Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
11DE~008
BAC~GROUND OF THE INVENTION
Field of the Invention
This invention relates to biological material. More
particularly, this invention relates to an immobilized
biological material (an immobilized biologicall~ active
material) having at least one primary or secondary amino
group per molecule and to a process for immobilizing
such material. Even more particularly, this invention
relates tb: (a) such biological material which has been
immohilized by contacting it with an excess of an isocyanate-
capped liquid polyurethane prepolymer to form a mixture
which is cured by contacting it (the mixture) with an
amount of a cùring agent (e.g., water or an appropriate
amine) effective for curing the biological material; and
(b) to the process whereby such biological material i5
immobilized.
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Des~ tion of the Prior Ar~
A review of enzyme technology was published in the
18 August 1975 issue of Chemical & Engineeri-ng News
~pp. 22-41). Chemical & Engineering News is published
by the American Chemical Society.
U. S. Patent No. 3,574,062 tl95/63, Sato) teaches
a method for preparing a bound protein (an enzyme) wherein
a polyester polyurethane is diazotized with a diazonium
salt of an amino acid and then coupled with a nonenzymatic
10 animal protein to form a diazotized polyurethane which is
reacted with an enzyme to form the ir~mobilized enzyme.
~ . S. Patent No. 3,705,084 (195~63, Reynolds~ teach~s
a flow-through enzyme reactor comprising; (a) a macro-
oorous reactor core; (b) a polymeric surface (~hich can
-be a polyurethane resin) on the reactor core; tcl an enzyme
adsorbed on the polymeric surface and cross-linked in place
thereon by a difunctional agent (e.g., a polyisocyanatel.
Reynolds prepares the immobilized enzyme for his
reactor by adsorbing an active enzyme on a polymeric
surface and further immobilizing the enzyme by cross-
linking it in place with a crosslinking agent such as
a monomeric polyisocyanate.
German Offenlegungsschrift No. 2,319,706 published
15 November 1973 teaches an enzyme bound to a polyurethane .
foam and a method for preparing such bound enzyme.
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10~38008
U. S. Patent No. 3,791,927 (195/63, Forgione et al2
teaches a water-insoluble bound protein (enz~me) entrapped
within the cells of a sel~-supporting reticulated cellular
material (which can be a pol~urethane foam~, the protein
~enzyme) being bound to the cellular m~terial.
U. S. Patent No. 3,672,955 (195/68, Stanley~ teaches
a process for preparing a ~o~nd protein Cenz~me~ comprising:
(a) emulsifying an a~ueous dl~spersion of the enzyme with
a solution of a poly~isoc~anate I`n a ~olatile water-
immiscible solvent (e.g., methylchlorcform); (b) admixingthe resulting emulsion with a solid particulate carrier;
and (c) evaporating the solvent therefrom. Stanley's
polyisocyanate can be an isocyanate-capped liquid poly-
urethane prepolymer.
'
It is noted that, in his Example 3, Stanley reports
the binding of an enzyme component (a peroxidase) of a
fermentation broth ~ admixing a portion of the broth with
a polyisocyanate dissolved in methylchloroform. It seems
probably that, under Stanley's reaction conditions, any
other enzymes which were present in the broth plus any
coenzymes which contained a primary or secondary amino
group and which were present in the broth would have been
immobilized (rendered insoluble).
Silman et al, Annual Review of Biochemistry, 1966,-
35 (~art 2), pages 873-908 present a revie~ of methods for
preparing water-insoluble derivatives of enzymes, antigens,
and antibodies.
Singer, Nat~re, 1959, 183, 1523-lS24 teaches a method
for reacting a protein with a diisocyanate (m-xylene
diisocyanate).
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The above-mentioned German Offenlegungsschrift
No. 2,319,706 in Claim 7 teaches a foamable composition
comprising: (a) an isocyanate-capped polyurethane prepolymer; -
(b) water; and (c) biostats, fungicides, or enzymes.
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Dean et al, U. S. Patent No. 3,904,478, teach a method
for immobilizing coenzymes wherein the coenzyme is immobi-
lized by: (a) forming a thio derivative of the coenzyme
in which an amino group of the coenzyme is replaced by a
thio group; (b~ reacting the thio group with: (i) a
bifunctional organic compound having a nucleophilic group
, so as to couple the bifunctional organic compound to the
coenzyme and then coupling the resultant coenzyme reaction
product to a water insoluble support material through the
unreacted functional group of the bifunctional organic
compound; or (ii) a water insoluble support material having
a plurality of pendant nucleophilic gr~ups so as to couple
the coenzyme to the support material.
Immobilized coenzymes are also taught by: (a) Mosbach
et al, Enzyme Eng., 2nd, 1973 (published in 1974~ which is
abstracted in Chemical Abstract~, 1975, 83, 39185c; (b)
McCormick, Methods Enzymol., 1974, 34(Affinity Tech.;
Enzyme Purif., Part B), 300-302, which is abstracted in
Chemical Abstracts, 1975, 83, 24325z; (c) Barker, Methods
20 Enzymol., 1974, 34(Affinity Tech.; Enzyme Purif., Part B),
479-491, which is abstracted in Chemical Abstracts 1975,
83, 39459v; (d) Morse et al, U. S. Patent No. 3,860,733,
which is abstracted in Chemical Abstracts 1975, 82, 138021g;
(e) French Patent No. 2,206,129 (to Merck and Co., Inc.),
which is abstracted in Chemical Abstracts 1975, 82, 138024k;
If) Chibata et al, Enzyme Eng., 2nd, 1973 (published 1974),
229-236, which is abstracted in Chemical Abstracts 1975,
83, 39356j; (g) Weibel et al, Enzyme Eng., 2nd, 1973
(published 1974) 203-208, which is abstracted in Chemical
30 Abstracts 1975, 83, 55279s; and (h) Molteni et al, Eur. J.
Med. Chem. - Chim. Ther. 19.74, 9(6), 618-620, which is
abstracted in Chemical Abstracts 1975, 83, 48130h.
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~B008
U. S. Patent No. 2,781,339 (260/211.5, Mitz et al)
teaches a process for preparing a coenzyme (coenzyme A)
comprising admixing a crude aqueous solution of the co-
enzyme with acid-conditioned charcoal to absorb the
coenzyme and thereafter eluting the coenzyme from the
charcoal.
. Immobilized antibiotics are taught by: (a) Wagman
et al, Antimicrob. Agents Chemother, 1975, 7(3), 316-319,
which is abstracted in Chemical Abstracts 1975, 83, 22169c;
and (b) Seela, Z. Naturforsch., C: Biosci., 1975, 30c(7-8),
544-545, which is abstracted in Chemical Abstracts 1975,
83, 74898j.
U. S. Patent No. 3,905,923 (260/2.5 AD, Klug) teaches
an immobilized enzyme system formed from an enzyme and a
hydrophilic poly(urea-urethane) foam, the foam surrounding,
entrapping, and supporting the enzyme in an active config-
uration. The hydrophilic foam is formed by the reaction of ;;
water with a hydrophilic isocyanate-capped polyoxyalkylene
prepolymer.
Isocyanate-capped polyurethane prepolymers are well
known to those skilled in the art. See, for example:
~a) the penultimate paragraph on page 854 of Volume 9 of
the Second Edition of the Kirk-Othmer "Encyclopedia of
Chemical Technology", John Wiley and Sons, Inc., New
York, N.Y.; or (b~ the third full paragraph in the left
hand ~first) column of page 872 of the Second Edition of
"The Encyclopedia of Chemistry", George L. Clark, Editor,
Reinhold Publishing Corporation, New York, N.Y.
~o~o~
It is noted that the cured isocyanate-capped liquid
polyurethane prepolymers of our invention (including those
comprising immobilized biological materials (i.e., immobilized
biologically active materials which are also referred to
- herein as "immobilized group member(s)") produced by foaming
an isocyanate-capped liquid polyurethane prepolymer with
water or by the reaction of said prepolymer with water under
nonfoaming conditions comprise poly(urea-urethane) products
which may be foams, films, discs, tubes, rods, spheres,
ox the like, and which can be hydrophilic.
SUMMARY OF THE INVENTION
In summary, this inven~ion is directed to a process
for immobilizing a biological' material (a biologically
: active material) selected from the group consisting of:
(a) a protein; (b) a coenzyme having at least one primary
or secondary amino group per molecule; (c) an admixture
of said coenzyme and an enzyme which is mediated by said
coenzyme; and (d) an antibiotic having at least one primary
or secondary amino group'per molecule, the process comprising:
A. contacting the group member and an excess
o an isocyanate-capped liquid polyurethane prepoly-
' mer to form a resulting mixture; and
B. curing the resulting mixture by contacting
it with an amount of a curing agent effective for
immobilizing said biological material under foam-
; forming or nonfoam-forming conditions, the resulting
mixture being shaped before curing where the biological
material consists of a protein
This invention is also directed to the product formed
by the aforesaid process.
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Thus, in accordance with the present teachings, a non-
foamed self-supporting shaped poly (oxyalkalene) urethane
product is provided which comprises an immobilized biologi
cally-active material dispersed throughout the interior of the
product and at the surface thereof and selected from: (a) a -
protein; (b) a coenzyme which has at least one primary or
secondary amino group per molecule; (c) an admixture of the
coenzyme and enzyme which is mediated by the coenzyme; and
(d) an antibiotic having at least one primary or secondary
amino group per alcohol.
Any liquid polyurethane prepolymer which contains at
least two free isocyanate groups per prepolymer molecule is
operable as an immobilizing agent in accordance with this
; invention. We prefer that the prepolymer contain an average
of two isocyanate groups per molecule. An even higher ratio ;`
can be used, for example, 2-8 isocyanate groups per poly-
urethane molecule. Ratios higher than this are operable, but
offer no advantage. Any excess isocyanate groups left in the
cured polyurethane will be destroyed by hydrolysis upon the
first contact with water, for example, during a washing step
preliminary to use of the immobilized biologically active
material.
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Brief Description of the Dra~ings
FIG. 1 shows a film (the film of Embodiment C discussed
hereinafter) supported on an inert support on which said film
was cured,
FIG. 2 shows the film of FIG. 1 after the film has been
stripped from the support,
FIG. 3 shows discs (the discs of Embodiment D as dis-
cussed hereinafter),
FIG. 4 is a lateral view of a tube (the tube of
Embodiment E as discussed hereinafter).with a part cut away.
FIG. 5 is a transverse section of the tube taken along
line 5-5 of FIG. 4,
FIG. 6 shows the tube of FIG. 4 in which a coating
(or film) consisting essentially of cured polyurethane prepolymer
.is b~und ~bonded) to the outer sur~ace of the tube comprising
immobilized group member,
FIG. 7 shows the tube of FIG. 4 in which a coating
.~ (or film) consisting essentially of cured polyurethane prepolymer
.! is bonded (bound) to the inner surface of the tube comprising
immobilized group member,
FIG. 8 is a transverse section of a pattern rod, (the
pattern rod of Embodiment F as discussed hereinafter);
FIG. 9 is a transverse section of the product rod of
FIG. 8 (Embodiment F as discussed hereinafter),
FIG. 10 shows spheres (Embodiment H as discussed herein-
after) packed in a column, the spheres being supported by a
packing support (glass wool);
FIG. 11 shows the spheres of FIG. 10;
FIG. 12 shows a film (the film of Embodiment C as
discussed hereinafter) in which said film is bonded (bound) to a
second film consisting essentially of cured polyurethane pre-
polymer,
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FIG. 13 shows discs (the discs of Embodimen~ D as
discussed hereinafter) in which each disc is bonded to a disc
consisting essentially of cured polyurethane prepolymer,
FIG. 14 is a transverse section of a rod (the rod
of Embodiment G as discussed hereinafter),
FIG. 15 is a transverse section of a rod (the rod of
Embodiment F as discussed hereinafter) in which the outer
surface of the film or coating comprising immobilized group
member is coated with a film or coating consisting essentially
of cured polyurethane prepolymer,
FIG. 16 shows a foamed section (the foam of Embodiment ;.
I as discussed hereinafter) and,
FIG. 17 shows, with parts cut away, a reactor packed
with foam comprising the immobilized biologically active material
(group member). Said reactor is adapted for utilizing the
immo~ilized biologically active material.
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Descr ption of Preferred Embodiments
~n one preferred embodiment ("Embodiment A") this
invention is directed to a process for immobilizing a
biological material selected from the group consisting of:
(a) a coenzyme having at least one primary or secondary
amino group per molecule; (b) an admixture of said coenzyme
and an enzyme which is mediated by said coenzyme; and (c)
an antibiotic having-at least one primary or secondary amino
group per molecule, the process comprising:
A. contacting the group member and an excess
of an isocyanate-capped liquid polyurethane prepolymer
` to form a resulting mixture; and
B. curing the resulting mixture by contacting
it with an amount of a curing agent effective for
immobilizing said biological material.
~his invention is also directed to the product formed
by the aforesaid process.
a In another preferred embodiment ~"Embodiment B") this
invention is directed to a process for immobilizing a
biological material selected from the group consisting of:
(a) a protein; (b) a coenzyme having at least one primary
or secondary amino group per molecule; (c) an admixture
of said coenzyme and an enzyme which is mediated by said
coenzyme; and (d) an antibiotic having at least one primary
or secondary amino group per molecule, the process comprising:
A. contacting the group member and an excess
of an isocyanate-capped liquid polyurethane prepolymer
to form a resulting mixture;
B. shaping the resulting mixture; and
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C. curing the shaped resulting mixture by
contacting it with a curing agent to form a cured
shaped article comprising the immobilized biological
material.
The shaped article comprising the immobilized
biological material (i.e., comprising the group member)
can be a film, a disc (which can be prepared by cutting
the film, e.g., with a cork bore or stamping apparatus),
a tube, a rod, a sphere, or a foam.
This invention is also directed to such shaped article.
The general method for preparing nonfoamed immobilized
enzymes recited in the aforesaid Stanley patent (US Patent
No. 3,672,955) can be used to immobilize (bind) the group
members of the above Summary, Embodiment A, or Embodiment B
in nonfoamed and biologically active form. The particulate
solid material (solid carrier) included in Stanley's method
can be included or omitted and the product can be a rod,
a tube, a self supporting film, a self supporting disc, a
sphere, or the like. Stanley's "essentially water-immis-
cible solvent" can be used or it can be replaced with a
water-soluble or substantially water-soluble solvent, a
volatile inert solvent is preferred.
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In another preferred embodiment ("Embodiment C")
this invention is directed to a self supporting cured
polyurethane film having ~wo surfaces, said film comprising
an immobilized biologically active material selected from
the group consisting of:
(a) a protein; (b) a coenzyme having at least one primary
or secondary amino group per molecule; (c) an admixture of
said coenzyme and an enzyme which is mediated by said
coenzyme; and (d) an antibiotic having at least one primary
or secondary amino group per molecule.
In another preferred embodiment ("Embodiment D"~
this invention is directed to a self supporting cured
polyurethane disc having two surfaces, said disc comprising
an immobilized biologically active material selected from
the group consisting of:
(a) a protein; (b) a coenzyme having a~ least one primary
or seconaary ~amino group per molecule; (c) an admixture of
.
said coenzyme and an enzyme which is mediated by said
coenzyme; and (d) an antibiotic having at least one primary
or secondary amino group pcr moleculc.
In another preferred embodiment ("Embodiment E"~
this invention is directed to a cured polyurethane tube
haying an inner surface and an outer surface, said tube
comprising an immobilized biologically active material
selected from the group consisting of: (a) a protein;
(b) a coenzyme having at least one primary or secondary
amino group per molecule; (c) an admixture of said coenzyme
and an enzyme which is mediated by said coenzyme; and
~d) an antibiotic having at least one primary or secondary
30 amino group per molecule.
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In another preferred embodiment ~"Embodiment ')
this invention is directed to a product rod comprising
an inert pattern rod having a longitudinal surface and ;,
two end surfaces, the longitudinal surface being coated
with a cured polyurethane film having an outer longitudinal
surface, said film comprising an immobilized biologically
active material selected from the group consisting of:
(a) a protein; (b) a coenzyme having at least one primary
or secondary amino group per molecule; (c) an admixture of
said coenzyme and an enzyme which is mediated by said
coenzyme; and (d) an antibiotic having at least one primary
or secondary amino group per molecule. Separating the pat-
tern rod from the film comprising the immobilized biologically
active material (group member) will form the tube of FIG. 5.
In one embodiment of the rod of Embodiment F a second
film consisting essentially of cured polyurethane can be
bound to the l~ngitudinal surface of the cured film comprising
the group member (immobilized biologically active material)
to form the rod of FIG. 14. Separating the pattern rod from
2Q the film comprising the immobilized group member (biologically
active material) will form the tube of FIG. 6.
In another preferred embodiment ("Embodiment G") this
invention is directed to a rod comprising an inert pattern rod
having a longitudinal surface and two end surfaces, the.longi-
tudinal surface being coated with a first film consisting
essentially of cured polyurethane, the first film having an
outer longitudinal surface, the outer longitudinal surface of
,the first film being coated with a film of cured polyurethane
comprising an immobilized biologically active material selected
from the group consisting of: (a) a protein; (b) a coenzyme
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ha-~ing a'. least one primary or sccon(lary amillo group per
molecule; (c) an adiniY.~Ilre of said coenzylne and an en7.y~e
~hic}l is me~diated by said coenzyme; and (d) an antibiotic
having a~ least one primary or secondary a~ino group ~er
2cul e, tne second film being bound to the irst film.
Separating the pattern rod from the film consisting
essentially of cured polyurethane will Lorm the tube of
- : ~IG. 7.
In another preferred embodiment ("Embodiment H")
this invention is directed to a cured polyurethane sphere
comprising an immobilized biologically active material
selected from the group consisting of: (a) a protein;
(b) a coenzyme havin~ at least one primary or secondary
amino group per molecule; (c) an admixture of said ccenzyme
and an enzyme which is mediated by said coenzyme; and
(d) an antibiotic having.at least one primary or secondary
amino group per molecule~ -
In another preferred embodiment ("Embodiment I")
this invention is directed to a cuxed self supporting
polyurethane foam comprising an immobilized biologically
active material selected from the group consisting of:
(a) a coenzyme having at least one primary or secondary
amino group per molecule; ~b) an admixture o.. said coenzyme
and an enzyme ~hich is mediated by said coenzyme; and
(c) an antibiotic having at least one primary or secondary
amino group per molecule.
In another preferred embodiment (llEm~odiment J")
this invention is direc~ed to a reactor comprising:
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~a) a eolumn having a lower end and an open
upper end,
(b) a eap having an upper portion and an open
lower portion, the lower portion of the cap eovering
ana eommunieating with the open upper end of the eolumn;
(e) an inlet line haviny an inlet port communi-
eating with the upper portion of the cap;
(d) an outlet line having an outlet port communi-
eating with the lower end of the column; and
(e) a cured self supporting polyurethane foam
: ~ comprising an immobilized biologically active material
seleeted from the group consisting of: (i) a protein,
(ii) a coenzyme having at least one primary or secondary
amino group per molecule; (iii) an admixture of said
eoenzyme and an enzyme which is mediated by said
eoenzyme; and ~iv) an antibiotic having at least one
primary or secondary amino group per molecule positioned
within the column, said foam being produced (foamed) and
formed (shaped) in place in the column (i.e;, in situ).
A ~alve can be inserted in the outiet line as shown
in FIG. 17. Said valve can be a stopcock.
In another preferred embodiment ("Embodiment K") this
invention is directed to a process for immobilizing a
biological material selected from the group consisting of:
(a) an admixture of an enzyme and an antibiotic having at
least one primary or secondary amino group per molecule;
and (b) an admixture of a coenzyme having at least one primary
or secondary amino group per molecule, an enzyme which is
mediated by said coenzyme, and an antibiotic having at
least one primary or secondary amino group per molecule,
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the process comprising:
A. contacting the group member and an excess
of an isocyanate-capped 'iquid polyurethane prepolymer
to form a resulting mlxture; and
B. curing the resulting mixture by contacti`ng
it with an amount of a curing agent effective for
immobilizing said biological material under foam-
forming or nonfoam-forming condit~ions.
This invention is also directed to the product prepared
by the process of said embodiment (Embodiment K). Said
product can be shaped (e.g., to form a shaped foam, a
self supporting film, a self supporting disc, a tube, a
rod, or a sphere.
Said product is useful for inhibiting or preventing
bacterial decomposition of a material undergoing enzymatic
reaction (e.g., to inhibit or prevent bacterial decompositior
of sucrose or~inert sugar during the hydrolysis of sucrose
with invertase to form inert sugar).
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- - Detailed Description of the Invention
- This invention is in the field of biological materials.
More particularly it is in the field of immobilized biological
materials including proteins (e.g., enzymes, antibiotics, and
antigens), coenzymes having at least one primary or second-
ary a~ino group per molecule, and antibiotics having at
least one primary or secondary amino group per molecule.
It is an object of this invention~to provide a method
for immobilizing a biological material selected from the
group consisting of: (a) a protein; (b) a coenzyme having
at least one primary or secondary amino group per molecule;
~c) an admixture of said coenzyme and an enzyme which is
mediated by said coenzyme; and (d) an antibiotic having at
least one primary or secondary amino group per molecule
without destroying or inactivating the biological activity
of the biological material.
It is a fùrther objective of this invention to immobilize
such biological material by contacting the biological material
with an excess of an isocyanate-capped liquid polyurethane
prepolymer to form a resulting mixture and then curing the
resulting mixture by contacting it with a curing agent
which can be a foam-producing curing agent (i.e., water)
or a nonfoam-producing curing agent (e.g., an amine such
as aniline, methylamine, diethylamine, or ammonia).
It is a further object of this invention to immobilize
the biological material by treating the-resulting mixture
with water under: (a) foam-forming; and (b) nonfoam-forming
conditions.
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- It is a further object of this invention to shape the
resulting mixture (before curing, during curing, or after
curing, or a combination thereof) to produce a shaped
object (e.g., a film, a tube, a rod, a disc, a sphere, or
the like) comprising the immobilized biological materi~al.
It is a further object of this invention to immobilize
a coenzyme and an enzyme which is mediated by the coenzyme
to form a product comprising the enzyme and the coenzyme in
immobilized and active form.
It is also an object of this invention to immobilize
the above-mentioned biological materials in active and
reusable form.
U.S. Patent 4,0g8,6~5 which is not prior art
with respect to the instant invention teaches: (a) a process
for immobilizing a protein which can be an enzyme, an
antibody, or an antigen; and (b) the product of such process,
the process comprising admixing the protein and an isocyanate-
capped liquid polyurethane prepolymer to form a resulting
product and foaming and polymerizing (curing) the resulting
product by admixing it with water under foam-forming conditions.
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Chemical Abstracts, 1975, 83, 174745a contains an
abstract reporting that Lipatova et al, Ukr. ~iokhm. Zh.,
1975, 47(4), 532-535 immobilized trypsin on a polyurethane
matrix. Said abstract states that:
Two methods were developed for the preparation
of trypsin immobilized on a polyurethane matrix.
The covalently bound trypsin was prepared by reacting
trypsin with toluene diisocyanate and reacting the
polyisocyanate obtained with polyoxypropylene glycol,
mol. wt. 1500, using 2,4,6-tris(dimethylaminomethyl)-
phenol as an accelerator of the reaction. The chem.
interaction between trypsin and the polymer was
confirmed by ir-spectroscopy and by the decrease in
the amt. of free isocyanate groups. The activity of
trypsin chem. bound to the polymer was preserved at
rooln temp., for several months. In the free trypsin
entrapped in a polymeric net formed by poly(diisocyanate)
under conditions in which no chem. interaction between
the polymer and enzyme occurred, the activity was also
preserved, its value in this case being dependent
on the relative content of enzyme in the polymer.
It was high when the relative content of the enzyme
was low~ which might indicate a role for the polymer
as a heterogenic catalyst or activator. The proteolytic
activity of the polymer with chem. bound trypsin change
very little during washing of the column for 24 hrs.
with 0.005M veronal buffer, whereas the polymer with
free trypsin under these conditions lost its activity
completely.
It is believed that the above-discussed Lipatova et al
publication is not prior art with respect to the instant
invention.
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A polyurethane prepolymer is produced in the known
way by the reaction of an excess of di- and tri- isocyanates
and other polyisocyanates (including mixtures of polyiso-
cyanates) with compounds containing active hydrogen,
particularly glycols, polyglycols, polyester polyols, poly-
ether polyols, other polyols, and mixtures of two or more
such polyols. This reaction produces an isocyanate-capped
liquid polyurethane prepolymer which can be used in the
pr~cess of the instant invention.
The above-mentioned U. S. Patent No. 3,672,955 teaches
that proteins (enzymes) can be bound to isocyanate-capped
polyurethanes. In the process of said patent the isocyanate-
capped polyurethane is dissolved in a water-immiscible
solvent. This solution is emulsified, using an emulsifying
agent in the presence of an active enzyme which is dispersed
in water.
Our process is similar in some respects to that of
Stanley's said U. S. Patent No. 3,672,955. We can use the
- same isocyanate-capped polyurethane prepolymers (which that
patent refers to as polyisocyanates) we can use the same
polyols (to prepare our prepolymer); we can use the same
enzymes; and we can use the same water-immiscible solvent.
As in that patent (although we do not wish to be bound to
any particular theory) the mechanism is apparently the
reaction of one or more amine and/or hydroxyl groups on the
biological material with one or more isocyanate groups on
the polyurethane prepolymer molecule.
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As used herein the term "liquid isocyanate-capped
polyurethane prepolymer" means a liquid polyurethane or
polyurea molecule containing at least about t~o free
isoc~anate groups per mol^c_ ~.
Representative examples of polyisocyanates which can
be reacted with an active hydro~en containing compound
~e.g., a glycol, polyol, polyglycol, polyester polyol,
polyether polyol, and the like) to make an isocyanate-capped
polyurethane in accordance with the inven.ion include:
toluene-~,4-diisocyanate
toluene-2,6-diisocyanate
commercial mixtures of toluene-2,4- and 2,6-
diisocyanates
ethylene diisocyanate
ethylidene diisocyanate
propylene-1,2-diisocyanate
cyclonexyiene-1, 2-c~iisocyanate
cyclohexylenc-1,4-diisocyanate
m-phenylene diisocyanate
3,3'-diphenyl-4,4'-biphenylene diisocyanate
4,4'-biphenylene diisocyanatc
~,3'-dichloro-4,4'-biphenylcne diisocyanate
1, 6-heY.?Imethylellediisocyanate
.. . . . . ..
- ,;, ,,'~,,',, , ~ '
~ : ~ , . .. ,. . ; . :
10~8008
1,4-tetramethylene-diisocyanate
l,10-decamethylenediisocyanate
1,5-naphthalenediisocyanate
cumene-2,4-diisocyanate
4-methoxy-1,3-phenylenediisocyanate
4-chloro-1,3-phenylenediisocyanate
4-bromo-1,3-phenylenediisocyanate
4-ethoxy-1,3-phenylenediisocyanate
2,4'-diisocyanatodiphenylether
5,6-dimethyl-1,3-phenylenediisocyanate
2,4-dimethyl-1,3 phenylenediisocyanate
4,4'-diisocyanatodiphenylether
benzidinediisocyanate
4,6-dimethyl-1,3-phenylenediisocyanate
9,10-anthracenediisocyanate -
4,4'-diisocyanatodibenzyl
3,3'-dimethyl-4,41-diisocyanatodiphenylmethane
2,6-dimethyl-4,4'-diisocyanatodiphenyl
2,4-diisocyanatostilbene
3,3'-dimethyl-4,4'-diisocyanatodiphenyl
3,3'-dimethoxy-4,4'-diisocyanatodiphenyl
1,4-anthracenediisocyanate
2,5-fluorenediisocyanate :~
1,8-naphthalenediisocyanate
2,6-diisocyanatobenzfuran
2,4,6-toluenetriisocyanate, and
p,p',p"-triphenylmethane triisocyanate.
:
- 23 -
- .. .
~0~ 8
A useful class of liquid isocyanate-capped polyurethane
prepolymers are those derived from polyether polyols and
polyester polyols. These compounds may be prepared, as
is well known in the art, by reacting a polyether (or poly-
ester) polyol with a polyisocyanate, using an excess of the
latter to ensure provision of free isocyanate qroups in
the product. A typical, but by no means limiting, example
is illustrated in idealized equation form below:
o {--C112--~}12--C112- C1~2--~ ~ I Polyether polyol
m
CH3
., I .
8 Polyisocyanate
7 3 1 ~3
OCN ~ NH- ~ -0 ~ ~12- CH2- CH2- C~T2- 0 ~ ~-NI ~ -NC0
' I50c~anate-ca~ped liquid
polyurethane Prepolvmer
(In the above formulas, m represents the number of
tetramethyleneether repeating units. This may range, for
example, about from 5 to 50.)
The compounds useful for the purposes OL the invention
may be prepared bi reacting any of the above-exemplified
polyisocyanates with any of a wide variéty of polyethex
polyols and polyes~er polyols, and representative examples
of ~hese polyols are described below.
- 24 -
: . . -
., . ~ . . . :
" 10;~8a~08
Among the polyether polyols which may be so used are
those prepared by reaction of an alkylene oxide with an initiator
containing active hydrogen groups, a typical example of the
initiator being a polyhydric alcohol such as ethylene glycol;
a polyamine such as ethylene diamine; phosphoric acid, etc.
The reaction is usually carried out in the presence of either
an acidic or basic catalyst. Examples of alkylene oxides which
may be employed in the synthesis include ethylene oxide, pro-
pylene oxide, any of the isomeric butylene oxides, and mixtures
of two or more different alkylene oxides such as mixtures of
ethylene and propylene oxides. The resulting polyether polyols
contain a polyether backbone and are terminated by hydroxyl
groups. The number of hydroxyl groups per polymer molecule is
determined by the functionality of the active hydrogen initiator.
For example, a difunctional alcohol such as ethylene glycol
(as the active hydrogen initiator) leads to polyether chains
in which there are two hydroxyl groups per polymer molecule.
When polymerization of the oxide is carried out in the presence
of glycerol, a trifunctional alcohol, the resulting polyether
molecules contain an average of three hydroxyl groups per mole-
cule. Even higher functionality--more hydroxyl groups--is
obtained when the oxide is polymerized in the presence of
such polyols as pentaerythritol, sorbitol, sucrose dipenta-
erythritol, and the like. In addition to those listed above,
other examples of polyhydric alcohols which may be reacted
with alkylene oxides to produce useful polyether polyols include:
~ 8008
propylene glycol
trimethylene glycol
1,2-butylene glycol
1,3-butanediol
1,4-butanediol
1,5-pentanediol
1,2-hexylene glycol
l,10-decanediol
1,2-cyclohexanediol
2-butene-l~4-diol
3-cyclohexene-1,1-dimethanol
4-methyl-3-cyclohexene-1,1-dimethanol
3-methylene-1,5-pentanediol :
diethylene glycol
(2-hydroxyethoxy)-1-propanol
4-(2-hydroxyethoxy)-1-butanol
5-(2-hydroxypropoxy)-1-pentanol
1-(2-hydroxymethoxy)-2-hexanol
1-(2-hydroxypropoxy)-2-octanol
3-allyloxy-1,5-pentanediol
2-allyloxymethyl-2-methyl-1,3-propanediol
[(4-pentyloxy)methyl]-1,3-propanediol
3-(o-propenylphenoxy)-1,2-propanediol
thiodiglycol
2,2'-[thiobis(ethyleneoxy)]diethanol
polyethyleneether glycol (molecular weight about 200)
2,2'-isopropylidenebis(p-phenyleneoxy)diethanol
1,2,6-hexanetriol
l,l,l-trimethylolpropane
3-(2-hydroxyethoxy)-1,2-propanediol
~ - 26 -
,,
``` 1~iiB008
3-(2-hydroxypropoxy)-1,2-propanediol
2,4-dimethyl-2-(2-hydroxyethoxy)methylpentanediol-1,5
1,1,1-tris[(2-hydroxyethoxy)methyl]ethane
1,1,1-tris[(2-hydroxypropoxy)methyl]propane
triethanolamine
triisopropanolamine
resorcinol
pyrogallol
phloroglucinol
hydroquinone
4,6-di-tertiarybutyl catechol -
catechol
orcinol -
methylphloroglucinol
hexylresorcinol
3-hydroxy-2-naphthol
2-hydroxy-l~naphthol
2,5-dihydroxy-1-naphthol
bis-phenols such as 2,2-bis(p-hydroxyphenyl)propane
and bis-(p-hydroxyphenyl)methane -
1,1,2-tris-(hydroxyphenyl)ethane
1,1,3-tris-(hydroxyphenyl)propane.
An especially useful category of polyether polyols
are the polytetramethylene glycols. They are prepared by
the ring-opening polymerization of tetrahydrofuran, and
; contain the repeating unit.
-cH2-cH2-cH2-cH2-o-
in the polymer backbone. Termination of the polymer chains
is by hydroxyl groups.
- 27 -
~` 10~008
Also especially desirable are the polyoxyethylene
polyols HO ( CH2CH2-O- )x H in which x is an average number
such that the polyol has an average molecular weight of up to
about lO00 (or about 2000 or somewhat higher).
The polyester polyols which may be employed as
precursors are most readily prepared by condensation poly-
merization of a polyol with a polybasic acid. The polyol and
acid reactants are used in such proportions that essentially
all the acid groups are esterified and the resulting chain of
ester units is terminated by hydroxyl groups. Representative
examples of polybasic acids for producing these polymers are
oxalic acid, malonic acid, succinic acid, glutaric acid,
adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic
acid, brassylic acid, thapsic acid, maleic acid, fumaric acid,
glutaconic acid, a-hydromuconic acid, ~-hydromuconic acid, a-
butyl-a-ethylglutaric acid, a , ~-diethylsuccinic acid, o-
phthalic acid, isophthalic acid, terephthalic acid, hemi-
mellitic acid, trimellitic acid, trimesic acid, mellophanic
acid, prehnitic acid, pyromellitic acid, citric acid, benzene-
pentacarbos~lic acid, 1,4-cyclohexane dicarboxylic acid,
diglycollic acid, thiodiglycollic acid, dimerized oleic acid,
dimerized linoleic acid, and the like. Representative examples
of polyols for forming these polymers include ethylene glycol,
1,3-propylene glycol, 1,2-propylene glycol, 1,4-butylene glycol,
1,3-butylene glycol, 1,2-butylene glycol, butene-1,4 diol,
1,5-pentane diol, 1,4-pentane diol, 1,3-pentane diol, 1,6-
hexane diol, hexene-1,6-diol, 1,7-heptane diol, diethylene
glycol, glycerine, trimethylol propane,
- 28 -
: . . , .: . .
- '
10~ 08
1,3,6-hexanetriol, trimethanolamine, pentaerythritol,
sorbitol, and any of the other polyols listed hereinabove
in connection with the preparation of polyether polyols.
It is believed that, on being intimately contacted
with a protein (or other biological material containing
at least one primary or secondary amino group per molecule)
such as an enzyme, antibody, antigen, or the like, an
isocyanate-capped polyurethane prepolymer becomes chemically
very active. Some of its free isocyanate groups of the
prepolymer react with the amine groups of the material,
and subsequently when water is present, some isocyanate
groups react with water to give carbon dioxide and to form
amine groups which react with free isocyanate groups of
polyurethane molecules to form urea type links. These
lat~er amine groups may react with free iso~yanate groups
on neighboring polyurethane molecules, and this reaction
(by forming a urea linkage) will cause further growth of
the polyurethane molecule and may also introduce cross
links between the polyurethane molecules. This further
preliminary growth and cross linking is essential for the
formation of a good polyurethane foam.
Other additives such as crosslinking agents (poly-
amines, polythiols, polyacids) surfactants, wetting agents,
antifoaming agents, dyes, antioxidants, fillers, etc., may
also be present during the curing and immobilization step.
It is, of course, the release of the carbon dioxide
that provides gas for foam formation where working under
foam-forming conditions.
The ratio of biological material to isocyanate-capped
liquid polyurethane prepolymer is not critical. However it
is important that said ratio be such that all of the
29
~0~108
isocyanate groups of the prepolymer are not consumed by
reaction with the protein, thereby to leave unreacted iso-
cyanate groups available to react with curing agent to
cure the above-defined resulting mixture and to immobilize
the biological material (the group member of the above Summary
which is also called a "biologically active material").~
: Where working under foam-forming conditions the ratio
of water to group member plus isocyanate-capped liquid
polyurethane prepolymer is not critical; however, we
generally prefer to use about 0.5-3 or 0.9-2 parts by
weight of water per part by weight of said prepolymer plus
group member.
Where using water as curing agent under nonfoaming
conditions we generally prefer to use about a stoichiometric
amount of water.
~ here using an amine as curing agent we generally
prefer to use at least about a stoichiometric amount of
amine. Amine curing agents do not produce foaming.
It is preferred t~at sufficient curing agent (whether
water or amine and whether under foaming or nonfoaming
conditions) be used to react with most (or all) of the
free isocyanates present on the prepolymer. However, the
final product can be wasted with water to "inactivate"
(react with) any free isocyanate groups on the final product
before using.
.
::. . ,.. , . : , -: .. .. :
:: , . :: :, . ., ,: : . :
, : :., .: . . ::,. . ~ :
:.. : .. . .. : .
- - . ::-: ...
10~008
ln the process and product of this invention:
1. The isocyanate-capped liquid polyurethane prepolymer
is prepared by reacting toiuene diisocyanate and a poly-
ethylene glycol.
2. The isocyanate-capped liquid polyurethane prepolymer
is prepared by reacting toluene diisocyanate and a poly-
ethylene glycol having a molecular weight of about 800-1,200.
3. The isocyanate-capped liquid polyurethane prepolymer
is prepared by reacting toluene diisocyanate and a member
selected from the group consisting of a polyoxybutylene
polyol polymer, ethylene glycol, diethylene glycol, a
polyoxyethylene polyol polymer, pentaerythritol, glycerol,
t~imethylolpropane, and a polyoxypropylene polyol polymer.
4. The isocyanate-capped liquid polyurethane prepolymer
is prepared by reacting toluene diisocyanate with an admixturP
of a polyethylene glycol having a molecular weight of
about 800-1,200`and trimethylolpropane, the trimethylolpropane
and the polyethylene glycol being provided in a mole ratio
of about 1:1-4 and.the toluene diisocyanate being provided
20 at a rate of about 0.85-1.25 mole per equivalent of -OH
provided by the polyethylene glycol plus the trimethylolpropane.
5. The cured product (the product.or article formed
by curing the isocyanate) which comprises the immobilized
group member is washed to remove nonimmobilized group
member and to hydrolyze any unreacted isocyanate groups.
6. The isocyanate-capped liquid polyurethane prepolymer
and the group member are admixed to form an intermediate
product which is cured and foamed by admixing it with an -
- 31 - .
-
~0~08
amount of water effective for curing and foaming the inter-
mediate product.
7. An aqueous solution of the group member and the
isocyanate-capped liquid polyurethane prepolymer are contacted
prior to a subsequent foaming step under foam-forming
condi-tions whereby the polyurethane foams and the~group ;~
member becomes immobilized. ~
8. The group member is a coenzyme having at least
one primary or secondary amino group per molecule, e.g.,:
nicotinamide-adenine dinucleotide,
nicotinamide-adenine dinucleotide phosphate,
flavin mononucleotide,
adenosine triphosphate,
flavin adenine dinucleotide r and
thiamin pyrophosphate.
9. The group member is an admixture of a coenzyme
A having at least one primary or secondary amino group per
molecule and an enzyme which is mediated by the coenzyme;
the following are illustrations thereof:
(a) the coenzyme is nicotinamide-adenine
dinucleotide and the enzyme is
alcohol dehydrogenase,
isocitric dehydro~enase,
~-glycerolphosphate dehydro~enase,
lactic dehyarogenase, or ,
~lyceraldehyde-3-phosphate dehydrogenase;
- 32 -
108~3008
(b) the coenzyme is nicotinamide-adenine
dinucleotide phosphate and the enzyme is
m~lic enzyme,
glucose-6-phosphate dehydrogenase,
5-dehydroshikimic ,reductase, or
glutathi,one xeductase;
~c) the coenzyme is flavin mononucleotide and
the enzyme is
glycolic acid oxidase,
yeast cytochrome c r'eductase,
- ' luciferase, or
nitro reductase ;
' (d) the coenzyme is adenosine triphosphate and
the enzyme is
glutam~l transferase,
glutathi.one synthetase,
g'lyco~cyamine 'phosphokinase,
hippuric acid synthetase, or
luci~erase;
(e) the coenzyme is flavin adenine dinucleotide
and the enzyme is
D-amino acid oxidase,
aldehyde 'oxidase, '
succinic dehydrogenase,
nitrate reductase,
xanthine oxidase,
lipoyl dchydrogenase, '~
diaphorase, ',
~lavin peroxidase, or
glycine oxidase; or
:
- 33 - ~
.,
10~008
.
~) the coenzyme is thiamin pyrophosphate and
the enzyme is
carboxylase, `
~-keto acid dehydrogenase, or
transketolase.
10. The group member is an antibiotic haYing at least
.
one primary or secondary amino group per molecule; the
following are examples of such antibiotics:
ampicillin,
bacitracin,
colistin, and
,~ .
neomycin.
11. The curing agent can be in liquid or vapor form
and it can be "neat" (undiluted) or it can be diluted with
a solvent where in the liquid state or with a gas where in
the vapor state. Typical curing agents include water or
an amine having zero to about 10 carbon atoms per molecule.
A Typical examples of such amine are
.ammonia, toluidine,
hydroxylamine, diethylamine,
hydrazine, ~eth~laniline,
diethylenetriamine dimethylamine,
propylamine, propylamine,
butylamine, dipropylamine,
ethylenediamine, dibutylamine,
aniline~ ahd the like.
Typical examples of inert gases useful for diluting
the curing agent where it is in the vapor state include
air, oxygen, nitrogen, hydrogen, carbon dioxide, methane,
argon, helium, xenon, and the like.
- 34 - -
- . . : . - ... : .
- . .
~OY38~OB
Typical examples of inert solvents useful for diluting
the curing agent are water (where the curing agent is an
amine), acetone, methyl ethyl ketone, methyl alcohol,
ethyl alcohol, a propyl alcohol, a butyl alcohol, dimethyl-
formamide, dimethyl sulfoxide, N-methyl-2-pyrrolidone~
methylchloroform, benzene, toluene, xylene, carbon tetra-
chloride, chloroform, hexane, petroleum ether, and the
li~e.
12. The group member is immobilizèd by a process in
which:
(a) the isocyanate-capped liquid polyurethane
prepolymer and the group member are admixed to form
an intermediate product; and
- (b) the intermediate product is cured and foamed
by admixing it with an amount of water effective
for curing and foaming the intermediate product.
13. The group member is immobilized by a process in
which an aqueous solution of the group member and the
isocyanate-capped liquid polyurethane prepolymer are
contacted prior to a subsequent foaming step under foam-
forming conditions whereby the polyurethane foams and the
group member becomes immobilized.
14. The resulting mixture can be cured under foam-
forming (foaming) or nonfoam-forming (i.e., nonfoaming)
conditions.
15. The curing agent can be water or an amine having 0-10
carbon atoms per molecule and at least one primary or
secondary amino group per molecule.
16. The resulting mixture can be cured under nonfoam-
forming conditions in which:
.
- 35 -
"; ,
~. ~ .. ~ ... ..
1~08
(a) the resulting mixture is admixed with an
inert volatile solvent and water to form an aqueous
system; and
(b) volatile components are evaporated from the
aqueous system.
The volatile solvent can be water soluble in
which case the aqueous system is a solution, or the
volatile solvent can be essentially water insoluble
in which case the aqueous system is an emulsion.
The aqueous system can be admixed with a solid carrier
in particulate form before evaporating volatile
components therefrom. Operable solvents include
acetone, methyl ethyl ketone, methyl alcohol, ethyl
alcohol, a propyl alcohol, a butyl alcohol, dimethyl-
formamide, dimethyl sulfoxide, N-methyl-2-pyrrolidone,
methylchloroform, benzene, toluene, xylene, carbon
tetrachloride, chloroform, hexane, petroleum ether,
and the like.
17. The group member can be immobilized under nonfoam-
forming conditions by admixing an aqueous solution of the
group member with an inert volatile solvent to form an
aqueous system and mixing the aqueous system with an excess
of the isocyanate-capped liquid polyurethane prepolymer to
immobilize the group member. I~here the inert volatile
solvent is water soluble the aqueous system is a solution
and where said solvent is essentially or substantially
; insoluble in water the aqueous system is an emulsion.
Operable solvents include but are not limited to acetone,
. . .
; methyl ethyl ketone, methyl alcohol, ethyl alcohol, a propyl
alcohol, a butyl alcohol, dimethylformamide, dimethyl
.
- 36 -
~0~008
sulfoxide, N-methyl-2-pyrrolidone, methylchloroform,
benzene, toluene, xylene, carbon tetrachloride, chloroform,
hexane, petroleum ether, and the like.
18. The protein can be:
~a) an enzyme, e.g.:
chymotryp~in 3.4.4.5, 3.4.4.. 6
ribonuclease, 2.7.7.16, 2.7.7.17
peroxida*e~ 1.1I.~.`7`
pepsin 3.4.4... 1
~enni~ 3 4 4 3
invertase 3.2.1.26
papain I 3.4.,4.10
asparaginase 3.5.1.1
pectinase ' 3.-2.1.15
pectin esterase 3.1.1.lI
penicill!~n,amidase 3.5.1.11
glucose isomerase- 5.3.1.5
lys~zyme, , 3.2.1.17
amino acid acylase 3.5.1... 14
pronase . 3.4.4.
aLcohol, dehydrogenase. 1.1.1.1., 1.1.1.2
a-amylase 3.2.1.1
~-amylase 3.2.. 1.. 2,
su~tiLisin 3.4.4:.16
amino acid oxidase 1.4.3.3, 1.4.3.2
, catalase 1.11.1.6 :
tannase 3.1.1.20
phenol.oxidase 1.10.3.1
glucoamylase 3.2.1.3
pullulanas~ 3.2.1.41
-,7-
.. I .
. ., ~
~b8
cellulase 3.2.1.4~
ficin 3.4.4.12
bromelain 3.4.4.24
pancreatin 3-4-4-4
isoamylase 3.2.1
lipase. 3.. 1.1.3
malic dehydrogenase 1.1.1.. 37
hexokinase 2.7.I.l
lactate dehydrogenase~ ~ 1.1.1.28:, 1.1.2.4
adenosine deaminase 3.5.4.4
uricas~e ~ 1.7.. 3.-3
galactose oxidase 1.1. 3.9
diaphorase 1.6.4.3
cholinesterase 3-.1.1.8
aldolase 4.1.2.7, 4.1.2.13
pyruvate carboxylase 6.4.1.1.
phosphorylase 2.4.1.1
cephalospQrin:amidase 3.5.1
isocitric dehydrogenase 1.1.. 1.41
a-glycerolphosphate dehydrogenase 1.1.99.5
glyceraldehyde!3-phosphate dehydrogenase 1.2.1.13
malic enzyme 1.1.1.38, 1.1.1.39r 1.1.1.40
glucose-6-phosphate dehydrogenase 1.1.1.49
S-dehydroshikimic. reductase 1.1.1.25
glutathione reductase 1.6.4.2 :
glycolic acld oxidase 1.1.3.1
yeast cytochrome-c reductase 1.6.99. 3
luciferase 1.. 2.3
nitrite reductase 1.6.6.4, 1.7.99. 3
glutamyl transferase 2.3.2.1
glutathione synthetase 6. 3.2.3
glycocyamine phosphokinase 2.3.2.1
-38-
,.,,~
,
-,.- ..
08
hippuric acid synthetase 6.3.2
aldehyde oxidase 1.2.3.1
succinic dehydrogenase 1.3.99.1
nitrate reductase 1.6.6.1, 1.9.6.1,
. 1.6.6.3, 1.6.6.2
xanthine oxidase 1.2.3.2
Lipoyl dehydrogenase 1.6.4.3
~ flavin peroxida3e - : 1.11.1 :
-~ ~ glycine~-oxidase 1,4.3
carboxyl~se 4.1.1.1
a-keto acid dehydroge~ase 1.4.3,
tran~ketolase. 2.2.1.1
(b) an antibody, e.g.: human`immunoglobulin G
or hepatitis antibody; or
- ~c): an antigen, e.g.: rheumatoid arthritis
.. . . . . .
factor. - - - .
19... The article produced ~product) can be a ~ilm
comprising the immobilized group member. Said film can be
prepared by a process in which:
(a) the resultiny mixture is applied to an
inert solid sur~ace and spread thereon to form a
,
--3q--
1~ ~
~1
- ;
1~8~808
film of the resulting mixture supported on the
inert solid surface; and
(b) the film of`resulting mixture is cured
by exposing it to an amount of the curing agent
effective for curing the resulting mixture and
immobilizing the group member.
The film can be stripped from the support
(inert solid surface), and if desired cut into
discs.
' 10 . If desired, a second film of the resulting
mixture can be applied to the cured film and
cured thereon and bonded thereto by exposing the
second film to an,amount of the curing agent
effective for curing the resulting mixture and
immobilizing the group member to lncrease the
thickness and strength of the cured film.
`Also, if desired, a film of isocyanate-
capped liquid polyurethane prepolymer can be
. applied to the,cured film and cured thereon and
bonded thereto (i.e., bound thereto) by exposing
the film of isocyanate-capped liquid polyurethane
prepolymer to an amount of ,the curing agent
effective for curing the film of isocyanate-
capped liquid polyurethane prepolymer to
strengthen the film of immobilized group member.
The resulting film can be stripped from the
support to yield a first laminate film comprising
a film of immobilized group member bound (bonded)
to a film of cured polyurethane prepolymer
which can be designated "Film A". A fully
equivalent film can be prepared by applying a
- 40 -
. :: :, :: :: ~ .
OQ8
film of the isocyanate-capped liquid polyurethane
prepolymer to the support surface~ curing the
prepolymer film, applying a film of the resulting
mixture to the film of cured prepolymer, curing
the film of resulting mixture to form a second
laminate film comprising a film of immobilized
group member bound to the film of cured prepolymer.
Stripping the second laminate film from the
support produces a film which can be designated
"Film B". Films A and B are fully equivalent and
... _ .....
the procedures by which they are made are fully
equivalent.
The inert solid surface used to prepare films comprising ;~
the immobilized group member can be any smooth inert solid
surface. Examples of such surfaces are pol-ymeric (plastic)
surfaces (e.g., polyolefin - including polyethylene and
polypropylene surfaces - Teflon surfaces, nylon surfaces,
polyvinyl chloride surfaces, polyvinyl acetate surfaces,
polyacrylonitrile surfaces, polystyrene surfaces, and the
like), glass surfaces, concrete surfaces, stone surfaces,
ceramic surfaces, metal surfaces (e.g., stainless steel
surfaces, mild steel surfaces, aluminum surfaces, zinc
surfacest copper surfaces, nickeI surfaces, chrome surfaces),
rubber surfaces, and the like.
. ~ . .
- 41 -
31.~ri~Q8
20. The resulting mixture and/or the isocyanate-
capped liquid polyurethane prepolymer can be diluted with
an inert volatile solvent which can be a water soluble
solvent or a solvent which is immiscible or substantially
immiscible in water. Typical solvents which can be used
for this purpose include acetone, methyl ethyl ketone, methyl
alcohol, ethyl alcohol, a propyl alcohol, a butyl alcohol,
; dimethylformamide, dimethyl sulfoxide, N-methyl-2-pyrrolidone,
methylchloroform, benzene, toluene, xylene, carbon tetra-
chloride, chloroform, hexane, petroleum ether, and the like.
21. The product (article produced) can be a tube
comprising the immobilized group member. Said tube can be
prepared by a process in which
(a) the resulting mixture is applied to the inner
surface of a pattern tube having an inner surface and
an outer surface to form a film of the resulting mixture
on the inne~ surface of the pattern tube; and
(b) the film of resulting mixture is cured by
exposing it to an amount of the curing agent effective
for curing the resulting mixture and immobilizing the
. . .
.: . , . .. : ~
. ,. ~ , ':, ," " .',
. , ,~ ,.
.: ~ .: .. , , . .: , . : :
:: .
-: :
. , ,, " : . ..
D08
group member to form the cured tube comprising the
immobilized group member, the cured tube having an
inner surface and an outer surface.
The tube comprising the immobilized group
member can be separated from the pattern tube,
e.g., by breaking the pattern tube if it (the
pattern tube) is made of glass, or by slipping
the tube comprising the immobilized group member
from the pattern tube.
If desired, a second film of resulting
mixture can be applied to the inner surface of the
cured tube comprising the immobilized group
member and cured thereon .and bonded thereto by
exposing the second film to an amount of the
curing agent effective for curing the resulting
mixture and immobilizing the group member to
increase the thickness and strength of the tube
comprising the immobilized group member.
Also, if desired, a film of isocyanate-
capped liquid polyurethane prepolymer can be applied
to the inner surface of cured tube comprising
the immobilized group member and cured thereon
and bonded thereto by exposing the film of
isocyanate-capped liquid polyurethane prepolymer to
an amount of the curing agent effective for curing
the prepolymer to increase the strength of the
tube comprising the immobilized group member.
22. The product can be a tube comprising the immobilized
group member. It can be prepared by a process in which:
_ 43 _
.
. .'
10l3~008
(a) the isocyanate-capped liquid polyurethane
is applied to the inner sur~ace of a ~irst tube which
is a pattern tube hàving an inner surf'ace and an
outer surface to form a film of the isocyanate-capped
liquid polyurethane prepolymer on the inner surface
of the first tube;
(b) the film of isocyanate-capped liquid poly-
urethane prepolymer is cured by exposing it to an '
amount of the curing agent effective for curing the
prepolymer to form a second tube comprising cured
prepolymer, the second tube having an inner surface ~
and an outer surface; ` ~,
(c) the resulting mixture is applied to the
inner surface of the second tube to form a film of ,
the resulting mixturë on the inner surface of the
second tube; and
(d) the film of resulting mixture is cured and
bonded to the inner surface of the second tube by
.
exposing the film of'resuIting mixture to an amount ' ~,
of the curing agent effective for curing the resulting
mixture and immobilizing the group member to form a
third tube comprising the immobilized group member ,
bonded to the inner surface of the second tube. ' ` '
The third tube can be separated from the -'
pattern tube.
The pattern tube (which can be a pipe) used to prepare
tubes comprising the immobilized group member can be a tube
o~ any inert material. Typical examples of such inert
materials are polyme'rs (plastics) such as polyolefins -
30 including polyethylene and polypropylene - Teflon, nylon,
polyvinyl chloride, polyvinyl acetate, polyacr,ylonitrile,
I '
~0~008
polystyrene, and the like, glass, ceramic materials, metals
(e.g., stainless steel, mild steel, copper, aluminum, zinc,
nickel, and tin), rubber, ànd the like.
- 23. The product can be a rod comprising the immobilized
group member. Such product rod can be prepared by a process
in which:
(a) the resulting mixture is applied to the
longitudinal surface of a pattern rod having a
longitudinal surface and two end surfaces to form
a film of the resulting mixture on the longitudinal
surface; and
(b) the film of resulting mixture is cured by
exposing it to an amount of the curing agent effecti~e
for curing the resulting mixture and immobilizing the
group member to form a product rod comprising the
pattern rod having on its longitudinal surface a coating
comprising the immobilized group member.
If desired, the coating comprising the
. immobilized group member can be separated from
the pattern rod, e.g., by breaking the pattern
rod if it is made of glass, or by slipping said
coating off the pattern rod to produce a tube
comprising the immobilized group member.
If desired, a second film of the resulting
mixture can be applied to the coating comprising
the immobilized group member and cured thereon
and bonded thereto by exposing the second film
to an amount of curing agent effective for curing
the resulting mixture and immobilizing the group
member to strengthen and increase the thickness
of the coating comprising the immobilized group
member.
- 45 -
!
" ~0~3~008
Also, if desired, a film of the isocyanate-
capped liquid polyurethane prepolymer ean be
applied to the coating comprising the immobilized
group member and eured thereon and bonded thereto
by exposing the film of isocyanate-capped liquid
polyurethane prepolymer to an amount of curing
agent effective for curing the isocyanate-eapped
liquid polyurethane prepolymer to strengthen
the coating comprising the immobilized group
member.
If desired, the eoating eomprising the im-
mobilized group member with the cured prepolymer
bound thereto or the coating comprising two
layers of immobiLized group member ti.e., tw~
layers of resulting mixture) can be separated
from the pattern rod to form a tube having an
A ' inner surface eomprising the immobilized group
member and an outer surface eomprising the cured
prepolymer or the immobilized group memb~er,
respeetively.
24. The product ean be a product rod eomprising the
immobilized group member prepared by a process in which:
(a) the isocyanate-capped liquid polyurethane
prepolymer is applied to a pattern rod having a
- longitudinal surface and two end surfaces to form
a film of isocyanate-capped liquid polyurethane
prepolymer on the longitudinal surface,
(b) the film of isocyanate~capped liquid polyure-
thane prepolymer is cured by exposing it to an amount
of the curing agent effective for curing the isocyanate-
- 46 -
.
1088008
capped liquid polyurethane prepolymer to form an inter-
mediate rod comprising the pattern rod having on its
lonqitudinal surface`a first coating of cured prepolymer, ;
the first coating having an inner surface adjacent to
the longitudinal surface of the pattern rod and an
outer surface; and
(c) a film of resulting mixture is applied to the
outer surface of the first coating and cured thereon
and bonded thereto by exposing the film of resulting
mixture to an amount of the curing agent effective
for curing the resulting mixture and immobilizing the
group member to form a product rod comprising the
pattern rod with a final coating thereon, the final
coating comprising the first coating with the second
coating bound thereto.
The product can be a tube comprising the immobilized
group member p~repared by separating the final coating from
the pattern rod to form said tube.
The pattern rod used to prepare rods comprising the
immobilized group member can be a rod or bar of any inert
material. Typical examples of such inert material are
polymers (plastics) such as polyolefins - including poly-
ethylene and polypropylene - Teflon, nylon, polyvinyl
chloride, polyvinyl acetate, polyacrylonitrile, polystyrene,
and the like, glass, ceramic materials, metals (e.g., stain-
less steel, mild steel, copper, and aluminum), rubber, and
the like. The rod can be hollow, e.g., it can be a tube or
pipe with or without capped ends or with one end capped.
.
- 47 -
10~ 08
25. The product can be a sphere prepared by a process
in which the sphere is formed and cured by feeding the
resulting mixture dropwise into a fluid comprising the curing
agent and permitting the resulting spherical drop to fall
freely through the fluid. The fluid can be a liquid or
a vapor, and it (the fluid) can: (a) consist of the curing
agentj or Ib~ consist essentially of the curing agent
d~ssolyed in an inert yolatile solvent (where the fluid
~s a li~uid); or (c) consist essentially of the curing agent
and an inert gas (where the fluid is a vapor (or gas)). If
desired, the resulting mixture can be diluted with an inert
volatile solvent to form a solution which is fed dropwise
into the fluid comprising the curing agent; where this is
done the inert volatile solvent can be vaporized while
curing the resulting mixture;
The drawings illustrate certain embodiments of the
instant invention.
FIG. 1 shows self supporting cured polyurethane film 1
comprising immobilized biologically active material (i.e.,
comprising the immobilized group member of the above Summary)
supported on inert solid surface 2 (a smooth surface).
FIG. 2 shows said film 1 after it (film 1) has been
stripped (separated) from said inert surface.
FIG. 3 shows self supporting discs 3 made of cured
polyurethane (cured polyurethane prepolymer) comprising the
immobilized biologically active material~
.FIG. 4 shows a tube made of cured polyurethane com-
prising immobillzed biologically active material 4.
FIG. 5 shows a transverse section of the tube of
FIG. 4, said tube bcing made of cured polyurethane com-
prising immobilized biologically active material 4.
- 48 ~
.. ..
lQ~08
FIG. 6 shows a tube made of cured polyurethane comprising
immobilized biologically active material 4 surrounded by
and laminated (i.e., bound or bonded) to a tube consisting
essentially of cured polyurethane 5 (i.e., cured polyurethane
prepolymer which does not comprise immobilized biologically
active material).
FIG. 7 shows a tube made of cured polyurethane com-
prising immobilized biologically a,-tive material 4 surround-
ing and bound (bonded or laminated) to a tube consisting
essentially of cured polyurethane 5.
FIG. 8 shows inert pattern rod 6.
FIG. 9 shows a rod comprising inPrt pattern rod 6
surrounded by a coating comprising cured polyurethane com-
prising immobilized biologically active material 4.
FIG. 10 is a column shown generally at 10 packed with
spheres 9 comprising cured polyurethane comprising immobilized
biologically active material. Spheres 9 rest on a bed of
glass wool 11.
FIG. 11 shows spheres 9 which are made of cured poly-
urethane comprising immobilized biologically active material.
FIG. 12 shows self supporting film 1 which is made ofcured polyurethane comprising immobilized biologically
active material bonded (bound or laminated) to film 12
which consists essentially of cured polyurethane.
FIG. 13 shows self supporting discs 3 which are made
of cured polyurethane comprising immobilized biologically
active material bonded to discs 13 which consist essentially
of cured polyurethane.
FIG. 14 shows lnert pattern rod 6 surrounded by a
first coating of cured polyurethane which comprises
- 49 -
~ 8008
immobilized biologically active material 4, the first
coating is surrounded by and bound to a second coating
consisting essentially of`cured polyurethane. 5.
FIG. 15 shows inert pattern rod 6 surrounded by a
first coating consisting essentially of cured polyurethane
5, the first coating is surrounded by and bonded to a
second coating of cured polyurethane comprising immobilized
biologically active material 4.
FIG. 16 shows foam 40 which is made of cured poly-
urethane comprising immobilized biologically active material.
FIG. 17 shows a reactor shown generally at 17.
Reactor 17 comprises:
(a) column 20 havin.g a lower end 22 and an open
. upper end 21;
(b) cap 23 having an upper portion 24 and an
open lower portion, the lower portion of the cap
covering and communicating with open upper end 21
A of column 20;
(c) inlet line.25 having an inlet port 26 com-
municating with upper portion 24 of cap 23;
(d) outlet line 30 having an.outlet port 31 t
communicating with lower end 22 of column 20; and
(e) cured self supporting polyurethane foam 29
comprising an immobilized biologically active material
selected from the group consisting of: (i) a protein;
(ii) a coenzyme having at least one primary or secondary
amino group per molecule; ~iii) an admixture of said
coenzyme and an enzyme which is mediated by said
coenzyme; and (iv) an antibiotic having at least one
primary or secondary amino group per molecule
positioned within the column, said foam beir~g foamed
in place in the column.
- 50 -
: . :. ..
: 10~08
.
Outlet line 30 can contain valve 32 (e.g., a stopcock).
Reactor 17 can be made of glass or of metal -(e.g.,
stainless steel or the like).
Foam 29 can be formed in place (in situ) in column 20
by:
1. Admixing the biological material (group member)
of the above Summary with an excess of the isocyanate-
capped liquid polyurethane prepolymer of said Summary to
form a resulting mixture (a first mixture or first admixture);
forming a second mixture by admixing the first mixture with
an amount of water effective for causing the second mixture
to foam and for immobilizing the biological material; and
placing the second mixture in column 20 while it (the
second mixture) is foaming.
2. By forming a mixture by admixing, in column 20,
the group member of said Summary, an excess of the isocyanate-
capped liquid ~polyurethane prepolymer of said Summary,
and an amount of water effective for causing the mixture to
foam and for immobilizing the group member.
3. By admixing the group member of the aforesaid
Summary with water to form an aqueous system and admixing
the aqueous system with the isocyanate-capped liquid poly-
urethane prepolymer of said Summary using a stoichiometric
excess of the polyurethane prepolymer and an amount of water
effective for causing foaming and for immobilizing the group
member. The aqueous system and the polyurethane prepolymer
can be admixed in column 20 or in an admixing apparatus
and transferred to column 20 while foaming.
Other methods for preparing foam 29 in situ in column
20 will, because of our disclosure, be rcadily apparent to
those skilled in the art.
- 51 -
` 10~8~08
Teehniques for using the product of this invention,
i.e., an immobilized biologically active material comprising
a foam, a film, a disc, a~tube, a sphere, or a rod (any of
whieh ean be made by the proeess of this invention) include,
but are not limited to, the following:
1. Passing an aqueous system comprising a reactant
through a foam comprising a cured polyurethane comprising
! ~ the immobilized biologieally aetive material or over a
surfaee eomprising a eured polyurethanè comprising the
. 10 immobilized biologically aetive material.
2. Adding to an aqueous solution eomprising a material
to be reaeted particles of foam eomprising cured polyurethane
compri~ing the immobilized biologieally aetive material,
or spheres, self supporting dises, partieles of tubing,
partieles of rod, or partieles of self supporting film
having a surface eomprising cured polyurethane comprising
the immobilized biologically active material.
Enzymes immobilized by the method of this invention
are useful in analytical chemistry and for synthesis
reactions. For example, urea can be determined by passing
a solution of urea through a column packed with urease
immobilized in cured polyurethane foam to quantitatively
eonvert the urea to ammonia which can be determined by
titration or by a colorimetric procedure. Spheres, dises,
tubes, or rods having surfaees comprising cured polyurethane
with urease immobilized thereon can be substituted for
the eured polyurethane foam having urease immobilized therein.
The apparatus (reactor) of Embodiment J can be used
where the immobilized urease comprises a cured polyurethane
foam.
~ 52 -
-- , ... ...
~: . . ~. ; . . .
~088()08
Sucrose can be converted to invert sugar by passing a
solution of sucrose over the cured polyurethane in which or
on which the urease has been replaced with invertase.
Similarly, these techniques can be used to hydrolize
organic esters by replacing the urea solution with an aqueous
solution comprising such ester and by replacing the immobilized
urease with immobilized lipase.
Where the enzyme is one that requires a coenzyme:
(a) both the coenzyme and the enzyme can be immobilized in
the cured polyurethane foam comprising the immobilized bio-
logically active material or on the surface of the cured
polyurethane article (film, disc, tube, sphere, or rod) com-
prising the immobilized biologically active material; or
(b) either the enzyme or the coenzyme can be immobilized and
the other can be present in the aqueous system containing
; the material (reactant) to be reacted.
For example, ethanol can be dehydrogenated by alcohol
dehydrogenase in the presence of the coenzyme ~-nicotine- ;
amide adenine dinucleotide ("~-NAD" which is also called
"NAD"). Thus: (a) both the enzyme (alcohol dehydrogenase)
and the coenzyme (NAD); or (b) the enzyme; or (c) the coenzyme
can be immobilized by the process of this invention and then
used to bring about the dehydrogenation of the ethanol.
Where both the enzyme and the coenæyme are immobilized
with the cured polyurethane a solution of ethanol is passed
over (or through) the cured polyurethane comprising the
immobilized enzyme plus the immobilized coenzyme. Where only
the enzyme is immobilized (bound) a solution comprising alcohol
and the coenzyme is passed over (or through) the cured poly-
urethane comprising the immobilized enzyme. Where only
10~3~08
the coenzyme is bound a solution comprising alcohol andthe enzyme is passed over (or through) cured polyurethane
comprising the bound coenYyme.
Immobilized (bound) antigens prepared by the method
. of this invention are useful for removing antibodies from
biological samples. For example, immobilized (bound)
human immunoglobuiin G (IgG) is useful for removing rheuma-
toid arthritis factor (an antibody) from human blood.
Immobilized (bound) antibodies prèpared by the method
of this invention are useful for removing antigens from
biological samples, For example, the antibody of hepatitis
can be immobilized (bound) according to the process of
this invention, and the resulting bound (immobilized)
antibody can be used to remove the hepatitis antigen from
blood (e.g., blood in blood banks). .
The bound (immobilized) biological material ~biologically
active material) of this invention has a long service life.
For example:
1. ~here the bound (immobilized) biological material
(the group member of the above Summary) is an enzyme, a
coenzyme having at least one primary or secondary amino
group per molecule or an admixture of such coenzyme and
an enzyme mediated by the coenzyme it (the immobilized
biological material) does not lose its activity even where
used for hundreds of hours
2. ~here the immobilized (bound) biolog~cal material
is an antigen (useful for removing an antibody from an
aqueous system) it will become spent ("saturated") when
it has taken up an equivalent amount of antibody. It
then becomes necessary to regenerate the bound antigen
- 5~ -
. :. . . : , .
lo~aos
~ i.e., to free it of antibody). This can be done by
washing with an aqueous regenerating solution ~e.g., by
passing such regenerating solution through a column packed
with the immobilized antigen and then washing the regener-
ating solution from the regenerated column). An aqueous
glycine hydrochloride solution (for example, 0.15-3 molar,
preferably about 0.5 molar) is an excellent regenerating
solution. Such glycine hydrochloride solution has a pH
of about 2.5.
3. Where the bound (immobilized) biological material
is an antibody (useful for removing an antigen from an
aqueous system) it will become spent (saturated) when it
has taken up an equivalent-amount of antibody. It then
becomes necessary to regenerate the bound protein (i.e., to
free it of antigen). This can be done by washing with an
aqueous regenerating solution such as the above described
glycine hydrochloride solution (e.g., by passing the regener-
ating solution through a packed column packed with the
immobilized antibody and washing the regenerated column
as above)-
4. Where the immobilized (bound) biological material
is an antibiotic it will become spent or partially spent
on extended use - i.e., it loses its antibiotic properties
or such properties become substantially reduced. Such
spent or partially spent immobilized antibiotlc can be
regenerated by washing with a sterile solvent such as
sterile water or sterile saline solution, or the like. For
example, sterile water can be passed through a column packed
with the antibiotic or rods, tubes, or films can be flushed
with sterile water.
- 55
.. ..
08
The instant invention will be bctter understood by
referring to the following specific but nonlimiting examples
and procedures. It is un~erstood that said invention is
not limited by these examples and procedures which are
offered merely as illustrations; it is also understood
that modifications can be made without departing from the
spirit and scope of the invention.
The examples were actually run.
The procedures, while not actually run, will illustrate
certain embodiments of our invention.
. . . . . . . . ,. . . . . . . .. : .. . . - .
., : , :. . : :: - .:
1~13008
'Example 1
(Preparation of Prepolymer)
An isocyanate-capped liquid polyurethane prepolymer
was prepared by reacting 2 milliequivalents (meq), ] g,
of a polyethylene glycol having an average molecular
weight of 1000 with 2.63 meq, 0.229 g, of toluene di~so-
cyanate. The resulting prepolymer was designated
"Prepolymer #1".
A replication of the above procedure was repeated
wherein said procedure was modified by using 20 meq of the
polyethylene glycol and 26.3 meq of the toluene diisocyanate
, _ . ,
to form a second lot of Prepolymer #1. Additional lots
were prepared as needed.
Example 2
(Preparation of Prepol~mer)
Two moles of a polyethylene glycol having an average
molecular weight of 1,000 (PEG 1,000) and one mole of
trimethylolpropane (T~P) were admixed and dried at 100-110C
under a pressure of 5-15 Torr to remove water. The resulting
dried mixture, which contained a total of 7 moles (119 g) of
reactive terminal hydroxyl t-OH) groups, was slowly added
(taking about an hour) to a vessel containing 6.65 moles
of toluene diisocyanate (TDI) while stirring the TDI and the
resulting mixture. The TDI and the resulting mixture in
the vessel were maintained at 60C. The resulting mixture
was stirred for 3 hours, while maintaining it at about
60C, after all of the PEG 1,000-TMP mixture had been added
to the reaction vessel. Then an additional 1.05 mole of
toluene diisocyanate was added and stirring was continued
for an additional hour while maintaining the stirred-mixture
- 57 -
10~ 8
at about 60C. Thus, a 10 molar percent excess o~ the TDI
was added to the PEG 1,000-T~P mixture. This assured that
all hydroxyl groups of the polyols (the PE5 1,000 plus the
20 TMP) were capped with isocyanate and that some chain e~tension
occurred because of crosslinking of the polyols with the
excess TDI.
The resulting liquid isocyanate-capped polyurethane
prepolymer which resulted was designated "Prepolymer ~2".
Additional lots of Prepolymer #2 were prepared as needed.
Example 3
(Preparation of Prepolymer)
An isocyanate-capped liquid polyurethane prepolymer
~-as prepared by reacting 31 g of glycerol (~lycerine)
with an amount of ethylene oxide to form 500 g of an
intermediate compound (a hydroxyl-capped polyether)
having an equivalent weight of about 500 (i.e., containing
~OUL 17 g of -OH ~roup per 500 g of intermediate compound).
Tnis intermediate compound was reacted with commercial
toluene diisocyanate using 1.05 mole of the toluenedi-
isocyana~e per 500 g of the intermediate compound. The
resulting isocyanate-capped liquid polyurethane prepolymer
was desi~nated "Prepolymer #3". Additional lots of Pre-
polymer #3 were prepared as needed.
Example 4
A solution of 10 mg Adenosine-5~Triphosphatc (ATP)
in 1.0 gm watcr was addcd to 1.0 ~m o~ Prcpolym~r #2.
The resultant solution was allowed to rcact at 280 while
stirring ~or about ~ive minutes. ~ftcr an additional
five minutes without stirring, the binding reaction and
~oam formation wcrc completc. Tl-e ~oam was washcd thor-
ougl~ly with watcr. The total wasll water was collccted
and analyzcd ~or ~TP by ultraviolct spectrophotomctry.
- 58 -
10~008
It was found that 9.5 mg of the ATP initially charged
was washed out of the ~oam by the wash water. In other
words, 5% of the ATP originally charged was bound to the
Dolyurcthane foam.
Exam~7le 5
A solution was made by mixing 20 mg of ATp with
2 ml of water for ten minutes until dissolved. A l.0 ml
portion o this solution was mixed with 1.0 gram of Pre-
polymer ~1. The resulting system began to foam and
within ten minutes foam formation was complete. The
resulting foam was thoroughly washed with water. The
wash water was collected and analyzed for ATP by ultra-
violet spectrophotometry. It was found that 4% of the
` ATP initially charged was bound to the polyurethane foam.
Example 6
A solution was prepared of 1.0 gram prepolymer #3
and 300 mg g-Nicotinamide-Adenine Dinucleatide (~-N~D).
After reacting ~or 15 minutes in a dry atmosphere, dis-
tilled wa~er was added and stirrcd for onc minutc. The
resulting product began to foam within 35 seconds and
foam ormation was complete within lO minu~cs. The re-
sulting foam was groun~ in a mortar with pestle until a
fine particle material was obtained. The particles were
then washed thoroughly in 100 ml of distilled watcr.
~ltraviolet absorption (260 m~) of the wash water showed
the prescnce of 218 mg NAD and,thcrcfore, 82 mg NAD was
bound to the foam. ~dditional washin~s of the foam with
small amounts of watcr showed no additional N~D was
washed out.
Ex~mple 7
An admixture was prcpared of 1.0 gram of Prepolymer ~2
with lO mg of Alcohol Dehydrogenase and 10 mg of
~-Nicotinamide Adenine Dinucleatide (~ D). ~fter react-
ing for 15 minutes in a dry atmosphere, 1.0 gm dis~illed
water was addcd to the product and stirred for one minute.
The product began to oam and within 10 minutcs foam or-
mation was complete. The rcsulting foam was thoroughly
washcd with water.
59
10~80C~8
The ~oamed system was then assayed a~ainst equal
portions of free enzyme and coenzyme by measuring the
decrease in concentration of an ethanol substrate by as-
saying aliquot samples from both reaction mixtures using
the dichromate titration method of Harger (J. Biol. Chem.
83, 197 [1950]).
It was found that the foamed enzyme-coenz~ne system
was enzymatically active, and the rate was 5% of that of
the free activity of the free system.
. Example 8
A mixture was prepared by mixing 1.0 gram of pre-
polymer #2 and lO mg of Adenosine-5-Triphosphate (ATP).
The resulting mixture was stirred while reacting at 250
~or 15 minutes. Water (1 gm) was added to the product
whi~ stirring. Thc resul~ing water-contailling systcm
began to ~oam and within 10 minutes foam formation was
completc. The foaM was then washcd thoroughly with water.
, The w~sh water was collectcd-and analyzed for ~TP by
ultraviolet spcctrophotometry. It was found that 7 mg
of the Al'P initially charged was washed out of the foam
by the wash wat;er. In othcr words, 30% of the ATP
originally charged was bound to the polyurethane foam.
Example 9
A mixture was prepared by mixing 10 mg o ATP with
1.0 gram of Prcpolymer #1 for 15 minutes in a dry atmos-
phere at ca. 250C. Then a l.0 gram portion of water was
added to the mixture at ca. 250C while stirring. The
resulting water-containing system began to foam and foam
formation was complete within 10 minutes. The foam was
thcn washed thoroughly with water. The wash water was
collect,ed and analyzed for ATP by ultraviolet spectro- -
photometry. It was found tllat 32% of the ATP initially
chargcd was bound to the polyurethane foam.
Example lO
A mixturc was prepared by mixing 1.0 gram of Pre-
pol~ner #l with 10 mg of Adenosinc-5-Diphosphate (ADP).
The resulting mixture was stirred for 15 minutcs in a
dry atmosphere (to prcvcnt foam formation due to atmos-
phcric moisture). Then a 1 ml portion of water was added
- 60 -
10~)08
while stirring. The resulting water-containing system
beg~n to foam, and within 10 minutes ~oam forma~ion was
complete. The resulting ~oam was cut up and thoroughly
washed with 100 ml o~ d H2O. The wash water was collected
and analyzed for ADP by ultraviolet spectrophotometry.
It was ound that 15% of ADP initially charged was bound
- to ~he polyurethanc ~oam.
Example ll
- Example lO was repeated using lO mg of Nicotinamide-
lO Adenine Dinucleotide Phosphate (NADP).
The foam had bound 20% of the initially charged
coenzyme.
Example 12
Example lO was repeated wherein said procedure was
modified by using lO0 mg of Thiamine Pyrophosphate and
0.9 grams of Prepolymer #l; and reacting at room moisture
for 15 minutes. The foam had bound 13~ of the initially
charged coenzyme.
Example 13
Example lQ was repeated except Flavin Adenine
Dinucleotide (FAD) was used as the coenzyme. O~ the
total coenzyme in the reactant step, 38% was bound after
washing.
Example 14
Example lO was repeated except Flavin Mononucleotide
(FMN) was used. A 33% binding level was achieved.
Example 15
A lO mg sample of ~-Nicotinamide-adenine dinucleotide
(~ -NAD) was mixed with one gram of Prepolymer #2 for 15
30 minutes in a dry atmosphere and then l ml of water was added
and mixed thoroughly. Foam formation was complete within lO
minutes. The foam was cut up and washed thoroughly. The
foam was assayed for activity using an ethanol solution
containing lO mg of free alcohol dehydrogenase.
The foam was found to be active in converting the
ethanol to acetaldehyde.
- 61 -
10~008
Example 16
A coenzyme bound to a non foamed cured polyurethane
was prepared in the follo~ing manner:
An admixture of two grams of Prepolymer ~2 with 27 mg
of the coenzyme Nicotinamide adenine dinucleotide was pre-
pared and reacted for 15 minutes. This material was then
mixed with two grams of acetone. The resulting solution
was poured into a petri dish and allowed to cure. The
resulting non foamed polyurethane bound coenzyme was washed
with water. The wash water was collected and analyzed for
Nicotinamide adenine dinucleotide by ultraviolet spectropho-
tometry. It was found that 28% of the Nicotinamide adenine
dinucleotide initially charged was bound to the non foamed
p~lyurethane.
Example 17
A coenzyme was bound to a non foamed polyurethane and
tested according to the general procedure of Example 16.
~owever, 15 mg of the coenzyme Flavin ~Iononucleotide was
used instead of Nicotinamide adenine dinucleotide.
It was found that 21~ of the Flavin Mononucleotide
initially charged was bound to the non foamed polyurethane.
ExamPle 18
A coenzyme bound to a non foamed polyurethane was
prepared in the following manner.
An admixture was prepared by mixing one gram of pre-
polymer as described in Example 16 with one gram of acetoneO
This mixture was then stir-added to an admixture consisting
of one gram of water and 20 mg of Flavin Mononucleotide. The
two liquids were mixed together until a clear liquid resulted
(1-2 min.). The liquid was then poured in a thin layer over
a glass plate and left to cure for 30 minutes. The non foamed
polyurethane was then removed from the plate~ cut up, washed,
and the wash water assayed for Flavin Mononucleotide by ultra-
violet spectrophometry.
It was found that 95~ of the Flavin Mononucleotide was
bound to the non-foamed polyurethane.
- 62 -
08
Example 19
A 40 mg sample of peroxidase was mixed with one gram
of Prepolymer #2. This sample was then added to 3 ml. of
acetone and mixed. The resulting solution was poured into a
petri dish, and the acetone was allowed to evaporate at room
conditions overnight while the prepolymer/enzyme sample cured
via room moisture. The material dried in a thin film. The
sample was washed and assayed for peroxidase activity using a
3~ peroxide solution containing p-phenylanaline diamine.
The sample was repeatedly active towards peroxide.
Example 20
A 1:4 acetone/Prepolymer #2 solution (weight basis)
was prepared by dissolving 4 parts of Prepolymer #2 in acetone.
This solution was poured into a petri dish and allowed to cure
for 16 hours by room moisture while the acetone evaporated.
A clear translucent sheet of polyurethane film was removed from
the plate. This film was then used to make polyurethane discs
by punching out circles on the film.
Example 21
Bound antibiotic discs were made in a similar manner
as Example 20. However, the prepolymer contained 10 mg of
ampicillin per 2 gm of prepolymer before being mixed with an
equal weight of acetone.
The discs were thoroughly washed and tested for
activity. The bound ampicillin discs and control were placed
on E. coli-inoculated agar plates. The E. coli growth spread
over the agar, however, not over or under the bound ampicillin
disc surface.
There was an absence of a zone of inhibition formed in
the agar due to the ampicillin being bound to the disc. There
was no growth on the surface of the disc due to surface bound
ampicillin. By contrast the control disc (no ampicillin) did
not inhibit growth of E. coli on the disc surface.
- 63 -
~0~ )8
,.
~xample 22
Bound urease polyurethane film and discs were made by
the following procedure: A 20 gram sample of Prepolymer ~2
was mixed with and dissolved ~n 20 grams of acetone to form
- a solution designated "Solution A".
A 100 mg sample of urease was mixed with one ml of
water until dissolved to form a solution designated
"Solution B". Solution B was added to Solution A at a
slow rate while stirring gently until all of Solution B
was added. The resulting mixture was then poured on a
glass plate, spread to form a film, and allowed to cure
for one hour. The resulting cured film was removed and
discs were punched from this film.
The discs were assayed for urease activity and found
to be active in a series of runs where using an aqueous
solution of urea as reactant. The urea was hydrolyzed.
- ~ . ~ ;. -.: : .
: . . ~ : : . ,.. ~ ;.
:
~: . . .
..
008
Example 23
A 10 gram sample of polyurethane prepolymer (Prepolymer ~2)
was admixed with one gram of Pluronic L-64 (a nonioni~
surfactant) to reduce viscosity. This mixture was then coated
on the inside of a 6" length of ~lass tubing - inter'or
diameter 4 mm - by use of a vacuum pulling the mixture into
the tube. The coating was cured hy pulling moist air
(relative humidity about 50%) through the tube by vacuum
suction~ The result was a glass tube with a cured nonfoamed
10 polyurethane coating covering its interior surface.
Example 24
The procedure of Example 23 was repeated except that
the Pluronic L-64 was omitted and a 10 gram portion of water
was mixed with a 10 gram portion of the prepolymer. While
foaming was taking place, the material was vacuum pulled
through a section of glass tubing. The result was a glass
tube that was completely coated on the inside with a thin
layer of cured polyurethane, with an open channel through
the tubing.
Example 25
The gener~l procedure of Example 23 was repeated;
however the Pluronic L-64 was used at a rate o~ 3 parts to
10 parts of prepolymer. Also, the tube was left to cure
slowly in a drying oven for 48 hours. The result ~7as a
glass tube which was completely coated on its inner surface
with c~red polyurethane, with an open channel througll the
tubing.
Exam~le~26
A sample of prepolymer, as described in Example 23
was vacuum-pulled through a glass tubing as described
in Example 23 leaving a thin layer of prepolymer coating
on the inner surface of the tubing. A stream of moist air
was pulled over the layer by vacuum for 18 hours. The
result was that a thin translucent film of polyurethane
was formed on the inner surface of the tubing.
10~8008
Example 27
The general procedure of Example 26 was repeated.
However, in this instance, after curing the prepolymer, a
second coating of prepolymer was applied to the first
(i.e., the cured) coat of prepolymer. This procedure was
repeated until a total of 6 coatings had been applied and
cured. On curing, each coat became bonded to (and was a
part of) the coat to which it had been applied. The glass
tube was broken and removed from the resulting tube consisting
essentially of cured polyurethane which was a translucent cured
polyurethane tube which could pass fluids without leakage.
Example 28
The general procedure of Example 26 was repeated.
However, a rubber tube having an inside diameter of 1/8
inch was used as a pattern tube. The product was a rubber
tube lined with a coat of cured polyurethane.
Example 29
The general procedure of Example 27 was repeated.
However, in this instance, the final (sixth) coat of
polyurethane prepolymer applied to the system contained
86 mg of jack bean urease. The resulting tube (the tube
consisting essentially of cured polyurethane (cured poly-
urethane prepolymer) which surrounded and was bonded to a
tube comprising cured polyurethane (cured polyurethane pre-
polymer) comprising immobilized jack bean urease was thoroughly
washed with water. A buffered (ph 7) urea solution, 80 mg~,
was passed through the tube. The effluent was titrated for
presence of ammonia. It was found that 33 ,u moles of NH3
were present per ml of effluent. This shows that urease was
bound (i.e., immobilized) in an active form.
Example 30 ;
Glucose oxidase was bound to polyurethane tubing by
:'
- 66 -
008
the general procedure of Example 29, except that 8 mg of
glucose oxidase was used in place of the urease of Example 29.
The tube was assayed for activity using a glucose solution,
90 mg %, and found to retain, through repeated testing, about
95~ of its initial activity where the glucose solution used
in the assay contained peroxidase.
- 66a -
008
Example -31
A 26 cm length of glucose oxidase/peroxidase bound
cured polyurethane tube made (with glucose oxidase/peroxidase
as biological material) by the general procedure of Example 29
was tested in an Autoanalyzer system for the determination
of glucose. Said tube was a tube of the type represented by
FIG. 5, i.e., it comprised an outer coat of cured poly-
urethane having an inner coat laminated thereto, the inner
coat comprising nonfoamed cured polyurethane comprising
immobilized glucose oxidase/peroxidase. Said tube was
found to be operable for the quantitative determination of
glucose in the Autoanalyzer system.
Example 32
An 8.5 length of peroxidase bound cured polyurethane
tubing (made by the general procedure of Example 30, but
with peroxidase as the biolosical material to be bound)
was assayed in an Autoanalyzer system, using free (unbound)
glucose oxidase. The tubing was found to be accurate for
the quantitative determination of glucose in the Autoanalyzer
20 system.
Example 33
A 120 cm length of glucose oxidase bound polyurethane
tubing was made by the general procedure of Example 30
except that the outercoat (i.e., the coat corresponding
to "5" in FIG. 6 which consisted essentially of cured
polyurethane (cured polyurethane prepolymer)) was made
from a mixture of prepolymer and acetone (1 part prepolymer
and 3 parts acetone). The last (fifth) coat consisting
essentially of isocyanate-capped liquid polyurethane
30 prepolymer (which was applied to and cured on the fourth
coat of cured polyurethane prepolymer) was admixed
with calcotone blue (a dye) before being applied. Said
fifth coat was cured and then coated with an isocyanate-
cappcd liquid polyurethane prepolymer comprising glucose
oxidase which was cured to form an inner coat (corresponding
to "4" of FIG. 6) comprising cured polyurethane (cured
polyurethane prepolymer) comprising i~nobilized glucose
oxidase. Said tube was tested and found to be operable for
the determination of glucose (where using free (i.e.,
40 nonbound (i.e., not immobilized)) peroxidase in the aqueous
solution comprising glucosc) in an Autoanalyzer system.
- 67 -
~0~08
Exa~ple 34
' A 60 cm length of urease bound polyurethane tube was
made by the general procedure of Example 29. In this
instance the tube was prepared by coating the interior
surface of a 60 cm length of glass tubing with 20 coats
of a prepolyme'r/acetone solution (one part prepoiymer +
three parts acetone)_ The solution was pulled through the
tubing to thoroughly coat. After applying each coat air
was continually passed through the tube to permit moisture
present in the air to cure the coat. After 20 coats were
applied and cured, a 2.7 mg portion of~ jack bean urease
was applied to the tubing in the final coat of isocyanate-
, capped liquid polyurethane prepolymer which was cured as
above to form a tube of the type represented by FIG. 6.
The resulting tube was washed and assayed for activity
in an Autoanalyzer system for the conversion of urea to
ammonia and found to be operable for the determination of
urea in the Autoanalyzer system.
Example 35
, A stainless steel rod (36" long; 1/16" diameter) was
dip-coated six times in 1:1 acetone/prepolymer solution
(Prepolymer #2). Each coat was allowed to cure in moist
air before the next coat was applied. The resulting
article was a steel rod coated with a coat consisting
essentially of cured polyurethane. The steel rod was
removed from the coating by soaking in water. The result
was a cured polyurethane tube (36" long with inside diameter
of 1/16").
Example 36
The same general procedure was followed as in
Example 35, except that a glass rod (12 cm long having
a 5 mm diameter) was usecl instead of a metal rod. The
resulting article was a glass rod coated with a coat
consisting essentially of cured polyurethane. The glass
rod was _eparated from the coating to form a tube con-
sisting essentially of cured polyurethane.
. .
- 68 -
1~88008
Example -37
The same genexal procedure was followed as in
Example 36 except that a quartz rod (30 cm long, 1.5 mm
diameter) was used instead of glass. The end product was
a 30 cm long cured polyurethane tube 1.5 mm inside diameter.
Example 38
A 36" glucose oxidase bound cured polyurethane tube
was made by the general procedure of Example 35. However,
before the 5 prepolymer/acetone coats are applied and
cured, a coat of prepolymer/enzyme material was applied in
a thin layer, covering the rod surface, and cured by
exposure to room moisture (i.e., moist air). The final
product (after removing the steel rod) was a tube of the
type shown in FIG. 6.
The tube was tested for activity in an Autoanalyzer
system using a glucose solution containing free peroxidase
and found to be operable~for determining glucose.
Example 39
A 36" peroxidase bound cured polyurethane tube was
made by the general procedu~e of Example 38. However, 7 mg
of peroxidase (rather than glucose oxidase) was included
in the first prepolymer~enzyme coat.
The product tube was tested for activity in an Auto-
analyzer syste~l using a glucose solution solution containing
free glucose oxidase and found to be active.
E~xample 40
A urease bound cured polyurethane tube was made in
the general procedure of Example 38, except the initial
prepolymer/enzyme coat contained urease at a 5% dry weight
basis.
A solution of 200 mg % urea was passed through the
tube at a rate of 1.75 ml/min.
The tube was found operable as a source of urease
for the determination of urea.
Example 41
A bound urease cured polyurethane tube . :,
was made by the general procedure of-Example 38, but
modified by using urease rather than.glucose oxidase.
~owever, after the polyurethane prepolymer/enzyme coat was
applied and cured, 14 coats ~rather than 5) of prepolymer/
acetone solution were applied and cured to make a thicker
- 69 -
.
10~3008
.
walled tube than in Example 38.
The product tube was found to be active towards a
urea solution (i.e., it hydrolyzed urea present in an aqueous
urea solution which was passed through it (the product tube)).
Example 42
A bound urease cured polyurethane tube was made by
the general procedure of Example 41. However, the enzyme/
prepolymer coat contained 25% urease on a dry basis.
The product tube was tested in a continuous run
which lasted for 480 hours by passing 34,000 ml of a
200 mg % urea solution through said tube. No loss of activity was
detected.
Example 43
Polyurethane prepolymer (Prepolymer #2) was dropped
from a size 20 syringe needle into a 4 foot high column filled
with a solution of 0.748 mg % ethylenediamine (EDA) in water.
The drop was allowed to fall while curing. Upon examination,
the product was spherically shaped.
Example 44
Two replications were run using the general method
of Example 43. However, said general method was modified
by using an aqueous solution containing: (a) 1.496 mg %
EDA in one replication; and (b) 2.992 mg % EDA in the other.
The results of these replications were indistinguishable
from those obtained in Example 43.
_ample 45
Polyurethane prepolymer (Prepolymer #2) was mixed
in a 50/50 weight ratio with acetone. This solution was
then dropped dropwise into a 4 foot long column reactor filled
with water. The solution clouded up near and on the top surface
of the water. From this cloud, small particles formed and
- 70 -
~8~08
fell slowly to the bottom of the reactor. The particles
were examined and found to be spherical in shape.
. ;~
- 70a -
~.$BOQ8
Example 46
A 10 mg sample of urease (Miles) was mixed with 1 gram
of Prepolymer #2. After 15 minutes 1 gram of acetone was
added thereto and the resùlting mixture was stirred until
a clear liquid resulted (ca. 1-2 minutes). The resulting
solution was dropped one drop at a time into a 4 foot
column filled with 0.748 mg % EDA in water. The top portion
of the column became cloudy and from this cloud streamers
of curing polyurethane prepolymer sank toward the bottom of
the column. As these streamers fell, the~ divided (broke
up1 into spherical particles. ~fter the particles settled
out, they were collected by vacuum filtration and washed
- with water. The spherical particles were then assayed,
after washing, for urease activity; a 20% activity retention
level was observed. The particles were then dried and
screened. The majority of the particles fell within a
0.5-1 mm range.
Procedure 1
An enzyme and an antibiotic can be bound (immobilized)
in the same article (cured polyurethane prepolymer comprising
a foam, a film, a disc, a tube, a rod, or a sphere); for
example:
A first admixture can be prepared by admixing 100 g
of an isocyanate-capped liquid polyuretnane prepolymer
(e.g., Prepolymer #1, Prepolymer #2, or Prepolymer #3),
5 g of invertase and 5 g of an antibiotic having at least
1 primary or secondary amino group per molecule (e.g.,
ampicillin, bacteracin, colistin, or neomycin). Said
first admixture can be cured to produce a foam comprising
the cured polyurethane prepolymer comprising immobilized
invertase and immobllized antibiotic - immobili7ed ampicillin,
immobilized bacitracin, immobilized colistin, or immobilized
neomycin - by admixing it (the aforesaid first admixture)
with water to form a second admixture using an amount of
- 71 -
.
~o~
water (e.g., 200 g) effective for foaming and curing the
prepolymer component of the second admixture and for forming a
cured polyurethane prepolymer foam comprising immobilized
invertase plus the immobilized antibiotic.
The thus produced foam, or a portion thereof, can be
packed into a column, or: (a) the first admixture can be
foamed in place in the apparatus of Embodiment J (Fig. 17);
or (b) the second admixture, while still foaming, can be
transferred to said apparatus (the apparatus of Embodiment J).
An aqueous sucrose solution (e.g., 100-10,000 mg%
sucrose) which has been contaminated with bacteria (e.g.,
e.g. E. coli) can be passed into the column which was packed
with the above described foam in an attempt to "contaminate"
or "innoculate" the apparatus, and allowed to stand therein
for 8-10 hours at about 25. Then sterile aqueous sucrose
solution (e.g., 100 - 10,000 mg % sucrose) can be passed through
the apparatus to Eorm invert sugar which will be substantially
free of (a) bacteria induced decomposition products; and (b)
said bacteria (E. coli.).
Where using a similar procedure with a similar foam
comprising immobilized invertase but not immobilized anti-
biotic the invert sugar product will be contaminated with
decomposition products (e.g., cellular debris, other sugars,
carbon dioxide, alcohols, etc.) and the bacteria (i.e., E. coli.).
Procedure 2
An ordinary sucrose solution which is slightly con-
taminated with bacteria such as E. coli. or bacillus subtilis
- 71a -
08
can be converted to invert sugar which is substantially
free of bacteria by passing such sucrose solution through
a column packed with cured polyurethane foam comprislng
immobilized invertase and one or more o~ the immobilized
antibiotics of Procedure 1.
;
, . . .. . ,, .:. .:::: ~i ~ -: :
.: - , -::, .
, . , . : . . :; , ,,; : :
- ,,: , : . ,. - :
.. : . .. . . :
. : :: . . : :: - : . i
. ~ . .. ~ : . : .. . - .
~0~008
A cured polyurethane prepol~mer film comprising an
immobilized antibiotic - the antibiotic being one which
tbefore being immobilized) had at least one primary or
secondary amino group per molecule) - can be used as a
dressing for wounds a~d abrasions on animals (including
mammals)to prevent or inhibit the development of infection
in such wounds or abrasions.
As used herein, the terms "immobilized" and "bound"
as applied to a biological material, i.e., a group member
as recited in the above Summary, means that the bound or
immobilized group member is no longer mobile where placed
in contact with a liquid in which it (the biological material)
is soluble in the free state and that it (the immobilized
biological material) is insoluble in said liquid. For
example, a group member dissolved in an excess of an iso-
cyanate-capped liquid polyurethane prepolymer exists in
the liquid stàte and is not immobilized; however, it becomes
immobilized when the liquid polyurethane prepolymer is
cured - e.g., by reaction with water or an amine. Likewise,
a group member dissolved in water is mobile and ~ot immobi-
lized. A pure water soluble enzyme, antibiotic, coenzyme,
antigen, or antibody - although in the solid state - is not
immobilized because, if l part of such solid group member
is added to about 5-lO0 or 10-50 parts of water such solid
enzyme, antibiotic~ coenzyme, antibody, or antigen will
dissolve and become mobile. A "bound" (immobilized) group
member (member of the group recited in said Summary) will
not ~issolve and become mobile if treated with such ratios
or quantities of water or with much larger quantities of
water. In the process of this invention the isocyanate-
capped liquid polyurethane prepolymer is the immobilizing
agent (binding agent) because it-brings about the immobili-
zation ~binding) of the biological material.
- 73 -
~L0~ 08
The term "mediated" as a~plied to an en~yme--coenzyme
reactall_(s) system means that the cc>cn~yr!e erfects, b~
acting as ~n intermediate between the enzyme and a rcactant
(or reac~a2lts), catalytic ac~ivity in the system ~7ith the
enzyme plus the coenæyme serving as the catal~st for a
- reaction involving the reactant(s).
The term "cure'.' as applied to an lsoc~T~nate-capped
lic~uid polyurethc~le prepo].~mer means tha~ s~ch prepolymer -
reacts with a mater;al called a "curing agent" to further
polymerize the prepolymer to produce a large water insoluble
nolecule ~7hich is a solid at 25. Water and. amines (primary
or secondary amines) are preferred c~lring agents.
.here immobilizing a biological material by the process
of this invention the reaction and manipulative steps are
conducted at temperatures below the denatura~ion temperature
of the biological materi.al or below 120, whichever is
lo.~er.
As used herein: (a) the symbol (~-) mea~s number;
(b) the symbol (") means inch or inches (thus 3" means
3 inches and 1/16" means 1/16 inch); (c) the symbol (%)
means percent; (d) the term tmg %) means milligram percent
i.e., 1 mg % = 10 ppm ); (e) (g or grn) means
gram~s); (f) ~mg) means mllligram(s); (a) (~l) means m.icron(s);
(h) (cm) r.eans centime~er(s); (i) (mm) means millimeter(s);
and (j) (m~) means millimicron(s)
~ 11 parts and rat].os are parts or rati~s by weic~ht
unless other~ise dcfined where used, and perc^r.t and
r.illigram percent are by weic3ht.
~ 11 temperatures are in degrces centigrade unlc~ss
otherwise defincd whcrc used. Thus 25 mcans 25 dcgrees
10~008
centigrade, and 120 means 120 degrees centrigrade.
As used herein the term "polyisocyanates" includes
diisocyanates.
The isocyanate-capped liquid polyurethane prepolymers
used in this invention contain at least two isocyanate groups
(reactive isocyanate groups) per molecule of prepolymer.
Isocyanate-capped polyurethane prepolymers are "liquid"
polyurethane prepolymers if they are free flowing liquids at .
40-70.
A foam which is not soluble in water is considered
to be a solid.
Unless another solvent is specified where describing
a solution, the solution is an aqueous solution (i.e., a
solution comprising water).
The term "free" where applied to an enzyme or co-
enzyme means that the enzyme or coenzyme is not bound (i.e., not
immobilizedj.
The films of polyurethane prepolymer, or such pre-
polymer plus biological material which are cured to form films
of cured prepolymer of films of cured prepolymer comprising
immobilized biological material are generally less than 5 mm
thick - generally about 0.5-1 mm thick.
As used herein the term "isocyanate-capped liquid
polyurethane prepolymer" means any isocyanate-capped
polyurethane prepolymer which is: (a) a free flowing liquid
at about 40-70C; or (b) can be dissolved in an inert solvent
to form a solution (containing about 1-99% by weight (or
about 10-90% by weight) of the prepolymer) which is a free
flowing liquid at about 40-70C.
Ppm (i.e. ppm) means parts per million by weight.
- 75 -
