Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~2~3592S
ADSORBENT FOR ~2-MICROGLOBULIN AND
IMMUNOGLOBULIN L-CHAIN
BACKGROUND OF THE INVENTION
The pre~ent invention relates to an adsorbent
suitable for use in an extracorporeal circulation
treatment, to remove ~2-microglobulin (hereinafter
referred to as "32-m") and immunoglobulin L-chain
thereinafter referred to as "L-chain") whic`n are
contained in a body fluid.
In recent years, many of patients who have been
treated by dialysis for a long piriod suffer from carpal
tunnel syndrome. "Carpal tunnel syndrome" is a disease
attended with symptons o~ paralysis of the median nerve
due to compression of the median nerve at the part of
carpal tunnel. Lately, it was found out that a ~-fibril-
like amyloid protein called "AH" was deposited on the
diseased part. Further, it was found out that a
precursor protein ¢orresponding to the amyloid is the ~2-
m which i8 contained in the patient's blood. However,
there hitherto has not been found an effective treatment,
especially drug treatment.
32-m~is a low molecular protein (M. W. 11,800)
consisting of one hundred of amino acids. The separation
method by means of membrane has been used to separate a
component in blood or plasma rather selectively according
to the size so ~ar. However, there are such
dlsadvantages in this method as use~ul protelns
except ~2-m may be removed and as the amount of
removed ~2-m i9 small. Therefore a more selective and
effeetive way~to remo~e a lot of ~2-m is desired.
The~meehod ~or removing ~2-m by means of
adsorbent ha8 hasdly been used so far. A few examples of
adsorbsnt ~o~r~purification of~B2-m are known, for
insta~nce, an~immunoadsorbent in which an anti ~2-m
antibody is immobilized onto its carrier, an adsorbent
~285925
- 2
based on a principle of a~finity chromatography in which
concanavalin A i~ immobilized as a compound showing an
affinity for ~2-m (hereinafter referred to as "ligand")
onto it9 carrier, and the like. Although these
adsorbents show a high selectivity ~or ~2-m, they are not
practical for a treatment because of the problems such as
expensive anti ~2-m antibody and ligands, for instance,
concanavalin A, an unstability in preservation and a
difficulty of sterilization.
On the other hand, there is a desease called
amyloidosis attended with serious troubles, for example,
insufficiency of organs such as heart and kidrey,
disturbance of impulse conducting system, progressive
dementia, cerebrovascular disease, nerve trouble and so
on. Amyloidosis i9 caused by a deposition of amyloid on
a blood vessel, a certain organ and so on. It is known
that there are some types o~ amyloidosis, i.e. primary
amyloidosis, secondary amyloidosis, familial amyloidosis,
senile amyloidosis, and so on, and that the composition
of proteins causing amyloidosis differs depending on the
type of amyloidosis.
Primary amyloidosis is caused by a protein
called "AH", and the precursor corresponding to deposited
amyloid is supposed to be L-chain. However, there
hltherto has not been found an e~fective treatment,
especially drug treatment. L-chaih is a low molecular
protein (M. W. 23,000) consisting of two hundreds of
amino acids in the ~orm of monomer.
Besides the primaly amyloidosis, there are some
diseases attended with an extraordinary product o~ L-
chain. The typlcal disea~es are multiple myeloma,
macroglobulinemia or malignant lymphoma and the highly
produced L-chain in these diseases is a cloned protein
called Bence Jones protein (hereinafter re~erred to as
"BJP"). Usually BJP is excreted into urine. At that
time, it inhibits the reabsorption of other protein~,
especially albumin, and causes a myeloma kidney
syndrone. Also, there are many ca~es to cause
,
..~ - . . - , - .
~285g2S
amyloidosis at heart, kidney and so on as a result of a
deposition of a lot of BJP in serum. Therefore, an
e~fective method for removing BJP in serum is wanted.
However, there has not been found a practical method for
removing 90 far, as same as that for primary amyloidosis.
The object of the present invention is to solve
the mentioned problems and to provide an inexpensive
adsorbent for use in an extracorporeal circulation
treatment which i9 able to adsorb and remove a lot of
both ~2-m and L-chain.
SUMMARY OF THE INVENTION
In accordance with the present invention, there
is provided an adsorbent for ~2-m and L-chain suitable
for use in an extracorporeal circulation treatment, which
comprises a porous water-insoluble carrier and a compound
immobilized onto said carrier, said compound satisfying
that the value of log P, in whi¢h P is a partition
coefficient in an octanol water system, 19 not less than
` 20 2.5.
8RIEF DESCRIPTION OF THE DRAWING
Fig. 1 is a graph showing a relation between a
flow rate and a pressure drop ~P obtained in Reference
Ixample.
DETAILED DESCRIPTION OF THE INVENTION
The adsorbent of the present invention
comprises a porous water-insoluble oarrier and a compound
~immobilized onto~said carrier, said compound satisfying
that the value of log P is not less than 2.5.
; The logarlthm of a partition coefficient in an
oo~tanol-water syst~em, i.e. log P, is a hydrophobic
;parameter of a compound. The partition coefficient P is
35~ obtained acco~rding to the ~following typical method.
First of all, a compound i 9 dissolved in an
octanol (or a water) and an equal volume of a water (or
an oc~tanal) is adde~d thereto. After shaking for 30
: . - . . . . .
.
.- : . . . . .
~;28592~;
minutes by Criffin flask shaker (made by Gri~fin & George
Ltd.), it is centrifuged for from 1 to 2 hours at 2000
rpm. Then concentrations of the compound in both octanol
and water layer can be measured by various methods such
as spectroscopic method and GLC, and the value of P is
obtained according to the following formula:
P - Coct/Cw
Coct: a concentration of a compound in
an octanol layer
Cw: a concentration of a compound in a
water layer
Before now, many investigators have measured
log P of various compounds and the found values were put
in order by C. Hansch et. al. ["PARTITION COEFFICIENTS
AND THEIR USES", Chemical Revlews, 71, 525 (1971)]. As
to the compounds whose found values are unknown, the
calculated values using~a hydrophobic fragmental constant
f (hereinafter referred to as "~f"), shown in "THE
HYDROPHOBIC FRAGMENTAL CONSTANT" (Elsevier Sci. Pub.
Com., Amsterdam, 1977) written by R. F. Rekker, can be a
~;~ 25 good guide.
A hydrophobic fragmental constant show the
hydrophobicity of various fragments, whlch are determined
by a statistioal management of many found values of log
P,~ and the total of f of each fragment, i.e. ~f which is
30~ a constitutent of one compound almost corresponds to log
In order to find out a compound whlch has an
;ef~fect on ad~o~rbing ~2-m and L-chaln, compounds havlng
varlous log~P~are~immobillzed onto a carrier. As a
35~ resu~l~t,~it~ found that~compounds, whose value of log P
are~not l~ess~than 2.50, ~are effective on adsorbing 32-m
and L-ch~ain and that compounds, whose value of log P are
esB t~han 2.5Q,;hardly show an adsorption ability of 32-m
.
.. . . ..
~8S925
and L-chain.
In a ca~e of alkyl amine being immobilized, for
example, an ad~orption ability of ~2-m and L-chain is
extraordinarily increa~ed by using n-octyl amine (log P =
2.90) instead of u~ing n-hexyl amine (log P - 2.06).
These re~ults indicate that an adsorption
of 32-m and L-chain on an ad~orbent of the present
invention is caused by the hydrophobic interaction
between atomic groups, which are introduced into a
carrier through immobilizing compounds who~e value of log
P are not les~ than 2.50J and ~2-m and L-chain, and that
compound~ who~e value of log P are less than 2.50 have no
ad~orption ability on an account of it~ too small
hydrophobicity.
In the present invention, compounds immobilized
onto a porou~ water-in~oluble carrier, ~atisfying that
the value of log P are not less than 2.50 can be u~ed
without no particular limitation. However, in a case of
immobllizlng a oompound onto a carrier by chemical bond,
a part o~ the compound is frequently eliminated. When
the elimination group greatly contributes to the
hydrophobicity o~ the c~ompound, i.e. when the ~f of the
atomic group immobilized onto a carrier by elimlnation is
smaller than 2.50, the compound is not ~uited for using
at the present lnvention ln vlew of the ob~ect of the
present invention. The typlcal example is that benzoic
acid isopentylester (~f - 4.15) is immobilized onto a
aarrier, having a hydroxy group, by an ester
interchange. In this case, the atomic group immobilized
30 ~ onto a carrie;r ls~C6H5C0- and ~f of this group i9 under
Thérefore, whether a compound is sufficient as a
compound used in the~present inventlon or not may be
determlned depending on whether the value of log P, when
an elimination part o~ the group i9 substituted by
35;~ hydr~ogen,~i~ les~ than 2.50 or not.
Among~the compounds whose~value of log P i~ not
Less than 2.50, compound~ having functional groups which
are available for i~mmobilization onto a carrier, e.g.
~ . . . i - ~ . ' . - ~' . . . .
1285925
unsaturated hydrocarbons, alcohols, amines, thiols,
carbonic acids and its derivatives, halides, aldehydes,
hydrazide~, isocyanates, compound~ having an oxirane ring
~uch as glycidyl ethers, halogenated silanes and so on
5 are preferable. The typical examples of these compounds
are amines such as n-heptylamine, n-octylamine,
decylamine, dodecylamine, hexadecylamine, octadecylamine,
2-aminooctene, naphtylamine, phenyl-n-propylamine and
diphenylmethylamine, alcohols such as n-heptylalcohol, n-
10 octylalcohol, dodecylalcohol, hexadecylalcohol, 1-octene-
3-ol, naphtol, diphenylmethanol and 4-phenyl-2-butanol,
glycidylethers thereof, carbonic acids such a~ n-octanoic
acid, nonanoic acid, 2-nonenoic acid, decanoic acid,
dodecanoic acid, stearic acid, arachidonic acid, oleic
15 acid, diphenylacetic acid and phenylpropionic acid,
carbonic acid derivatives thereof such as acid halides,
ester and amide, halides such as octylchloride,
octylbromide, decylchloride and dodecylchloride, thiols
~uch as octanethiol and dodecanethiol, halogenated
20 silanes such as n-octyltrichlorosilane and
octadecyltrichlorosilane, and aldehydes such as n-
octylaldehyde, n-caprinaldehyde and dodecylaldehyde.
Moreover, among the above-mentioned compounds
whose hydrogen in their hydrocarbon moiety is substituted
25 by substituents having hetero atoms such as hoalogen,
nitrogen, oxygen and sulfur, other alkyl groups and 90
on, the compounds whose log P are not less than 2.50 can
be used. And the compounds shown on the tables of from
page 555 to page 613 in the above mentioned reference,
30 "PARTITION COEFFICIENTS AND THEIR USES", satisfying that
the value of log P are not less than 2.50 can also be
used. However, the present invention is not limited
thereto.
These compound may be used alone, in
35 combination thereof~or even together with a compound
having a value of log P of less than 2.5.
The typical examples of the water-insoluble
carriers in the present invention are inorganic carrlers
: :
: :
. - ~ . . .
.
.
~;~8592~i
such as glas~beads and silicagel, organic carriers made
- of synthetic polymers such as cross linked
polyvinylalcohol, cross linked polyacrylate, aross linked
polyacrylamide, cro~s linked polystyrene and
polysaccharides ~uch as crystal cellulose, cross linked
cellulose, cross linked agaro~e and cross linked dextran,
and complex carrier obtained by combining them, e.g.
organic-organic carrier, organic-inorganic carrier and so
on. Especia11y, hydrophilic carriers are preferable
since non-specific adsorption is rather small and the
adsorption selectivity for ~2-m and L-chain is good.
The term "hydrophilic carriers" means the
carriers which contact with water at an angle of not more
than 60 when compounds constltuting the carriers are
made plane. Typical examples of these carriers are made
of cellulose, polyvinylalcohol, saponi~icated compound of
ethylene-vinyl acetate vinyl copolymer, polyaarylamide,
polyacrylic acid, polymethacrylic acid, polymethyl
methacrylate, acrylic acid grafted polyethylene,
acrylamide grafted polyethylene, glass and 90 on. In
particular, the porous cellulose gels are preferred for
the reasons that (1) they are hard to be broken into
pieces or fine powder by operation such as agitation
beoause of a relatively high mechanical strength and
toughness, and also even if a body fluid is passed
through~ a column packed therewith at a high flow rate,
oonsolidation and choking do not ocour and, thererore, it
19 pQssible to pass a body fluld at a high flow rate, and
further the pore structure is hard to suffer change upon
high pressure steam sterilization, (2) the gels are
hydrophilic because Or being made of cellulose and plenty
of~hydroxyl group utilizable for bonding the sulfated
palysaccharide, and also nonspecific adsorption is a
lL~ttle,~(3) an adsorption capacity comparable to that of
35~the soft gel4 i8 obtained, because the strength i9
maintained relatively~high even if the porosity volume i~
incr;easej and (4)~the safety i9 high as compared with the
ynthetic polymer gel~. However, the present invention
- ~ ~
~2~5
- 8
is not limited thereto. The above-mentioned carriers may
b~e used either alone or in combination thereof
volantarily.
The mo~t essential property for a water-
insoluble carrier of the pre~ent invention is to containa lot of pore~ having a preferable size, i.e. to have a
porous structure. ~2-m and L-chain, both of which are
objects to be removed by the adsorbent of the present
invention, are proteins having molecular weights 11,800
and 23,000 respectively as mentioned above. And it is
preferable for an effective adsorption of these proteins
that ~2-m and L-chain can enter the pores of adsorbent at
a certain high probability, but on the other hand, that
other proteins can hardly enter the pores. There are
many methods for measuring pore size. Though mercury
porosimetry is most widely used now, sometimes it cannot
be applied to the porous water-insoluble carrier of the
present invention. In such case, it i9 proper to use the
molecular weight of the excluslon limit as a measure of
the pore size.
The term "the molecular weight of the exclusion
limit" means, for instance, as described in the
literature such as "Jikken Kosoku Ekitai Chromatography
(Experimental High Performance Liquid Chromatography)"
written by Hiroyuki Hatano and Toshihiko Hanai, pùblished
by Kabu~hiki Kaisha Kagaku Do~in, the minimum molecùlar
weight of the molecule which cannot permeate into a pore,
i.e. which i~ excluded, in a gel permeation
chromàtography.
The moleoular weight of the exclusion limit is
generally well~studied about globular protein, dextranj
polyethylene~-glycol and 80 on and, in case o~ the carrier
of the present inventi~on, it is proper to use the value
obtained by globular protein.
3~5~ As-~the r~esult of an investigation using
oarriers having various molecular welghts of the
exclusion limit, it is shown that the preferable range of
pore size indicated~by molecular weight of the exclusion
~..2~S925
limit i~ ~rom 1 x 104 to 6 x 105. That is, it is ~ound
that a carrier having a molecular weight of the exclusion
limit of less than 1 x 104 can hardly adsorb the ~2-m and
L-chain and i~ not suited for practical use and that a
carrier having a molecular weight of exclusion limit of
over 6 x 105 adsorbs a large amount of proteins other
than ~2-m and L-chain, mainly albumin, and is not suited
for practical u~e in the point of selectivity.
Therefore, the moleculr weight of the exclusion limit of
the carrier employed in the present invention is
preferably from 1 x 104 to 6 x 10 5, more preferably from
2 x 104 to 3 x 105.
With respect to the porous structure of a
carrier, a structure uniformly having pores at any part
of the carrier is more preferable than a structure having
pores only on the surface from the viewpoint of
adsorption capacity per unit volume. And it is preferred
that the porosity of a ¢arrier is not less than 20 ~ and
the specific surface area is not less than 3 m2/g.
The 9hape of a carrier can be optionally
selected from such shapes as particle, fiber and hollow
fiber.
In the present invention, it is preferable that
there are some functional group9 which are available for
immobilizing a ligand onto the surface of the carrier.
Typical examples of the above functional groups are
hydroxyl group, amino group, aldehyde group, carboxyl
group, thiol group, silanol group, amide group, epoxy
group, halogen group, succinimide group, acid anhydride
30~ group and the like.
Both a~soft carrier and a hard carrier may be
employed~in the~ pre9ent invention. However, when the
carrier is used as an adsorbent for an extracorporeal
c~irc~ulation treatment, it i9 important that the carrier
35~;~ has a sufficlent~mechanical strength 90 as to prevent
consolidation when a body fluid is passed through a
column packed with the adsorbent.
Therefore~, it is preferable that a carrier
:: . : ~ .: , ; , ' . -
~2~35925
employed in the present invention is a hard carrier. The
term "hard carrier" means, as shown in the Reference
Example herein below, that the relation between a
pressure drop ~P and a flow rate determined by passing an
aqueous fluid through a cylindrical column uniformly
packed with the gel, such as particle gel, keeps a linear
relationship until the pressure drop is increased to 0.3
kg/cm .
The adsorbent of the present invention is
obtained by immobilizing a compound, whose value of log P
is not less than 2.50, on a porous water-insoluble
carrier and there are many known methods available for
immobilization without any particular limitation.
However, it i9 important in view of the safety to
minimize the elimination and elution of ligands during a
sterilization or a treatment because the adsorbent of the
present invention is employed for an extracorporeal
circulation treatment. Therefore, it is most preferable
that a compound is immobilized onto a carrier by a
covalent bond.
There are many ways of using the adsorbent of
the present invention in a treatment. For the most
simple example, the adsorbent of the present invention
can be used as follows: i.e. partient's blood is
introduced outside of hiq body so as to be put into a
blood bag and then mixed with the adsorbent of the
present invention to remove ~2-m and L-chain, followed by
removing the adsorbent through filter. Consequently, the
blood treated in this way is returned back to the patient
.
himself. Though this method does not need an intricate
apparatus, there are some defects such as a small amount
of a treated blood at one time, a lot of time for
treatment and a complicated operation.
For another method, a column packed with the
3~5~ adsorbent of the present invention is incorporated into
an ex~tracorporeal circulation clrouit, and then removal
o~ 32-m and L-chain by adsorption is taken by on-line
sy~stem. There are two tre~atment methods: i.e. one is
~. : ,:
.
. , . . .. , . .. - . . . .
i~85925
1 1 --
that whole blood i5 directly perfused and another is that
only plasma separated from the blood is passed through
the colu~n.
The adsorbent of the present invention can be
used in any of above method~ and, as mentioned above, on-
line system is most preferable.
Present invention is more specifically
described and explained by the following Re~erence
Example, Examples and Comparative Examples. It is to be
understood that the present invention is not limited to
the Reference Example, Examples and Compar~tive Examples
and various changes and modifications can be made without
departins Lrom the scope and spirit of the present
invention.
eference Example
A relation between a flow rate and a pressure
drop ~P was determined by passing water by means of a
peristaitic pump through cylindrical glass columns (inner
diametar: 9mm, column length: 150 mm) equipped at both
ends with filters having a pore size of 15 ~m, in which
an agarose gel (Biogel A5m made by Biorado Co., particle
size: 50 to 100 mesh) a vinyl polymer gel (Toyopearl HW-
65 made by Toyo Soda Manufacturing Co., Ltd., particle
size: 50 to 100 ~m, and a cellulose gel (Cellulofine GC-
~; 700m made by Chisso Corporation, particle size: 45 to
105 ~m) were packed respectively. The results are shown
~0 in Fig. 1.
As shown in Fig. 1, an increase o~ a flow rate
~::: ::
i9 nearly proportional to that of a pressure drop in case
of Toyopearl HW-65 and Cellulofine GC-700m, whereas in
case of Biogel A-5m, consolidation occurrs and a flow
rate does not increase even if a pressure drop
increases. In the present invention, the term "hard gel"
means a gel having the relation between a pressure
drop ~P and a ~low rate keeps a liner relationship until
the pressure drop is increased to 0.3 kg/cm2, as in the
former case of Toyopearl HW-5 and Cellulofine GC-700m.
* Trade-mark
~28592S
12
Example 1
After water was added to 170 mQ of a porous
cellulose hard gel Cellulofine GC-200m (made by Chisso
Corporation, exclusion limit of globular proteins:
120,000) to make the whole volume 340 mQ, 90 mQ of 2M
aqueous solution of sodium hydroxyde is added thereto and
the temperature of reaction mixture was adjusted to
40C. Then there was added 31 mQ of epichlorohydrin and
the reaction mixture was reacted with stirring for 2
hours at 40 C. After the reaction, the resultant was
thoroughly washed with water to give an epoxy-activated
gel.
To 10 mQ of the obtained epoxy-activated gel
was added 200 mg of n-octylamine (log P - 2.90). After 6
days reaction by allowing it to stand in a 50 % by volume
aqueous solution of ethanol at 45C, the resultant was
throughly washed with a 50 % by volume aqueous solution
of ethanol, ethanol, again a 50 % by volume aqueous
solution and then water to give an n-octylamine-
immobilized gel.
To 0.5 m~ of the obtained n-octylamine-
immobilized gel was added 2 m~ of serum of a patient who
is under dialysi~ treatment, the serum containing ~2-m in
a concentration of 65 ~g/mQ and the mixture was incubated
for 2 hours at 37C. The concentrations of 3 -m and
: albumin in the supernatant were measured to obtain the
amount of adsorbed 32-m, ratio of adsorbed 32-m based
on:32-m added first and amount of adsorbed albumin.
The results are shown ln Table 1.
30::~
Example 2
: The procedures in Example 1 were repeated
except that 3;15~:mg of dodecylamine (~f - 5.10) was used
lnstead of n-oe~tylamlne to glve a dodecylamlne-
`35~ immobilized~gel:.:
~ ;-
The~adsorption of the~obtained dodecylamine-
immobilized gel: was~examined by means of the same
: procedure:s as:in Example 1.
.. ,~ , . .. .. ..
-,
~2~3592S
- 13
The result is ~hown in Table l.
Example 3
Tne procedure~ in Example 1 were repeated
except that 170 mQ of a porous cellulose hard gel
Cellulofine GC-700m (made by Chisso Corporation,
exclu~ion limit of globular proteins: 400,000) was used
as a carrier instead of Cellulofine GC-200m to give an n-
octylamine-immobilized gel.
The adsorption of the obtained n-octylamine-
immobilized gel was examined by means of the same
procedures as in Example 1.
The re~ult is shown in Table 1.
Example 4
The procedure~ in Example 3 were repeated
except 315 mg of that dodecylamine was used in~tead of n-
octylamine to give a dodecylamine-lmmobilized gel.
The adsorptlon of the obtained dodecylamine-
immobiliæed gel was examined by means of the ~ame
procedures as in Examplle 1.
The results is shown in Table 1.
Example 5
The procedures in Example 3 were repeated
except that 410 mg of cetylamine (~f - 7.22) was used
instead of n-ootylamine and that ethanol was u~ed as a
.:
solvent in immobilizatlon instead o~ a 50 S by volume
aqueous ~olution of ethanol to give a cetylamine-
immobilized gel.
The adsorption of the obtained cetylamine-
immobilized gel was examined by mean~ of the same
procedures in Example l.
The result is shown in Table 1.
; Example 6
The procedures in Example 2 were repeated
except that 170 m~ of a porous cellulo~e hard gel
.,
,, ~ . ~ . . . . - - . ~ , . - .
~2~35925
- 14
Cellulofine GCL-300m (made by Chisso Corporation,
exclu~ion limit of globular protein~: 90,000) was used as
a carrier instead of Cellulofine GC-200m to give a
dodecylamine-immobilized gel.
The ad~orption of the obtained dodecylamine-
immobilized gel was excamined by means of the qame
procedureq as in Example 1.
The result i_ shown in Table 1.
Example 7
The procedures in Example 1 were repeated
except that 1 T mQ of a porouq cellulo_e hard gel
Cellulofine GC-lOOm (made by Chisso Corporation,
exclusion limit of globular protein~: 60,000) was used as
a carrier instead of Cellulofine GC-200m to give an n-
octylamine-immobilized gel.
The adsorption of the obtained n-octylamine-
immobilized gel wa9 examined by mean9 of the same
procedure~ as in Example 1.
' The result i 9 shown in Table 1.
Example 8
The prooedures ln Example 1 were repeated
exoept that 170 mQ of a porous cellulose hard gel
Cellulofine GCL-9Om (made by Chi~qso Corporation,
excluqion limit of globular proteins: 35,000) waq used as
; a~carrler instead of Cellulofine GC-200m to give an n-
octylamine-immobilized gel.
M ~ ~ ~ The ad~orption of the obtained n-octylamine-
30~ lmmobilized~gel was~-~examined by mean_ o~ the same
p~rocedures~as in~Example 1.
The~reqult is qhown in Table 1.
Example 9
35~ The~procedures in; Example 1 were repeated
e~xoept~that 1~7~0~mQ~or~a porous vlnyl polymer hard gel
Toyo~pearl HW-SO~o~oarse ~made;by Toyo soda Manufacturing
Co.~ td.,'~exolusion limit of~globular proteins: 80,000)
.... .. .. . . . . .... .. ..
~2~35925
-- 1 5
was used as a carrier instead of Cellulofine GC-200m to
give an n-octylamine-immbolized gel.
The adsorption of the obtained n-octylamine-
immobilized gel was examined by means of the same
procedures a~ in Example 1.
The result is shown in Table 1.
Example 10
The procedures in Example 1 were repeated
except that 170 mQ o~ a porous cellulose hard gel
Cellulofine GCL-300m was used as a carrier instead of
Cellulo~ine GC-200m and that 200 mg of dodecylamine was
used instead of n-octylamine to give a dodecylamine-
immobilized gel.
To 0.5 mQ of the obtained dodecylamine-
immobilized gel was added 3 mQ of plasma of a IgA myeloma
patient, the plasma containing Bence Jones protein which
i9 the immunoglobulin L-chain in a concentration of 200
~g/mQ, and the mixture was incubated for 2 hours at
3~C. The concentrations of 8JP and albumin in the
supernatant were measured to obtain the amount of
adsorbed BJP, ratio of adsorbed BJP based on BJP added
first and amount o~ adsorbed albumin.
The~results are shown in Table 2.
- 25
Example 11
The procedures in Example 10 were repeated
except that 410 mg o~ cetylamine was used instead of
dodecylamine to give a oetylamine-immobilized gel.
30~ The ad~sorption of the obtained oetylamine-
immobilized ge~l wa9~examined by means of the same
pro~cedure~s~as~in~Example 10.
The~result is ~shown in Table 2.
35~ Example 12
The~procedures in Example 10 were repeated
exo~ept~that~170~mQ~of a~porous oellulose hard gel
ellu1ofi~nè~OC-700~m~was used as a oarrler instead of
~Z~3592~;.
Cellulofine &CL-300m and that 220 mg of n-octylamine was
used instead of dodecylamine to give an n-octylamine-
immobilized gel.
The adsorption of the obtained n-octylamine-
immobilized gel was examined by means of the sameprocedures as in Example 10.
The re~ult is shown in Table 2.
Example 13
The procedures in Example 12 were repeated
except that 315 mg of dodecylamine was used instead of n-
octylamine to give a dodecylamine-immobilized gel.
The adsorption of the obtained dodecylamine-
immobilized gel was examined by means of the same
procedures as in Example 10.
The result is shown in Table 2.
Example 14
The procedures in Example 12 were repeated
eXcept that 410 mg of cetylamine was u9ed instead of n-
octylamine to give a cetyIamine-immobilized gel.
The adsorption of the obtained cetylamine-
mmobilized gel~was examined by means o~ the same
prooedure~ as in Example 10.
2~5 The result is~3hown in Table 2.
; Example 15
The procedures in Example 10 were repeated
exoept that 170~mQ of a porous oellulose hard gel
Cellulofine GC-200m wa~ u9ed a9~a carrier instead of
C~el;lulofine GCL-300m to give a dodecylamine-immobilized
The~adsorption of the obtalned dodecylamine-
immobl1l~zed~gel wa~s examined by means o~ the same
35 ~procedures~as in~Example 10.
The~result 1s shown~in Table 2.
Example 16
:~ :~ . - , . , . - . , . : - .
- 17
The procedures in Example 15 were repeated
except that 410 mg of cetylamine was used instead of
dodecylamine to give a cetylamine-immobilized gel.
The adsorption of the obtained cetylamine-
immobilized gel wa~ examined by means o~ the sameprocedures as in Example 10.
The result is shown in Table 2.
Comparative Example 1
The procedures in Example 3 were repeated
except that 172 mg of n-hexylamine (log P - 2.06) was
u~ed instead of n-octylamine to give an n-hexylamine-
immobilized gel.
The adsorption of the obtained n-hexylamine-
immobilized gel was examined by means of the same
procedures as in Example 1.
The result is shown in Table 1.
.
Comparative Example 2
: ~ 20 The pro¢edures in Example 3 were repeated
except that 125 mg of n-butylamlne (log P - 0.97) was
used instead of:n-o¢tylamlne and that water was used as a
solvent in immobilization instead of a 50 % by volume
aqueous so1ution of ethanol to give an n-butylamine-
immobilized g~e1.
The adsorption of the obtained n-butylamine-
immobilized gel wa9 examlned by mean9 of the same
procedures as in Example 1.
The re3u1t 19 shown in Table 1.
Comparative Example 3
The;p:rocedures :ln ExampIe 10 were repeated
exc~ept that 170~mQ of a porous cellulose hard gel
. `:`Celluloflne~GC-7QOm~wa9~u9ed as a car~rler lnstead of
35,~ :C~ellùlofine~:GCL-300m and~that 500 ~Q of 70 % by welght
a~que~ous:so:1utio'n~of ethyla~mlne (log P - -0.13) was used
in:s~t,ead of~d:odecylamine to give an ethylamine-lmmobillzed
~2~3~92
-- 1 8
The adsorbent of the obtained ethylamine-
immobilized gel was examined by means of the same
procedures as in Example 10.
The result i9 shown in Table 2.
Comparative Example 4
The procedure~ in Comparative Example 3 were
repeated except that 125 mg of n-butylamine was used
instead of ethylamine to give an n-butylamine-immobilized
gel.
The adsorbent of the obtained n-butylamine-
immobilized gel was examined by means of the same
procedures as in Example 10.
The result i9 shown in Table 2.
~35æ5
1 9
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-- 20
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- 21
As ~hown in Tableq 1 and 2, the adsorbent of
the present invention e~fectively adsorbs both ~2-m and
L-chain, while albumin is hardly adsorbed.
In addition to the ingredients used in the
Examples, other ingredients can be used in the Examples
a~ set forth in the specification to obtain substantially
the same re~ults.
, . : , .: ,. .
