polypeptide
N-glycanase (recombinant N-glycosidase from Roche, Germany
employing 25 units/mg EPO protein at 37.degree. C. for 16 hours);
analysis by SDS-PAGE resulted in a typical shift of the EPO protein
to a migration position of the N-glycosidase treated unmodified EPO
of approximately 20 KDa.
[0629] Modification of the single desialylated and galactose
oxidase treated EPO O-glycan at Ser 126 was demonstrated by
SDS-PAGE migration of the de-N-glycosylated product by detection of
its migration position compared to nonreacted de-N-glycosylated
EPO. If required, modified EPO was fractionated by RP-HULK on a
C8-phase before SDS-PAGE analysis. HAS O-glycan modification of EPO
was also analyzed by .beta.-elimination of the O-glycan and
detection of the de-O-glycosylated form of EPO in Western blots
using a polyclonal antibody raised against recombinant human
EPO.
Example 15
Quantitation of EPO and Modified EPO Forms
[0630] EPO forms where quantitated by UV measurements as described
in Ph. Eur (2000, Erythropoietini solutio concentrata, 1316,
780-785) and compared to the international BRP reference EPO
standard. Alternatively, EPO concentrations were determined by a
RP-HPLC assay using a RP-C4-column and absorption at 254 nm
employing 20, 40, 80 and 120 .mu.g of the BRP standard EPO
reference preparation for calibration.
Example 16
In-Vitro Biological Activity of HES-Modified Recombinant Human
EPO
[0631] Purified HES-modified EPO is tested for activity using the
erythropoietin bioactivity assay as described by Krystal [Krystal,
1984, Exp. Heamatol., 11, 649-660].
[0632] Anemia is induced in NMRI mice by treatment with
phenylhydrazine hydrochloride and spleen cells are collected and
used as described in [Fibi et al., 1991, Blood, 77, 1203 ff.].
Dilutions of EPO are incubated with 3.times.10.sup.5 cells/well in
96-well microtiter plates. After 24 hours at 37.degree. C. in a
humidified atmosphere (5% CO.sub.2) cells are labeled for 4 hours
with 1 .mu.Ci of .sup.3H-thymidine per well. Incorporated
radioactivity is determined by liquid scintillation counting. The
International reference EPO standard (BRP-standard) is used for
comparison.
[0633] Alternatively, EPO bioactivity may be measured by an in
vitro assay using the EPO-sensitive cell line TF-1 (Kitamura et.
al., [J. cell Phys., 140. 323-334]. Exponentially growing cells are
washed free of growth factors and are incubated in the presence of
serial dilutions of the EPO for further 48 hours. Proliferation of
the cells is assessed by using the MTT reduction assay as described
by Mosmann [Mosmann, 1983, J. Immunol. Methods, 65, 55-63].
Example 17
In-vivo activity determination of EPO and HAS-modified EPO
forms
(HCO Fragen)
[0634] In vivo activity determinations are performed in
normocythemic mice by measuring the increase of reticulocytes after
4 days after animals received the foreseen dose of EPO or modified
EPO forms. Assays are performed using the BRP EPO standard which is
calibrated against the WHO EPO standard in the polycythemic mouse
assay. EPO samples are diluted in phosphate buffered saline
containing 1 mg/ml of bovine serum albumin (Sigma).
[0635] 0.5 ml of the EPO test solution in Dulbecco's buffered
saline (corresponding to an EPO protein equivalent of a 100, 80, 40
or 20 IU/ml of the BRP standard EPO) are infected subcutaneously
per animal. Blood samples are taken after 4 days after injection
and reticulocytes are stained with acridine orange; quantitation of
reticulocytes is performed by flow-cytometry by counting a total of
30,000 blood cells within 5 hours after the blood sample was taken
(see Ph. Eur, 2000, Erythropoietini solutio concentrata, 1316,
pages 780-785) and European Pharmacopoeia (1996/2000, attachment
2002).
Example 18
In-Vivo Half-Life Determinations
[0636] Rabbits are injected intravenously with specified amounts of
unmodified or HAS-modified EPO forms. Blood samples are obtained at
specified times, and serum is prepared. Serum erythropoietin levels
are determined by in vitro bioassay or by an EPO-specific
commercial ELISA.
Example 19
In Vivo Pharmacokinetics
[0637] In mice: Each animal receive 300 IU EPO/kg subcutaneously.
Seven days after the post-treatment hematocrit of each animal is
determined. A substantial increase in hematocrit is observed in all
animals treated with modified EPO.
[0638] In rabbits: Rabbits are treated with a single dose of
unmodified or HAS-modified EPO corresponding to 200 or up to 800
ng/kg body weight. After 2, 6, 16, 24 and 48 hours blood samples
are analyzed by using a commercial EPO-specific ELISA for
determination of plasma concentrations. Mean plasma EPO
concentrations are determined and the average initial half-lives
(.alpha.-phase) and the terminal half-lives (.beta.-phase) are
calculated from the ELISA values as described: (Zettlmissl et al.,
1989, J. Biol. Chem., 264, 21153-21159).
Literature:
[0639] Sytkowski, Lunn, Risinger, and Davis, 1999, An
Erythropoietin Fusion Protein Comprised of Identical Repeating
Domains Exhibits Enhanced Biological Properties, J. Biol. Chem.,
274, 24773-24778.
Example 20
Assessment of the In Vitro Biological Activity of HES-Modified
Recombinant Human IL-2
[0640] Modified IL2 is recovered by gel filtration on Ultrogel AcA
54. Aliquots of corresponding fraction are sterile filtrated and
IL2 bioactivity is determined by using the IL2 dependent murine
CTLL-2 cell line [Gillis, Ferm, On, and Smith, 1978, J. Immunol.,
120, 2027-2032]. Activity is related to the international reference
IL2 standard preparation.
TABLE-US-00003 TABLE 1 Abreviation Chemical Name Type AMAS
N-(.alpha.-Maleimidoacetoxy) succinimide ester E ##STR00088## BMPH
N-(.beta.-Maleimidopropionic acid) hydrazide.cndot.TFA A
##STR00089## BMPS N-(.beta.-Maleimidopropyloxy) succinimide ester E
##STR00090## EMCH N-(.epsilon.-Maleimidocaproic acid) hydrazide A
##STR00091## EMCS N-(.epsilon.-Maleimidocaproyloxy) succinimide
ester E ##STR00092## GMBS N-.gamma.-Maleimidobutyryloxy-succinimide
ester E ##STR00093## KMUH N-(.kappa.-Maleimidoundecanoic acid)
hydrazide A ##STR00094## LC-SMCC Succinimidyl 4-(N-maleimidomethyl)
cyclohexane-1-carboxy- (6-amido-caproate) E ##STR00095## LC-SPDP
Succinimidyl 6-(3'-[2-pyridyl-dithio] propionamido)hexanoate F
##STR00096## MBS m-Maleimidobenzoyl-N- hydroxysuccinimide ester E
##STR00097## M.sub.2C.sub.2H 4-(N-Maleimidomethyl)-cyclohexane-
1-carboxyl-hydrazide.cndot.HCl.cndot.1/2 dioxane A ##STR00098##
MPBH 4-(4-N-Maleimidophenyl)-butyric acid hydazide.cndot.HCl A
##STR00099## SATA N-Succinimidyl S-acetylthio-acetate H
##STR00100## SATP N-Succinimidyl S-acetylthio- propionate H
##STR00101## SBAP Succinimidyl 3-(bromoacetamido) propionate D
##STR00102## SIA N-Succinimidyl iodoacetate C ##STR00103## SIAB
N-Succinimidyl(4-iodoacetyl) aminobenzoate C ##STR00104## SMCC
Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate E
##STR00105## SMPB Succinimidyl 4-(p-maleimidophenyl) butyrate E
##STR00106## SMPH Succinimidyl-6-(.beta.- maleimidopropionamido)
hexanoate E ##STR00107## SMPT 4-Succinimidyloxy-carbonyl-
methyl-.alpha.-(2-pyridyldithio)toluene F ##STR00108## SPDP
N-Succinimidyl 3-(2-pyridyldithio) propionate F ##STR00109##
Sulfo-EMCS N-(.epsilon.-Maleimidocaproyloxy) sulfosuccinimide ester
E ##STR00110## Sulfo-GMBS N-.gamma.-Maleimidobutryloxy-
sulfosuccinimide ester E ##STR00111## Sulfo-KMUS
N-(.kappa.-Maleimidoundecanoyloxy)- sulfosuccinimide ester E
##STR00112## Sulfo-LC-SPDP Sulfosuccinimidyl 6-(3'-[2-pyridyl-
dithio]propionamido) hexanoate F ##STR00113## Sulfo-MBS
m-Maleimidobenzoyl-N- hydroxysulfosuccinimide ester E ##STR00114##
Sulfo-SIAB Sulfosuccinimidyl(4-iodoacetyl) aminobenzoate C
##STR00115## Sulfo-SMCC Sulfosuccinimidyl 4-(N- maleimidomethyl)
cyclohexane-1-carboxylate E ##STR00116## Sulfo-SMPB
Sulfosuccinimidyl 4-(p- maleimidophenyl) butyrate E ##STR00117##
Sulfo-LC-SMPT Suflosuccinimidyl 6-(.alpha.-methyl-.alpha.-
[2-pyridyldithio]-toluamido) hexanoate F ##STR00118## SVSB
N-Succinimidyl-(4- vinylsulfonyl)benzoate G ##STR00119##
TABLE-US-00004 TABLE 2 Monosaccharide compositional analysis of
glycans from HES-modified EPO and control samples II. VI. I.
Glycans III. III. IV. V. Cystein Glycans from Glycans Glycans
Glycans Glycans modified **Mono- from EPO-GT- from from from EPO-
from EPO pro- saccharide A2 1A K2 A2 GT-1A K2 tein* fucose 1,935
3,924 2,602 2,246 4,461 2,601 2,181 mannose 6,028 11,020 9,198
6,379 11,668 6,117 6,260 galactose 8,886 19,935 14,427 10,570
16,911 11,555 10,386 glucose 17,968 -- -- 21,193 trace trace 33,021
GlcNAc 7,839 21,310 14,440 11,360 15,953 10,503 10,498 GlcHe1 5,583
-- -- 5,926 -- -- 14,857 GlcHe2 1,380 -- -- 1,552 -- -- 3,775
NeuNAc 5,461 822 4,504 3,895 4,871 13,562 13,003 inositol 1,230
2,310 1,620 2,050 1,320 1,134 1,087 *the equivalent of
Cys-HES-modified EPO protein was subjected to compositional
analysis; the EPO protein was isolated from the HES-incubation
mixture by chromatography on a Q-Sepharose column as described
above and was desalted by centrifugation using a Vivaspin 5
separation device. **Monosaccharide determinations were performed
from single GC runs of the pertrimethylsilylated methylglycosides;
the electronical integration values of peaks are given without
correction for losses during the derivatisation procedure and
recoveries of each compound.
TABLE-US-00005 TABLE 3 Calculated specific activity of EPO sample
Sample (based on A280 nm No. Sample description and RP-HPLC
determination) 850247 1. HES-modified EPO A2 344,000 U/mg 850248 2.
EPO-GT-1-A 82,268 U/mg 850249 3. Control EPO K2 121,410 U/mg 850250
4. BRP EPO standard 86,702 U/mg 850251 1. diluted with 4 volume of
PBS 309,129 U/mg 850252 2. diluted with 4 volume of PBS 94,500 U/mg
850253 3. diluted with 4 volume of PBS 114,100 U/mg 850254 4.
diluted with 4 volume of PBS 81,200 U/mg 850255 1. diluted with 4
volume of PBS 230,720 U/mg
* * * * *