Note: Descriptions are shown in the official language in which they were submitted.
~L2~8~
BAC~GR~UND OF T~E I~VENTION
Field of the Invention
This invention relates to a filter with a definite
pore size comprising fibrin and a process for the prepara-
tion thereof from fibrinogen. This invention also relates
to a size selective process employing the filters of the
invention.
DISCUSSION OF PRIOR ~RT
Formation of fibrin gels by contacting fibrinogen
with a coagulation enzyme has long been known~ It has been
observed that when a liquid is passed over the gel,
permeation of the gel increasingly becomes difficult -
sometimes to the point where permeation and passage of the
liquid are rapidly diminished or cease. It was believed
that the gel infrastructure was extremely fragile and that
the gel consisted of networks of channels and pores of
varying size which were highly changeable and highly
dependent upon and variable with liquid or liquid mixtures
passed thereover, especially one having solid particles.
It was therefore thought that such fibrin gel was
not useful in separating components where the separation was
effected solely on the basis of particle size.
Specifically, when investigating the fibrin
formation from fibrinogen the interest was directed to the
flow properties of fibrin gels. It has e.g. been shown
before that the flow properties through silica gel as well
as through agar and gelatin gels are such as for a viscous
-- 1 --
mlslLCM ~.r
~2~g~
flow, It has also been shown earlier that the flow through
a fibrin gel is dependent upon the ionic strength and
fibrinogen concentration in the preparation. In the
investigations made, the permea~ility coefficient (Ks) of
the fibrin gels was determined by Poiseulle's law as
follows:
(Darcy coefficient) ~s = Q . L . n (1)
t . A .~p
wherein Q is the flow through the gel in cm3, A is the gel
surface in cm2, ~p is the pressure difference in dynes/cm2
(=0.1 N/m2= 0.1 Pa), t is the time in seconds, L is the
length of the gel in cm and n is the viscosity in poise
(~0.1 Pa . 9). Moreover, Kozeny-Carman has shown that the
following relationship applies in a viscous or laminar flow
in a capillary system:
m = ~ . Ks (2)
CoS2 ~
0 wherein m is the hydraulic radius ( wettable surfaee
wettable cireumferenee
in cm, Ko is a factor decided by the geometry of the
capillaries, and 0 is the orientation (angle) of the
capillaries to the direction of flow, is the partial
share of liquid in the gel and r is the radius of the
capillaries in cm. ~ can be calculated by means of the
protein concentration and with a knowledge of the partial
specific volume of the fibrinogen which is 0.72. For gels
mls/LCM
.,f~
~LZ3~4~
of the type concerned here ~O and Cos 0 cannot be
calculated. In the theoretical calculations it has been
assumed here that the capillaries are cylindrical and
parallel to the direction of flow, which according to Madras
et al. brings the indicated formula to the following:
~ S
r = 2 m = ~- ~ (3)
The theoretical pore size is therefore 2r. By effec-tive
pore size we mean: the size at which particles of smaller
size pass through the pores and particles of larger siæe are
re~ained~ It has appeared from the tests that the clotting
t~me (time of gel formation) of the thrombin-fibrinogen
mixture, here called Ct, is directly proportional to the
flow (Q) through the gel. The flow (Q) has further been
found to be inversely proportional to the fibrinogen
concentration (C). Provided Q = 0, when 1 = 0
and Ct = 0, the equation (1) will have the following form:
- KS-c A~ P . t
wherein k is a constant which is dependent on pH, ionic
strength and calcium concentration and, moreover, is
characteristic of the enzyme used in the gel formation, and
Ct is the clotting time in seconds. The other symbols are
the same as in equation (1). The term t is omitted when the
flow is expressed in cm3/s. According to this equation the
~ls/LCM
~Z~8~
permeability coefficient ~s is thus directly proportional to
the clotting time Ct and inversely proportional to the
fibrinogen concentration.
By varying the pH between 6 and 10 the ionic
strength between 0.05 and 0.5, the calcium ion concentration
between 0 and 20 mM and/or ~he concentration of enzyme (e.g.
thrombin, "Batroxobin" or "Arvin") between 0.01 and 10 NIH-
units (or the corresponding units of other enzymes) per ml
solution and the fibrinogen concentration from 0.1 and up to
10 40 g/l, preferably between 1 and 10 g/l, gels with Ks-values
[calculated according to the equation (1)] between 10-7 and
10-12, preferably between 10-8 and 10-11, can be prepared.
Calculated according to the equation (3), the corresponding
average radii will be 0~03 - 9 ~m, preferably 0.09 - 2.8 ~m.
I~ FXIXI (a transamidation enzyme) and calcium ions are
present in the gel formation the stability of the gels will
be increased as covalent crosslinkings will arise between
the chains in the subunits o~ the gel matrix.
Thus, now it has been found according to the
invention that these fibrin gels can be used as a ~ilter.
The filter acc-ording to the invention is characterized in
that it is built of fibrin and the fibrin gel is in
association with a shape-retaining means which retains the
shape of at least one surface of said gel against
deformation when contacted by a flowing liquid.
The filter of the invention has substantially
uniform pores. By that i9 meant that the standard deviation
of pore size is less than 15 percent~ preferably less than
-- 4 --
mls/LCM
,.~'
~Z~8~
10 percent and in some instances less than 5 percent.
The pore size of the gel has9 moreover, been found
to be a function of the clotting parameters used in the
gels' preparation, i.e., the pore size is varied by changing
said parameters. The pore size is then proportional to the
clotting time.
It has now been discovered, in accordance with the
invention, that fibrin in gel form can be used as a filter
if means are provided to retain the shape of at least one
surface of the gel against deformation when the gel is
contacted by a flowing medium such as a flowing liquid
medium containing components to be separated. It has also
been discovered, quite surprisingly, that the gel has
~ubstantially uniform pore sizes and that these pore sizes
can be regulated simply by altering the process parameters
employed for the formation of the gel.
Specifically, it has been discovered if the gel is
in some way stabilized by a shape-retaining means, that the
gel structure is preserved and the uniform pores therein
function ideally as a filter mediùm.
Generally speaking,-the gel is brought in contact
with a shape-retaining means. The shape-retaining means can
be a foraminous member such as a foraminous sheet member and
is preferably disposed on or in association with an upper
surface of the gel, preferably in contact with the gel
either directly or through an adhesive or a graft. Since
the foraminous member serves to preserve the shape and
structure of the upper surface of the gel when the medium to
mls/LCM
~2~89~
be filtered contacts the same, the gel does not collapse,
thereby allowing the uniform pores thereof to function
ideally as a filter medium.
Foraminous members functioning as shape-retaining
means can have virtually any size and shape, although they
are preferably in the form of a sheet and preferably are
substantially co-extensive with the upper surface of the
gel. The foraminous sheet members can be in the form of a
fibrous network such as in the form of a woven or non-woven
or knitted fabric, the fibers of which can be natural or
synthetic.
When the fibers or a foraminous sheet member are
natural, they can be, for example, made of silk, wool,
cotton, cellulose, hemp, jute or the like.
~9 synthetic Eibers, there are contemplated in
particular fibers made of nylon, polyester, polyolefin,
fibers made of vinyl polymers, acrylics such as
polyacrylonitrile, rayon, to name a few.
The fibers generally have a thickness between 1 ~m
20 and 1000 ~m, preferably between 10 and 20 ~m, and are
disposed in relationship to one another to define openings
therebetween of between 0.01 and 5 mm, preferably between
0.05 and 1 mm, it being understood that the size of the
openings between the fibers of the foraminous sheet is not
especially critical, provided i~ allows passage therethrough
of the medium to be filtered. It is preferred that as much
fiber be in contact with or adhere to the gel as possible so
as to ensure maximum structural integrity of the surface of
the gel initially to come in contact with the medium to be
mls/LCM
..
~L2~ 0
filtered.
Instead of using a fibrous foraminous member, one
can use one made of wires, such as wires made of copper,
tin, zinc, aluminum, glass, boron, titanium, steel, stain-
less steel, etc. The wires function analogously to the
function performed by the fibers in providing structural
integrity to at least one surface of the gel, preferably the
upper surface or surface which is to be initially brought in
contact with a mixture to be filtered. The interstices
between the wires are of the same magnitude as the
interstices between the fibers of a woven, non-woven or
knitted Eabric serving as a foraminous sheet member. The
wires can be in the ~orm o~ a screen, wire mesh or an
expanded wire sheet and are preferably co-extensive with at
least one side of the gel, preferably the upper surface.
The gel has uniform pores but owing to the manner
by which the gel can be formed, can have uniform porPs over
a wide range. Preferably, the substantially uniform pores
of the fibrin gel have a theoretical pore size or diameter
20 in the range of about 0.003 to 1 ~m, more preferably 0.009
to 0.3 ~m.
The gel is formed by contacting fibrinogen with an
enzyme, especially a coagulation enzyme. Particularly
contemplated enzymes for use in forming a fibrin gel include
thrombin, Batroxobin, Arvin, Eccarin, Staphylocoagulase,
Papain, Trypsin, caterpillar venom enzyme, etc.
Generally speaking, the gel formation is effected
at room temperature, although temperatures from -3C up to
mls/LCM
~`
~l2~8~0
58C can be employed. Preferably, the temperature is in the
range of 0 to 40C.
It is preferred that the gel be formed by
contacting the fibrinogen with an enzyme in the presence of
calcium ions. The calcium ion concentration can be up to 20
mM. The presence of calcium ions is not required in all
instances. Where thrombin is employed as the coagulation
enzyme, the gel can be formed in the absence of a calcium
ion.
In forming the gel, there is generally employed
0.1 to 10-5 enzyme units per gram fibrinogen, preferably 10
to 10- enzyme units per unit weight fibrinogen. Following
~orlnation of the gel whose coagulation time is a function of
~he relative amount o enzyme to fibrinogen as well as the
concentration of calcium ion, the gel is preferably hardened
or set by crosslinking the components thereof by contacting
the gel with a crosslinking agent. Crosslinking agents
contemplated include bis-imidates such as suberimidate.
azides like tartryl di(~ -amino carproylazide), aryl
dihalides like 4,4-difluoro-3, 3'-dinitrophenyl sulfone,
glutardialdehyde, nitrenes, N,N'(4-azido-2-nitrophenly)--
cystamine dioxide, cupric di(1,10-phenanthroline), dithio
bis-(succinimidyl propionate), N,N'-phenylene dimaleimide as
well as polyethyleneimides and other bifunctional compounds.
especially those known to crosslink with epsilon lysine,
alpha amino groups, carboxy groups of aspartic and glutamic
acids, and hydroxyl groups of amino acids in the protein
chain (e.g. threonine and serine).
mls/LCM
41D
Bis-imidates which can be used include those of
the formula
O O
11 11
N3 - C - (C~2)n ~ C N3
wherein n - 3 to 15 especially 3 to 10.
Azides which can be used include substituted and
unsubstituted azides of the formula
~) N~12 ~ 2 (~
Il 11
CH30 - C ~(C~l2)a_2C - OC1~3
10 wherein n - 1 to 20 especially 1 to 15. Azides contemplated
include those having a hetero atom in the chain, especially
nitrogen. Also contemplated are hydroxy substituted azides.
Aryl dlhalides which can be used include those
having mono, poly and fused rings as well as rings joined by
a direct bond or through a methylene bridge or a sulfo
bridge. The haIogen of the halide can be fluorine,
chlorine, or bromine. The compounds can be substituted by
inert or functional groups such as nitro, or disulfide.
Contemplated compounds include those where a functional
group has replaced one of the halo substituents, e.g. nitro.
Compounds contemplated include ~0 ~ 2
F-y
N02
F
mls/LCM 9 N2-~ NH cll2c~l2-s-s-cH2c~l2-~l2
~b~,` ' N02
.~ ,
~2~89~
Especially contemplated is glutardialdehyde.
Generally speaking, the crosslinking agent is
employed in an amount of between 0.001% and 8% by weight,
preferably between 1 and 2~ by weight of the gel for 1-120
; minutes. Crosslinking is effected at temperatures of
between 10 and 40C, preferably 20 to 25C. After the
hardened or crosslinked structure is obtained, the gel is
usually washed free of extraneous material.
The gel in such hardened form is useful as a
filter, i.e., without any foraminous sheet material.
Preferably, however, the gel is formed on or in association
with a shape-retaining means such as a net, wire mesh or
other sheet material and while in contact with such shape-
retaining means is hardened by the use of a hardening or
crosslinking agent.
Preferably, the gel is supported on its upper and
lower surfaces by a shape-retaining means such as a
foraminous sheet or the like, whereby to insure that the gel
retains its shape during use as a filter.
This invention further contemplates a process for
separating~a first substance having a theoretical size of
0.003 to 1 ~m from a second substance having a larger size
which comprises passing a mixture of said first and second
substances over a filter comprising fibrin in gel form and
having pores of substantially uniform size, said filter
having means for retaining the shape of at least one surface
of said gel against deformation when contacted by a flowing
medium, wherein the effective pore size of said fibrin gel
-- 10 --
mls/LCM
~21~
is larger than the particle size of said first substance
and smaller than the particle size of said second
substance. Preferably, the pores of the gel have a
theoretical size of 0.009 to 0.3 mm.
-~ The filters of the inventiOn are important, as
they permit the separation of bacteria and viruses from
mixtures containing the same. The ability to regulate the
pore size and to achieve a gel of uniform pore si~e is an
important and critical characteristic of the filters of the
invention. These filters permit the separation of blood
components, the separation of components of blood plasma,
the removal of platelets from blood, the fractionation of
cells and cell fragments and the separation of high
molecular weigl1t protein aggregates. In addition, a variety
of particles such as latex, silica, carbon and metallic
particles may be separated over these filters. Components
which can be separated include those shown in the table
below:
TABLE
- _ ________________________
Material A Material from How Separ~ted Effective
Separated Which Mate-rial Pore Size
"A" is Separated Retained Eluted Range for
Filter
____________________________________________________________
Blood platelets Blood plasma X Below 1 ~m
Red blood cells " " X 1 ~m and below
Sendai virusCulture medium X 0.1 ~m and
below
" " " " X 0.2 ~m and
above
Liver mito-
chondriaCyto plasma X 0.5 ~m and
below
-- 11 --
mls/LCM
Table A Contd. 2 ~z~94~
Material A Material ~rom How Separated Effective
Separated Which Material Pore Siæe
"A" is Separated Retained Eluted Range for
Filter
____________________________________________________________
" " " " X0.5 ~m and
above
Adeno virus Culture medium X 0.05 ~m and
below
" " " XO.l ~m and
above
E. coli bacteria " " X l ~m and
below
FVIII complex High molecular X X0.05 ~lm and
weight material (h.m.w) (l.m.w) below
(h.m.w.) separ-
ated from low mo-
lectllar weight ma-
terial (l.m.w)
Blood leucocytes Blood plasma X l ~m and
below
Blood lymphocytes " " X l ~m and
below
Fibrin gel filters have above all the advantages
over other gel filters that ~he pore size can be simply
varied as desired. Moreover9 the present filters have high
flow rates at such pore sizes as can be used to remove very
small particles, such as virus particles. In this respect,
the filters of the invention are more suitable than known
membrane filters and filters of polyacrylamide gels. The
absence of absorption of protein on the filters is also an
advantage as compared with certain other filters~
DESCRIPTION OF SP~CIFIC EMBODIM~NTS
The process according to the invention for the
- 12 -
mls/LCM
'1-'
r
9~0
preparation of gel filters is as described above,
characterized in that a fibrinogen solution with
preadjusting clotting parameters is mixed with a coagulation
enzyme and the resulting mixture is made to clot in a form
intended for the filter. It may be convenient to strengthen
the fibrin gel fo}med during or after clotting by a shape-
retaining means of greater strength than the gel which is
preferably applied to the upper surface of the gel to be
prepared and preferably to both the upper and lower surfaces
thereof.
The shape-retaining means (reinforcing meshes) are
preferably in the form of a net which is applied at the
lower and preferably also at the upper surface in the mold
in whlch the ~ibrino~en mixture is poured (cast). This mix-
ture can preferably penetrate the net (foraminous sheet
material) e.g. have its surface 10 ~m-5mm, preferably 0.5-2
mm, from the net. The net can have a mesh width, for
example, 10 ~m to 5 mm, preferably 50 ~m-lmm and the wire
diameter can be, for example, 0.01-1.0 mm, preferably 0.1-
0.5 mm, where wires are employed as a shape-retaining means.
In addition to the metallic wires noted above, wires of
natural fibers and plastics can also be employed. The
filter can also be reinforced in other places than at the
surfaces. It can, for example, be built on a foam material,
such as a plastic foam, which can support part of the entire
filter.
The filters of the invention, especially in a non-
hardened or crosslinked form, should not be subjected to
- 13 -
mls/LCM
23L~39~
temperatures in excess of 100C, as such heat sterilization
tends to destroy the gel structure. It is, therefore,
necessary in utili~ing the filters for biological processes
to prepare them sterilely from the beginning. On the other
hand7 it has been found possible to harden or crosslink the
filter during the preparation by carrying out the gel
formation in the presence of Factor FXIII and calcium ions.
Where Factor FXIII is to be present, it is preferably
present, it is preferably present in an amount of at least 5
units per gram fibrinogen, preferably at least 50 units per
gram. Calcium is present in a concentration of at least 20
mMO
A still stronger filter is obtained by effecting
crosslinking with one of the above-mentioned crosslinking
a8ents especially a dialdehyde and particularly one of the
Eormula OCH-R-CHO, wherein R is an alkylene group of 1 to 8
carbon atoms, such as glutardialdehyde. The filter obtained
in this way can be heat treated in an autoclave and
consequently sterilized.
The clotting parameters are above all the enzyme
concentration, e.g. between 100 and 3000 NIH units/L for
thrombin and for the fibrinogen concentration between 0.1
and 70 g/l, preferably between l and 10 g/l, increased
concentration giving a tighter gel. A tighter gel has a
smaller pore size. Increased ionic strength also provides a
tighter gel as weIl as a higher pH. It is preferred to
carry out the gel preparation using a gel mixture having a
pH o between 5.5 and ll, preferably between 6 and 9, and an
- 14 -
ml~/LCM
~ , .p~
,~
. ~ , ~
~2~39~
ionic strength between 0.05 and 0.5. Gels formed at calcium
ion concentrations between 0 and 20 mM are tighter.
Pore size is also affected by the temperature at
which the clotting (gelation) is effected. A lower
temperature of gelation means an increased clotting time,
which in turn means that the resul~ant gel has a larger pore
size. As a result of its larger pore size, it provides a
greater rate of flow.
The gel of the invention can be used other than as
a filter. One can dispose catalytically active substances
such as catalytically active enzymes or catalytically active
meta]s withill the pores and thus use the pores' structure as
a catalyst. The ~ilter, therefore, can act more or less as
a catalyst support for the catalytically active agent dis-
posed therein. When the catalytically active agent is dis-
posed within the pores, the resultant structure can be
employed as a siæe selective catalyst converting only those
components whose size is such as to freely pass through the
pores of the catalyst support. Those materials retained in
the surface of the gel are not catalytically converted.
By such a filter,- one can conveniently effect
enzymatic conversions, especially when the enzyme is
immobilized within the filter covalently, ionically or
otherwise~ Since the gel structure is formed by the use of
an enzyme, the filter of the invention's chemical components
is compatible with the enzyme being employed as an enzyme
catalyst. Thus, one can use the filter of the invention for
any of the following enzyme conversions when the same con-
mls/LCM
.~'
~L218~
tains the appropriate enzyme to effect that enzymaticcatalysis: for reactions involving various oxido-
reductases, transferases, hydrolasesl lyases, isomerases and
ligasis (synthetases). Hydrolases which have capacity of
degrading the protein strands in the gels cannot be used.
The method by which the enzyme or other catalytic
component is disposed within the filter, i.e., within the
pores of the filter, depends upon the nature of the enzyme.
Preferably it is disposed by the use of a known enzyme
immobilizing agent followed by washing of the filter to
remove extraneous materials.
One can also dispose reactive cellular components
within the pores of the gels. Upon reaction, low molecular
welght components may be released and subsequently eluted
from the gels. An example of such a type of reaction ls
production of interferon by leukocytes after their reaction
with Sendai virus. As shown in this invention, both of
these components can be disposed within the pores of the
gels.
BRI~F DES~IPTION OF DRAWINGS
The invention is described more in detail with
reference to the enclosed drawing, in which:
Fi~. 1 a shows molding of a filter according to
the invention;
Fi~. 1 b shows the filter arranged for filtering;
Fi~s. 2 a and 2 b show graphs of the flow as a
function of the coagulation time at different pH with
thrombin and Batroxobin, respectively;
- 16 -
mls/LB
~.' ..
Fi~, 3 shows the turbidity of the fibrinogen
solution (fibrinogen) as a function of the time after
addition of thrombin.
Fi~s 4 a and 4 b show the flow as a function of
the coagulation time at different ionic strength of thrombin
and Batroxobin, respectively;
Figs. 5 a and 5 b are graphs showing the relation-
ship between protein concentration in the gel forming system
and flow-rate.
Figs. 6 a and 6 b show the temperature plotted
against the coagulation time and the flow, respectively, as
a function of the coagulation time at different
temperatures;
Fig. 7 shows the t~rbidity of the effluent
(turbidity in the effluent) in % as a function of the pore
diameter in ~m;
Figs. 8 a, 8 b and 8 c are graphs plotting
activation of fibrinogen and clotting time (Ct).
In order to more fully illustrate the invention
and the manner of practicing the same, the following
examples are presented.
E~AMPLE 1
M~THODS AND MAT~RIALS
Human fibrino~en, ~raction 1-4 (7) was obtained from IMC0,
Stockholm, Sweden. The preparation, either a freeze-dried
powder or a wet paste, was 97-100% clottable (as determined
spectrophotometrically). A solution being Q.3 M in NaCl and
2% in protein was prepared. This solution (50ml) was
- 17 -
mls/LB
~`'` ' .
:~2~L8~
dialyzed agains~ 0.3 M NaCl at 4 for 3 hrs. with changes of
outer fluid (5 litre) every hour. This dialyzed solution
was further diluted with deareated Tris-imidazole buffer (8)
of pH between 6.5 and 8.2 to pro~ein concentrations between
1.2 and 5.0 g/l. In the final dilutions the concentrations
of each Tris and imidazole was 0.02 M. When necessary
increase in ionic strength was achieved by inclusion of
sodium chloride in the buffer. In order to inhibit any
trace of plasmin which may be generated, Trasylol (Bayer AG,
Germany) was added to a concentration of 5 KIE/ml to all
buffers and dialysis fluids.
In the gelation experiments the following
procedures was employed: To 3.65 ml of fibrinogen solution
in a plastic tube was added 70 ~l o~ 1 M CaCl2 solution,
immediately followed by 50 ~l of thrombin or Batroxobin
solutions of varying concentrations. This mixture is
called Reaction Mixture. The tubes are rapidly inverted
twice and transfer}ed to the gel cup or to the
spectrophotometer cell within 10 seconds after addition of
enzyme. The further handling is described under separate
paragraphs.
Thrombin. In most experiments a bovine preparation prepared
as previously described (9) was used. Specific activity:
100-200 NIH units per mg. Control experiments with highly
purified specific activity: about 2000 NIH units per mg
human thrombin ~10) was performed in some instances.
Batroxobin (from Bothrops marajoensis) was obtained from
Pentapharm AG, Basel, Switzerland. Specific activity: 505
BU per mg.
- 18 -
mls/LB
, ~ .
.. . .
Hirudin was also obtained from Pentapharm AG. Specific
activity: 1000 ATU per mg.
Rea~ents. All reagents used were of analytical grade.
PRLPARATI~N O~ G~L COLUMN
A solution of thrombin is added to a solution of
fibrinogen in a tris-imidazole buffer containing calcium
salts with a p~l of between 6.5 and 8.2 and an ionic strength
between 0.1 and 0.3 so that the final concentration is
between 0.05 and 2.5 NIH-units per ml. In other tests
Batroxobin is used to obtain gel formation in a
concentration between 0.27 and 3.6 BU per ml. The
concen~ration of "Tris" and imidazole salts is each 0.02 M
and the concentration of calcium salt is also 0.02 M. The
variation in ionic strength is obtained by addition of NaCl.
Gels are also prepared at calcium ion
concentration between 0 and 20 mM. With reduced calcium ion
concentration the opacity of the gels is increased. When
thrombin is used to achieve the gel formation the clotting
time (Ct~, also in the absence of calcium ions is directly
proportional to the flow rate and thus also to Ks. When
"Batroxobin" is used for the gel formation, the stability of
the gels in the absence of calcium ions is unsatisfactory,
which makes flow measurements more difficul-t.
After addition of an enzyme such as thrombin or
Batroxobin, the solution is rapidly mixed and then poured
into a cup, e.g.,such as one shown in Fig. la. It is made
of acrylic plastic and has an inside diameter of about 14 mm
-- 19 --
mls/LB
;'~
8~
and a height of about 27 mm. The plastic cup is shown in
Fig. la and the lower part of the cup is provided with a
nylon filter having a mesh size of 80 x 80 ~m. This filter
is fastened by a plastic guard ring. A film layer~ e.g.,
"Parafilm ~", is preferably applied at the lower portion so
that liquid is prevented from leaking out of the cup.
Immediately after introducing the solution into the cup, a
silk net with the mesh size 150 x 180 ~m is adapted at the
upper end and is fastened with a guard ring. The liquid in
the gel cup can e.g., be about 1 mm over the net surface.
The cup is left at room temperature for at least 2 hours for
complete gel formation, preferably in a place free of
vlbrat:lon 8 .
After this time, the film is removed at the lower
portion and the cup with its contents of gel is placed in
the holder A according to Fig. lb. A holder B is applied
over the upper end of the cup 1. At the upper end of the
holder B there is an opening as well as at the lower end of
the holder A. The holder B is filled with liquid (buffer or
water) and a rubber cork provided with a tube, which is
connected with a rubber hose is inserted into the opening.
The rubber hose is connected with a container for permeation
solution which is allowed to fill the rubber hose without
air bubbles. The container (not shown in the drawing) is
placed at such a height that a suitable 1OW is (other
materials can also be used such as nylon and polystyren)
- 20 -
mls/LB
~Z~ 41~
obtained through the gel. The hydrostatic pressure is
varied at different tests between 4 and 40 x 103 d~nes/cm2.
The fibrinogen used for the preparation of the
gels contains trace amoun~s of factor XIII, which is a
transamidase. In the presence of this enzyme and calcium
ions, covalent intermolecular cross linkages between chains
in the molecule units of the fibrin gel are Eormed. This is
especially the case when thrombin is present as thrombin
activates factor XIII.
An electrophoretic analysis of reduced fibrin from
various gels in the presence of sodium-dodecyl sulphate
shows that a complete cross-linking of the ~ and ~ -chains
of the fibrin takes place in the presence of thrombin. A
partial cross-linking takes place in the presence of
Batroxobin. The covalent cross-linkings formed in the
presence of factor XIII contributes to the stabilization of
the gel structure.
The silk net applied to the top of ~he gel cup and
wnich is in intimate contact with the gel matrix is of great
importance for the mechanical stability of the gels. With-
out this net or some other means for preventing collapse of
the gel, the gel compound is destroyed in the flow tests,
the gel collapsing in the central portion and a conical
inward bend arising.
The silk net can, of course, be replaced with
other nets, e.g., o~ cotton, nylon, iron or copper, which
~ 1~ 2
also stabilize the gel structure at pressures up to 40/~ynestcm .
Turbidity measurements
-
mls/L~M
., ~,
~2~9~
In parallel to the flow s~udies, the turbidity
profile of the system was determined under identical
conditions. In these experiments the Reaction Mixture (see
under Fibrinogen) was poured into a cuvette (5 ml) ofa
recording spectrophotometer (Beckman Acta III) and the
turbidity (optical density) recorded at 450 nm. After a
lag-phase there was a rapid increase in turbidity (cf. Fig.
8) which was accompanied by gelation. A tangen~ was drawn
to the steepest part of the sigmoidal curve. Its
intersection with the time axis is defined as the gelation
or clotting time (Ct). (Ct is about the same as the time
~or visually observed turbidity increase in the gel cup.)
In addition to Ct also maximum turbidity (OD-max) and rate
of turbidity increase ( ~ OD/min) was recorded. The time
required for gelation to reach completion was judged from
the turbidity curve. This time ran8ed from 1 hr. to 2 hrs.
for the high and the low enzyme concentrations
respectively.
Determination of fibrinopeptides and cross-linkin~
2Q Reaction Mixtures (see under Fibrinogen) were
prepared in several identical tubes. One of them was use~d
for turbidity measurement as described above. The other
tubes contained each 1 ml of Reaction Mixture. The reaction
in the latter tubes was quenched at different times by
addition of hirudin (2 ATU/ml) and an equal volume of 8 M
urea. Thereafter the fibrin (ogen) was precipitated by
addition of an equal volume of chilled ethanol. The
mixtures were kept on ice-bath for 2 hrs. and thereafter the
- 22 -
mls/LCM
~' .
.
~2~
precipitates were secured by centrifugation, dissoLved in
urea and used for SDS-gel electrophoresis. The supernatants
were used for radiolmmunoassay (RIA) of FPA, FPB and B ~ 15-
42 was assayed using the recently developed method of Kudryk
et al.
Viscosit~ was determined with a viscometer type Ubbelohde,
having a flow time for water of about 290 sec at 25C. It
was calibrated against a standard (CNI. Cannon Instrument
Company, Pa~ USA). Density was determined with a 5 ml
pyknometer.
Pore size. The equation for calculation of average pore
size of membranes (18) and acrylamide polymer gels (4) was
applied:
r = ~ ~ (2)
where r is the average pore radius (in cm), and îs the
fractional void volume of the gel, i.e., the fractional
volume of liquid in the gel. iS calculated on the basis
of protein concentration assuming a partial specific volume
for fibrinogen of 0.72 (l9), iS in this case the
fractional voi~ volume for gels in which no water is bound
to ~he gel matrix. However~ the degree of hydration of
fibrinogen in solution has been reported as high as 6 g per
g protein (20). Assuming that this water is retained by the
mls/LCM
~i"; ~.,.
. ~,
9~
gel matrix we also calculated for such hydrated gels.
Diffusion coefficient. The apparent diffusion coefficient
of water in the gel was calculated from Ks acco~ding to
Ticknor (21) and White (4).
D R T ~s
~.v. n
where D is the diffusion coeEficient (in cm2/sec.). R is
the gas constant (in ergs/mole-degree), T is the absolute
temperature (in ~) and V is the molar volume of the
permeflnt (in cm3/mole).
Ionic stren~ths were calculated on basis of the molarity of
the electrolytes. Activity coefficients and degree of
calcium binding to protein were not taken into account.
Least square analysls was used for calculation of
correlation coefficients, slopes and intercepts. All lines
shown in figures were drawn ac`cordingly.
~ESULTS
Preparation and stabilit~ of ~els.
The flow studies were performed on gels which had
been ormed at ambient temperature. The average temperature
was 24 ~ 2. However, in each series of experiments the
variation in temperature never exceeded 2. Preliminary
experiments suggested that this variation in temperature has
a negligable effect on the Ct of the system. In the
permeation experiments, when not otherwise stated, the flow-
rates were corrected to 25.
- 24 -
mls(LCM
~LZgL8940
The silk net at the upper end of the gels
stabilizes the gel structures. Without support of the silk
net, the gels will yield to flow at the pressure applied
(about 7 x 103 dynes /cm2~. The yielding is only noted at
the center of the gel, since the gel matrix adheres firmly
to the walls of the plastic cup.
The nets in the column do not significantly reduce
the flow-rate of liquid in columns without gels. We,
therefore, assume that also when the nets are in contact
with gels they do not restrict the area available for flow.
Before a flow experiment was star~ed, the extent
of incorporation of fibrinogen into the gel matrix was
~etermined. This was done by determining the protein
content in the void volume of the column (about 4 ml). The
amount of protein, as measured spectrophotometrically using
the extinction coefficient of fibrinogen (22), ranged
between 1 and 3% of the total protein used for gelation.
When deemed necessary the non-clottable portion was taken
into account in calculations of the fibrin content of gels.
The effect of changing permeant on the flow-rate
of gels was studied in some experimentsO A representative
series of experiments is shown in Table I. The gel was
first percolated with buffer of ionic strength 0.21
(experiment I). On changing the permeant to water
(experiment II) an increase in flow-rate occurred, which is
larger than expected on the basis of the viscosity change of
the permeant. On return to the original permeant
(experiment III) the flow-rate decreased, but not completely
- 25 -
mls/LCM
.
9~
to the original value (experimen-t I)o When buf~er of ionic
strength 0.36 was percolated through the gel (experiment IV)
a small decrease occurred which was almost as expected on
the basis of the difference in viscosity be~ween the two
buffers. When the permeant was again changed to water
(experiment V) the flow-rate increases to almost the same
value as after the first change to water (experiment II).
These results suggest that the final gel structure is not
influenced by moderate changes in permeant composition, but
10 changes may occur on drastic changes in ionic environment
and these are not completely reversible.
,1
.~ TABLE I
J Flow Properties of Fibrin Gels with Different
Permeants.
Gel formation: Tris-imidazole buffer pH 7.4, ionic strength
0~21, thrombin 0.8 NIH-units/ml, temperature 21 and
j fibrinogen concentration 2 mg/ml.
i Permeation: at 22 - 23.5.
~ 20 E~periment Permeant Flow, ml/hr %
____________________________________________________________
I Tris-imidazole 3.177 100
pH 7.4 ~ 2 0.21
II ~2 3.708 117
III Tris-imidazole 3.385 106
pH 7.4, r/2 0.21
IV Tris-imidazole 3.271 103
pH 7.4, r/2 0.36
V H20 3.649 115
- 26 -
mls/LCM
.~ ~
4~
Flow pattern throu~ fibrin gels
Viscous flow. In order to test if the flow obeyed
Poiseuille's law, the flow-rate at different pressures (4.5
- 5.6 x 103dyne/cm2) for gels formed at pH 7.4, ionic
strength 0.21 was determined~ at three different thrombin
concentrations (0.1-0.8 NIH units/ml). The ~s-range for
these gels was 10-8 to lO-l. Permeation was in one
experiment with the same buffer as above and in the other
cases with water. In all cases the drop-rate decreased
linearly with decreasing pressure. As shown in Table II for
one of the gels, the flow-rate per unit pressure was almost
independent of total pressure.
In another series of experiments the flow-rates
were determined with permeants of different viscosities.
The gels used in these experiments were formed at pH 7.4,
ionic strength 0.21, at four fibrinogen concentrations.
Thrombin as well as Batroxobin were used as inducers of gel
formation. Permeation was performed at five different
temperatures between 4.5 and 40. In all cases there was a
linear relationship between the inverse viscosity of the
permeant and the flow-rate. These experiments suggest that
the flow through the gels is viscous. In addition Reynold's
number was calculated and found to be within the laminar
region for all gels.
Diffusive flow. It was pointed out by Ticknor, J. Phys.
Chem. 62, 1483-5 (1958) that the equation for viscous flow
is identical in form to equations for diffusive flow, when
the relationship between diffusion coefficient (D) and
- 27 _
mls/LCM
: .
;. ~
,. .
viscosity according to Johnson and Babb, Chem. Revs. 56,
387-453 (1956) is taken into consideration. T~e relation
between Ks and D is given in ~quation 3. In flow experi-
ments using water as permeant we calculated the apparent
diffusion coefficient for water at 22 - 23. Even for
the tightest gels (Ks 10-l), the calculated D-values were
6-orders of magnitude larger than the reported self-
diffusion coefficient of water at 25 (2.8 x 10-5 cm2/sec.).
This supports the above conclusion that the flow through the
fibrin gels is predominately viscous.
T~BLE II
Relationship Between Pressure and Flow-Rate.
Gelformation: pH 7.4J ionic strength 21, thrombin 0.1 NIH
unit/~nl, temperature 23.5 and fibrinogen concentration
2 mg/ml.
Permeation: H20, temperature 23.5 Ks = 9 x 10-9
____________________________________________________ _______
Pressure Flow Flow per dyne per cm2
dyne/cm2 % ml/hr ml/hr x 103 %
- -- --_---_-___________________________
5531 100 11.280 2.0394 10~
5319 96.210.817 2.0337 99.7
5127 921710.418 2.0320 99.6
4874 88.1 9.859 2.0228 99.2
4576 82.19.148 1.9991 98.0
Gel Permeability and clotting time (Ct~
There is a correlation between clotting time (Ct)
of fibrinogen and enzyme concentration. We explored the
- 28 -
mls/LB
,~
:~2~8~
relationship between Ct and permeability of the final gels.
Therefore, at the same time as gels were prepared for
permeability studies, the Ct of the gel forming system was
determined in parallel experiments by turbidity measurements
(see Methods).
PH. At a constant fibrinogen concentration and
ionic strength, the flow rates for both thrombin and
Batroxobin gels were directly related to the Ct of the gel
forming system over a wide range of Ct (17 sec - 500 sec).
10 This applied to three different pH's (6.5, 7.4 and 8.2) as
exemplified in Fig. 2. At all pH's there was a difference
in slope between curves for thrombin as compared to those
~or Batroxobin.
At each pH, the correlation coefficients (r) for
9iX different Ct versus flow-rate curves (4 experimental
points in each) were calculated. The mean r-values and
their standard deviations (SD) were as follows: at pH 6.5,
0.9709 + 0.0184; at pH 7.4, 0.9721 ~ 0.0394; at pH 8.2,
0.9599 + 0.0434. There was no significant difference in r-
values for thrombin and Batroxobin curves.
Ionic stren~th. In another series of experiments the ionicstrength of the gel forming system was, at constant protein
concentration, varied between 0.21 and 0.31. At all pH's
(6.5, 7.4, 8.2) an increase in ionic strength from 0.2 to
0.3 resulted in a decrease in flow-rate by roughly one order
of magnitude. This applied to both thrombin and Batroxobin
gels.
Ct were prolonged with increasing ionic strength
- 29 -
mls/LB
:;
~Z~B',~ ~
at all enzyme concentra~ions and pH's. At each ionic
strength, however, there was for both thrombin and
Batroxobin gels a linear relationship between Ct and flow-
rate. The results at pH 7.4 is shown in Fig. 4. At all
ionic strengths a difference in slope between curves for
thrombin as compared to those for Batroxobin was noted.
At two ionic strengths, regardless of pH, r-
values for six differen-t Ct versus flow-rate curves (4
experimental points in each) were calculated. Mean r-values
10 and SD were as follows: at ionic strength 0.21, 0.9851 +
0.0208 and at ionic strength 0.26, 0.9511 + 0.0470. There
was no significant difference in r-values for thrombin and
Batroxobin curves.
Gel per~eability and fibrino~n concentration
In a series of experiments we showed that the
relationship between Ct and flow-rate applied to a wide
range of protein concentrations in the gel forming system.
These experiments were only perfor~ed at pH 7.4 and ionic
strength 0.21. When Ct at a given protein concentration
were plotted against flow-rates a linear relationship,
similar to that shown in Fig. 3 (pH 7.4), was demonstrated
at all fibrinogen concentrations (1.5 - 5.0 g/l). The plots
for both thrombin and Batroxobin converged towards an
intercept near the origin with decreasing clossing times.
Like in the experiments shown in Fig. 2, the slopes for
Batroxobin curves were steeper than those for thrombin at
all protein concentrations. The r-values for 8 different Ct
versus flow-rate f_urves (8 points in each) were calculated.
- 30 -
mls/LB
"
~ .. ~
9~
Mean r-values and SD were as follows: for thrombin, 0.4800
+ 0.0157 and for Batroxobin, 0.9830 + 0.0187.
Table III shows Ct at different protein and enzyme
concentrations in one series of experiments. In case of
Batro~obin, increasing protein concentrations did not
markedly influence Ct. However, in the case of thrombin
there is a smal 1 prolongation of Ct with increasing
fibrinogen concentrations.
The relationship between protein concentration in
the gel forming system and flow-rate was next studied. Fig.
5 shows the result of one series of experiments. It is
evident that there exists, at different enzyme
concen~rations, a linear relationship between Elow-rate and
inverse protein concentration. The curves for thrombin and
~atroxobin gels converge to a more or less common intercept
near ~he origin with increasing protein concentration. The
r-values for 15 different l/C versus flow-rate curves (4-8
experimental points in each) were calculated. Mean r-
values and SD were as follows: for thrombin, 0.9738 +
0.0308 and for Batroxobin, 0.9711 + 0.0356.
It is apparent from Fig. 2 how the flow rate Q,
at a constant hydrostatic pressure, is directly proportional
to the coagulation time (Ct) of the thrombin-fibrinogen
mixture at different pH. The coagulation time of the system
is determined spectrophotometrical ly in a separate test
under otherwise identical conditions. The optical density
OD at 450 nm is determined. At gel formation the turbidity
of the solution increases rapidly as shown in Fig. 3. The
-- 31 --
mls/LB
.
~L8~
tangent of the steepest portion of the curve intersects the
time axis at a distance designated as coagulation time Ct.
As there is a direct relation between Ct and flow rate Q ,
Ks is also directly correlated with Ct according to equation
1. .
It is apparent from Fig. 4 that the flow rate of
gels formed at different ionic strength is always directly
correlated to the Ct of the enzyme-fibrinogen solution used
in the gel preparation. In addition the great influence on
the flow rate at a change of the ionic strength is pointed
out.
As is apparent from Fig. S, the flow rate Q is
inversely proportional to the fibrin concentration
t~ibrinogen concentration) in the gel. Thus, according to
equation 1, Ks will also be inversely proportional to the
fibrinogen concentration.
The flow is dependent on the temperature, as
according to equation 1, the flow is inversely proportional
to the viscosity of the permeation solution. The
temperature in gel formation is also of importance as a
constant enzyme concentration, Ct is reduced at a higher
temperature. This is apparent from Fig 6a. However, the
flow rate in gels formed at different temperatures is
directly proportional at Ct at the relative temperature, as
is evident from Fig. 6b.
The columns prepared in the way schematically
illustrated in Fig. l are of small dimensions (1.5 cm2 x 2.6
cm). Similar qualitative results are observed wi-th gel
~ 32 -
mls/LB
,, ,
~z~
columns of grea~er dimensions (5 cm2 x 12 cm). When nothing
else is indicated, the smaller type of column is used in the
tests.
Example _
Standardization of pore size with late2 particle_ of a known
__. _ _
size.
Spherical latex particles of diameters between
0.085 + 0.0055 (SD) ~m (SD - standard deviation) and 0.198 +
0.0036 (SD) ~m from Dow Chemicals, USA, were used in the
tests. a number of gels formed at pH 7.4 and at two dif-
ferent ionic strengths, were used. In the tests, ~t varied
from 23 to 314 seconds. The theoretical radius was
calculated for each gel according to equation 3 assuming
that a cylindrical vertical capillary system was present.
Fig. 7 shows two series of tests. In series I the
ionic strength was 0,23 during the gel formation and in
series II 0.21. The gel columns were equilibriated with
water. After this suspensions of particles were applied to
the gels. In series I the particle size was 0.085 ~m and
20- -i-n series II 0.198 ~m. The particles were slurried in
water to a concentration of 0.1% (weight/volume). The
turbidity at (450nm) of the effluent was determined. It was
then possible to establish by means of the turbidity values
if the latex particles had passed the filter.
In Fig. 7 the turbidity has been expressed in % of
maximum turbidity of the effluent. As is apparent from Fig.
7 the turbidity of the effluent increases above a certain
theoretical pore size of the gel. At an additional increase
- 33 -
mls/LB
~8~
in pore size more particles will pass through the gel
(filter) and over a certain pore size a constant amount of
particles permeate the gels. The difference in theoretical
pore size between no and complete permeation is a measure of
the sum of pores and particle variation. The pore size at
50% permeation is an expression of the average size of pores
and particles. If the total variation in pore size is
within the range of the average particle size + 3 SD it can
be assumed that the pore size in the gel is uniform~
In Fig. 7 the variation in particle size (average
size + 3 SD) has been shown with a horizontal line 50%
permeation. It is apparent that the total variation can, to
a large extent, be explained by the particle variation. It
can be concluded from this that the pores in the gels are
rather uniform. It is also apparent from Fig. 7 that the
theoretical average pore size is about of an order (one ten
power) greater than the real effective particle size. Thus,
calculation of the pore size according to equation 3 only
gives relative values for the pore size.
Example 3
-
Passa~e of proteins and dextran thro~h fibrin ~els
Various protein solutions were (applied) to fibrin
gels prepared in the way described in Example 1. The gel
formati-on was carried out at room temperature (21 - 25C).
In most cases, the buffer used in gel formation had the same
composition as the buffer used for permeation. The
filtration tests took place at room temperature (22 - 25C).
When nothing else is indicated, the volume of the gels was
- 34 _
mls/LB
lZ~ 4~
1.47 cm2 x 2.48 cm = 3.65 ml. The tests were carried out on
different days and with different fibrinogen preparations.
Thus, it is not possible to make a comparison as to the
pore size between different tests. Table 1 shows the
proteins tested with respect to filtering ability through
fibrin gels of different porosities. It is apparent from
the table, that proteins, including those having a very high
molecular weight, are filtered even through gels having
small pore sizes. A high molecular weight polysaccharide
("Blue Dextran") shows the same filtration properties as the
proteins. The Table shows that the yield of proteins in the
eluate is high, from which it appears that at least at room
temperature the interaction between gel matrix and proteins
is small. This also applied to such proteins as fibrinogen,
fibronectin and the factor VIII complex.
- 35 -
mls/LCM
. .., j,
lZ~ 4~)
~U~
r ~1
N E-~ E~ E~ I::q E-l ~ E-l ~
~1 ~ O
~ ~ ~0 ~ ~ ~0 O ~ ~ ~ ~ ~
,~[ ~ I~ `O I~ ~ ~ ; \~ ~ 0 'U~
h ~ ~: O O O O O O O O ~JV )
. C~ O ~0 C`l ~D U') r~ ~ 0 o ~ E~ ~
h ~ O ~ ~1 C~l ~ ~o u l ~ ~ O
cJ c~ta ~1 O ~ ~ O O ~
~ ~ ~O t`l ~ ~ ~t ~ ~
~ '~ ~ ~D ~ ~1 ~ ~ ~
1~~1 o ~ I co _ . r l . r-~~ r-l O O O ~ V V
0~~4 ~1 ' O ~t ~I ~I .... . ~ ~
~rl O O O O ~ O r-l oh ,~
O~ CO r-iCO CO C~ CO (~ r H
4~Ro p ~l ~ r1 C~ Cl~ O CS~ ~0 H '1~
d~rJ 0 R C)
~rr-l r r-l O r; O r; r; O O ~
C/~ ~r ~ _
b ~ + ~ ~ ~ ~ ~ ~1 ~ V U
H C~ H~.) H t~ H C~ 1~ H ~ H C~ O V
r; v E-~ +E~ I O O O ~ ~Drl ~ ? tq
E~ E03 ~C o o Oo X oh C
t~ r~ l l O O O l X O ~ O ~:1
rl~ ~D ~ ;t ~ ~I ~ ~ r~~I r~l
0 3 O O r-i E3 ~ r~ ~ 3 ~ O
~ E3 ~ 13 rl r-i rl r-l O rl c~ Clh H ~rl c rl ~C
.IJ t~ ~ ~ u~ ~ ~ S ~ ) r~ r-l J ) H S.
U~ ~ ~1 ~i td ~ ~ CC a d ~ ~ Y ~ w r~ c) ~ h ~
E-~ ~ u~ ~ P~ ~:i Cd~ _ F4 ~ ~ c c a~ h h ~ ~ _~ _
ch/ \~ - 36 -
~z~
N E-~ E-' E~ E~ ~ ~ E~ E~ E~
::
~0 ~ ~0 ~0
r~ r~ ~ I~ 1~ 1-
`~CI~ ~ ~ ~ ~1 ~ ~ ~D
~,~ O O O O O 0 00 O
~OOC u~ O ~`I ~1 Oo. ~1 X 0~) ~D
P~ ~ ~ ~ C~ ~ ~ ~ ~ ~
~ C~a o~ u) ~ ~ ~ ~ ~ ~ ~00
H ~ ~ ~ _~ r~ _~ ~ _~
~ ~ ~o ,1 o ,~ ~ ~o o u~ oo o ~ oo
P~ ~ !3 '`! . ~ ~ co ~ ~ ~ ~ ~ ~ o ~ ~ o
~ ~ oo ~o ~o ~o ~_, ooc~lo~o oo
~ r~ ~ coo
e e
e ~ ' e " e ~ + ,~ , ~ H ~ + J~,~
H ~ ~ ~ H C~ ~ E-l I $ ~ X H y H C~ S~ H
. ~q E ~ ;~
~( JJ 'D 1~ )3 ~ ~ ~:1 . oo .~ ~
~ ~o ~ U~ ~ o o 0 o ~3
'~ X ~ ~ o.~ O ~ ~
~ o ~2 l ~ --~ ~ S~ ~ ~ ~ ~1 ~
JJ 0~ ~0~ ~ ~ ~ ~ ~ 3"
~q 13 R ~1 u7 E3 0 ~ X 13 ~d ~ ~. ~ R ~q E ~ H ~
~ ~ ~ ~ o 3 ~ ~ ~ ~ ~ u~ ~ R-- ~ R
ch/ \~
~2~ 4~
EXAMPLE _
Filtration of suspensions of red blood corpuscles through
fibrin gels
Fibrin gels were prepared in the way described in
Example I and the conditions of gel formation is shown in
Example 3. A small amount (0.2 ml) of human blood was
applied to a gel column. Continued filtration was carried
out at room temperature (22 - 25C) under the conditions
shown in Example 3. The blood corpuscles did not pass
through the fibrin gel. This was expected as the diameter
of the red blood corpuscles(7-8 ~m) is much larger than the
effective pore diameter of the fibrin gel.
- 38 -
mls/LB
~2~
EXAMPLE 5a
Filtration of plasma rich in platelets throu~ fibrin gels
Plasma rich in platelets (PRP) was prepared from
blood by centrifuga-tion for 4 minutes at 120g, the blood
being drawn in citrate solution to prevent coagulation. It
was centrifuged at 2000 g for 5 minutes-to remove the
remaining red blood corpuscles and EDTA at a concentration
of 10 mM was added to the PRP, 0.5 ml of the PRP was applied
to a fibrin gel column prepared in the way described in
Example I. The conditions of gel formation is shown in
Example 3 and filtration was continued under the conditions
shown in Example 3. To prevent aggregation of the platelets
and their adhesion to the gel matrix, EDTA (10 mM) was added
not only to PRP but also to the solution which was filtered.
No platelets could be demonstrated in the eluate from the
~ibrin gel column. This was expected as the diameter of the
platelet lies between 2 and 4 ~m, which is considerably more
than the effective pore size of the gel.
EXAMPLE 5b
Separation of mitochondïa from fragments of liver cell by
filtration through fibrin ~
Liver cells of a rat were homogenized in a
homogenizator according to Potter-Elvehjem. Separation of
cell fragments was achieved by differential centrifugation
in known manner. The mitochondria were slurried in a buffer
solution containing Na-EDTA (10 mM) and succrose (0.25M).
0.3 ml of the resulting suspension was applied to a fibrin
gel column prepared in the way described in Example 1. The
- 39 -
mls/LB
~,,` .
conditions of gel formation is shown in Table I and
filtration was continued under the conditions indicated in
Table 1. No mitochondria could be demonstrated in the
eluate, which was as expected, since their diameter is about
0.5 ~m; thus considerably bigger than the effective pore
size of the gel.
EXAMPLE _
Separation of Sendai-virus b~ filtration throu~h fibrin gels
Sendai virus is a virus specific to mice which is
used for preparation of interferon in human lymphocyte
cultures. A partially purified virus preparation (640
hemagglutination units/ml) was used in the tests.
0.2 ml of the virus suspension was applied to each
of three ~ibrin gel columns prepared in the way described in
Example 1. The conditions of the gel formation appear from
Table 1 and filtration was continued under the conditions
shown in Table l. No hemagglutination activity could be
demonstated in the eluate from column l; 50% of
hemagglutination activity were demonstrated in the eluate
from column II and 95% of the hemaggultination activity of
the virus particles was demonstrated in the eluate from
column III (see Table 1).
After filtation the silk nets at the upper part of
the three columns were washed with a buffer solution
(containing 1% of borine serum albumin, BSA) and the
hemagglutination activity of the washings was analyzed. In
the washing liquid from columns 1 100% of the
hemagglutination activity was found; in the washing liquid
- 40 -
mls/LCM
~z~
from column II 25% of the activity was found and in the
washing liquid from column III no activity was found.
The particle diameter of Sendai virus is stated to
be about 0.15 ~m. The tests show that when the effective
pore radius is more than 0.15 ~ the virus particles pass
through the gel. When the effective pore radius of the gel
is less than 0.15 ~m a retention of the particles will, on
the other hand, occur.
EXAMPLE 7
Separation of Eschericia Coli (E. coli) by filtration
throu~h fibrin ~el.
E. coli is an elongQted intestinal bacterium of
the approximate dimensions 0.8 x 1.2 ~Im. A suspension of E.
coll in trisimida~ole-buffer, free of calcium and the pH 7.4
~nd lonic strength 0.21, was prepared (see Example 1). The
suspension contained between 107 and 108 bacteria/ml. 45 ml
of the suspension were supplied to a fibrin gel column of
the dimensions 5 cm2 x 11 cm prepared in the way described
in Example 1. The conditions of the gel formation and the
filtration are shown in Table 1. The flow rate was 31 ml/h.
No bacteria passed through the gel, determined by turbidity
measurements of the eluate from column. The flow rate at
constant pressure was less at the end of the test than at
its beginning. Assuming an unchanged Ks the reduction of
surface corresponding to the reduction in flow can be
calculated according to equation l. According to this
calculation the surface had been reduced to 58%. Thus, one
might expect that the bacteria were enriched on the upper
- 41 -
mls/LCM
gel surface. By washing the silk net attached to the upper
part of the gel with buffer solution 99% of the bacteria
applied to the gel were found in the washing liquid.
The test shows that E. coli cannot pass through
gels having a pore diameter which is considerably less than
the smallest diameter of the bacteria.
EXAMPLE 8
Preparation of gels with reinforcement of porous plastic
In the foregoing examples nets of silk, plastic or
metal adapted to the upper and lower portions of the gel
have served as stabilizing structure of the fibrin gels. A
corresponding stability can also be obtained in such a way
that the fibrin gel is cast into a porous plastic, e.g.
polyurethane, polyester or some similar porous plastic
material, preferably one which is wettable by water.
In this example a foam plastic of polyurethane
("Regilen 40 AG") of a pore size 0.4 mm has been used. The
gels were cast in a special apparatus. This consisted of a
cylindrical plastic chamber in which the porous plastic had
been introduced; the plastic was accommodated in a ring of
acrylic plastic (height 2 cm and diameter 9 cm). The
apparatus (chamber) had an opening at the upper and lower
end, respectively. One opening was connected to a vaccum
pump and the other opening was kept closed. The chamber was
evacuated by means of the vacuum pump. After this the valve
connecting the chamber with the vacuum pump was shut off. A
fibrinogen thro~bin solution was subsequently allowed to
fill the chamber rapidly through the valve in the opposite
- 42 -
mls/LCM
~ .....
`~: g
.
~ ~ ~ .
~%~
opening, The valve was thereafter closed and the chamber
was left for 2 hours, so that the fibrinogen solution in-the
porous plastic material should be completely converted to a
fibrin gel. The clotting parame~ers of the thrombin-
fibrinogen mixture was shown in Table 20 For comparison a
gel was also prepared in the way described in Example 1, In
Table 2 the ~s-value of this latter gel is also shown.
After complete gel formation the chamber was opened and the
plastic cake with fibrin gel (including its plastic frame)
was taken out. I~ was transferred to a special filter
chamber~ The framed ring, in which the plastic material and
the gel were accomodated, fitted ~ightly to the edges of the
~ilter chamber through two 0-rings. The upper lid of the
chamber was provided with an inlet for the liquid to be
filtered and a ventilating valve to let out the air above
the gel surface. In the lower portion of the chamber there
was an outlet for collecting the filtered liquid. A buffer
solution with the composition shown in Table 2, was filtered
through the gel cake. The ~s value was calculated accordlng
to equation 1 (Table 2). As is apparent from the table the
Ks-value of the gel, cast in plastic is of the same order as
the gel prepared according~to Example 1. The partial
specific volume of the plastic material in the gel cake is
0,03 which means that the plastic matrix reduces the surface
available for flow only to a small extent.
EXA~PLE 9
Preparation of ~els in a cellulose matrix
Cellulose materials can also be used as reinfor-
- 43 -
mls/LCM
,, ~. ?
~Z~899~
cing agent (supporting substance). In this example, a
porous cellulose compound (I'Wehex cloth") is used as re-
inforcing agent of the fibrin gel. It had a thickness of
0.2 - 0.3 cm. Circular pieces of a radius of about 3 cm
were wetted with a thrombin-fibrinogen solution. The cel-
lulose pieces then swelled to about double thickness. The
partial specific volume of the swollen cellulose compound
was 0.04. Immediately after swelling which lasted for about
2 - 4 seconds the pieces were placed on the filter disc of a
Buchner funnel. Measures were taken so that the pieces
fitted tightly to the edges of the funnel. The openings of
the funnel were covered with "Parafilm" and the funnel was
left at room temperature for 2 hours in order to obtain a
complete fibrin formation in the pores of the cellulose.
Buffer solutions, the composition of which is shown in Table
1, were filtered through the gels. The ~s-value of the gels
which are cast in cellulose is of the same order of
magnitude as control gels prepared wi~hout reinforcing
substance.
EXAMPLE 10
Preparation of fibrin ~els in thin layers for filtration
In this example it is shown that fibrin gels in
thin layers with reinforcement only on the lower surface can
be used for filtration. About 10 ml of fibrinogen solution
intris-imida201e buffer with pH 7.4 were mixed with a
thrombin solution. The mixture was thereafter poured into a
Petri cup the bottom of which was covered by a damp silk
cloth. The cup was covered with a lid and was left for 2
~ 44 -
mls/LCM
~2~
hours for a complete gel formation. The thickness of the
gel layer was 2 mm. The clotting parameters of the gel is
shown in Table 2. The filter was thereafter attached to a
"Millipore'~ filter support provided with a funnel. The
funnel was filled with bufer solution and the flow rate was
determined. As is apparent from Table 2 the ~s value is of
the same order or magnitude for a corresponding fibrin gel
prepared according to Example 1. However, the filter showed
in course of time gradually diminishing ~s-values, which
presumably is due to compression of the gel matrix during
the flow.
~XAMPL~ 1 1
Stabilization of ~els by treatment with dialdehyde
In this example it is shown that gels prepared
according to Examples 1, 8 and 10 can be stabilized by
treatment with dialdehyde.
A. A gel prepared according to Example 1 was
first equilibrated with water and then brought into
equilibrium with 0.014 M phosphate buffer solution with pH
20 7.2 in 0.15 M NaCl (phosphate buffered saline solution PBA).
2 - 4 column volumes of a 1% glutaraldehyde solution were
then allowed to filter through the gel in the course of 10
minutes - 2 hours. After this the gel was washed with
several column volumes of PBS and then with water. The
column was finally equilibrated wi~h tris-imidazole buffer
and flow measurements were carried out. The ~s-value is
almost unchanged after treatment with 1utar dialdehyde.
After the flow measurements, the gel was taken out and
- 45 -
mls/LCM
~z~
treated for 72 hours with 8 M urea containing 1% of sodium
dodecyl sulphate (SDS). The gel was then reduced with 1%
dithiotreitol in a way known per seO Polyacrylamide gel
electrophoresis in the presence of SDS showed in comparison
with non-stabilized gels the absence of free fibrin chains
~fibrinogen chains), which can be interpreted as a proof
that glutar dialdehyde had cross-linked the chain units of
the fibrin structure.
B. A gel prepared in porous plastic according to
Example was first washed with a tris-imidazole buffer
solution free of calcium and was then brought into
equilibrium with a 0.014 M phosphate buffer solution with pH
7.2 in 0.15 M NaCl (PBS). Two column volumes oE a 1~ glutar
dlal~ehyde solution were then passed through the gel cake
(column) in the course of 10 minutes. The gel cake was then
washed with several column volumes of PBS and then with
water. Finally the column was brought into equilibrium with
tris-imidazole buffer and flow measurements were carried
out. These are shown in Table 2. As is apparent from the
Table the ~s-value is only slightly changed after the
treatment wi~h glutardialdehyde and is of the same order of
magnitude as a gel prepared according to Example 1. The gel
stabilized with glutardialdehyde was then autoclaved at
120C for 20 minutes at a pressure of 1.4 atm. After
autoclaving the flow of buffer solution was again tested
through the gel cake. As is apparent from Table 2, auto-
claving has influenced the flow properties of the gel only
to a small extent. Cracks in the gel would have caused
- 46 -
mls/LCM
..' '
drastic increase, of the flow through the gel.
C. A fibrin gel prepared according to Example 10
was transferred to a cup with 500 ml water ~o remove buffer
salts by diffusion. AftPr 2 hours the gel was transferred
to a cup with a new portion of water. After 2 hours the gel
was transferred to a cup with 500 ml phosphate buffer
solution with pH 7.2 in 0.15 M NaCl (PBS) and was left over
night. The gel was then transferred to a Petri cup
containing 50 ml of 1% glutar dialdehyde. After 2 hours the
glutardialdehyde solution was exchanged for a new portion of
the same liquid. After additional 2 hours the gel was
transferred to a cup with water and washed in the way
described above. After washing with water the gel was
transferred to a cup with tris-imidazole buffer solution.
After 2 hours the washing liquid was exchanged for a new
portion and after additional 12 hours the gel was
transferred to a "Mullipore"-filter carrier with a funnel.
As a comparison measurements were carried out with a gel
prepared in the same way except the treatment with
gl-utardialdehyde. Directly after casting, this gel was
transferred to a "Millipore" filter container for flow
measurements. As is apparent from Table 2 the flow through
the vulcanized filter was comparable with that through the
non-vulcanized filter at the start of the flow measurement.
However,at the end of the measuring period, the ~s-value of
the nonstabilized ~ er had been reduced to a large extent
which was not the case with the vulcanized filter. After
the flow measurement the filters were sterilized through
- 47 -
mls/LCM
~z~
autoclaving of the filter (and the filter apparatus~ at
120C for 20 minutes at the pressure 1.4 atm. Flow
measurements were carried out after the heat treatment and
the ~s-values are shown in Table 2.
No flow could be demonstrated through the non-
stabilized filter. On the other hand the vulcanized filter
showed ~s-values of the same order before as well as after
autoclaving.
5 ml of a suspension of Sendai-virus were supplied
to the vulcanized ~ilter. Filtration was carried out by
means of a water suction. When the liquid had passed the
~lter additional 5 ml of buffer solution were passed
through the filter. This was repeated twice. The filtrate
was tested for hemagglutination activity. Inconsiderable
hemagglutination activi-ty could be demonstrated. The upper
surface of the filter was washed with several portions of
buffer solution. The washing liquid was opalescent and its
hemagglutination activity corresponded to a yield of virus
particles of almost 100%.
These examplés show that the filters can be
stabilized with a dialdehyde such as glutardialdehyde.
- 48 -
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