Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Process of improvin~ the compatibilit~ of gamma globulins
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The present invention relates to a prscess of improving the
intravenous compatibility of gamma globulins precipitated
from blood or blood products.
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- Blood is a liquid which consists of solid and liquid com-
ponents. The solid components include the red and white
blood corpuscles as well as the blood platelets---~r throm-
bocytes. The plasma, i.e. the liquid portion of the blood,
contains about 90% of water and 10% of solids. The sub-
stances dissolved in the plasma include, among other sub-
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stances, the gamma globulin which is used for the therapy
and prophylaxe of infections. For the isolation of gamma
- globulin, all of the other substances contained in the
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plasma must be removed as far as possible, in order that
as pure as possible a gamma globulin is provided.
For the precipitation and isolation of gamma globulin from
blood, there is used particularly a process which is known by
the name "COHN-Method." (COHN et al., J. Amer. Chem. Soc.,
-; Vol. 68, pp. 459... 475, and Vol. 72, pp. 465... 474). This
process starts with a plasma mixed from various blood
samples. Principally, this involves a ~ractionated pre-
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~ cipitation under various conditions. In a first stage, ~
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as the first sediment there is initially separated mainly
fibrinogen at a temperature of minus 3~C and at a pH of
7.2 with an ethanol addition of 8%. The supernatant
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liquid is precipitated in a second stage with about 19% of
ethanol at a pH of 5.6. The precipitate contains mainly
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gamma globulin. For the purification of this precipitate
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~designated as COHN-Fraction II-XII), this precipitate is
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~ 30 re-dissolved and thereafter initially re~recipitated at a
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pH of 5 and with 8% of ethanol. The remaining supernatant
phase is thereafter again precipitated with 25~ of ethanol, '
and at a pH of 7.2 b The resul~ing precipitate comprises at
least 90% of gamma globulin. The precipi~ate is received
by a suitable buffer solution, and after the sterile
filtration this precipitate is ready for application to
human beings.
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It has been found that in numerous instances the gamma
globulin preparations which have been obtained in the above-
indicated or in another manner9 are of anticomplementary
activity such that complications result from corporeal
re-actions in the case of intravenous application.
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Accordingly, it has already been attempted to increase or
improve the intravenous compatibility of gamma globulins.
As examples, the following methods or processes known from
literature may be mentioned:
1) Treatment with suitable enzymes;
Z) hydrolysis at high hydrogen ions concentration (e.g.
at a pH of 4.0);
3~ modification by means of beta-propiolactone.
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However, it has been found that the known processes act to
~` modify *he gamma globulin molecules_and to vary them in
~heir chemical structu~e to such degree that their activity
~ is reduced and their average retention time in th~ organism
`:~ is shortened.
~ Therefore 9 the present invention has.as its object the
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r.," provision~of a novel process for increasing or improving
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. the intravenous compa~ibility of gamma globulins, which
` ~ process, on the one hand, avoids the disadvantages of the
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conventional processes and, on the other hand, provides the
following improvements:.
- The st~ucture of the existing gamma globulin molecules
is varied to minimum degree,
- the anticomplementary activity should be greatly reduced
as compared wi~h conventional gamma globulin preparations;
- the retention period in ~he organism should be increased9
whereby the preparation should be more compatible than
conventional preparations.
These objects are solved by a process for impro~ing the
intravenous compatibility, wherein the gamma globulin
precipitated from blood or from blood products is introduced
into an aqueous solution in the presence of macromolecular
substances which shield the globulin molecules from each
other and which displace these molecules from the solution,
such as hydroxyethyl starch (HES), gelatine, destranes, ~ t
albumin, polyalcohols, poli~inyls, whereupon the gamma
globulin is precipitated and re-dLissolved, particularly in
a phisiologic~l normal saline solution.
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Acco~ding to present knowledge, the effecti~ity of HES is
at an optimum; however, it is likew*se possible to obtain
~;~ a similar effect by the adtition of the other substances
specified above.
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Preferably, the precipitate is introduced into a buffered
- aqueous HES solution having a pH of from 3.5 to 8Ø A
`; particularly fa~orable practical value has been found to --
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be within the range of pH from 6.5 to 609.
~¦ The contents of HES in the aqueous solution may range ;~
between 1 ~nd 30~; a particula~ly good practical Yalue ~ ~ ;
amounts to f~om 8 ~o 10% of H~S.
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~L058~) 75
Preferably, it is operated with HES having a molecular weight
of between 1,000 and 900,000.
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Upon redissolving of the gamma globulin precipitate in the
buffered HES solution - is added to the mixture (10~ of
polyethylene glycol). The mixture is made free of unwanted
precipitan~s by centrifugation. In the place of the poly-
ethylene glycol as the precipitation agent~ other polymerized
polyvalent alcohols may be used well. The remaining super-
natant phase after centrifugation is mixed with 20~ of poly-
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ethylene glycol at a pH of from 7.Q to 7.2 and then centri-
fuged anew. Expediently, the thus obtained precipitate is
dissolved in a physiological normal saline solution and
adjusted to a concentration of about 5% of albumin or
~, protein. After the sterile centrifugation, the solution is
ready for therapeutical use.
r'; Below, the individual process steps are explained again in
; an Example.
` Separation of the gamma globulin:
;i T~e process starts with a collected plasma which is blended
,; 20 with 8~ of ethanol and precipitated at a pH of 7.2 at a
temperature o minus 3C. Hereby, Fraction I is separated.
The recovered liquid is thereafter mixed with 19% of ethanol
a~ a temperature of minus 5C and a pH of 5.8. Hereby,
Fraction II-III is separated which consists of gamma globulins.
The precipitate is re-dissolved and again precipitated at a
pH of 5 with 8% of ethanol. The remaining supe~natant phase
is then again preclpitated with 25~ of ethanol at a pH of
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~ 7.2. The precipitatè obtained in this stage (= Fraction II)
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~; comprises at least 90% of gamma globulin. ~
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Reduction of t~e anti`complementary activity:
The gamma globulin precipita~e is resolved in a buffered
- aqueous solution at a pH of 6.7, in a concentration of
about 6%, whereby the aqueous solu~ion has added thereto
- about 10~ of hydroxylethyl starch ~HES). The HES renders
` possible a separation of already existing aggregates and
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i simultaneously protects non-aggregated gamma globulins.
Introduction into a Physiolo~ical normal saline solution:
After the addition of 10~ of polyethylene glycol 7 the
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mixture is centrifuged. The remaining supernatant phase is
mixed with 20% of polyethylene glycol at a pH of 7.2, and
centrifuged. The thus obtained precipitate is adjusted to
a concentration of 5.2~ of albumin in a physiological
; normal saline solution, and subsequently filtered under
sterile conditions. Thereupon, the precipitate is ready
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~ for therapeutical use.
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~ The accompanying drawing shows in Figure 1 thereof the
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structural formula of hydroxyethyl starch (HES), and in
Figure 2 thereof immune electrophoresis diagrams ~IEP) of
various gamma globulins or of normal sera, respectively,
. which have been prepared in accordance with the conventional ~ -
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: and novel processes.
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', The upper half of the diagrams of fig. 2 each shows IEP
diagrams of normal blood sera, whereas the lower half shows
diagrams of:
(a) standard gamma globulin~
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;~1 (b) proteolytically modified gamma globulin, ~
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`' ~c~ beta-p~opiolactone-modified gamma globulin,
j (d~ gamma globuline as prepared in accordance with the
: . 30 noveI process. . ~ -
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Samples (a) to (c) each represent a commercially available
modified preparation. In addition to the gamma globulin
line (elongated crescen~-shaped line in the right hand
portion of the diagram), ~hese samples also show additional
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albumin or protein components of the human blood. Owing
to the chemical modification, the gamma globulin lines of
samples (a) and (b) are shown blurred. Sample (c) exhibits
a varied position as compared with the gamma globulin line
of the cont TO 1 S erum.
In contrast, it is clearly evident that sample (d) consists
substantially exclusively of pure gamma globuline as the
crescent-shaped line of the spectrum is distinctly present
in the normal.serum.
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9 It can be concluded from the diagrams that the gamma
globulin obtained by the novel process has a purity of
substantially 100% so as to completely correspond to the
' gamma globulin of the original blood serum. The la~ter
fact means that the molecules are not modified or chemically
~ Yaried. These characteristics result in the further advan-
; 20 tageous properties of the gamma globulin according to the
; novel process which have also been verified by tests, namely
`` its absolutely positive intravenous compatibility and its
greatly reduced anticomplementary activity to intracorporeal
; antibodies, which properties could be clearly demonstrated
;~, in in-vitro tests.
As another advantage, storage tests nave shown a particularly
high stability of gamma globulins prepared in the above
disclosed manne~, using the steps of invention.
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