Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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T}le presellt inv~ntien relates to a pTocess for
increasing the intravenolls compatability oE gamma
g:lobulil~ p ~ci~ cl rrom blood or Erom b:loocl products,
wherein the ganlma glubu:lin precipitate is ie cliss~l~o~l
in an aquaeolls solution containîng macromolecular
substances~ preferably hydroxyet]lyl starch, active to
shield the globuline molecules from each other and
displacing them from the solution, according to Patent
(patent application).
In the process entitled'Process for Improving
the Compatability of Gammas Globulin",f:iled in Canadc
on the 30th day of December, 1975, as serial number
242734, the portion of undesirable gamma globluins,
is removed by re-introducing them into an aqueous ~i
solution o- particu'larly hydroxyethyl starch in a
concentration of from 8 to 10%. The mixture is
centrifuged after the addition of polyethylene glycol
(PEG). The settling precipitate contains the undesirable
portion of gamma globluin, whereas the desirable portion
is recovered in another centrifuging step at a pH of
7.2 and with 20% of P~iG. ~ ,
Even if the gamma globulin obtained by the
above-described process meet every requirement of medical
practice, nevertheless it has been found to be desirable
to increase the yield and to provide a still higher
quality and purity of the desired gamma globulin. ~'
According to the present invention, these ,`
objects are solved in such a process by additionally ~;~
mixing an aqueous solution containing gamma globulin
with a slurry or suspension of micaceous stratified -
silicates in a concentration in the rage o~ from 0.2
to 10 pcrcent, by weight; alld, a~ter allowing a
sufEicient period of reaction to pass and adding a
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sufficient perio(l oF reaction to pass and ad(ling a
precipitclnt sllch as polyet}lylelle glycol, rcmov;ng the
gamma globuLin portion to he separatcd or disposed o~
toge-ther with said stratifie(l silicates.
The inverltion thcrefore contemplates a
process of increasing the intravenous compatability of
gamma globulins precipitated from blood or rom blood
products, wherein the gamma globulin pr~cipitate is
re-dissolved in an aqueous solution containing
macromolecular substances, preerably hydroxyethyl
starch, active to shield the globuline molecules from
each other and displacing them from the solution,
cha~acterized by additionally mixing said aqueous
solution containing gamma globulin with a slurry or
suspension of micaceous stratified silicates in a
concentration in the range o rom 0.2 to 10 percent by
weight; and, after allowing a sufficient period o
reaction to pass and adding a precipitant such as polyethylene
glycolj removing the gamma globulin por~ion to be separate
or disposed of together with said stratified silicates.
The term 'rmicaceous stratifiedsilicates"
refers to silicates the composition of which is expressed
by the following average formulae:
) M +Y {(A13~ Cr3+ Fe2* Mg2*,Zn2 ,Ni )2 3~o~l)2~si4-xAlxo)lo}
and/or
b) Me~Y {A13~,Cr3,Fe ,Fe jMg ,Zn ,Ni ,Li )( 3 ) ~0H)2~Si4
The ~gtions Me may be replaced zeolitically by other,
even organic cations. As the swelling liquid Y9 any
desired molecules, with the exception of saturated
hydrocarbons, may be introduced. The quantity of the
swelling liquid may be varied within broad limits. In
view o~ the fact that thc quantity x of the monova:Lent
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cation~ ructural Ull;t o~ (Sl, ~1) 410~ namely the
stratificatioll or s~ratifiecl charge, varies, too, an
extremely great variety results.
Extensive research work has been conclucted
particularly with respec~ to the montmorillonite ~compare
in this connection the bibliography according to "Organische
Derivate der glimmerartigen Schichtsilikate" by WBISS;
"Angewandte Chemie" 1963; pages 113 et seq.). Mont~
morillonite compr:ises two-dimensionally in-finite macro
anions being electrosatically reticulated through the
intermediate cations. The energy -for the swelling action,
i.e.,~or increasing the stratum distance by spacing the
positive and negative charges from each other, is
provided by the hydratization or solvatation of the ;~
cations and anion strata. Since the solvatation energy
in general is substantially smaller than the hydratization
energy, only molecule species having high dielectric ;
constants or having the capability of forming hydrogen
; links can be incorporatedO
Amino acids, peptides and proteins may be
incoTporated into micaceous stratified silicates. At a low
pH, proteins are exchanged almost quantitatively. With ;~
increasing pH value, the number of the cationic positions
within the proteins decreasesg and the exchange comes to
be incomplete.
lbumins, globulins and prolamines are likewise
bonded. With serum globulines, the reaction appears to
proceed from the edge of the silicate crystals into the
interior thereo~ to a depth of only ~rom 20 to 30 R
(~ngstro:m). Albumins are spread between the silicate
strata. The Stratum spacing of from 14.5 to 15.0 R
shows that a stratum thickness of a maximum o-f 5.5 to - -~
6 R is available to the spread protein.
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In viel~l of tlle f.lct thclt the quclntity oE
gamma glubuliTI introduccd colltains both dal1lagcd ~injured~
and undalllaged or intact molecules, it must bc attempted
to bond as far as possible the damagc~l molecules in
one way or other so as to render them chcmically distin~uish-
able from the physiologically per~ect mole cules. The
damaged molecules are capable of Eorming hydrogen link
bonas. -Therefore, they are deposited and bonded to
greater degree within the silicate strata, whereas the
intact gamma globulin molecules are left in the solution.
In their state as bonded within the strata of the
stratified silicate, the damaged gamma ~lobulin molecules
can easily be removed subsequently, e.g. by centrifuging.
The cation exchange capacity being desirable
according to the invention, is exhibited according to
HOFMANN (Z. Kristallogr. ~A) 98 ~1937) 31; Chemie
55 (1942) ~283) also by kaolin and related substances
(for instance, halloysite, A12O3.2SiO2.4H2O) and certain
micaceous minerals being related thereto with respect
to their lattice structure.
These substances also include vermiculite and
the related batavite (= iron-ree vermiculite). According
to WEISS and HOFFMANN (Z.Naturf., 6b, 405) the structural
formula of the vatavite is as follows:
82.64 0.33~ ¦(OH)1.9 ~ ¦ Si2 99A11 ¦} S8
~Mgo . 34~2 ~34H20 .
It has been found that this material, same as
the montmorillonite, exhibits intercrystalline swelling
capacity and a high capacity of cation exchange. -
Investigations ~compare the abo~e references? showed
that batavite absorbs up to 5 percent by weight of
human albumin, with the protein being deposited between
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the silicat~ stra-ta. Likewise, verl~iculite having the
approximatc structural formula
{~Mg3,A12,~e~ r:2~ O~I) [si3~3sAlo~tj5olo~} ,65
exhiblts the desired properties regarcling the strata
formation and swellability. Vermiculite corresponds to
a tri-octahedral montmorillonite, but its crystals are
by far greater and the lattice is shaped more regularly.
Summarizinga it may therefore be concluded
that such minerals are suitable for the purification
process which have a stratiEied structure, which show
cations between the silicate strata, and which tend to
swelling. According to experimental experience, minerals
of this type act to retain the defect gamma globulin
molecules - apparently due to their strong cation ~ -
activation - whereas intact molecules are not bonded.
It is hereby necessary to maintain specific limits of ~ ;~
concentration and of the pH value. ;~
According to the present invention, particularly
the minerals montmorillonite, batavite or vermiculite
are employed. Depending on the dissolved quantity of gamma
globulin du~ing -the re-dissolution~ from 0.2 to about 5
.
percent by weight of silicates are added. According to ;~
experimental experience, the reaction takes place within
a termperature range of from 2 to 20C, and the ~ ~
process is carried out at a pH of between 4 0 and 7~6~ ~-
The time or reaction should range between 3 to 10
hours. Preferably, the process is carried out overnig~lt -;
~since the rçaction proceeds relativcly slowly and an-~
exkended period of time provides improves results. -~
Discharge or separation of the silicates is perfol~med
preferably by centrifuging step. llowever, otller methods
known from general process techniques may be used, too,
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such as e.g. IIlu~!ial ~iltratioll.
Prior to givin~ cx~lmples of the embodiments,
it should bc noted that calculatiorls arld measurcments
by immunc electrophoresis diagralns al]Lo~ for
the follo~ing determination; namely, that the
yield oE pure gamma globulin is incre~sed by applying
the process according to the present invention. The
gamma globulin recovered is not modifiied nor chemically
varied. It is absolutely compatable when administered
intravenously, and does not show, substantially, any
anti-complementary properties or characteristics.
Furthermore, the substance appears to possess a high
degree of stability.
The following Examples are intended to further
explain the invention.
Example 1
~a) Separation of the gamma globulin:
The process commences with a collected plasma
which is mixed with 8% of ethanol, and which has been
p~ecipitated at ~ pH of 7.2 and at a temperature of minus
3 C. Hereby, raction I is separated. Thereafter,
the supernatant is mixed with 19% of ethanol at a
temperature of minus 5 C and at a pH of 5.8. In
this step, fraction II-III is separated which comprises
gamma globulins. The precipitate is re-dissolved
and again precipitated at a pH of 5 and with g% o-f
ethanol. The remaining supernatant material is then
again precipitated with 25% of ethanol at a pH of 7.2.
The re~ulting precipitate (= fraction II) compri~es
at least 90% of gamma globulin.
(b) Reduction of the anti-complement,ary activity:
The gamma globulin precipitate is introduced
into a buffered aqueous solution having a pH of 7.0, in
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75Z~ 7 -
a concentration of about 6%, with about 10% of hydroxyethyl
starch being added to the aqueous solution. Further, an
aqueou~ slurry or suspen~iorl of bentonite SF (main
,
components: montmorillonite; grain ~ize less than 80
microns; manufacturer: Feinbiochemika, Heidelberg) i~ added
to the solution. Thereupon, the solution is mixed with 2.5
percent by weight of bentonite, and the mixture i8 agitated
thoroughly. Then, the ~olution i~ allowed to stand for 6
hours at a temperature of 15 X + 2. ~ollowing this, the
bentonite together with the unde~irable cornponents is
removed by centrifuging after the addition of 10% of
polyethylen0 glycol.
(c) Conversion into a phy3iological normal saline
solution:
The ~upernatant material obtained by the
centrifuging ~tep contains the desirable gamma globulin
constituent~ in the form of a solutio~. The supernatant
liquid i8 adjusted to a pH of 7.2 by means of 0.1 NaOH and
mixed with ~0 percent by weight of PEG. Hereby a
pr~cipitate of pure gamma globulin ~et~les. The precipi-tate ~-
is recovered by centrifuging, adjusted to a concentration of
5.2% of protein in a physiological normal saline ~olution,
and thereafter 3ubjected to ~terile filtration. Then, this
material i ready for therapeutical u~e. ~ ~
~XAMPLE 2 `~ -
Step~ (a) and (c) are identical to the re6pective
step~ according to Example 1. Step (b) i~ modified as
~ollows:
The gamm~ globulin precipit~te i~ introduced into
a buffered aqueous ~olution having a pH of 7.4, in a
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concentration of about 4 percent by weight, with
approximately 8% of hydroxyethyl starch being added
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to the a~ eous s~lutiorl. Purtllcrmore, the solutio
is added an aqueous slurry or sllspension of Einely
divided (p~rticle size lcss than 80 microns)
vermiculite until a proportion of 5 pelcellt by weight
is reached. The whole composition is agitated
vigorously. Thereupon, the solution :is a:llowed to
stand foT 8 hours at a temperature of 15 - 2 C.
About 10 percent by weight o~ hydroxyethyl staTch
are then added to the solution. Fina:Lly, the
~ermiculite is removed by centrifuging together with
the undesirable componenets.
This operation is followed by method step (c).
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