U.S. patent application number 17/165958 was filed with the patent office on 2021-11-11 for method for preparing functionalized polymers from polymer alcohols.
The applicant listed for this patent is Nektar Therapeutics. Invention is credited to J. Milton Harris, Antoni Kozlowski, Samuel P. McManus.
Application Number | 20210347941 17/165958 |
Document ID | / |
Family ID | 1000005728089 |
Filed Date | 2021-11-11 |
United States Patent
Application |
20210347941 |
Kind Code |
A1 |
McManus; Samuel P. ; et
al. |
November 11, 2021 |
METHOD FOR PREPARING FUNCTIONALIZED POLYMERS FROM POLYMER
ALCOHOLS
Abstract
The present invention provides, among other things, methods for
preparing functionalized and other polymers from polymer alcohols
such as poly(ethylene glycol)s. In addition, polymer compositions,
conjugates, polymeric reagents, are also provided.
Inventors: |
McManus; Samuel P.;
(Huntsville, AL) ; Kozlowski; Antoni; (Huntsville,
AL) ; Harris; J. Milton; (Huntsville, AL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Nektar Therapeutics |
San Francisco |
CA |
US |
|
|
Family ID: |
1000005728089 |
Appl. No.: |
17/165958 |
Filed: |
February 3, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16165903 |
Oct 19, 2018 |
10941248 |
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17165958 |
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15359344 |
Nov 22, 2016 |
10144804 |
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16165903 |
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14074421 |
Nov 7, 2013 |
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15359344 |
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10897386 |
Jul 22, 2004 |
8604159 |
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14074421 |
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60489583 |
Jul 22, 2003 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C08G 65/33337 20130101;
C08G 65/33306 20130101; C08G 2650/38 20130101; C08G 65/322
20130101; C08G 65/48 20130101; C08L 2203/02 20130101; C08G 65/33365
20130101; C08G 65/3322 20130101; C08G 65/329 20130101; C08G 65/00
20130101; C08G 65/323 20130101; A61K 47/60 20170801; C08G 65/30
20130101; B01D 15/361 20130101 |
International
Class: |
C08G 65/333 20060101
C08G065/333; C08G 65/322 20060101 C08G065/322; C08G 65/329 20060101
C08G065/329; A61K 47/60 20060101 A61K047/60; C08G 65/00 20060101
C08G065/00; C08G 65/323 20060101 C08G065/323; C08G 65/48 20060101
C08G065/48; B01D 15/36 20060101 B01D015/36; C08G 65/30 20060101
C08G065/30; C08G 65/332 20060101 C08G065/332 |
Claims
1.-48. (canceled)
49. A method of forming a functionalized polymer, comprising: (a)
providing a polymer comprising a formula HO-POLY-OH, wherein POLY
is a water-soluble and non-peptidic polymer; (b) optionally,
converting HO-POLY-OH to a mixture comprising HO-POLY-OH, HO-POLY-Z
and Z-POLY-Z, wherein Z is a leaving group, under conditions
effective to form no more than about 45 percent of Z-POLY-Z; (c)
reacting HO-POLY-OH of step (a) or the mixture of step (b) with a
functionalizing reagent comprising the structure X--L.sub.0,1--Y,
wherein X is a group that reacts with a hydroxyl, optionally in
anionic form, or with a carbon atom to which the hydroxyl or
leaving group is attached, L.sub.0,1 is an optional linker, and Y
is an ionizable group, optionally as a protected ionizable group,
to form a mixture comprising HO-POLY-OH, HO-POLY-L.sub.0,1--Y, and
Y--L.sub.0,1-POLY-L.sub.0,1--Y, under conditions effective to form
preferably no more than about 45 percent of
Y--L.sub.0,1-POLY-L.sub.0,1--Y; (d) optionally, alkylating the
mixture from step (b) or step (c); and (e) after deprotection of
the functional group Y, if necessary, purifying the mixture from
step (c) or step (d) by ion exchange chromatography to provide
substantially pure polymer comprising a single --L.sub.0,1--Y
group.
50. The method of claim 49, wherein X is a halogen or a sulfonate
ester.
51. The method of claim 49, wherein said purifying step comprises:
passing the mixture from step (c) or step (d) through a first ion
exchange column to provide an eluate, wherein said passing the
mixture step is carried out under conditions effective to adsorb
substantially all polymer species comprising two --L.sub.0,1--Y
groups onto the first column; passing the eluate through a second
ion exchange column under conditions effective to adsorb
substantially all of polymer species having a single --L.sub.0,1--Y
group onto said second column; washing the second column with a
solution having low ionic strength to remove only polymer species
containing no --Y groups; and passing a solution having high ionic
strength through the second column to desorb polymer species having
a single --L.sub.0,1--Y group.
52. The method of claim 51, wherein the optional linker, L.sub.0,1,
is present and is hydrolytically stable.
53. The method of claim 51, wherein the optional linker, L.sub.0,1,
is present and has a structure --(CR.sub.1R.sub.2).sub.m--, wherein
R.sub.1 and R.sub.2 are each independently H or alkyl, and m ranges
from 0-10.
54. The method of claim 51, wherein Y is an amine or carboxylic
acid.
55. The method of claim 51, wherein said alkylating step comprises
treating the mixture with an alkylating agent selected from the
group consisting of dialkylsulfate, alkyl sulfonate, diazoalkane,
alkyl halide, N,N'-dimethylformamide dialkyl acetal,
3-alkyl-1-p-tolyltriazene, trimethylanilinium hydroxide,
trialkyloxonium fluoroborate, trimethylsulfonium
hexafluorophosphonate and alkyl trichloroacetimidate.
56. The method of claim 51, wherein said alkylating step is
effective to convert hydroxyl groups in said mixture to --OR',
wherein R' is selected from the group consisting of
C.sub.1-C.sub.20 alkyl, substituted C.sub.1-C.sub.20 alkyl,
C.sub.1-C.sub.20 alkylene-aryl, and substituted C.sub.1-C.sub.20
alkylene-aryl.
57. The method of claim 56, wherein R' is selected from the group
consisting of methyl, ethyl, propyl, butyl, pentyl, hexyl, and
benzyl.
58. The method of claim 51, wherein X is a nucleophile effective to
displace --Z.
59. The method of claim 58, wherein Z is a leaving group selected
from the group consisting of halogen and sulfonate esters.
60. The method of claim 53, wherein X is selected from the group
consisting of leaving groups, substituted vinyl groups, and
unsubstituted vinyl groups, m is 1-3, and Y is a protected
carboxylic acid.
61. The method of claim 60, wherein the protected carboxylic acid
group is selected from the group consisting of nitrile, amide,
alkyl ester and ortho ester.
62. The method of claim 49, wherein X is selected from the group
consisting of leaving groups, substituted vinyl groups, and
unsubstituted vinyl groups, m is 1-3, and Y is a carbonitrile.
63. The method of claim 51, wherein X is a halogen, m is 0, and Y
is p-tolylsulfonyl, methylsulfonyl, trifluoromethylsulfonyl, or
trifluoroethylsulfonyl.
64. The method of claim 53, wherein the functionalizing reagent is
selected from the group consisting of
X'--(CR.sub.1R.sub.2).sub.m--C(O)--O--Rp,
CH.sub.2.dbd.CY'--C(O)--O--Rp, X'--(CR.sub.1R.sub.2).sub.m--Z,
CH.sub.2.dbd.CY'--(CR.sub.1R.sub.2).sub.m--Z,
X'--(CR.sub.1R.sub.2).sub.m--C.dbd.N, and
CH.sub.2.dbd.CY'--C.dbd.N, wherein X' is halogen or sulfonate
ester, Z is an ortho ester, Y' is H, halogen, alkyl, substituted
alkyl, alkoxy, or substituted alkoxy, and Rp is alkyl or
substituted alkyl.
65. The method of claim 49, wherein said converting comprises
reacting HO-POLY-OH with X'--SO.sub.2--R.sub.3, wherein R.sub.3 is
alkyl, substituted alkyl, aryl, or substituted aryl, and X' is Br
or Cl.
66. The method of claim 65, wherein R.sub.3 is p-tolyl, methyl,
trifluoromethyl, or trifluoroethyl.
67. The method of claim 49, further comprising, after said
purifying step, transforming Y of the substantially pure polymer to
a different reactive moiety.
68. The method of claim 49, wherein said reactive moiety comprises
a functional group selected from the group consisting of hydroxyl,
active ester, active carbonate, ortho ester, acetal, aldehyde,
aldehyde hydrate, ketone, ketone hydrate, oxime, alkenyl, acrylate,
methacrylate, nitrile, primary or secondary amide, imide,
acrylamide, active sulfone, amine, hydrazide, thiol, carboxylic
acid, isocyanate, isothiocyanate, maleimide, succinimide,
vinylsulfone, dithiopyridine, vinylpyridine, amidate,
2-substituted-1,3-oxazoline, 2-substituted
1,3-(4H)-dihydrooxazines, 2-substituted-1,3-thiazoline,
2-substituted 1,3-(4H)-dihydrothiazines, hydroxylamine,
iodoacetamide, orthopyridyl disulfide, epoxide, glyoxal, dione,
mesylate, tosylate, and tresylate.
69. The method of claim 49, wherein Y is a protected functional
group and said method further comprises deprotecting the functional
group prior to said purifying.
70. A method of separating a mixture of polymer species by ion
exchange chromatography, comprising: (a) providing a mixture of
water-soluble and non-peptidic polymers, said mixture comprising a
neutral polymer absent an ionizable functional group, a
monofunctional polymer comprising a single ionizable functional
group, and a difunctional polymer comprising two ionizable
functional groups; (b) passing the mixture through a first ion
exchange column to provide an eluate, wherein said passing the
mixture step is carried out under conditions effective to adsorb
substantially all of the difunctional polymer onto the first
column; (c) passing the eluate through a second ion exchange column
under conditions effective to adsorb substantially all of the
monofunctional polymer onto said second column; (e) washing the
second column with water or a low ionic solution to remove neutral
polymer absent an ionizable functional group in a wash solution;
and (f) passing a solution having high ionic strength through the
second column to desorb the monofunctional polymer.
71. The method of claim 70, wherein said second ion exchange column
is connected in series to one or more additional ion exchange
columns, and said washing step further comprises washing said
second and one or more additional ion exchange columns and said
passing the eluate step further comprises eluting adsorbed
monofunctional polymer from said second and one or more additional
columns.
72. The method of claim 70, further comprising passing a solution
having sufficient ionic strength through the first column to desorb
the difunctional polymer.
73. The method of claim 70, wherein the first column is of a size
sufficient to adsorb substantially all of the difunctional polymer
and a fraction of the monofunctional polymer.
74. The method of claim 73, wherein the fraction of monofunctional
polymer adsorbed on the first column is less than about 70
percent.
75. The method of claim 71, further comprising monitoring the wash
solution from the second and one or more additional columns during
said washing step, to determine that substantially all of the
neutral polymer has been removed.
76. The method of claim 75, wherein said monitoring step comprises
analyzing the wash solution by a method selected from the group
consisting of ion exchange chromatography, size exclusion
chromatography, and high performance liquid chromatography, thin
layer chromatography, and infrared analysis.
77. The method of claim 75, wherein said polymer species are PEG
and said monitoring step comprises treating a sample of said wash
solution with 1% polyacrylic acid (Mn 250,000) in 1 N HCl to
determine the presence of polymer.
78. The method of claim 70, wherein said washing step is carried
out with distilled water.
79. The method of claim 70, wherein the polymer mixture in step (a)
is provided in an aqueous solution having low ionic strength to
allow adsorption of functionalized polymer on the ion-exchange
gel.
80. The method of claim 70, wherein the neutral polymer absent an
ionizable functional group has the structure, HO-POLY-OH or
R'O-POLY-OR'; the monofunctional polymer has the structure,
HO-POLY-L.sub.0,1--Y or R'O-POLY-L.sub.0,1--Y; and the difunctional
polymer has the structure, Y--L.sub.0,1-POLY-L.sub.0,1--Y, wherein
L is an optional linker, Y is a functional group and R-- is
selected from the group consisting of C.sub.1-C.sub.20 alkyl,
substituted C.sub.1-C.sub.20 alkyl, C.sub.1-C.sub.20 alkylene-aryl,
and substituted C.sub.1-C.sub.20 alkylene-aryl.
81. The method of claim 64, wherein the mixture in step (a)
comprises CH.sub.3O-POLY-OCH.sub.3, in combination with
H.sub.3CO-POLY-L.sub.0,1--Y, and Y--L.sub.0,1-POLY-L.sub.0,1--Y,
wherein L is an optional linker and Y is a functional group.
82. The method of claim 81, wherein the mixture comprises no more
than about 40% by weight of HO-POLY-OH and
Y--L.sub.0,1-POLY-L.sub.0,1--Y in combination.
83. The method of claim 82, wherein the mixture comprises no more
than about 10% by weight of HO-POLY-OH and
Y--L.sub.0,1-POLY-L.sub.0,1--Y in combination.
84.-86. (canceled)
87. An ion exchange chromatography apparatus, comprising: a supply
of solution of a water-soluble and non-peptidic polymer mixture
comprising a neutral polymer absent ionizable functional groups, a
monofunctional polymer comprising a single ionizable functional
group, and a difunctional polymer comprising two ionizable
functional groups; a first ion exchange column comprising a first
inlet, a first outlet, and a first ion exchange media, said first
inlet being in fluid communication with said supply; a second ion
exchange column comprising a second inlet, a second outlet, and a
second ion exchange media, said second inlet being in fluid
communication with said first outlet; and at least one product
recovery vessel in fluid communication with said second outlet,
adapted to receive eluent exiting from said second ion exchange
column.
88. The apparatus of claim 87, wherein said first ion exchange
column is of a size sufficient to adsorb substantially all of the
difunctional polymer.
89. The apparatus of claim 87, further comprising a product
recovery vessel in fluid communication with said first outlet,
adapted to receive eluent exiting from said first ion exchange
column.
90. The apparatus of claim 87, further comprising a salt solution
supply in fluid communication with at least one of said first and
second inlets.
91. The apparatus of claim 87, further comprising a neutral
solution supply in fluid communication with at least one of said
first and second inlets.
92. The apparatus of claim 87, further comprising one or more
additional ion exchange columns connected in series, and in fluid
communication with said second outlet.
93. A compound of the following structure: ##STR00016## wherein (n)
is from 2 to 3,000, R.sub.4 is H or an organic radical, and (n') is
3 to 10.
94. The compound of claim 93, wherein (n) is from 10 to 2,000.
95. The compound of claim 93, wherein R.sub.4 is alkyl.
96. The compound of claim 95, wherein alkyl is methyl or
benzyl.
97. The compound of claim 93, wherein (n') is 3.
98.-102. (canceled)
103. A composition comprising monomethoxy end-capped polyethylene
glycol) ("mPEG-OH"), wherein the composition has a polyethylene
glycol) diol content of less than 0.3 wt. %.
104. The composition of claim 103, wherein the composition has a
polydispersity less than about 1.025.
105.-112. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/165,903, filed Oct. 19, 2018, now allowed,
which is a continuation of U.S. patent application Ser. No.
15/359,344, filed Nov. 22, 2016, now U.S. Pat. No. 10,144,804,
which is a continuation of U.S. patent application Ser. No.
14/074,421, filed Nov. 7, 2013, now abandoned, which is a
divisional of U.S. patent application Ser. No. 10/897,386, filed
Jul. 22, 2004, now U.S. Pat. No. 8,604,159, which claims the
benefit of priority to U.S. Provisional Patent Application No.
60/489,583, filed Jul. 22, 2003, the disclosures of which are
incorporated herein by reference in their entireties.
FIELD OF THE INVENTION
[0002] Among other things, this invention relates to
functionalized, water-soluble and non-peptidic polymers, and in
particular, to methods for making, purifying, and utilizing such
polymers.
[0003] Covalent attachment of the hydrophilic polymer,
poly(ethylene glycol), abbreviated "PEG," to molecules and surfaces
is of considerable utility in areas such as biotechnology and
medicine. PEG is a polymer that possesses many beneficial
properties. For instance, PEG is soluble in water and in many
organic solvents, is non-toxic and non-immunogenic, and when
attached to a surface, PEG provides a biocompatible, protective
coating. Common applications or uses of PEG include (i) covalent
attachment to proteins, e.g., for extending plasma half-life and
reducing clearance through the kidney, (ii) covalent attachment to
small molecules for improving water solubility and ease of
formulation, and to reduce the rate of kidney clearance, (iii)
attachment to surfaces such as in arterial replacements, blood
contacting devices, and biosensors, (iv) as a soluble carrier for
biopolymer synthesis, and (v) as a reagent in the preparation of
hydrogels.
[0004] In many if not all of the uses noted above, it is necessary
to first activate the PEG by converting its hydroxyl terminus to a
functional group capable of readily reacting with a functional
group found within a desired target molecule or surface, such as a
functional group found on the surface of a protein. For proteins,
typical reactive amino acids include lysine, cysteine, histidine,
arginine, aspartic acid, glutamic acid, serine, threonine,
tyrosine, the N-terminal amino group and the C-terminal carboxylic
acid.
[0005] The PEG used as a starting material for most PEG activation
reactions is typically an end-capped PEG. An end-capped PEG is one
where one or more of the hydroxyl groups, typically located at a
terminus of the polymer, is converted into a non-reactive group,
such as a methoxy, ethoxy, or benzyloxy group. Most commonly used
is methoxyPEG, abbreviated as mPEG. End-capped PEGs such as mPEG
are generally preferred, since such end-capped PEGs are typically
more resistant to cross-linking and aggregation. The structures of
two commonly employed end-capped PEG alcohols, mPEG and monobenzyl
PEG (otherwise known as bPEG), are shown below,
##STR00001##
[0006] wherein n typically ranges from about 10 to about 2,000.
[0007] Although the use of mPEG is preferred in many respects,
there are also some serious disadvantages associated with the use
of mPEG as a starting material. Commercially available mPEG is
often contaminated with PEG diol
(HO--(CH.sub.2CH.sub.2).sub.n--OH), where values of n are typically
as stated above. Although some manufacturers produce low-diol mPEG,
some of the diol impurity is always present, and content can range
as high as 10-15%, or in some cases, even greater. PEG diol arises
from the presence of trace amounts of water contamination during
the base catalyzed polymerization of ethylene oxide to form mPEG.
Due to a lower concentration of methoxide initiator in the
preparation of high molecular weight PEGs, e.g., exceeding 20
kilodaltons (K) or so, water contamination and hence diol formation
can present a more serious problem. For high molecular weight PEG,
diol contamination can reach or even exceed 30%. Further, because
the diol chain can grow at each end, the contaminating diol
typically has a higher average molecular weight than the desired
mPEG.
[0008] One characteristic of PEGylation chemistry is that, in most
cases, the diol and corresponding difunctional or di-activated PEG
resulting from PEG diol are not removed. In such cases, the
conjugate product will contain a certain amount of cross-linked
product, and additionally possess an increased polydispersity due
to polymer diol and diol-derived contaminants. This is highly
undesirable for a pharmaceutical product, since the presence and
amounts of such contaminants can be highly variable, thus leading
to irreproducibility of the product.
[0009] Different approaches have been employed to date in an
attempt to overcome these problems. In one approach to reduce the
amount of diol impurity in mPEG starting materials, monofunctional
PEG alcohols have been manufactured by polymerization of ethylene
oxide under strictly anhydrous conditions using an alcohol
initiator in the form of a sodium or potassium salt (Odian,
Principles of Polymerization, 3.sup.rd edition, Wiley, 1991; F. E.
Bailey, Jr. & J. V. Koleske, in Poly(ethylene oxide), Academic
Press, New York, 1976). Although resulting in mPEGs having somewhat
reduced diol content, this approach does not lend itself to
commercial scale syntheses, due to the sensitivity of the process
to moisture and associated problems in controlling the molecular
weight and polydispersity of the product. Moreover, the process is
rather complicated and expensive to operate, especially for the
manufacture of the relatively small quantities of higher molecular
weight polymeric reagents needed for many pharmaceutical
applications. Further, the explosive reactivity of the monomer
requires additional safety precautions that add to the cost of
manufacturing.
[0010] In another approach to dealing with diol contamination,
crude benzyloxy PEG containing diol impurity is methylated and then
hydrogenated to remove the benzyl group (See U.S. Pat. No.
6,448,369). As a result, PEG diol present in the composition is
converted to the inert dimethyl ether. However, this process can be
disadvantageous in several respects. First, this approach adds to
the total number of synthetic steps that must be carried out to
prepare a final activated PEG reagent or product. Secondly,
although inert, this approach still leads to the formation of an
impurity in the activated PEG composition.
[0011] Alternatively, an activated PEG product may be purified to
remove difunctional material, however, such purifications are
typically extremely laborious and time-consuming, as well as
difficult to accomplish. For example, gradient-based
chromatography, a frequently employed separations approach,
requires the analysis of multiple eluate fractions, utilizes a
large volume of solvent, and is poorly suited for commercial scale
processes. Moreover, gradient-based separation techniques rarely
achieve acceptable purity levels, particularly when separating
higher molecular weight polymer species.
[0012] In sum, the present methods for preparing activated PEGs,
particularly monofunctional activated PEGs, are unsatisfactory in
many respects. For the most part, the current methods rely on the
use of relatively expensive mPEG starting material, which often
contains large amounts of the undesirable contaminant, PEG diol.
Current synthetic approaches to avoid diol formation are
complicated, requiring multiple additional reaction steps, and can
still result in the formation of detectable amounts of PEG diol or
PEG-diol derived byproducts. Finally, existing separations
approaches, particular chromatographic methods, are unsatisfactory
for the reasons discussed above.
[0013] The Applicants have realized a continuing need in the art
for new methods for preparing activated PEGs that (i) do not rely
on expensive monofunctional polymer starting materials, (ii) do not
require multiple additional cumbersome reaction steps, and (iii)
overcome the problems associated with the presence of PEG diol by
providing high purity polymer reagents having a low diol content.
In response to these and other needs, the Applicants have, among
other things, developed new methods for forming activating PEGs
which overcome many of the shortcomings noted above.
SUMMARY OF THE INVENTION
[0014] In one aspect, the invention provides a method for forming a
functionalized polymer, the method comprising the steps of: (a)
providing a water-soluble and non-peptidic polymer comprising two
hydroxyl groups (i.e., a water-soluble and non-peptidic polymer
having two or more hydroxyl groups); (b) reacting the water-soluble
and non-peptidic polymer comprising two hydroxyl groups, in one or
more reaction steps, with one or more functionalizing reagents to
effect the introduction of a functional group, Y, to form a mixture
comprising (i) unsubstituted water soluble and non-peptidic polymer
from step (a), (ii) a monosubstituted polymer comprising a single Y
group, and (iii) a disubstituted polymer comprising two Y groups,
under conditions effective to form either no more than about 45
percent of the disubstituted polymer; and (c) purifying the mixture
from step (b) to provide a monosubstituted polymer substantially
free from the unsubstituted and disubstituted polymer species.
Functionalized polymers, as well as monosubstituted polymers,
prepared in accordance with this method represent additional
aspects of the invention. The method optionally comprises the
further step of alkylating the non-peptidic polymer comprising two
hydroxyl groups prior to step (b), or alkylating the mixture formed
in step (b) prior to or subsequent to the purification step (c).
This optionally step can be used to convert unreacted hydroxyl
groups to alkoxy groups.
[0015] In another aspect, the invention provides a method for
forming an alkylated functionalized polymer, said method comprising
the steps of: (a) providing a water-soluble and non-peptidic
polymer comprising two hydroxyl groups; (b) alkylating the
water-soluble and non-peptidic polymer to form a mixture comprising
(i) unalkylated water-soluble and non-peptidic polymer from step
(a), (ii) a monoalkylated polymer comprising a single alkoxy group,
and (iii) a dialkylated polymer comprising two alkoxy groups, under
conditions effective to form at least about 25 mol percent of the
dialkylated polymer; (c) reacting the mixture from step (b), in or
more reaction steps, with one or more functionalizing reagents to
effect the introduction of a functional group, Y, to form a mixture
comprising (i) unalkylated polymer comprising two Y groups, a
monoalkylated polymer polymer comprising a single Y group, and a
dialkylated polymer comprising no Y groups, (d) purifying the
mixture from step (c) to provide a monoalkylated polymer
substantially free from the unalkylated and dialkylated polymer
species. Alkylated functionalized polymers, as well as
monoalkylated polymers substantially free from the unalkylated and
dialkylated polymers species, prepared in accordance with this
method represent additional aspects of the invention.
[0016] In still another aspect, the invention provides a method of
forming a functionalized polymer, the method comprising the steps
of: (a) providing a polymer comprising a formula HO-POLY-OH,
wherein POLY is a water-soluble and non-peptidic polymer; (b)
optionally, converting HO-POLY-OH to a mixture comprising
HO-POLY--OH, HO-POLY-Z and Z-POLY-Z, wherein Z is a leaving group,
under conditions effective to form no more than about 45 percent of
Z-POLY-Z; (c) reacting HO-POLY-OH of step (a) or the mixture of
step (b) with a functionalizing reagent comprising the structure
X--L.sub.0,1--Y, wherein X is a group that reacts with a hydroxyl,
optionally in anionic form, or with a carbon atom to which the
hydroxyl or leaving group is attached, L.sub.0,1 is an optional
linker, and Y is an ionizable group, to form a mixture comprising
HO-POLY-OH, HO-POLY-L.sub.0,1--Y, and
Y--L.sub.0,1-POLY-L.sub.0,1--Y, under conditions effective to form
preferably no more than about 25 percent of
Y--L.sub.0,1-POLY-L.sub.0,1--Y; (d) optionally, alkylating the
mixture from step (b) or step (c); and (e) purifying the mixture
from step (c) or step (d) by ion exchange chromatography to provide
substantially pure polymer comprising a single --L.sub.0,1--Y
group. Functionalized polymers, as well as substantially pure
polymer comprising a single --L.sub.0,1--Y group, prepared in
accordance with this method represent additional aspects of the
invention.
[0017] In yet an additional aspect, the invention provides a method
of separating a mixture of polymer species by ion exchange
chromatography, said method comprising the steps of: (a) providing
a mixture of water-soluble and non-peptidic polymers, said mixture
comprising a neutral polymer absent an ionizable functional group,
a monofunctional polymer comprising a single ionizable functional
group, and a difunctional polymer comprising two ionizable
functional groups; (b) passing the mixture through a first ion
exchange column to provide an eluate, wherein said passing the
mixture step is carried out under conditions effective to adsorb
substantially all of the difunctional polymer onto the first
column; (c) passing the eluate through a second ion exchange column
under conditions effective to adsorb substantially all of the
monofunctional polymer onto said second column; (e) washing the
second column with water or a salt solution having low ionic
strength to remove substantially only neutral polymer absent an
ionizable functional group in a wash solution; and (f) passing a
solution having sufficient ionic strength through the second column
to desorb the monofunctional polymer.
[0018] In still another aspect, the invention provides an ion
exchange chromatography apparatus, comprising: a supply of solution
of a water-soluble and non-peptidic polymer mixture comprising a
neutral polymer absent ionizable functional groups, a
monofunctional polymer comprising a single ionizable functional
group, and a difunctional polymer comprising two ionizable
functional groups; a first ion exchange column comprising a first
inlet, a first outlet, and a first ion exchange media, said first
inlet being in fluid communication with said supply; a second ion
exchange column comprising a second inlet, a second outlet, and a
second ion exchange media, said second inlet being in fluid
communication with said first outlet; and at least one product
recovery vessel in fluid communication with said second outlet,
adapted to receive eluent exiting from said second ion exchange
column. The first and second ion exchange media can either be the
same or different, and the apparatus includes instances where the
first and second ion exchange media are the same and instances
where the first and second ion exchange media are different.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Having thus described the invention in general terms,
reference will now be made to the accompanying drawings,
wherein:
[0020] FIG. 1 graphically illustrates the statistics of
substitution of a PEG diol in a nucleophilic substitution reaction.
This plot demonstrates the relative concentrations of diol, mono-
and di-substituted product in a reaction mixture at any point
during such a reaction;
[0021] FIG. 2 illustrates an embodiment of the ion exchange
chromatography system of the invention in which two columns are
employed; and
[0022] FIG. 3 illustrates a multiple column embodiment of the ion
exchange chromatography system of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0023] Before describing the present invention in detail, it is to
be understood that this invention is not limited to the particular
polymers, synthetic techniques, active agents, and the like as such
may vary. It is also to be understood that the terminology used
herein is for describing particular embodiments only and is not
intended to be limiting.
[0024] It must be noted that, as used in this specification, the
singular forms "a," "an," and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to a "polymer" includes a single polymer as well as two
or more of the same or different polymers, reference to a
"conjugate" refers to a single conjugate as well as two or more of
the same or different conjugates, reference to an "excipient"
includes a single excipient as well as two or more of the same or
different excipients, and the like.
I. Definitions
[0025] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions described below.
[0026] "PEG," "polyethylene glycol" and "poly(ethylene glycol)" are
used herein to mean any water-soluble poly(ethylene oxide).
Typically, PEGs for use in the present invention will comprise one
of the two following structures: "--O(CH.sub.2CH.sub.2O).sub.n--"
or
"--CH.sub.2CH.sub.2O(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--,"
where n is 3 to 3000, and the terminal groups and architecture of
the overall PEG may vary. "PEG" means a polymer that contains a
majority, that is to say, greater than 50%, of subunits that are
--CH.sub.2CH.sub.2O--.
[0027] One commonly employed PEG is end-capped PEG. When PEG is
defined as "--O(CH.sub.2CH.sub.2O).sub.n--," the end-capping group
is generally a carbon-containing group typically comprised of 1-20
carbons and is preferably alkyl (e.g., methyl, ethyl or benzyl)
although saturated and unsaturated forms thereof, as well as aryl,
heteroaryl, cyclo, heterocyclo, and substituted forms of any of the
foregoing are also envisioned. When PEG is defined as
"--CH.sub.2CH.sub.2O(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--,"
the end-capping group is generally a carbon-containing group
typically comprised of 1-20 carbon atoms and an oxygen atom that is
covalently bonded to the group and is available for covalently
bonding to one terminus of the PEG. In this case, the group is
typically, alkoxy (e.g., methoxy, ethoxy or benzyloxy) and with
respect to the carbon-containing group can optionally be saturated
and unsaturated, as well as aryl, heteroaryl, cyclo, heterocyclo,
and substituted forms of any of the foregoing. The other
("non-end-capped") terminus is a typically hydroxyl, amine or an
activated group that can be subjected to further chemical
modification when PEG is defined as
"--CH.sub.2CH.sub.2O(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--."
In addition, the end-capping group can also be a silane.
[0028] Specific PEG forms for use in the invention include PEGs
having a variety of molecular weights, structures or geometries
(e.g., branched, linear, forked PEGs, multifunctional, and the
like), to be described in greater detail below.
[0029] The end-capping group can also advantageously comprise a
detectable label. When the polymer has an end-capping group
comprising a detectable label, the amount or location of the
polymer and/or the moiety (e.g., active agent) to which the polymer
is coupled to is of interest can be determined by using a suitable
detector. Such labels include, without limitation, fluorescers,
chemiluminescers, moieties used in enzyme labeling, colorimetric
(e.g., dyes), metal ions, radioactive moieties, and the like.
[0030] The polymers of the invention are typically polydisperse
(i.e., number average molecular weight and weight average molecular
weight of the polymers are not equal), possessing low
polydispersity values--expressed as a ratio of weight average
molecular weight (Mw) to number average molecular weight (Mn),
(Mw/Mn)--of generally less than about 1.2, preferably less than
about 1.15, more preferably less than about 1.10, still more
preferably less than about 1.05, yet still most preferably less
than about 1.03, and most preferably less than about 1.025.
[0031] As used herein, the term "ionizable functional group" and
variations thereof is a functional group that may gain or lose a
proton by interaction with another ionizable species of functional
group in aqueous or other polar media. Ionizable functional groups
include, but are not limited to, amines, carboxylic acids, aldehyde
hydrates, ketone hydrates, amides, hydrazines, thiols, phenols,
oximes, dithiopyridines, and vinylpyridines.
[0032] As used herein, the term "carboxylic acid" is a moiety
having
##STR00002##
functional group [also represented as a "--COOH" or --C(O)OH], as
well as moieties that are derivatives of a carboxylic acid, such
derivatives including, for example, protected carboxylic acids.
Thus, unless the context clearly dictates otherwise, the term
carboxylic acid includes not only the acid form, but corresponding
esters and protected forms as well. Reference is again made to
Greene et al., "PROTECTIVE GROUPS IN ORGANIC SYNTHESIS" 3.sup.rd
Edition, John Wiley and Sons, Inc., New York, 1999.
[0033] "Activated carboxylic acid" means a functional derivative of
a carboxylic acid that is more reactive than the parent carboxylic
acid, in particular, with respect to nucleophilic acyl
substitution. Activated carboxylic acids include but are not
limited to acid halides (such as acid chlorides), anhydrides,
amides and esters.
[0034] The term "reactive" or "activated", when used in conjunction
with a particular functional group, refers to a reactive functional
group that reacts readily with an electrophile or a nucleophile on
another molecule. This is in contrast to those groups that require
strong catalysts or highly impractical reaction conditions in order
to react (i.e., a "nonreactive" or "inert" group).
[0035] The terms "protected" or "protecting group" or "protective
group" refer to the presence of a moiety (i.e., the protecting
group) that prevents or blocks reaction of a particular chemically
reactive functional group in a molecule under certain reaction
conditions. The protecting group will vary depending upon the type
of chemically reactive group being protected as well as the
reaction conditions to be employed and the presence of additional
reactive or protecting groups in the molecule, if any. Protecting
groups known in the art can be found in Greene et al., supra.
[0036] As used herein, the term "functional group" or any synonym
thereof is meant to encompass protected forms thereof.
[0037] The term "spacer" or "spacer moiety" is used herein to refer
to an atom or a collection of atoms optionally used to link
interconnecting moieties such as a terminus of a water-soluble
polymer portion and a functional group. The spacer moieties of the
invention may be hydrolytically stable or may include a
physiologically hydrolyzable or enzymatically degradable
linkage.
[0038] "Alkyl" refers to a hydrocarbon chain, typically ranging
from about 1 to 20 atoms in length. Such hydrocarbon chains are
preferably but not necessarily saturated and may be branched or
straight chain, although typically straight chain is preferred.
Exemplary alkyl groups include ethyl, propyl, butyl, pentyl,
1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, and the like. As used
herein, "alkyl" includes cycloalkyl when three or more carbon atoms
are referenced.
[0039] "Lower alkyl" refers to an alkyl group containing from 1 to
6 carbon atoms, and may be straight chain or branched, as
exemplified by methyl, ethyl, n-butyl, iso-butyl, tert-butyl.
[0040] "Cycloalkyl" refers to a saturated or unsaturated cyclic
hydrocarbon chain, including bridged, fused, or spiro cyclic
compounds, preferably made up of 3 to about 12 carbon atoms, more
preferably 3 to about 8.
[0041] "Non-interfering substituents" are those groups that, when
present in a molecule, are typically non-reactive with other
functional groups contained within the molecule.
[0042] The term "substituted" as in, for example, "substituted
alkyl," refers to a moiety (e.g., an alkyl group) substituted with
one or more non-interfering substituents, such as, but not limited
to: C.sub.3-C.sub.8 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and
the like; halo, e.g., fluoro, chloro, bromo, and iodo; cyano;
alkoxy, lower phenyl (e.g., 0-2 substituted phenyl); substituted
phenyl; and the like.
[0043] "Substituted aryl" is aryl having one or more
non-interfering groups as a substituent. For substitutions on a
phenyl ring, the substituents may be in any orientation (i.e.,
ortho, meta, or para).
[0044] "Alkoxy" refers to an --O--R group, wherein R is alkyl or
substituted alkyl, preferably C.sub.1-C.sub.20 alkyl (e.g.,
methoxy, ethoxy, propyloxy, benzyloxy, etc.), preferably
C.sub.1-C.sub.8.
[0045] "Aryl" means one or more aromatic rings, each of 5 or 6 core
carbon atoms. Aryl includes multiple aryl rings that may be fused,
as in naphthyl or unfused, as in biphenyl. Aryl rings may also be
fused or unfused with one or more cyclic hydrocarbon, heteroaryl,
or heterocyclic rings. As used herein, "aryl" includes
heteroaryl.
[0046] "Heteroaryl" is an aryl group containing from one to four
heteroatoms, preferably N, O, or S, or a combination thereof.
Heteroaryl rings may also be fused with one or more cyclic
hydrocarbon, heterocyclic, aryl, or heteroaryl rings.
[0047] "Electrophile" refers to an ion or atom or collection of
atoms, that may be ionic, having an electrophilic center, i.e., a
center that is electron seeking or capable of reacting with a
nucleophile.
[0048] "Nucleophile" refers to an ion or atom or collection of
atoms, that may be ionic, having a nucleophilic center, i.e., a
center that is seeking an electrophilic center or capable of
reacting with an electrophile.
[0049] A "physiologically cleavable" or "hydrolyzable" or
"degradable" bond is a relatively weak bond that reacts with water
(i.e., is hydrolyzed) under physiological conditions. The tendency
of a bond to hydrolyze in water will depend not only on the general
type of linkage connecting two central atoms but also on the
substituents attached to these central atoms. Appropriate
hydrolytically unstable or weak linkages include, but are not
limited to, carboxylate ester, phosphate ester, anhydrides,
acetals, ketals, acyloxyalkyl ether, imines, orthoesters, and
oligonucleotides.
[0050] An "enzymatically degradable linkage" means a linkage that
is subject to degradation by one or more enzymes.
[0051] A "hydrolytically stable" linkage or bond refers to a
chemical bond, typically a covalent bond, that is substantially
stable in water, that is to say, does not undergo hydrolysis under
physiological conditions to any appreciable extent over an extended
period of time. Examples of hydrolytically stable linkages include
but are not limited to the following: carbon-carbon bonds (e.g., in
aliphatic chains), ethers, amides, urethanes, and the like.
Generally, a hydrolytically stable linkage is one that exhibits a
rate of hydrolysis of less than about 1-2% per day under
physiological conditions. Hydrolysis rates of representative
chemical bonds can be found in most standard chemistry
textbooks.
[0052] "Multifunctional" or "multisubstituted" in the context of a
polymer of the invention means a polymer having 2 or more
functional groups contained therein, where the functional groups
may be the same or different. Multifunctional polymers of the
invention will typically contain from about 2-100 functional
groups, or from 2-50 functional groups, or from 2-25 functional
groups, or from 2-15 functional groups, or from 3 to 10 functional
groups, or will contain 2, 3, 4, 5, 6, 7, 8, 9 or 10 functional
groups within the polymer backbone.
[0053] A "difunctional" or "disubstituted" polymer means a polymer
having two functional groups contained therein, either the same
(i.e., homodifunctional) or different (i.e.,
heterodifunctional).
[0054] A "monofunctional" or "monosubstituted" polymer means a
polymer having a single functional group contained therein (e.g.,
an mPEG based polymer).
[0055] A basic or acidic reactant described herein includes
neutral, charged, and any corresponding salt forms thereof.
[0056] The term "patient," refers to a living organism suffering
from or prone to a condition that can be prevented or treated by
administration of a conjugate, and includes both humans and
animals.
[0057] "Optional" or "optionally" means that the subsequently
described circumstance may or may not occur, so that the
description includes instances where the circumstance occurs and
instances where it does not.
[0058] Unless otherwise noted, molecular weight is expressed herein
as number average molecular weight (M.sub.n), which is defined
as
N .times. i .times. M .times. i Ni , ##EQU00001##
wherein Ni is the number of polymer molecules (or the number of
moles of those molecules) having molecular weight Mi.
[0059] Each of the terms "drug," "biologically active molecule,"
"biologically active moiety," "active agent" and "biologically
active agent", when used herein, means any substance which can
affect any physical or biochemical properties of a biological
organism, including but not limited to viruses, bacteria, fungi,
plants, animals, and humans. In particular, as used herein,
biologically active molecules include any substance intended for
diagnosis, cure mitigation, treatment, or prevention of disease in
humans or other animals, or to otherwise enhance physical or mental
well-being of humans or animals. Examples of biologically active
molecules include, but are not limited to, peptides, proteins,
enzymes, small molecule drugs, dyes, lipids, nucleosides,
oligonucleotides, polynucleotides, nucleic acids, cells, viruses,
liposomes, microparticles and micelles. Classes of biologically
active agents that are suitable for use with the invention include,
but are not limited to, antibiotics, fungicides, anti-viral agents,
anti-inflammatory agents, anti-tumor agents, cardiovascular agents,
anti-anxiety agents, hormones, growth factors, steroidal agents,
and the like.
[0060] As used herein, "non-peptidic" refers to a polymer backbone
substantially free of peptide linkages. However, the polymer
backbone may include a minor number of peptide linkages spaced
along the length of the backbone, such as, for example, no more
than about 1 peptide linkage per about 50 monomer units.
[0061] The term "conjugate" is intended to refer to the entity
formed as a result of covalent attachment of a molecule, e.g., a
biologically active molecule, to a reactive polymer molecule,
preferably poly(ethylene glycol).
[0062] "Eluate" refers to a solution that has passed through a
chromatography column (i.e., an effluent stream).
[0063] "Eluent" refers to the mobile phase utilized during a
chromatographic separation.
[0064] "Pre-column" and "first column" are used interchangeably
herein and refer to a single chromatography column, as well as two
or more columns connected in series that serve as the "pre-column"
or "first column." In addition, the terms "main column" and "second
column" are used interchangeably herein and refer to a single
chromatography column, as well as two or more columns connected in
series that serve as the "main column" or "second column."
II. Method of Preparing Functionalized Polymers Using Polymeric
Polyol Starting Material
[0065] In one aspect, the present invention provides a method of
forming functionalized polymeric reagents, particularly
monofunctional polymeric reagents, using polymeric polyol starting
materials, such as dihydroxy PEG, instead of the expensive,
difficult to purify mPEG starting materials known in the art. The
method of the invention involves reacting the polymeric polyol
starting material with a functionalizing reagent comprising a
functional group, --Y. The functionalizing reagent is capable of
reaction, in one or more steps, with the polyol to form a plurality
of substituted polymers, each comprising a varying number of --Y
groups. The reaction is typically carried out under conditions
effective to produce a mixture of an unsubstituted polymer (i.e.,
the original polymeric polyol), a monosubstituted polymer (i.e., a
polymer having a single Y group), and one or more multi substituted
polymers (e.g., a disubstituted polymer having two Y groups)
characterized by a relatively wide difference in content of the
monosubstituted product and the multi substituted product(s).
[0066] The mixture of polymer products is subjected to a
purification step in order to separate the mixture components and
provide a monosubstituted polymer substantially free from the
unsubstituted and multisubstituted polymer species. By performing
the purification/separation process while the desired
monosubstituted polymer and the multisubstituted polymer species
are present at differing concentrations, separation is made easier
and formation of highly pure monofunctional polymeric reagents is
possible. In essence, controlling the extent to which the
functionalizing reaction is allowed to proceed is used as a means
to enhance and simplify separation of the polymeric species formed
in the reaction. The approach of the present invention is
particularly well suited for use with functionalizing reagents that
attach ionizable functional groups to the polymer and separation
processes adapted for separation based on differences in
charge.
[0067] For purposes of illustrating one or more advantages of the
invention, the use of a dihydroxy PEG staring material is
considered. Commencement of a reaction of the dihydroxy PEG with a
functionalizing reagent comprising a protected amine or protected
carboxylic acid will result in formation of a monosubstituted
polymer species (e.g., a polymer having a single protected or free
amine or protected or free carboxylic acid group) and a
disubstituted polymer species (e.g., a polymer having two protected
amine or protected carboxylic acid groups). As the number of moles
of the mono- and disubstituted polymers increases, the number of
moles of the original PEG diol starting material will decrease
concomitantly. The theoretical yield of monosubstituted and
disubstituted polymer species expressed as a % of substitution
(i.e., mole percent) is shown in FIG. 1. As shown, the
monosubstituted product reaches a theoretical maximum of 50% and
then declines as the percentage of disubstituted product
continually increases. The amount of unsubstituted PEG diol
starting material continually declines as the reaction
proceeds.
[0068] In one or more embodiments of the present invention, the
reaction is allowed to proceed until a certain predetermined amount
of the monosubstituted and disubstituted polymer species is formed.
This predetermined amount is selected based on the disparity in
concentration of the monosubstituted product and the disubstituted
product. By stopping the reaction at a point characterized by a
large difference in concentration of the monosubstituted product
and the disubstituted product (e.g., when the reaction mixture
comprises 25.5% monosubstituted product and only 2.25%
disubstituted product), separation or purification of the polymer
mixture is easier. As noted above, this is particularly true when
ionizable groups are utilized that allow separation of the polymer
mixture based on differences in charge.
[0069] Once the functionalizing reaction ends (e.g., by quenching
or reagent exhaustion) at the desired point, separation of the
mixture can take place and the purified monosubstituted polymer can
then be used, optionally after further functionalization, for any
one of a number of purposes (e.g., to form a conjugate with a
biologically active agent). Further functionalization can be
carried out by subjecting the purified monosubstituted polymer to
additional reaction steps to form other useful active polymeric
reagents, such as the formation of active esters from carboxylic
acid terminated polymers or the formation of maleimides from amine
terminated polymers.
[0070] Examples of suitable functional groups that can be formed on
the final purified polymer include hydroxyl, active ester (e.g.,
N-hydroxysuccinimidyl ester and 1-benzotriazolyl ester), active
carbonate (e.g., N-hydroxysuccinimidyl carbonate, 1-benzotriazolyl
carbonate, and p-nitrophenyl carbonate), acetal, aldehyde having a
carbon length of 1 to 25 carbons (e.g., acetaldehyde,
propionaldehyde, and butyraldehyde), aldehyde hydrate, alkenyl,
acrylate, methacrylate, acrylamide, active sulfone, amine,
hydrazide, thiol, alkanoic acids having a carbon length (including
the carbonyl carbon) of 1 to about 25 carbon atoms (e.g.,
carboxylic acid, carboxymethyl, propanoic acid, and butanoic acid),
acid halide, isocyanate, isothiocyanate, maleimide, vinylsulfone,
dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal,
dione, mesylate, tosylate, and tresylate. Exemplary functional
groups are discussed in the following references: N-succinimidyl
carbonate (see e.g., U.S. Pat. Nos. 5,281,698, 5,468,478), amine
(see, e.g., Buckmann et al. Makromol. Chem. 182:1379 (1981),
Zalipsky et al. Eur. Polym. J. 19:1177 (1983)), hydrazide (See,
e.g., Andresz et al. Makromol. Chem. 179:301 (1978)), succinimidyl
propionate and succinimidyl butanoate (see, e.g., Olson et al. in
Poly(ethylene glycol) Chemistry & Biological Applications, pp
170-181, Harris & Zalipsky Eds., ACS, Washington, D C, 1997;
see also U.S. Pat. No. 5,672,662), succinimidyl succinate (See,
e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) and
Joppich et al., Makromol. Chem. 180:1381 (1979), succinimidyl ester
(see, e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see,
e.g., U.S. Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et
al. Eur. J. Biochem. 94:11 (1979), Elling et al., Biotech. Appl.
Biochem. 13:354 (1991), oxycarbonylimidazole (see, e.g., Beauchamp,
et al., Anal. Biochem. 131:25 (1983), Tondelli et al. J. Controlled
Release 1:251 (1985)), p-nitrophenyl carbonate (see, e.g.,
Veronese, et al., Appl. Biochem. Biotech., 11:141 (1985); and
Sartore et al., Appl. Biochem. Biotech., 27:45 (1991)), aldehyde
(see, e.g., Harris et al. J. Polym. Sci. Chem. Ed. 22:341 (1984),
U.S. Pat. Nos. 5,824,784, 5,252,714), maleimide (see, e.g., Goodson
et al. Bio/Technology 8:343 (1990), Romani et al. in Chemistry of
Peptides and Proteins 2:29 (1984)), and Kogan, Synthetic Comm.
22:2417 (1992)), orthopyridyl-disulfide (see, e.g., Woghiren, et
al. Bioconj. Chem. 4:314 (1993)), acrylol (see, e.g., Sawhney et
al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g., U.S.
Pat. No. 5,900,461). All of the above references are incorporated
herein by reference.
[0071] If a monofunctional end-capped polymer is desired, the
method of the invention can also include an alkylation step, which
can occur either before or after the polymer polyol staring
material is reacted with the functionalizing agent. Preferably, the
optional alkylation step occurs after the functionalizing reaction
so that the functionalizing step remains the controlling step in
the process that determines the relative concentrations of the
monosubstituted polymer product as compared to the disubstituted or
other multi substituted polymer species. If the alkylating step is
performed before the reaction with the functionalizing reagent,
then the alkylating step becomes the controlling reaction that
determines the desired disparity in monosubstituted polymer content
versus disubstituted polymer content. Alternatively, the alkylation
step can be avoided by utilizing a polymeric mixture of a polymeric
diol and its monoalkylated form (e.g., mPEG) if such mixtures
having a proper balance of the two components are readily
available.
[0072] As discussed in greater detail below, the functional group,
Y, is preferably an ionizable functional group. Exemplary ionizable
functional groups include amine and carboxylic acid groups.
Examples of other suitable functional groups include aldehyde
hydrate, ketone hydrate, amide, hydrazine, hydrazide, thiol,
phenol, oxime, other alkanoic acids having a carbon length
(including the carbonyl carbon) of 1 to about 25 carbon atoms
(e.g., carboxymethyl, propanoic acid, and butanoic acid),
dithiopyridine, and vinylpyridine.
A. Polyol Starting Materials
[0073] A polymeric polyol can be used in the present invention and
can comprise any water soluble and non-peptidic polymer having at
least two hydroxyl groups covalently attached thereto. Preferably,
the polymeric polyol is a diol (i.e., a polymer having two hydroxyl
groups attached thereto); however, polyols containing greater than
2 hydroxyl groups can be utilized, such as polyols comprising about
3-100 hydroxyl groups, or from 3-50 hydroxyl groups, or from 3-25
hydroxyl groups, or from 3-15 hydroxyl groups, or from 3 to 10
hydroxyl groups, or will contain 3, 4, 5, 6, 7, 8, 9 or 10 hydroxyl
groups attached to the polymer. Although the hydroxyl groups are
preferably attached to the termini of the polymer, the hydroxyl
groups may also be attached to the polymer as side chains in
pendant fashion.
[0074] The polymer should be non-toxic and biocompatible, meaning
that the polymer is capable of coexistence with living tissues or
organisms without causing harm. When referring to the polymeric
polyol, it is to be understood that the polymer can be any of a
number of water soluble and non-peptidic polymers, such as those
described herein as suitable for use in the present invention.
Preferably, poly(ethylene glycol) (i.e., PEG) is the polymeric
polyol. The term PEG includes poly(ethylene glycol) in any of a
number of geometries or forms, including linear forms, branched or
multi-arm forms (e.g., forked PEG or PEG attached to a polyol
core), pendant PEG, or PEG with degradable linkages therein, to be
more fully described below.
[0075] Multi-armed or branched PEG molecules, such as those
described in U.S. Pat. No. 5,932,462, which is incorporated by
reference herein in its entirety, can also be used as the PEG
polymer. Generally speaking, a multi-armed or branched polymer
possesses two or more polymer "arms" extending from a central
branch point (e.g., C in Formula II below). For example, an
exemplary branched PEG polymer has the structure:
##STR00003##
wherein PEG.sub.1 and PEG.sub.2 are PEG polymers in any of the
forms or geometries described herein, and which can be the same or
different, and L' is a hydrolytically stable linkage. An exemplary
branched PEG of Formula I has the structure:
##STR00004##
wherein: poly.sub.a and poly.sub.b are PEG backbones, such as
methoxy poly(ethylene glycol); R'' is a nonreactive moiety, such as
H, methyl or a PEG backbone; and P and Q are nonreactive linkages.
In a preferred embodiment, the branched PEG polymer is methoxy
poly(ethylene glycol) disubstituted lysine.
[0076] The branched PEG structure of Formula II can be attached to
a third oligomer or polymer chain as shown below:
Formula III
[0077] wherein PEG.sub.3 is a third PEG oligomer or polymer chain,
which can be the same or different from PEG.sub.1 and
PEG.sub.2.
[0078] The PEG polymer may alternatively comprise a forked PEG.
Generally speaking, a polymer having a forked structure is
characterized as having a polymer chain attached to two or more
functional groups via covalent linkages extending from a
hydrolytically stable branch point in the polymer. An example of a
forked PEG is represented by PEG-L-CHY.sub.2, where Lisa linking
group and Y is a functional group. Each Y group is linked to CH by
a chain of atoms of defined length. U.S. Pat. No. 6,362,254, the
contents of which are incorporated by reference herein, discloses
various forked PEG structures capable of use in the present
invention. The chain of atoms linking the Y functional groups to
the branching carbon atom serve as a tethering group and may
comprise, for example, an alkyl chain, ether linkage, ester
linkage, amide linkage, or combinations thereof.
[0079] As noted above, the PEG polymer may comprise a pendant PEG
molecule having reactive groups, such as hydroxyl, covalently
attached along the length of the PEG backbone rather than at the
end of the PEG chain. The pendant reactive groups can be attached
to the PEG backbone directly or through a linking moiety, such as
an alkylene group.
[0080] In addition to the above-described forms of PEG, the polymer
can also be prepared with one or more hydrolytically stable or
degradable linkages in the polymer backbone, including any of the
above described polymers. For example, PEG can be prepared with
ester linkages in the polymer backbone that are subject to
hydrolysis. As shown below, this hydrolysis results in cleavage of
the polymer into fragments of lower molecular weight:
-PEG-CO.sub.2-PEG-+H.sub.2O-PEG-CO.sub.2H+HO-PEG-
[0081] Other hydrolytically degradable linkages, useful as a
degradable linkage within a polymer backbone, include carbonate
linkages; imine linkages resulting, for example, from reaction of
an amine and an aldehyde (see, e.g., Ouchi et al., Polymer
Preprints, 38(1):582-3 (1997), which is incorporated herein by
reference); phosphate ester linkages formed, for example, by
reacting an alcohol with a phosphate group; hydrazone linkages
which are typically formed by reaction of a hydrazide and an
aldehyde; acetal linkages that are typically formed by reaction
between an aldehyde and an alcohol; ortho ester linkages that are,
for example, formed by reaction between a formate and an alcohol;
and oligonucleotide linkages formed by, for example, a
phosphoramidite group, e.g., at the end of a polymer, and a 5'
hydroxyl group of an oligonucleotide.
[0082] It is understood by those skilled in the art that the term
poly(ethylene glycol) or PEG represents or includes all the above
forms of PEG.
[0083] Any of a variety of other polymeric polyols comprising other
non-peptidic and water soluble polymer chains can also be used in
the present invention. The polymeric polyol can be linear, or can
be in any of the above-described forms (e.g., branched, forked, and
the like). Examples of suitable polymers include, but are not
limited to, other poly(alkylene glycols), copolymers of ethylene
glycol and propylene glycol, poly(olefinic alcohol),
poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),
poly(hydroxyalkylmethacrylate), poly(saccharides),
poly(.alpha.-hydroxy acid), poly(acrylic acid), poly(vinyl
alcohol), polyphosphazene, polyoxazoline,
poly(N-acryloylmorpholine), such as described in U.S. Pat. No.
5,629,384, which is incorporated by reference herein in its
entirety, and copolymers, terpolymers, and mixtures thereof.
[0084] Different polymers can be incorporated into the same polymer
backbone. For example, one or more of the PEG molecules in the
branched structures shown in Formulas I-III can be replaced with a
different polymer type. Any combination of water soluble and
non-peptidic polymers is encompassed within the present
invention.
[0085] The molecular weight of the polymeric polyol will vary
depending on the desired application, the configuration of the
polymer structure, the degree of branching, and the like.
Generally, polymers having a molecular weight of about 100 Da to
about 180,000 Da are useful in the present invention, preferably
about 500 Da to about 60,000 Da, and more preferably about 5,000 Da
to about 40,000 Da. Exemplary polymer embodiments have a molecular
weight of approximately 1,000 Da, 2,000 Da, 3,000 Da, 4,000 Da,
5,000 Da, 7,500 Da, 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da,
30,000 Da, 35,000 Da, and 40,000 Da.
[0086] The polymeric polyol is typically dissolved in water or an
organic solvent prior to the functionalizing reaction discussed
below. Any organic solvent compatible with polymers of the type
used in the present invention can be utilized, such as toluene,
xylene, benzene, dichloromethane, chloroform, acetonitrile,
tetrahydrofuran, or acetone. Mixtures of the above solvents or
other similar solvents known in the art also can be used.
B. Functionalizing Reaction
[0087] The reaction step or steps used to react a functionalizing
reagent with the polymeric polyol can vary depending on a number of
factors, including the type of functional group involved, the type
and configuration of the polymer, and so forth. The exact nature of
the reaction sequence is not critical to the present invention and
any known method of functionalizing polymers of the type used in
the present invention can be utilized without departing from the
invention.
[0088] As noted above, in one embodiment, the functionalizing
reaction is only allowed to proceed under conditions effective to
produce a product mixture characterized by a wide difference in the
concentrations of the monosubstituted product and the di- or other
multi substituted products. Preferably, the reaction is also
conducted under conditions effective to produce a relatively low
content of multi substituted product. To achieve the desired
content disparity, the reaction between the polyol starting
material and the functionalizing reagent can be stopped or quenched
at the appropriate time using any method known in the art, such as
by rapidly changing process parameters (e.g., temperature or degree
of mixing) or by carefully controlling the amount of reactants,
thereby controlling the reaction on a stoichiometric basis. The
appropriate time for stopping or quenching the reaction can be
determined by obtaining periodic samples of the reaction mixture
and determining the amount of species present (e.g., by
chromatographic methods, NMR methods and so forth) or by measuring
a parameter (e.g., pH) known to correlate with the amount of
species present. Alternatively, if a significant deficiency of the
functionalizing reagent is charged, the reaction will only proceed
to partial conversion of the diol. In this instance, the reaction
may be allowed to proceed to completion. In such cases, knowing the
stoichiometry of the reactants allows for the estimation of the
final compositional components when reference is made to FIG.
1.
[0089] The reaction is generally performed under conditions
effective to form no more than about 45 percent of the
disubstituted polymer. Reactions allowed to continue past this
point result in disubstituted polymer being present in an amount
greater than monosubstituted polymer, with the result that
separation becomes increasingly inefficient. While no more than
about 45 percent of the disubstituted polymer is typically allowed
to form, it is often preferred that the percent of disubstituted
polymer formation is encompassed in one or more of the following
ranges: no more than about 40 percent; no more than about 35
percent; no more than about 30 percent; no more than about 25
percent; no more than about 20 percent; no more than about 15
percent; no more than about 12 percent, and nor more than about 10
percent. In certain embodiments, no more than about 8 percent,
preferably no more than about 5 percent, more preferably no more
than about 2 percent, and most preferably no more than about 1
percent of the disubstituted polymer is formed. In certain
embodiments, the functionalizing reaction results in a ratio of
monosubstituted polymer to disubstituted polymer from about 2:1 to
about 40:1, preferably about 4:1 to about 20:1, and more preferably
about 10:1 to about 18:1.
[0090] Typically, the final functionalized polymer mixture will
comprise about 8 percent to about 50 percent of the monosubstituted
polymer, preferably about 8 to about 45 percent, and more
preferably about 8 to about 30 percent. The final functionalized
polymer mixture will typically comprise about 1 to about 45 percent
of the disubstituted polymer, preferably about 1 to about 12
percent, and more preferably about 1 to about 5 percent. Generally,
the final functionalized polymer mixture will comprise about 10 to
about 91 percent of the original unsubstituted polymeric polyol,
preferably about 43 to about 91 percent, more preferably about 65
to about 91 percent.
[0091] The functionalizing reaction typically comprises a
nucleophilic substitution reaction or a nucleophilic addition
reaction (e.g., a Michael addition reaction), wherein the
nucleophile can be present on the polymer or the functionalizing
reagent. For example, the reaction can involve reaction of a
hydroxyl group of the polymeric polyol, or an anion thereof, as a
nucleophile with a suitable electrophilic group. Alternatively, the
hydroxyl groups of the polymeric polyol can be converted into good
leaving groups, such as sulfonate esters, and reacted with a
functionalizing reagent containing a nucleophilic group.
[0092] The functionalizing reagent will typically comprise a
reactive group, X, that is either an electrophilic group reactive
with a hydroxyl group or anion thereof on the polymeric polyol or,
if some or all of the hydroxyl groups of the polyol have been
converted to good leaving groups, a nucleophilic group. The
functionalizing reagent will also comprise the functional group, Y,
that is intended to be covalently attached to the polymer.
Optionally, the functionalizing reagent will further comprise a
spacer moiety linking the reactive group, X, with the functional
group, Y. Exemplary spacer moieties include --C(O)--, --C(O)--NH--,
--NH--C(O)--NH--, --O--C(O)--NH--, --C(S)--, --CH.sub.2--,
--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2, --O--CH.sub.2--,
--CH.sub.2--O--, --O--CH.sub.2--CH.sub.2--,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--CH.sub.2--O--,
--O--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--O--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--O--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--O--,
--O--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--O--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--O--CH.sub.2--,
--CH.sub.2--CH.sub.2--C H.sub.2--CH.sub.2--O--,
--C(O)--NH--CH.sub.2--, --C(O)--NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--C(O)--NH--CH.sub.2--, --CH.sub.2--CH.sub.2--C(O)--NH--,
--C(O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--,
--C(O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--,
--C(O)--O--CH.sub.2--, --CH.sub.2--C(O)--O--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--O--CH.sub.2--,
--C(O)--O--CH.sub.2--CH.sub.2--, --NH--C(O)--CH.sub.2--,
--CH.sub.2--NH--C(O)--CH.sub.2--,
--CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.2--,
--NH--C(O)--CH.sub.2--CH.sub.2--,
--CH.sub.2--NH--C(O)--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.2--CH.sub.2--,
--C(O)--NH--CH.sub.2--, --C(O)--NH --CH.sub.2--CH.sub.2--,
--O--C(O)--NH--CH.sub.2--, --O--C(O)--NH--CH.sub.2--CH.sub.2--,
--NH--CH.sub.2--, --NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--NH--CH.sub.2--, --CH.sub.2--CH.sub.2--NH--CH.sub.2--,
--C(O)--CH.sub.2--, --C(O)--CH.sub.2--CH.sub.2--,
--CH.sub.2--C(O)--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--NH--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--NH--C(O)--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--NH--C
(O)--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--NH--C(O)--C-
H.sub.2--CH.sub.2--,
--O--C(O)--NH--[CH.sub.2].sub.h--(OCH.sub.2CH.sub.2).sub.j--,
bivalent cycloalkyl group, --O--, --S--, an amino acid,
--N(R.sup.6)--, and combinations of two or more of any of the
foregoing, wherein R.sup.6 is H or an organic radical selected from
the group consisting of alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, aryl and
substituted aryl, (h) is zero to six, and (j) is zero to 20. Other
specific spacer moieties have the following structures:
--C(O)--NH--(CH.sub.2).sub.1-6--NH--C(O)--,
--NH--C(O)--NH--(CH.sub.2).sub.1-6--NH--C(O)--, and
--O--C(O)--NH--(CH.sub.2).sub.1-6--NH--C(O)--, wherein the
subscript values following each methylene indicate the number of
methylenes contained in the structure, e.g., (CH.sub.2).sub.1-6
means that the structure can contain 1, 2, 3, 4, 5 or 6 methylenes.
Additionally, any of the above spacer moieties may further include
an ethylene oxide oligomer chain comprising 1 to 20 ethylene oxide
monomer units [i.e., --(CH.sub.2CH.sub.2O).sub.1-20]. That is, the
ethylene oxide oligomer chain can occur before or after the spacer
moiety, and optionally in between any two atoms of a spacer moiety
comprised of two or more atoms. Also, the oligomer chain would not
be considered part of the spacer moiety if the oligomer is adjacent
to a polymer segment and merely represent an extension of the
polymer segment.
[0093] In one or more embodiments, the functionalizing reagent has
the following structure:
X--(CR.sub.1R.sub.2).sub.m--Y Formula IV
wherein: X is a group reactive with a hydroxyl group or anion
thereof, or a good leaving group, in a nucleophilic substitution or
nucleophilic addition reaction; R.sub.1 and R.sub.2 are each
independently selected H or alkyl; m is 0-10 (e.g., 0, 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10), preferably 1-3; and Y is a functional group,
optionally in protected form, and preferably selected from the
group consisting of such as aldehyde hydrate, ketone hydrate,
amide, amine, hydrazine, hydrazide, thiol, carboxylic acid,
dithiopyridine, vinylpyridine, phenol, and oxime.
[0094] The X reactive group is preferably a good leaving group,
such as halogen (e.g., bromo or chloro) or a sulfonate ester (e.g.,
p-tolylsulfonyl, methylsulfonyl, trifluorosulfonyl, or
trifluoroethylsulfonyl), or a substituted or unsubstituted vinyl
group. The substituting group or groups attached to the vinyl group
carbon atoms are typically alkyl, substituted alkyl, alkoxy,
substituted alkoxy, or halogen.
[0095] In one or more embodiments, X is halogen, m is 0, and Y is
p-tolylsulfonyl, methylsulfonyl, trifluorosulfonyl, or
trifluoroethylsulfonyl. Other exemplary functionalizing reagents of
Formula IV include X'--(CR.sub.1R.sub.2).sub.m--C(O)--O--Rp,
CH.sub.2.dbd.CY'--(CR.sub.1R.sub.2).sub.m--C(O)--O--Rp,
X'--(CR.sub.1R.sub.2).sub.m--Z,
CH.sub.2.dbd.CY'--(CR.sub.1R.sub.2).sub.m--Z,
X'--(CR.sub.1R.sub.2).sub.m--CN, and
CH.sub.2.dbd.CY'--(CR.sub.1R.sub.2).sub.m--CN, wherein X.sup.1 is
Br or Cl, Z is an ortho ester, Y.sup.1 is H, halogen, alkyl,
substituted alkyl, alkoxy, or substituted alkoxy, and Rp is alkyl
or substituted alkyl. If the functional group, Y, of the
functionalizing reagent is in protected form, the method of the
invention further comprises deprotecting the functional group. For
example, if the Y group is a protected carboxylic acid (e.g., an
ortho ester or an alkyl ester), the deprotecting step comprises
hydrolysis of the protecting group to form the carboxylic acid. An
exemplary protected carboxylic acid group has the structure
--C(O)--O--Rp, wherein Rp is an alkyl or substituted alkyl group.
Protected carboxylic acids include: esters, such as methyl ester,
methoxymethyl ester, methylthiomethyl ester, tetrahydropyranyl
ester, benzyloxymethyl ester, phenyacyl ester, n-phthalimidomethyl
ester, 2,2,2-trichloroethyl ester, 2-haloethyl ester,
2-(p-toluenesulfonyl)ethyl ester, t-butyl ester, cinnamyl ester,
benzyl ester, triphenylmethyl ester, bis(o-nitrophenyl)methyl
ester, 9-anthrylmethyl ester, 2-(9,10-dioxo)anthrylmethyl ester,
piperonyl ester, trimethylsilyl ester, t-butyldimethylsilyl ester
and S-t-butyl ester; thiolesters, such as methylthiol, ethylthiol,
phenylthiol, p-snitrophenylthiol, benzylthiol and t-butylthiol;
amidates such as O-alkyl-N-alkyl, O-aryl-N-alkyl, O-alkyl-N-aryl,
O-aryl-N-aryl, 2-substituted-1-3-oxazolines,
2-substituted-1-3-(4H)-dihydrooxazines; thioamidates, such as
S-alkyl-N-alkyl, S-aryl-N-alkyl, S-alkyl-N-aryl, S-aryl-N-aryl,
2-substituted-1,3-thiazolines,
2-substituted-1,3-(4H)-1,3-dihydrothiazines; amides and hydrazides
such as N,N-dimethylamide, N-7-nitroindoylamide, hydrazide,
N-phenylhydrazide, N,N'-diisopropylhydrazide.
[0096] If the Y group is a protected amine (e.g., a carbonitrile
group), the deprotecting step can comprise reducing the
carbonitrile group to form the amine. Alternatively, one can
consider the carbonitrile group as a protected carboxylic acid and
deprotection would involve hydrolysis. Protected amines include:
carbamates such as 9-fluorenylmethyl, 9-(2-sulfo)fluorenylmethyl,
9-(2,7dibromo)fluorenylmethyl,
17-tetrabenzo[a,c,g,i]fluorenylmethyl, 2-chloro-3-indenylmethyl,
benz[f]inden-3-ylmethyl,
2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl,
1,1-dioxobenz[b]thiophene-2-ylmethyl, 2,2,2-trichloroethyl,
2-trimethylsilylethyl, 2-phenylethyl,
1-(1-adamantyl)-1-methylethyl, 2-chloroethyl,
1,1-dimethyl-2-haloethyl, 1,1-dimethyl-2,2-dibromoethyl,
1,1-dimethyl-2,2,2-trichloroethyl, 1-methyl-1-(4-biphenylyl)ethyl,
1-(3,5-di-t-butylphenyl)-1-methylethyl, 2-(2' and 4'-pyridyl)ethyl,
2,2-bis(4'-nitrophenyl)ethyl,
N-(2-pivaloylamino)-1,1-dimethylethyl,
2-[(2nitrophenyl)dithio]-1-phenylethyl,
2-(N,N-dicyclohexylcarboxamido)ethyl, t-butyl, 1-adamantyl,
2-adamantyl, vinyl, allyl, cinnamyl, 2-3'-pyridyl-prop-2-enyl,
8-quinolyl, N-hydroxypiperidinyl, alkyldithio, p-methoxybenzyl,
p-nitrobenzyl, p-chlorobenzyl, 2,4-dichlorobenzyl,
4-methylsulfinylbenzyl, 9-anthrylmethyl, diphenylmethyl,
2-methylthioethyl, 3-methylsulfonylethyl,
2-(p-toluenesulfonyl)ethyl, [2-(1,3-dithianyl)]methyl,
4-methylthiophenyl, 2,4-dimethylthiophenyl, 2-phosphonioethyl,
1-methyl-1-(triphenylphosphonio)ethyl, 1,1-dimethyl-2-cyanoethyl,
2-dansylethyl, 2-(4-nitrophenyl)ethyl, 4-phenylacetoxybenzyl,
4-azidobenzyl, 4-azidomethoxybenzyl, m-chloro-p-acyloxybenzyl,
p-(dihydroxyboryl)benzyl, 5-benzisoxazolylmethyl,
2-(trifluoromethyl)-6-chromonylmethyl, w-nitrophenyl,
2,5-dimethoxybenzyl, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl,
.alpha.-methylnitropiperonyl, o-nitrobenzyl, and
3,4-dimethoxy-6-nitrobenzyl; urea type derivatives such as
phenothiazinyl-(10)-carbonyl derivative, N'-p-toluenesulfonyl
aminocarbonyl, N'-phenylaminothiocarbonyl; amides such as N-formyl,
N-chloroacetyl, N-trichloroacetyl, N-trifluoroacetyl,
N-phenylacetyl, N-3-phenylpropionyl, N-4-pentenoyl, N-picolinoyl,
N-3-pyridylcarboxamido, N-benzoylphenylalanyl derivative,
N-p-phenylbenzoyl, N-o-nitrophenylacetyl, N-o-nitrophenoxyacetyl,
N-3-(o-nitrophenyl)propionyl,
N-2-methyl-2-(o-nitrophenoxy)propionyl, N-3-methyl-3-nitrobutyryl,
N-o-nitrocinnamoyl, N-o-nitrobenzoyl,
N-3-(4-t-butyl-2,6-dinitrophenyl)2,2-dimethylpropionyl,
N-o-(benzoyloxymethyl)benzoyl,
N-2-methyl-2-(o-phenylazophenoxy)propionyl, N-4-chlorobutyryl,
N-acetoacetyl, N-3-(p-hydroxyphenyl)propionyl,
(N'-dithiobenzyloxycarbonylamino)acetyl, N-acetylmethionine
derivative, and 4,5-diphenyl-3-oxazolin-2-one; cyclic imide
derivatives such as N-phthaloyl, N-tetrachlorophthaloyl,
N-4-nitrophthaloyl, N-dithiasuccinoyl, N-2,3-diphenylmaleoyl,
N-2,5-dimethylpyrrolyl, N-2,5-bis(triisopropylsiloxy)pyrrolyl,
N-2,5-bis(triisopropylsiloxy)pyrrolyl,
N-1,1,4,4-tetramethyldisilyazacyclopenane adduct,
N-1,1,3,3-tetramethyl-1,3-disilaisoindolyl, 5-substituted
1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted
1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted
3,5-dinitro-4-pyridonyl, 1,3,5-dioxazinyl. These and other
protective groups are described in detail in Greene el al.,
supra.
[0097] As noted above, in one or more embodiments, the hydroxyl
groups of the polyol, or some fraction thereof, are converted to a
good leaving group prior to reaction with the functionalizing
reagent. For example, the hydroxyl groups can be converted to a
leaving group of structure --Z, wherein Z is halogen or a sulfonate
ester, by reacting the polyol with a reagent having, for example,
the structure X'--SO.sub.2--R.sub.3, wherein R.sub.3 is alkyl or
substituted alkyl and X' is Br or Cl. Preferred R.sub.3 groups
include p-tolyl, methyl, trifluoromethyl, and trifluoroethyl. In
this embodiment, the conversion of the hydroxyl groups to good
leaving groups can serve as the controlling step used to produce
the desired disparity in concentration between the monosubstituted
polymer product and the multi substituted polymer species. For
instance, the reaction to convert the hydroxyl groups to good
leaving groups can be performed under conditions effective to form
no more than about 25 percent of the disubstituted polymer (i.e.,
the polymer species having two hydroxyl groups converted to leaving
groups) and typically no more than about 12 percent of the
disubstituted polymer. In certain embodiments, no more than about 8
percent, preferably no more than about 5 percent, more preferably
no more than about 2 percent, and most preferably no more than
about 1 percent of the disubstituted polymer is formed. The
reaction converting hydroxyl groups to leaving groups typically
results in a ratio of monosubstituted polymer to disubstituted
polymer of about 2:1 to about 40:1, preferably about 4:1 to about
20:1, more preferably about 10:1 to about 18:1.
C. Optional Alkylation Step
[0098] If a monofunctional, end-capped polymer is desired, the
process of the invention can include an alkylation step, either
prior to or after the above-described functionalizing reaction.
Preferably, the alkylation step occurs after the functionalizing
reaction so that the alkylation reaction can be allowed to go to
completion without the need to control the reaction
stoichiometrically as described more fully below. Typically, the
alkylation step will occur before any deprotecting step, if
needed.
[0099] If the alkylation step is conducted prior to the
functionalizing reaction, then the alkylating reaction becomes the
controlling reaction step that determines the ratio of the
monosubstituted polymer to the disubstituted polymer products. In
this embodiment, the polyol starting material is subjected to an
alkylating step, thus forming a mixture comprising an unalkylated
polymer, a monoalkylated polymer comprising a single alkoxy group,
and a dialkylated polymer comprising two alkoxy groups, under
conditions effective to form at least about 25 mol percent of the
dialkylated polymer. This polymer mixture is then reacted with a
functionalizing reagent as described above to form a mixture
comprising an unalkylated polymer comprising two Y groups, a
monoalkylated polymer comprising a single Y group, and a
dialkylated polymer comprising no Y groups. This polymer mixture
can then be purified to provide a monoalkylated, monofunctional
polymer substantially free from the unalkylated and dialkylated
polymer species. In certain embodiments, the alkylation reaction is
allowed to proceed until at least about 25 mol percent of
dialkylated polymer is produced, preferably at least about 65 mol
percent, more preferably at least about 40 mol percent, and still
more preferably at least about 90 mol percent.
[0100] The alkylation step converts hydroxyl groups to alkoxy
groups of formula --OR', wherein R' is an alkyl or substituted
alkyl group, such as C.sub.1-C.sub.20 alkyl, substituted
C.sub.1-C.sub.20 alkyl, C.sub.1-C.sub.20 alkylene-aryl, and
substituted C.sub.1-C.sub.20 alkylene-aryl. Preferred R' groups
include methyl, ethyl, propyl, butyl, pentyl, hexyl, and
benzyl.
[0101] Preferably, the alkylation reaction comprises treating the
polymeric polyol (if the alkylation step occurs prior to
functionalization) or the polymeric mixture (if the alkylation step
occurs after functionalization) with any known alkylating agent in
the art, such as dialkylsulfate, alkyl sulfonates (such as alkyl
p-toluenesulfonate, alkyl methanesulfonate, alkyl trifluoromethyl
sulfonate, and alkyl trifluoroethyl sulfonate), diazoalkane, alkyl
halide, N,N'-dimethylformamide dialkyl acetal,
3-alkyl-1-p-tolyltriazene, trimethylanilinium hydroxide,
trialkyloxonium fluoroborate, trimethylsulfonium
hexafluorophosphonate, or alkyl trichloroacetimidate.
D. Exemplary Reaction Schemes
[0102] To further illustrate certain embodiments of various aspects
of the invention, exemplary reaction schemes are provided below.
These schemes are meant to be representative; details for the
following particular transformations and purifications are provided
in the Examples section. The schemes provided below can be extended
to any of the polymers, functionalizing reagents, leaving groups,
protecting groups, and purification modes described herein.
[0103] Scheme I demonstrates the use of PEG diol as a starting
material (rather than mPEG) to prepare a monofunctionalized mPEG.
The introduction of an ionizable group, in this case a carboxyl,
renders the PEG material suitable for an ion exchange
chromatographic separation as described in detail herein, to
provide monofunctional mPEG that is essentially free of diol and
diol-derived impurities. Generally speaking, the purified
monofunctionalized polymers of the invention, whether end-capped or
not, contain less than 0.3 wt % of a difunctional polymer. The
purified mPEG monofunctionalized material, depending upon the
nature of the functional group, is of a purity suitable for
conjugation to a protein, small molecule, surface, or the like, or
for any other pharmaceutical application, or may be further
functionalized to prepare a desired polymer reagent.
##STR00005##
[0104] In Scheme I, PEG diol is used as a starting material. In
I(i), the PEG diol is functionalized using an exemplary
functionalizing agent, bromo-acetic acid methyl ester. The
functionalizing reagent reacts with the anionic form of the PEG
diol to displace the halogen, and form the corresponding methylene
methyl ester. As illustrated in Scheme I(i), one of the PEG
hydroxyl groups is converted to the corresponding methylene methyl
ester. Although shown as a simple reaction scheme in which only one
terminus of the polymer is functionalized, as has been described in
great detail herein, the product of the functionalization reaction
is really a mixture of unsubstituted PEG diol starting material,
the desired mono-substituted PEG-OH product, and the disubstituted
PEG ester. The progress of the reaction can be monitored to ensure
the reaction is stopped or quenched at the desired time, although
using a known amount of starting materials and a limited amount of
the functionalizing reagent will stop the reaction automatically as
a result of exhaustion of the functionalizing reagent. Again,
routine experimentation will provide the amount of functionalizing
reagent that results in the desired amounts of products.
[0105] With reference to FIG. 1, it can be seen that formation of
25% monosubstituted product corresponds to about 72% unreacted PEG
diol and about 3% disubstituted product. Upon formation of 50%
monosubstituted PEG product (the maximum amount of monosubstituted
product that can be formed from diol starting material), the crude
product mixture contains 25% of each PEG diol and PEG disubstituted
product. FIG. 1 also demonstrates that as the amount of
disubstituted PEG product in the reaction mixture exceeds 25%, the
amount of monosubstituted product concomitantly decreases. Thus,
the reaction ends (e.g., as a result of depletion of
functionalizing reagent) or is quenched upon formation of 25% or
less disubstituted product. The progress of the reaction can be
monitored using any one of a number of analytical techniques, such
as .sup.1H NMR or HPLC.
[0106] In returning to Scheme I, in I(ii), the hydroxyls in the PEG
mixture from I(i), namely those present in the PEG diol starting
material and the monosubstituted product, are methylated with a
protecting group, e.g., tosylate or any other suitable protecting
group, followed by conversion of the functional group, in this case
a methyl ester, to an ionizable group, --COOH, designated generally
herein as Y. The use of methyl as an alkylating group is preferred
when mPEG functional materials are desired. At this point, the
reaction mixture contains neutral dimethoxy PEG (resulting from the
alkylation step), monofunctional mPEG,
CH.sub.3O(CH.sub.2CH.sub.2O).sub.n--CH.sub.2COOH (also referred to
as "mPEG carboxymethyl acid"), and difunctional PEG,
HOOCCH.sub.2(OCH.sub.2CH.sub.2).sub.nOCH.sub.2COOH, also referred
to as "bis-carboxymethyl PEG"). The mixture is then purified to
remove neutral PEG, which is nominally dimethoxy PEG but
potentially with neutral impurities such as PEG diol or mPEG-OH and
difunctional PEG.
[0107] While any of a number of suitable purification techniques
may be employed, generally, chromatography is preferred, and in
particular, ion exchange chromatography. The separation of PEG diol
(optionally in it alkylated form, typically referred to herein as
neutral PEG) and disubstituted PEG (also referred to herein as
difunctional PEG) from the desired monofunctional material is
preferably done in a sequential fashion.
[0108] In Scheme I, the purified acid is then reduced to the
corresponding alcohol to provide mPEG-OH that is extremely pure,
that is to say, which, in some cases contains less than 1% diol.
Although lithium aluminum hydride is shown as the reducing agent,
any suitable reducing agent can be used. Examples include sodium
borohydride, sodium cyanoborohydride, Eh/palladium, lithium
triethylborohydride, HI, alkali metals in liquid ammonia, and zinc
with acid or base. In some cases, the amount of diol or
difunctional material present in the purified material is below
current limits of detection. Although Scheme I illustrates
reduction of the carboxylic acid to the corresponding alcohol to
provide mPEG-OH of ultra-high purity, this transformation is meant
to be illustrative, and any of a number of alternative subsequent
transformations of the polymer acid may be carried out.
[0109] Scheme I can also be referred to when considering this
aspect of the invention more generally. Moreover, detailed
descriptions of the variables and examples thereof are extendable
to every aspect of the invention to which they apply. For instance,
any water-soluble non-peptidic polymer can be used in place of PEG.
Such a polymer is represented generally as HO-POLY-OH, where POLY
is the water-soluble and non-peptidic polymer portion of the
molecule. Although not shown in Scheme I, the method may optionally
include a step wherein the hydroxyl groups in the polymer diol are
converted to a better leaving group, Z. Leaving groups include
halogens such as iodide, bromide, and chloride, as well as
sulfonate esters, --N.sub.2.sup.+. Preferred leaving groups are
groups that are better leaving groups than --OH. As previously
described for methylating PEG in FIG. 1, the conversion to a better
leaving group produces a mixture of products, unreacted,
unsubstituted polymer diol starting material, monosubstituted
polymer having a single Z group, HO-POLY-Z; and disubstituted
polymer comprising two Z groups, Z-POLY-Z. Again, such
transformations are typically carried out under conditions
effective to form no more than about 25 percent of the
disubstituted polymer.
[0110] In a subsequent step, the polymer diol (or the polymer
mixture produced in an optional preceding step to convert hydroxyls
to leaving groups) is reacted, in one or more reaction steps, with
a functionalizing reagent. The functionalizing reagent reacts with
the polymer in a nucleophilic substitution or nucleophilic addition
reaction. The functionalizing reagent is useful for introducing
into the polymer a functional group, most preferably an ionizable
group, or a precursor to an ionizable group, or an ionizable group
in protected form.
[0111] In one or more particular embodiments, the functionalizing
reagent comprises the structure X-L--Y, wherein X is a group that
allows the functionalizing reagent to react with the polymer in a
nucleophilic addition or substitution reaction. Preferably, X is a
group that reacts with a hydroxyl, optionally in anionic form, or
with a carbon atom to which the hydroxyl is attached, or is
displaced by a hydroxyl. L is an optional linker that is interposed
between X and Y. L.sub.0 indicates the absence of a linker and Li
indicates the presence of a linker, and L encompasses both.
Preferably L is hydrolytically stable, and is made up of inert or
non-reactive atoms or groups of atoms each of the moieties
described above with respect to the spacer moiety describe above
can be an L.
[0112] In one or more embodiments, L has a structure
--(CR.sub.1R.sub.2).sub.m--, where R.sub.1, in each occurrence, is
independently H or an organic radical selected from the group
consisting of alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl;
and R.sub.2, in each occurrence, is independently H or an organic
radical selected from the group consisting of alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
aryl, and substituted aryl, and m ranges from 0-15. For example, m
may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. In one
or more embodiments, R.sub.1 and R.sub.2 are each independently H
or alkyl, and m ranges from 0-10. Typically, the alkyl group is
straight chain lower alkyl or branched lower alkyl such as methyl,
ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, pentyl,
etc., with straight chain being generally preferred. One
particularly preferred alkyl substituent is methyl. In one or more
embodiments thereof, R.sub.1 and R.sub.2 are each independently H
or lower alkyl. In yet one or more additional embodiments, R.sub.1
and R.sub.2 are each H, and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
In addition, L can be --(CR.sub.1R.sub.2).sub.m-- where R.sub.1 on
the carbon proximal to Y is alkyl, and in all other occurrences,
R.sub.1 and R.sub.2 are H. In one particular embodiment of the
preceding, R.sub.1 is methyl or ethyl or propyl. Alternatively, L
is --(CR.sub.1R.sub.2).sub.m-- where R.sub.1 on the carbon beta to
Y is alkyl, preferably methyl or ethyl or propyl of isopropyl, and
in all other occurrences, R.sub.1 and R.sub.2 are H. Although any
of the exemplary spacer moieties described supra can be an L
moiety, preferred L moieties in some embodiments possess a
structure selected from --CH.sub.2--, --CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--CH(CH.sub.3)--,
--CH.sub.2--CH(CH.sub.2CH.sub.3)--, --CH(CH.sub.3)CH.sub.2--,
--CH.sub.2--CH(CH.sub.3)--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--CH(CH.sub.3)--,
--CH.sub.2--CH.sub.2--CH(CH.sub.3)--CH.sub.2--,
--CH.sub.2--CH(CH.sub.3)--CH.sub.2--CH.sub.2--,
--CH(CH.sub.3)--CH.sub.2--CH.sub.2--CH.sub.2-- --O--CH.sub.2--,
--CH.sub.2--O--, --O--CH.sub.2--CH.sub.2--,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--CH.sub.2--O--, --O
--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--O--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--O--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--O--,
--O--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--O--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--O--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--O--.
[0113] Preferably, Y is an ionizable functional group. Ionizable
functional groups are particularly well suited for the
chromatographic purifications of the invention, and can be
exploited as a means to separate unsubstituted, monosubstituted,
and disubstituted polymer species contained in a mixture. Relative
amounts of the various polymer species are ideally carried through
the various transformations, thereby forming a mixture comprising a
neutral polymer of formula HO-POLY-OH, a monosubstituted polymer of
formula HO-POLY-L--Y, and a disubstituted polymer of formula
Y--L-POLY-L--Y, where ideally, no more than about 25 percent of the
disubstituted polymer species relative to the other polymer species
is present.
[0114] Optionally, the method may further comprise alkylating the
polymer mixture produced in either the first or second steps to
convert the remaining hydroxyl groups to alkoxy groups. Thereafter,
the resulting polymer mixture can be purified using, for example,
ion exchange chromatography, to provide a substantially pure
monosubstituted polymer comprising a single --L--Y group, and in
the case where an alkylation has been conducted, to provide a
substantially pure monosubstituted methoxy-terminated polymer.
[0115] Scheme II provides another representative embodiment of the
method of the invention. In Scheme II, PEG diol is first methylated
to provide a polymer mixture as previously described (II(i)). The
alkylated polymer mixture is then reacted with a functionalizing
agent, in this case,
1-(3-bromo-propyl)-4-methyl-2,6,7-trioxa-bicyclo[2.2.2]octane
(II(ii)). As can be seen, the functionalizing agent possesses a
group, in this case, Br.sup.-, that is displaced by the counter
anion of hydroxide. The Br.sup.- is an example of the variable, X.
The functionalizing agent also contains Y, in this case a protected
carboxylic acid. Specifically, Y is an ortho ester,
4-methyl-2,6,7-trioxabicyclo[2.2.2.]octane, but can be any carboxyl
protecting group. The linker portion, L, in the functionalizing
agent, is --(CH.sub.2).sub.2--. The protecting group is then
removed, in this instance by hydrolysis, to produce a PEG
carboxylic acid (II(iii)). As described previously, although shown
as the mPEG-acid, the mPEG acid is really present in a mixture
comprising neutral PEG, dimethoxy PEG, the desired monofunctional
mPEG-acid, as well as the difunctional material,
HOOC--CH.sub.2CH.sub.2--(OCH.sub.2CH.sub.2).sub.2--O--CH.sub.2CH.sub.2COO-
H. The monofunctional PEG carboxylic acid is then purified, e.g.,
by ion exchange chromatography. Scheme II can also be modified to
conduct the alkylation step following the introduction of the ortho
ester (i.e., after reaction with the functionalizing reagent).
##STR00006##
[0116] In illustrative Scheme III below, a Michael-type addition
reaction is used to functionalize a PEG diol by introduction of a
carbonitrile group. Generally speaking, the functionalizing reagent
in the method of the invention in this instance is a Michael type
reagent. In (III(iii)), the functionalizing reagent is a
carbonitrile. In this case, the functionalizing agent contains a
functional group, Y, where Y is a nitrile, and is part of a Michael
type reagent. The nitrile is a precursor to an ionizable group. In
Scheme III, X is --CH.sub.2, and the linker, L, in the polymer
nitrile product mixture is --CH.sub.2CH.sub.2--. The nitrile is
then reduced to an amine using a reducing agent, e.g., Eh over a
metal catalyst such as rhodium-containing catalysts, nickel,
palladium or platinum. Scheme III demonstrates a diol as the
precursor for the Michael addition reaction, however, if desired,
the PEG diol can first be alkylated, or alternatively, can be
alkylated subsequent to the Michael addition. The polymer
amine-mixture is then purified, e.g., by ion exchange
chromatography. Introduction of an ionizable group such as an amine
makes this approach particularly suitable for an ion exchange-based
separation to yield essentially pure monoamine, absent neutral and
disubstituted polymer species.
##STR00007##
[0117] Other Michael-type reagents can be substituted for the
carbonitrile shown in Scheme III. For example, the following
reagents can be used as Michael Type reagents,
##STR00008##
wherein X'' is halo or alkyl and X''' is H, halo, or alkyl.
[0118] The CP amine shown in Scheme III can be used to prepare a
variety of heterobifunctional derivatives by further
functionalizing either the amino terminal or the terminal --OH
group. Moreover, by using a chromatographically purified material
such as the CP amine, any such heterobifunctional polymer prepared
therefrom will be essentially free of polymeric contaminants such
as the neutral and disubstituted polymer species described
herein.
[0119] One such representative reaction scheme is provided as
Scheme IV below. In Scheme IV, the CP amine from Scheme III is
converted into a maleimide by transformation of the amino group.
This synthetic approach is advantageous since maleimide-terminated
polymers are particularly useful for conjugation to
thiol-containing moieties, such as the cysteines in proteins.
Moreover, the heterobifunctional maleimide amino-terminated polymer
can then be further transformed, if desired, by further
functionalization at the hydroxyl terminus.
##STR00009##
[0120] In Scheme IV, the CP amine is converted to the maleimide by
reaction with N-methoxycarbonylmaleimide
2,5-dioxo-2,5-dihydro-pyrrole-1-carboxylic acid methyl ester.
Additional maleimidyl polymer derivatives that can be prepared from
the chromatographically purified starting materials described
herein, as well as methods that can use the chromatographically
purified starting materials described herein, are disclosed in U.S.
Pat. No. 6,602,498, the contents of which are incorporated herein
by reference. This reaction approach can be employed with any
chromatographically purified polymer amine prepared by the methods
described herein. Again, as with all of the illustrative schemes
herein, the scheme below is applicable to any of the herein
described polymers, functionalizing reagents and purified
monofunctional polymers.
[0121] In Scheme IV (as well as each of Schemes I, II, III and V),
(n) is a positive integer, typically in at least one of the
following ranges: from 2 to 3,000; from 10 to 2,000; and from 100
to 1000. In addition, each hydrogen of the hydroxyl groups shown in
Scheme IV can optionally be an organic radical, typically an alkyl
(such as lower alkyl) including benzyl. The amine terminated
polymer in Scheme IV is a useful starting material to form the
polymeric reagent bearing a terminal maleimidic group (as shown in
Scheme IV) that can be used, for example, in a conjugation reaction
with a biologically active protein.
[0122] Scheme V represents yet another particular embodiment of the
method of the invention. Generally, hydroxyls on a polymer diol (or
on an end-capped polymer alcohol) are first transformed to a better
leaving group. An exemplary leaving group shown here is mesylate or
methanesulfonyl, although any suitable leaving group may be used.
In the first step of this approach, conversion is preferably held
to about 20% so as to reduce the ultimate amount of difunctional
amine formed. Again, although a single monofunctionalized polymer
species is shown in V(i), the monofunctionalized polymer is really
present in a reaction mixture containing unreacted starting
material and polymer di-mesylate. The polymer mesylate, HO-POLY-Ms,
can then be reacted with a variety of different functionalizing
agents. In this approach, the functionalizing reagents react with
the polymer via a nucleophilic substitution reaction, such that the
--OMs group on the polymer is displaced. In reaction V(ii-a), the
functionalizing reagent can be described generally as X-L--Y, where
X is --O, L is --(CH.sub.2).sub.3--, and Y is --NH.sub.2. In
reaction V(ii-b), the functionalizing reagent is ammonia, which
again acts to displace --OMs. The third reaction V(ii-c)
illustrates a functionalizing reagent having a four carbon linker
rather than a three carbon linker as in V(ii-a). Each of these
reaction mixtures is particularly suited for purification by ion
exchange chromatography as described herein, to provide essentially
pure monofunctionalized, monosubstituted polymers. These polymers
may be used directly, e.g., to prepare functionalized active
agents, hydrogels, or any other such suitable application, or may
be further functionalized as described above.
##STR00010##
E. Purification Step
[0123] The process of functionalizing a polymer diol or polyol
starting material results in a mixture of products, including a
monosubstituted polymer and one or more multi substituted polymer
species (e.g., a disubstituted polymer). Thus, in order to make the
method of invention of the utmost practical utility, the product
polymer mixture is purified to separate the monosubstituted polymer
from the di- or multi-substituted polymer, as well as any remaining
unreacted polymer diol, polyol, or other neutral polymer species.
Any of a number of purification techniques can be used.
[0124] In a preferred embodiment of the invention, in particular
where the polymer mixture contains polymer species having ionizable
functional groups, ion exchange chromatography is employed to
separate the various polymer constituents of the product mixture
based on their differences in charge. In one aspect, the present
invention provides an improved ion exchange chromatography approach
that overcomes the problems associated with other commonly
employed, e.g., gradient, ion exchange methods used to separate
polymers. This process is referred to as gradient polymer elution
chromatography, and differs from the method of the invention in
many respects.
[0125] Gradient based chromatography involves changing the ionic
strength of the mobile phase or eluent to drive differently charged
molecules off an ion exchange column at different intervals.
Generally, in a gradient chromatography, a gradient is applied that
changes from a poor or low eluting strength solvent to a good or
high eluting strength solvent, based upon the relative affinity of
the column versus the mobile phase for a particular polymer.
[0126] In a typical gradient separation, a sample is applied to a
column and a low eluting strength solvent is employed, so as not to
allow any separation to occur initially. Rather, the mixture
components are collected at the top of the column, in a
concentrating step. The gradient is then progressed and the ionic
strength of the solvent is gradually increased until "good" or high
eluting strength solvent conditions are achieved such that sample
components begin their separation and begin to migrate. Charged
substances are separated via column materials carrying an opposite
charge. Species with a higher charge are bound to an ion exchange
column more strongly, while the less highly charged species elute
more rapidly. The strength of the eluent is typically altered by
changing pH, buffer, and/or salt concentration (ionic strength).
Techniques that rely upon gradient separation are tedious,
time-consuming, use large volumes of solvent, and require analysis
of multiple fractions. Thus, gradient type methods are poorly
suited for commercial-scale processes. Moreover, gradient-based
separation techniques also rarely achieve high purity levels of any
given polymer (e.g., in reference to the number of various polymer
species present and the polydispersity of the purified polymer
product), particularly when separating higher molecular weight
polymer species.
[0127] The ion exchange separation process of the invention
provides superior separation and purification of polymer mixtures
that contain multivalent anions or cations. More specifically, the
method is well suited for polymer mixtures that contain uncharged
and charged substances differing in charge, e.g., polymer that are
uncharged, singly charged, doubly charged, triply charged, and so
on (that is, to say, two or more species having ionizable groups
that under certain pH conditions, carry different charges). One
such example is a polymer mixture containing a neutral polymer
(i.e., a polymer diol or polyol or a mono- or di-alkylated polymer
absent an ionizable functional group), a monosubstituted polymer
having a single ionizable group, such as an amine or carboxylic
acid group, and a di- or multi-substituted polymer having two or
more ionizable functional groups. Separation is achieved by relying
upon differences in charge, and, in certain embodiments,
differences in molecular weight. Rather than eluting species having
different charges from a single column (or a number of single
column chromatograph separations) by changing the ionic strength of
the eluate in a stepwise, gradient fashion, the present method
involves the use of discrete columns and discrete eluates.
Generally, a solvent having a constant or static concentration as
it is fed into a column is used. That is to say, the solvent feed
as is enters the column is of a constant, non-gradient composition.
The ionic strength and/or pH of the solvent is adjusted to suit the
polymer species being eluted from the column.
[0128] Specifically, the method of the invention involves the use
of more than one ion exchange column to achieve ultra high purity
mono-substituted polymers, e.g., typically containing less than
0.3% by weight difunctionalized or multi-functionalized polymer
impurities.
[0129] The first column(s) or pre-column(s) are sized to adsorb
substantially all, and most preferably, all, of the disubstituted
polymer and other multi substituted polymer species that are
present in a polymer mixture. Typically, determination of an
appropriate size for the first column(s) or pre-column(s) involves
the step of establishing column capacity. Column capacity is
experimentally determined and typically involves passing a solution
containing an excess amount of standard solution of one type of
species of polymer [e.g., a solution of
HO(O)CH.sub.2O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2C(O)OH or
H.sub.2N--CH.sub.2CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2C-
H.sub.2CH.sub.2--NH.sub.2] known to adsorb to the stationary phase.
This standard is added so as to saturate the column, often verified
by detecting the polymer species in the eluate retrieved from the
column. Thereafter, any nonadsorbed species are washed out of the
column, typically by passing distilled water through the column.
Next, all polymer species adsorbed on the column are eluted
(generally by means of passing a salt solution), extracted with
organic solvent and then weighed after removal of solvent. This
amount corresponds to the column capacity. To the extent that two
or more columns are provided in series, the overall column capacity
of the system is equivalent to the added column capacities of the
individual columns.
[0130] Having established column capacity, only column(s)
sufficiently sized to adsorb substantially all of the di- or multi
substituted polymer species (e.g., disubstituted polymer, polymer
species comprising two --L.sub.0,1--Y groups, or difunctional
polymer) desired to be removed form a mixture will be used in an
initial purification step. A column is sufficiently sized in this
regard when it has a column capacity greater than the amount of the
di- or multi substituted polymer species to be retained from a
mixture. As discussed previously, the amount of the polymer species
in any mixture can be determined by analyzing a sample of the
mixture, by having reference to FIG. 1, or any other art-known
method.
[0131] Thus, the column capacity of pre-column(s) used in a first
eluting step can be one or more of at least a 10%, at least a 20%,
at least a 30%, at least a 40%, at least a 50%, at least a 60%, at
least a 70%, at least a 80%, at least a 90%, at least a 100%, and
at least a 110% increase of the total amount of the polymer species
in the mixture to be purified. For example, with respect to a first
step in a method for purifying of a mixture containing 5 g of
disubstituted polymer, a first ion exchange column having capacity
to adsorb 10 g of the disubstituted polymer can be used (thereby
representing 100% increase of the total amount of the difunctional
polymer species to be adsorbed on the first column). In addition, a
mixture containing 25 g of disubstituted polymer, a first ion
exchange column having capacity to adsorb 35 g of the disubstituted
polymer can be used (thereby representing a 40% increase of the
total amount of the polymer species to be adsorbed on the first
column).
[0132] With respect to the second column(s) or main column(s) used
in the purification step, it is sufficient to have a column
capacity substantially equivalent to the amount of monofunctional
polymers within the polymer species to be retained from the mixture
(e.g., monosubstituted polymer, polymer species comprising one
--L.sub.0,1--Y groups, or monofunctional polymer). Second or main
column(s) having greater column capacities can also be used to
prevent any losses of monofunctional polymer(s).
[0133] Having identified appropriate columns, purification can take
place. Advantageously, the polymer mixture equilibrates with the
solid phase media in the precolumn as the mixture flows through the
column to allow the strongest binding material (e.g., those species
bearing the greatest number of the charges to which the column is
directed) to be retained. Slower rates of adding the mixture
correspond to an increased extent of equilibration.
[0134] In one or more embodiments, a plurality of "precolumns"
(e.g., 2, 3, or 4 precolumns) connected in series is used to remove
the multi substituted polymer species, the plurality of precolumns
being sized to collectively adsorb all of the disubstituted polymer
and other multi substituted polymer species. Typically, some amount
of monosubstituted polymer species will be adsorbed as well, but to
a lesser extent since only one ionized species is associated with
the monosubstituted polymer species.
[0135] Advantageously, the purification method does not require the
use of a distillation step to concentrate solutions such as the
eluate. Furthermore, the purification method described herein is
suited to purify not only relative small molecular weight polymers
(e.g., 2,000 Da), but can be used to purify molecular weight
polymers having higher molecular weights as well. Thus, the
purification method is suited for purifying molecular weights in
the following ranges: from about 100 Da to about 180,000 Da; from
about 3,000 Da to about 120,000 Da; from about 5.000 Da to about
100,000 Da; from about 8,000 Da to about 100,000 Da; from about
10,000 Da to about 100,000 Da; from about 12,000 Da to about
80,000; and from about 15,000 Da to about 80.000 Da. In addition,
the equipment used in the purification process does not rely on
gradients, thereby reducing the need for obtaining many very
diluted eluate fractions, which, in turn, requires a multitude of
collection vessels. Furthermore, the present method uses
substantially less volumes of eluent compared to prior art methods,
typically on the order of less than about 50% eluent, preferably
less than about 75% eluent, more preferably less than about 85%
eluent, still more preferably less than about 90% eluent, with
eluent amounts of less than about 95% relative to prior art methods
being most preferred. Consequently, the methods described herein
require only a single collection vessel, and do not require a
distillation step to concentrate eluate to enable extraction of
purified product. In addition, the apparatuses described herein do
not require more than a single collection vessel and do not require
a means for distillation.
[0136] The eluate from the first column, which contains the
monosubstituted polymer and the neutral polymer, is then passed
through the second (or main) ion exchange column or columns
connected in series. The monosubstituted polymer is absorbed onto
the second (or main) column(s), which are sized in order to retain
preferably all of the monosubstituted polymer. The neutral polymer
passes through all of the columns and can be collected and possibly
recycled for reuse in the method of the invention. It is generally
preferred to wash each column with a solution having low ionic
strength (e.g., deionized water) to remove any remaining neutral
polymer thereon.
[0137] Solutions having the requisite low ionic strength for any
particular system are known to those having ordinary skill in the
art. In addition, solutions having the requisite low ionic strength
can be determined through routine experimentation by passing a
candidate solution (typically, although not necessarily, a very
weak salt solution or buffered solution) through column(s) known to
have both charged and neutral polymer species contained therein,
collecting the candidate solution that has passed through the
column(s), and then testing the collected solution for the presence
of any charged polymer species. A candidate solution having passed
through the column(s) with no or substantially no (e.g., less than
1%) charged polymer species content represents a solution having a
low ionic strength for that particular system.
[0138] Retrieval of charged polymer species (whether they be singly
charged polymer species or di- or multiply charged polymer species)
adsorbed onto the ion exchange columns is typically requires
desorbing. Desorption typically involves passing salt solution
having high ionic strength through the column(s), thereby desorbing
charged polymer species. For instance, the second (or main)
column(s) containing monosubstituted polymer can be washed with a
salt solution having high ionic strength, such as a NaCl solution,
to remove and collect a substantially pure monosubstituted polymer
product.
[0139] Salt solutions having the requisite high ionic strength for
any particular system are known to those having ordinary skill in
the art. In addition, solutions having the requisite high ionic
strength can be determined through routine experimentation by
passing a candidate solution through the column(s) having a known
amount of charged polymer species adsorbed therein, collecting the
candidate solution that has passed through the column(s), and then
testing the collected solution for the presence of charged polymer
species. A candidate solution having passed through the column(s)
with at least about 85%, more preferably at least about 90%, still
more preferably at least about 95%, and most preferably at least
about 99% of the known amount of charged polymer species contained
therein represents a solution having a high ionic strength for that
particular system. This procedure can be used to identify a
solution having sufficient ionic strength so that the solution will
desorb difunctional polymer through the first column or
precolumn.
[0140] Since the differently charged polymer species have been
separated by adsorption on separate columns, there is no need to
use a salt solution gradient to recover each polymer species
separately. Instead, a salt solution having a constant ionic
strength can be used to elute the desired product from each
column.
[0141] If desired, the multi substituted polymer species absorbed
on the precolumn(s) can also be collected by passing a salt
solution through the precolumn to drive desorption of the polymer.
Typically, the precolumn(s) are sized so as to ensure absorption of
all of the multi substituted polymer in the feed stream, meaning
that some monosubstituted polymer will also be absorbed on the
precolumn. Thus, purity of the multi substituted product eluate is
typically lower as compared to the monosubstituted product eluted
from the one or more additional columns. Preferably, the product
eluted from the precolumn(s) contain no more than about 70 weight
percent monosubstituted polymer, more preferably no more than about
50 weight percent, and most preferably no more than about 30 weight
percent. If the product eluted from the precolumn(s) contain
multiple multicharged polymer species (e.g., doubly-charged and
triply-charged), then a second pass through the ion exchange system
can be used to further separate the polymer mixture by retaining
the higher charged species in the precolumns (e.g., the
triply-charged species) and retaining the less highly charged
species (e.g., doubly-charged) in the second column.
[0142] Analytical determination, using an HPLC column that responds
to both charge and molecular weight, can be used to determine how
much of each species is present in a sample, both before being run
through a column and after. By "substantially pure" is meant that
the monosubstituted polymer contains less than about 5 weight
percent polymer impurities, such as multi substituted polymer or
unsubstituted (i.e., neutral) polymer, preferably less than about 3
weight percent, more preferably less than about 2 weight percent,
and most preferably less than about 1 weight percent. A preferred
composition will comprise monomethoxy end-capped poly(ethylene
glycol) ("mPEG-OH"), wherein the composition has a poly(ethylene
glycol) diol content of less than 0.3 wt. %.
[0143] If it is desired to narrow the molecular weight range (i.e.,
polydispersity) of the monosubstituted polymer product, a series of
two or more columns (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 columns)
following the precolumn can be used to attenuate the molecular
weight range of the monosubstituted polymer absorbed on each
column. Monosubstituted polymer of smaller molecular weight will
absorb first, meaning the average molecular weight of the polymer
material absorbed on each successive column will increase. Thus, by
increasing the number of columns, one can not only separate the
monosubstituted polymer from the higher charged species, but also
lower polydispersity. In certain embodiments, the polydispersity of
the monosubstituted polymer is reduced by at least about, 0.01
preferably at least about 0.02, more preferably at least about
0.03, and most preferably at least about 0.05. In an alternative
embodiment, if lower molecular weight monosubstituted polymer is
the desired product, one can simply undersize the second or main
column such that all of the monosubstituted polymer cannot be
adsorbed thereon. Since lower molecular weight species will
selectively bind first, the desired lower molecular weight
monosubstituted polymer will absorb on the column. In addition or
alternatively, one can use several columns and collect lower
molecular weight monofunctional polymer from the first column in
the series of columns following the precolumn.
[0144] If the original polymer mixture that is subjected to
functionalization contains a relatively small amount of polyol,
such as in the case of a polymer starting material comprising an
impure mPEG (e.g., an mPEG contaminated with less than about 20% by
weight PEG diol), then the resulting polymer mixture requiring
purification may contain no neutral polymer or only a negligible
amount of neutral polymer if the functionalization reaction is
allowed to proceed to completion. In this case, the ion exchange
purification system can comprise only a single column sized to
absorb all of the multifunctional polymer species (i.e., a
precolumn). The eluate from the precolumn will then contain only
the desired monofunctional product.
[0145] Following purification, if desired, the substantially pure
monosubstituted polymer can be further modified to convert the
ionizable functional group to a second functional group, such as
hydroxyl, active ester, active carbonate, ortho ester, acetal,
aldehyde, aldehyde hydrates, ketone, ketone hydrate, alkenyl,
acrylate, methacrylate, nitrile, primary or secondary amide, imide,
acrylamide, active sulfone, amine, hydrazide, thiol, carboxylic
acid, isocyanate, isothiocyanate, maleimide, substituted
succinimide, vinylsulfone, dithiopyridine, vinylpyridine, amidate,
2-substituted-1,3-oxazoline, 2-substituted
1,3-(4H)-dihydrooxazines, 2-substituted-1,3-thiazoline,
2-substituted 1,3-(4H)-dihydrothiazines, hydroxylamine,
iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, and
tresylate.
[0146] During the ion exchange process, the eluate from each column
can be monitored using techniques known in the art, such as by
measuring the conductivity of the eluate, analyzing the eluate by
ion exchange chromatography, size exclusion chromatography, high
performance liquid chromatography, or thin layer chromatography, or
by detecting the presence of PEG in the eluate by treating a drop
of eluate with a drop of 1% polyacrylic acid (Aldrich, Mn 250,000)
in 1 N HCl ("PAA test"). Presence of PEG is indicated by the
immediate appearance of a white precipitate of PEO/PAA complex.
This test is very specific to the poly ether backbone of PEG and
not influenced by end group modifications of the polymer, molecular
weight, or the presence of inorganic ions in the analyzed solution.
Monitoring of the eluate streams is particularly important during
the washing step to determine when substantially all of the neutral
polymer has been removed from the columns.
[0147] As would be understood in the art, the ion exchange columns
utilized in the present invention can be any ion exchange columns
conventionally used to separate a mixture based on charge (Ion
Exchange Chromatography. Principles and Method. Pharmacia Biotech
1994; "Chromatography: a laboratory handbook of chromatographic and
electrophoretic techniques." Heftman, E (Ed.), Van Noostrand
Rheinhold Co., New York, 1975). Each column comprises an ion
exchange media and a mobile phase, or eluent, that passes through
the ion exchange media. Ion exchange columns suitable for use in
the present invention include POROS.RTM. ion exchange media made by
Applied Biosystems and SEPHAROSE.RTM. ion exchange media made by
Pharmacia.
[0148] The ion exchange media, which is typically a polymeric resin
(e.g., dextran, agarose, cellulose, styrene-divinylbenzene
copolymer) containing charged groups, is selected based on a number
of factors, including the charge and pKa value of the ionizable
functional group present on the polymers to be separated.
Typically, the ion exchange media is selected so as to provide a
sufficient difference in pKa value between the ionizable functional
group and the ion exchange media to favorably drive absorption of
the polymer, preferably a difference of at least 4-5 units. As
would be understood, the ion exchange media will comprise
negatively charged groups (i.e., a cation exchanger) if the
ionizable functional group is positively charged and will comprise
positively charged groups (i.e., an anion exchanger) if the
ionizable functional group is negatively charged. Exemplary
negatively charged groups that may be used include carboxymethyl
(CM), sulphopropyl (SP), and methyl sulphonate (S). Exemplary
positively charged groups include triethylammoniumethyl (TMAE),
diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and
quaternary ammonium (Q). Typically, the media in each column will
be the same, but different media could be used in each column
without departing from the present invention.
[0149] A two column embodiment of the ion exchange system of the
invention is shown in FIG. 2. As shown, the ion exchange system 10
comprises a feed tank or vessel 12 that contains a supply of the
solution of the crude polymer mixture to be separated. Typically,
the polymer mixture will be dissolved in deionized water or a
neutral aqueous solution having very low ionic strength. As noted
above, the polymer mixture will often include a neutral or
unsubstituted polymer species, such as a polymer having the
structure HO-POLY-OH or R'O-POLY-OR', a monosubstituted polymer of
formula HO-POLY-L--Y or R'O-POLY-L--Y, and a disubstituted polymer
of formula Y--L-POLY-L--Y, wherein Y, R', L, and POLY are as
defined above.
[0150] The feed tank 12 is in fluid communication with a first ion
exchange column or precolumn 16 sized to trap higher charged
species (i.e., a disubstituted polymer). The outlet of the
precolumn 16 is in fluid communication with the inlet of the second
or main ion exchange column 18, which is appropriately sized to
retain all of the monocharged polymer species. The outlet of each
column is in fluid communication with one or more product recovery
or receiving vessel 20, each vessel adapted to receive eluate from
one or more of the columns. The salt solutions and neutral
solutions used to wash the columns and/or recover the absorbed
polymer species can be housed in one or more solvent vessels 22,
which are in fluid communication with the inlet of one or more of
the columns.
[0151] FIG. 3 illustrates an embodiment comprising a precolumn 16
and a plurality of second or main columns 18 that can be used to
narrow the molecular weight range of the desired monosubstituted
polymer product as explained above.
F. Exemplary Products
[0152] As previously discussed, the methods described herein can be
used in the preparation of substantially pure polymeric reagents.
Examples of such products existing as substantially pure
compositions with little or no poly(ethylene glycol) diol species
include the following:
##STR00011## ##STR00012## ##STR00013##
wherein (n) is a positive integer, typically falling within at
least one of the following ranges: from 2 to 3,000; from 10 to
2,000; from 100 to 1,000, and each mPEG is
CH.sub.3--(OCH.sub.2CH.sub.2).sub.n--, wherein (n) is as previously
defined.
[0153] The corresponding functional group with optional spacer
moiety (e.g., L.sub.0,1--Y) are evident from the above exemplary
polymeric structures.
[0154] All articles, books, patents, patent publications and other
publications referenced herein are hereby incorporated by reference
in their entireties.
EXAMPLES
[0155] It is to be understood that while the invention has been
described in conjunction with certain preferred specific
embodiments thereof, the foregoing description as well as the
examples that follow are intended to illustrate and not limit the
scope of the invention. Other aspects, advantages and modifications
within the scope of the invention will be apparent to those skilled
in the art to which the invention pertains.
[0156] All PEG reagents referred to in the appended examples are
commercially available unless otherwise indicated, e.g., from
Nektar Therapeutics, Huntsville, Ala. All .sup.1HNMR data was
generated by a 300 or 400 MHz NMR spectrometer manufactured by
Bruker. High Performance Liquid Chromatography (HPLC) was performed
using Agilent 1100 HPLC system (Agilent), gel permeation or ion
exchange column, aqueous phosphate buffer as a mobile phase, and
refractive index (RI) detector.
Example 1
mPEG (20,000 Da)-Butanoic Acid
[0157] A. 4-Bromobutyrate ester of 3-methyl-3-hydroxymethyloxetane
(MW=251.12)
##STR00014##
[0158] 3-Methyl-3-hydroxymethyloxetane (10.2 g, 0.1 mole)
[Sigma-Aldrich Corporation of St. Louis, Mo.] was dissolved in
anhydrous dichloromethane (200 ml) and pyridine (9.8 ml, 0.12
moles) was added. The solution was cooled to 0.degree. C. and
4-bromobutyryl chloride (18.5 g, 0.1 mole) [Sigma-Aldrich
Corporation of St. Louis, Mo.] dissolved in anhydrous
dichloromethane (50 ml) was added dropwise over 20 minutes. The
mixture was stirred overnight under an argon atmosphere. Next, the
reaction mixture was washed with water and dried with anhydrous
magnesium sulfate. The solvent was then distilled off under reduced
pressure. Yield 23.6 g. NMR (de-DMSO): 1.26 ppm (s, 3H), 2.07 ppm
(m, 2H), 2.51 ppm (t, 2H), 3.56 ppm (t, 2H), 4.14 ppm (s, 2H), 4.24
ppm (d, 2H), 4.38 ppm (d, 2H).
[0159] B,
1-(3-Bromopropyl-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane
(MW=251.12)
[0160] (4-Bromobutanoic Acid OBO Ester)
##STR00015##
[0161] Crude 4-bromobutyrate ester of
3-methyl-3-hydroxymethyloxetane (20.1 g, 0.08 moles) was dissolved
in anhydrous dichloromethane (100 ml), the solution was cooled to
0.degree. C. and boron trifluoride diethyl etherate (2.5 ml, 0.022
moles) was added. The mixture was stirred for 4 hours at 0.degree.
C. Triethylamine (12 ml) was added, the mixture was stirred for 15
minutes, and the solvent was distilled off under reduced pressure.
The crude product was dissolved in ethyl ether (180 ml) and the
solution was filtered to remove the solid impurities. Next, ether
was distilled off and the product was distilled under reduced
pressure (kugelrohr, 110-115.degree. C., 0.05 mm Hg). Yield 15.0 g.
NMR (d.sub.6-DMSO): 0.74 ppm (s, 3H), 1.68 ppm (m, 2H), 1.88 ppm
(m, 2H), 3.52 ppm (t, 2H), 3.81 ppm (s, 6H).
[0162] C. PEG(20.000 Da)-Butanoic Acid
[0163] A solution of commercially available PEG (Mn=20,300 Da,
polydispersity 1.040) (50.0 g, 0.005 equivalents) in toluene (300
ml) was azeotropically dried by distilling off 50 ml toluene. 1.0M
solution of potassium tert-butoxide in tert-butanol (5.0 ml, 0.005
moles) and
1-(3-bromopropyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane (0.65
g, 0.0025 moles) were added and the mixture was stirred overnight
at 70.degree. C. under an argon atmosphere. Next, 1.0M solution of
potassium tert-butoxide in tert-butanol (18.0 ml, 0.018 moles) and
methyl p-toluenesulfonate (4.4 g, 0.0239 moles) were added and the
mixture was stirred overnight at 45.degree. C. under an argon
atmosphere. The solvent was distilled off under reduced pressure
and the residue was dissolved in distilled water (450 ml). The pH
of the solution was adjusted to 2 with 5% phosphoric acid and the
solution was stirred for 15 minutes at room temperature. Next, the
pH was readjusted to 12 with 1M sodium hydroxide and the solution
was stirred for 2 hours keeping the pH at 12 by periodic addition
of 1M sodium hydroxide. The pH was adjusted to 3 with 5% phosphoric
acid and the product was extracted with dichloromethane. The
extract was dried with anhydrous magnesium sulfate and added to
ethyl ether. The precipitated product was filtered off and dried
under reduced pressure. Yield 44.7 g. NMR (d.sub.6-DMSO): 1.72 ppm
(q, CH.sub.2--CH.sub.2--COO--) 2.24 ppm (t, --CH.sub.2 --COO--),
3.24 ppm (s, --OCH.sub.3), 3.51 ppm (s, PEG backbone).
[0164] D. Purification
[0165] The product from (C) above was determined to be a mixture of
polymer species. An HPLC chromatogram revealed that the polymer
mixture product from C included PEG(20,000 Da)-dibutanoic acid
(26.1%), mPEG(20,000 Da)-butanoic acid (50.4%), and PEG(20,000 Da)
dimethyl ether (23.5%). A purification was carried out in
accordance with the invention to obtain purified mPEG(20,000
Da)-butanoic acid.
[0166] The above mixture of products from C was dissolved in
distilled water (4470 ml) and the resulting solution was passed
through a first chromatographic column (precolumn) filled with 300
ml of anion exchange gel: DEAE SEPHAROSE.RTM. Fast Flow
(Pharmacia). This amount of anion exchange gel was only able to
retain about 35% of PEG acids present in the polymer mixture from
step C. Column capacity was previously determined in small scale
laboratory experiments. The anion exchange chromatogram revealed
that the eluate contained only mPEG(20,000 Da)-butanoic acid and
PEG(20,000 Da) dimethyl ether. PEG(20,000 Da)-dibutanoic acid and a
portion of mPEG(20,000 Da)-butanoic acid were absorbed by the gel
in the precolumn.
[0167] Next the eluate collected from the precolumn was applied to
a second column (main column) containing 1000 ml of DEAE
SEPHAROSE.RTM. Fast Flow gel. The amount of anion exchange gel in
the column was sufficient to retain all mPEG(20,000 Da)-butanoic
acid present in the eluate from the first column. Column capacity
was previously determined in small scale laboratory experiments. An
anion exchange chromatogram showed that the eluate from the second
or main column contained only PEG(20,000 Da) dimethyl ether. Anion
exchange chromatography showed that the only polymer adsorbed on
the second column was mPEG(20,000 Da)-butanoic acid, which was
eluted using 5% NaCl solution (1500 ml).
[0168] The pH of the eluate from the second or main column was
adjusted to 3 by addition of 5% phosphoric acid and the product was
extracted with dichloromethane. The extract was dried over
anhydrous magnesium sulfate, the drying agent removed, and the
dried solution was added to ethyl ether to precipitate the purified
product. The precipitated product was filtered off and dried under
reduced pressure. Yield 14.4 g. NMR (d.sub.6-DMSO): 1.72 ppm (q,
CH.sub.2--CH.sub.2--COO--) 2.24 ppm (t, --CH.sub.2--COO--), 3.24
ppm (s, --OCH.sub.3), 3.51 ppm (s, PEG backbone).
[0169] Anion exchange chromatography: mPEG(20,000 Da)-butanoic acid
100%. (no other polymer species detected). Gel permeation
chromatography: Mn=20,700 Da, polydispersity 1.010.
[0170] The product eluted from the first column (precolumn)
contained both some mPEG(20,000 Da)-butanoic acid and the
PEG(20,000 Da)-dibutanoic acid.
Example 2
mPEG (20,000 Da)-Amine
[0171] A solution of commercially available PEG (Mn=20,300 Da)
(50.0 g, 0.005 equivalents) in toluene (300 ml) was azeotropically
dried by distilling off 50 ml toluene. Dichloromethane (60 ml),
triethylamine (1.40 ml, 0.0100 moles), and methanesulfonyl chloride
(0.35 ml, 0.00452 moles, 90.4% of stoichiometric amount) were added
and the mixture was stirred overnight at room temperature under
argon atmosphere. The mixture was filtered and the solvents were
distilled off under reduced pressure. The residue was dissolved in
anhydrous toluene (250 ml) and sodium methoxide (25% solution in
methanol, 23.0 ml) was added. The mixture was stirred overnight at
70.degree. C. under an argon atmosphere, filtered and the solvents
were distilled off under reduced pressure. The crude product was
dissolved in 500 ml distilled water. NaCl (40 g) was added and the
pH of the solution was adjusted to 7.2 with 5% phosphoric acid. The
product was extracted with dichloromethane. The extract was dried
over anhydrous magnesium sulfate, filtered, and the solvent was
distilled off under reduced pressure. Yield 43.3 g. NMR analysis
showed that 79% of PEG-OH groups were converted to PEG-OCH.sub.3
groups.
[0172] The obtained partially methylated PEG(20,000 Da) (20.0 g)
was dissolved in toluene (150 ml) and the solution was
azeotropically dried by distilling off 50 ml of toluene.
Dichloromethane (25 ml), triethylamine (0.60 ml), and
methanesulfonyl chloride (0.30 ml) were added and the mixture was
stirred overnight at room temperature under n argon atmosphere. The
mixture was filtered and the solvents were distilled off under
reduced pressure. The product was then dissolved in dichloromethane
(30 ml) and 500 ml of isopropyl alcohol was added. The precipitated
product was filtered off and dried under reduced pressure giving
19.5 g of product. NMR analysis showed that the product contained
79% of PEG-OCH.sub.3 groups and 21% of PEG-methanesulfonate
groups.
[0173] The product was then dissolved in 350 ml of ammonium
hydroxide (30%) and the solution was stirred for 70 hours at room
temperature. The resulting mixture of methoxy-PEG-amine products
was extracted with dichloromethane. The extract was dried with
anhydrous magnesium sulfate and the solvent was distilled off under
reduced pressure. The product was re-dissolved in dichloromethane
(30 ml) and precipitated with 500 ml of isopropyl alcohol. Yield
15.2 g.
[0174] Analysis of relative proportions of polymer species by
cation exchange chromatography: PEG(20,000 Da)-diamine 4.7%,
mPEG(20,000 Da)-amine 30.4%, PEG(20,000 Da) dimethyl ether
64.9%.
[0175] The above mixture was dissolved in distilled water (1500 ml)
and the resulting solution was passed through a first
chromatographic column (precolumn) filled with 40 ml of cation
exchange resin POROS.RTM. 50 HS (Applied Biosystems). This amount
of cation exchange gel was only able to retain about 10% of PEG
amines present in the polymer mixture. Column capacity was
previously determined in small scale laboratory experiments.
[0176] Cation exchange chromatography showed that the eluate from
the precolumn contained only mPEG(20,000 Da)-amine and PEG(20,000
Da) dimethyl ether. PEG(20,000 Da)-diamine and part of mPEG(20,000
Da)-amine were adsorbed by the resin in the precolumn.
[0177] Next, the eluate from the precolumn was applied to the
second column (main column) containing 300 ml of POROS.RTM. 50 HS
resin. The amount of cation exchange gel in the column was
sufficient to retain all mPEG(20,000 Da)-amine present in the
eluate from the first column. Column capacity was previously
determined in small scale laboratory experiments. Cation exchange
chromatography showed that the eluate from the second (or main)
column contained only PEG(20,000 Da) dimethyl ether, leaving solely
the desired monofunctionalized polymer on the second (or main)
column. The mPEG(20,000 Da)-amine, adsorbed on the second (or main)
column, was then eluted using a 5% NaCl solution (600 ml).
[0178] The pH of the second (or main) column eluate was adjusted to
11 with 0.5 M sodium hydroxide and the product was extracted with
dichloromethane. The extract was dried over anhydrous magnesium
sulfate, filtered, and added to ethyl ether. The precipitated
product was isolated by filtration and dried under reduced
pressure. Yield 3.1 g.
[0179] NMR (de-DMSO): 2.64 ppm (t, --CH.sub.2 --NH.sub.2), 3.24 ppm
(s, --OCH.sub.3), 3.51 ppm (s, PEG backbone).
[0180] Analysis by cation exchange chromatography revealed that the
collected, dried product contained 100% m-PEG(20,000 Da)-amine,
free from detectable amounts of neutral or difunctionalized
polymer.
Example 3
mPEG (20,000 Da)-Carboxylic Acid, Sodium Salt
[0181] A solution of commercially available PEG (Mn=20,300 Da,
polydispersity 1.040) (50.0 g, 0.005 equivalents) in toluene (300
ml) was azeotropically dried by distilling off 50 ml toluene.
Dichloromethane (60 ml), triethylamine (1.30 ml, 0.0093 moles), and
methanesulfonyl chloride (0.30 ml, 0.00388 moles, 77.5% of
stoichiometric amount) were added and the mixture was stirred
overnight at room temperature under an argon atmosphere. The
mixture was filtered and the solvents were distilled off under
reduced pressure. The residue was dissolved in anhydrous toluene
(250 ml) and sodium methoxide (25% solution in methanol, 21.0 ml)
was added. The mixture was stirred overnight at 70.degree. C. under
an argon atmosphere. The mixture was then filtered and the solvents
were distilled off under reduced pressure. The crude product was
dissolved in 500 ml distilled water, NaCl (40 g) was added and the
pH of the solution was adjusted to 7.2 with 5% phosphoric acid. The
product was extracted with dichloromethane. The extract was dried
with anhydrous magnesium sulfate and the solvent was distilled off
under reduced pressure. Yield 44.1 g. NMR analysis showed that 66%
of PEG-OH groups were converted to PEG-OCH.sub.3 groups.
[0182] The partially methylated PEG (20,000 Da) (40.0 g) obtained
above was dissolved in toluene (150 ml) and the solution was
azeotropically dried by distilling off 50 ml of toluene. Next a
1.0M solution of potassium tert-butoxide in tert-butanol (8.2 ml,
0.0082 moles, 6.0 fold excess) was added to the above reaction
mixture. Ethyl bromoacetate (1.13 ml, 0.0102 moles, 7.5 fold
excess) was then added and the mixture was stirred overnight at
50.degree. C. under an argon atmosphere. The solvent was distilled
off under reduced pressure and the residue was dissolved in
distilled water (500 ml). The pH of the solution was adjusted to
12.10 with 1M sodium hydroxide and the solution was stirred
overnight, keeping the pH at 12.10 by periodic addition of 1M
sodium hydroxide. The pH was adjusted to 1.0 with 1M hydrochloric
acid and the product was extracted with dichloromethane. The
extract was dried with anhydrous sodium sulfate, concentrated, and
added to ethyl ether. The precipitated product was filtered off and
dried under reduced pressure. Yield 34.7 g. NMR (d.sub.6-DMSO):
3.21 ppm (s, --OCH.sub.3), 3.51 ppm (s, PEG backbone), 4.01 ppm (s,
--CH.sub.2-- COO--).
[0183] Analysis by anion exchange chromatography: PEG(20,000
Da)-dicarboxylic acid (11.5%), mPEG(20,000 Da)-carboxylic acid
(45.0%), PEG(20,000 Da) dimethyl ether (43.5%). NMR analysis showed
that the product contained 66% of PEG-OCH.sub.3 groups and 34% of
PEG-carboxylic acid groups.
[0184] The above mixture was dissolved in distilled water (3500 ml)
and the resulting solution was eluted through the first
chromatographic column (precolumn) packed with 200 ml of anion
exchange gel: DEAE SEPHAROSE.RTM. Fast Flow (Pharmacia).
[0185] Anion exchange chromatographic analysis revealed that the
eluate contained only mPEG(20,000 Da)-carboxylic acid and
PEG(20,000 Da) dimethyl ether. All of the PEG(20,000
Da)-dicarboxylic acid and part of the mPEG(20,000 Da)-carboxylic
acid were adsorbed (retained) by the gel in the precolumn.
[0186] Next, the solution was applied to the second column (main
column) containing 800 ml of DEAE SEPHAROSE.RTM. Fast Flow gel.
Anion exchange chromatography revealed that the eluate from the
column contained only PEG(20,000 Da) dimethyl ether. The
mPEG(20,000 Da)-carboxylic acid, adsorbed (retained) on the column,
was eluted using 5% NaCl solution (1100 ml).
[0187] The pH of the eluate was then adjusted to 7 and the product
was extracted with dichloromethane. The extract was dried with
anhydrous magnesium sulfate and added to ethyl ether to precipitate
the purified monocarboxylic acid polymer. The precipitated product
was filtered off and dried under reduced pressure. Yield 13.2 g.
NMR (d.sub.6-DMSO): 3.21 ppm (s, --OCH.sub.3), 3.51 ppm (s, PEG
backbone), 4.01 ppm (s, --CH.sub.2--COO--).
[0188] Anion exchange chromatography revealed the recovered product
to be 100% mPEG(20,000 Da)-carboxylic acid, without any detectable
amounts of PEG(20,000 Da) dimethyl ether or PEG(20,000
Da)-dicarboxylic acid.
Example 4
Diol-free mPEG-20,000 Da
[0189] mPEG(20,000 Da)-carboxylic acid, sodium salt, (10.0 g,
0.00050 moles) was dissolved in 150 ml toluene and the solvent was
distilled off to remove traces of water. The dried product was
dissolved in anhydrous tetrahydrofuran (100 ml) at 40-45.degree. C.
and lithium aluminum hydride (1.0M solution in tetrahydrofuran, 1.5
ml, 0.0015 moles) was added.
[0190] The mixture was stirred at 45.degree. C. overnight under
argon atmosphere. Ethyl acetate (0.5 ml) was added, the mixture was
stirred 30 min, then the mixture was cooled to about 30.degree. C.
and water (0.06 ml) was added and the mixture was stirred 10 min.
Next sodium hydroxide (15% solution in water, 0.06 ml) was added,
the mixture was stirred 10 min, and finally water (0.18 ml) was
added, the mixture was stirred 15 min, and then was filtered to
remove precipitated aluminum salt. To the filtrate, isopropyl
alcohol (500 ml) was added, and the precipitated product was
filtered off and dried under reduced pressure. Yield 8.7 g. NMR
(d.sub.6-DMSO): 3.21 ppm, (s, 3H, --OCH.sub.3); 3.51 ppm (s, PEG
backbone); 4.57 ppm (t, 1H, --OH).
[0191] HPLC analysis showed that the product is 100% pure
mPEG-20,000 Da and free of diol. Gel permeation chromatography:
Mn=20,800 Da, polydispersity 1.018.
Example 5
PEG (10,000 Da)-.alpha.-hydroxy-.omega.-propylamine
[0192] A mixture of PEG of molecular weight 10,000 Da (20.0 g;
0.00400 equivalents), distilled water (20.0 g) and potassium
hydroxide (0.4 g) was cooled to 0-5.degree. C. in an ice bath.
Acrylonitrile (0.5 g; 0.00942 moles) was added slowly, and the
solution was stirred for 2 hours at 0-5.degree. C. NaCl (2 g) was
added and the pH of the solution was adjusted to 7.0 with
phosphoric acid. Next, the reaction product was extracted with
dichloromethane and the solvent was distilled off under reduced
pressure giving 18.7 g of white solid product. NMR analysis showed
that the product contained 75% of PEG-OH groups and 25% of
PEG-OCH.sub.2CH.sub.2CN groups. The product was dissolved in 150 ml
of ethyl alcohol and palladium catalyst (10 wt % on activated
carbon; 2 g) was added. The mixture was hydrogenated at 65.degree.
C. under 800 psi of hydrogen. The mixture was then filtered and the
solvent was removed under vacuum giving 16.4 g of white product.
NMR analysis showed that the product contained 77% of PEG-OH groups
and 23% of PEG-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2 groups.
[0193] Analysis by cation exchange chromatography: PEG(10,000
Da)-dipropylamine 5.3%, PEG (10,000
Da)-.alpha.-hydroxy-.omega.-propylamine 35.4%, and PEG(10,000 Da)
59.3%.
[0194] The above mixture was dissolved in distilled water (1500 ml)
and the resulting solution was filtered through a first
chromatographic column (precolumn) filled with 30 ml of cation
exchange resin POROS.RTM. 50 HS (Applied Biosystems).
[0195] Cation exchange chromatographic analysis showed that the
filtrate contained only PEG (10,000
Da)-.alpha.-hydroxy-.omega.-propylamine and PEG(10,000 Da). All of
the PEG(10,000 Da)-dipropylamine and part of the PEG (10,000
Da)-.alpha.-hydroxy-.omega.-propylamine were adsorbed (retained) by
the resin in the precolumn.
[0196] Next, the solution was applied to the second column (main
column) containing 250 ml of POROS.RTM. 50 HS resin. Cation
exchange chromatography showed that the eluate from the column
contained only PEG(10,000 Da). PEG (10,000
Da)-.alpha.-hydroxy-.omega.-propylamine, adsorbed on the column,
was then eluted using 5% NaCl solution (350 ml). The pH of the
eluate was adjusted to 11 with 0.5 M sodium hydroxide and the
product was extracted with dichloromethane. The extract was dried
with anhydrous magnesium sulfate and added to ethyl ether. The
precipitated product was filtered off and dried under reduced
pressure. Yield 4.6 g. NMR (de-DMSO): 1.76 ppm (m,
--CH.sub.2CH.sub.2NH.sub.2), 2.80 ppm (t, --CH.sub.2--NH.sub.2).
3.51 ppm (s, PEG backbone), 4.58 ppm (t, --OH).
[0197] Cation exchange chromatography: PEG (10,000
Da)-.alpha.-hydroxy-.omega.-propylamine 100%.
Example 6
PEG (10,000 Da)-.alpha.-hydroxy-.omega.-propylmaleimide
[0198] PEG (10,000 Da)-.alpha.-hydroxy-.omega.-propylamine from the
Example 5 (4.0 g, 0.0004 moles) was dissolved in saturated aqueous
solution of NaHCO.sub.3 (30 ml) and the mixture was cooled to
0.degree. C. N-methoxycarbonylmaleimide (0.3 g) was added with
vigorous stirring. After stirring for 10 minutes, water (10 ml) was
added and the mixture was stirred an additional 50 minutes. The pH
was adjusted to 3.0 with 0.5 N sulfuric acid and about 15 wt % NaCl
was added. The reaction product was extracted with dichloromethane,
the extract was dried with anhydrous magnesium sulfate, and the
solvent was distilled off under reduced pressure to dryness. The
crude product was dissolved in 6 ml of dichloromethane and
precipitated with 100 ml of isopropyl alcohol giving 3.6 g of white
powder after drying under reduced pressure. NMR (d.sub.6-DMSO):
1.88 ppm (m, --CH.sub.2CH.sub.2--maleimide. 3.51 ppm (s, PEG
backbone), 4.58 ppm (t, --OH), 7.03 ppm (s, CH.dbd.CH
maleimide).
Example 7
Diacid-Free mPEG (20,000 Da)-propionic Acid, N-hydroxysuccinimidyl
Ester
[0199] A mixture of methoxy-PEG (or M-PEG-OH) of molecular weight
20,000 Da containing 6 wt % of PEG-diol having molecular weight
about 40,000 Da (HO-PEG-OH) (25.0 g), distilled water (25.0 ml) and
potassium hydroxide (0.5 g) was cooled to 0-5.degree. C. in an ice
bath. Acetonitrile (3.4 g) was added slowly, and the solution was
stirred for 2.5 hours at 0-5.degree. C. The pH of the solution was
adjusted to 7.0 by addition of phosphoric acid. The product was
extracted with dichloromethane (200, 70, and 50 ml). The organic
layer was dried over magnesium sulfate and added to cold ethyl
ether. The precipitated product was removed by filtration and dried
under vacuum giving 23.5 g of mPEG(20,000 Da) nitrile. NMR
(d.sub.6-DMSO): 2.74 ppm (t, --CH.sub.2CN), 3.21 ppm (s,
--OCH.sub.3), 3.51 ppm (s, PEG backbone).
[0200] A mixture of mPEG nitrile from the above step (23.5 g) and
concentrated hydrochloric acid (117.5 g) was stirred at room
temperature for 36 hours. The solution was diluted with one liter
of water and extracted with dichloromethane (200, 150, and 100 ml).
The combined organic extracts were washed twice with water, dried
over sodium sulfate, filtered, and concentrated to dryness by
rotary evaporation. Yield of mPEG amide 21.5 g. NMR (d.sub.6-DMSO):
2.26 ppm (t, --CH.sub.2CONH.sub.2), 3.21 ppm (s, --OCH.sub.3), 3.51
ppm (s, PEG backbone).
[0201] mPEG amide from the above step (16.0 g) was dissolved in
1150 ml of distilled water, 100 g of potassium hydroxide was added,
and the solution was stirred for 22 hours at room temperature.
Sodium chloride (150 g) was added, and the solution was extracted
with dichloromethane. The combined organic extracts were washed
with 5% phosphoric acid, water (twice), and dried over sodium
sulfate. The solution was concentrated and the product precipitated
by addition to ethyl ether. The product, largely mPEG(20,000 Da)
propionic acid, was collected by filtration and dried over vacuum.
Yield of acid 14.0 g. NMR (d.sub.6-DMSO): 2.43 ppm (t,
--CH.sub.2COOH), 3.21 ppm (s, --OCH.sub.3), 3.51 ppm (s, PEG
backbone).
[0202] Anion exchange chromatography showed that the product
contained: PEG(40,000 Da)-dipropionic acid (6%), mPEG(20,000
Da)-propionic acid (91%), and mPEG(20,000 Da) (3%).
[0203] The above mixture was dissolved in distilled water (2,000
ml) and the resulting solution was filtered through the first
chromatographic column (pre-column) filled with 50 ml of anion
exchange gel: DEAE SEPHAROSE.RTM. Fast Flow (Pharmacia).
[0204] Anion exchange chromatographic analysis showed that the
filtrate contained only mPEG(20,000 Da)-propionic acid and
PEG(20,000 Da). All of the PEG(40,000 Da)-dipropionic acid and part
of the m-PEG(20,000 Da)-propionic acid were adsorbed (retained) by
the gel.
[0205] Next, the solution was applied on the second column (main
column) containing 600 ml of DEAE SEPHAROSE.RTM. Fast Flow gel.
Anion exchange chromatography showed that the eluate from the
column contained only PEG(20,000 Da). mPEG(20,000 Da)-propionic
acid, adsorbed on the column, was eluted using 5% NaCl solution
(1100 ml). The pH of the eluate was adjusted to 7 and the product
was extracted with dichloromethane. The extract was dried with
anhydrous magnesium sulfate and added to ethyl ether. The
precipitated product was filtered off and dried under reduced
pressure. Yield 12.0 g. NMR (d.sub.6-DMSO): 2.43 ppm (t,
--CH.sub.2COOH), 3.21 ppm (s, --OCH.sub.3), 3.51 ppm (s, PEG
backbone).
[0206] Anion exchange chromatography: mPEG(20,000 Da)-propionic
acid 100%. No PEG(40,000 Da)-dipropionic acid was detected.
[0207] Diacid-free mPEG (20,000 Da) propionic acid (4.0 g, 0.20
mmol) was dissolved in dichloromethane (20 ml) and
N-hydroxysuccinimide (0.21 mmol) was added. The solution was cooled
to 0.degree. C., a solution of dicyclohexylcarbodiimide (0.20 mmol)
in 4 ml dichloromethane was added dropwise, and the solution was
stirred at room temperature overnight. The reaction mixture was
filtered, concentrated, and precipitated by addition to ethyl
ether. Yield of final product: 3.8 g. NMR (de-DMSO): 2.81 ppm (s,
NHS), 2.92 ppm (t, --CH.sub.2--COO--), 3.21 ppm (s, --OCH.sub.3),
3.51 ppm (s, PEG backbone).
[0208] Many modifications and other embodiments of the invention
will come to mind to one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing description. Therefore, it is to be understood that the
invention is not to be limited to the specific embodiments
disclosed and that modifications and other embodiments are intended
to be included within the scope of the appended claims. Although
specific terms are employed herein, they are used in a generic and
descriptive sense only and not for purposes of limitation.
* * * * *