Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
6~4
Process of preparing a serum protein composition for
intravenous application
The present invention relates to a process o preparing a
serum protein composition for intravenous application or
administration, in which process, starting from a human blood
protein solution, a stab~ized, universally applicable
preparation or composition is prepared which contains dissol-
ved in an aqueous isotonic solution, such proteins in which
the ratio of globulins and albumins corresponds substantially
to that of the native blood serum.
. , I :
- ,. . .
; Serum protein preparations or compositions represent valuable ~
, . .
~ blood derivatives. The albumin contained in such compositions
. .
functlons as a transporting protein. These compositions
; contain further functional proteins, such as transferrin,
eruloplasmin, ~ -antikripsyn and particularl~ the immune
globulins. Of importance ara the antibodies which are present
in the ~ -globulin. Due to the passive immunization caused
by these substances, they are suitable for prophylactic
:
therapy, and they strengthen the body weakened by loss of
blood.
., ' .
A serum protein composition which should be suitable to be
intravenously administer d to a human patient has to comply
with two strict requkements:
~' :
_3~
-- 2 --
1 1) The composition must form a storable product;
¦~ 2) infection carriers (vixuses or bac-terla) should be
! neutràlized reliably. This applies particularly to the
carrier of virus hepatltis.
Serum protein compositions are normally provided in the form
of an aqueous solution of the stable proteins
` ~; having an electrolyte
content that is toler~ble to the ~ody. The total protein
content is normally 5 percent by weight ~i.e. 5,000 milli-
I grams per 100 milliliters)~
. ~
The l~terature shows to ~e known a process for the
preparation of a serum protein composition suitable for
- intravenous application or administration, which composition
is obtained directly from human blood in a number of
process steps (W. Stephan r "Hepatitis-Free and Stable
Human Serum for Intravenous Therapy"; XXIVth Sc. Meeting
;~ of the Blood Research Institute, Vox Sanguin, V. 20,
(p. 422 - 457));
The conventional process i5 performed in the followin~
steps:
, ~
(a~ Recovery of a natural serum from donor blood upon
removal of the solid constituents ~blood corPuscles,
blood platelets) and recovery of a serum having a
protei~ concentrat~on of about 7.5 % and a ~H of from
7.2 to 7.8.
3269~
- 3
1,
.
(b) For the removal of the lipoproteins which can be
easily denaturalized~ 2.0 g of Aerosil 2491~380
(Aerosil: registered trademark of the firm Degussa,
for a colloidal silicic acid) are added per 100 ml
of serumO The suspension is agltated for 4 hours at
45C, cooled, and the sediment is removed ~y
centrifuging. The lipoproteins are therebv
~ quantitatively bonded to the Aerosil so as to be
: removed from the blood serum.
: i
(c~ FilterinYof the solutlon through a microfilter for
~;~ the removal of any suspended particles still present
~- in the so~ution~ :
(d) For sterilization, the solution is mixed with 0.3 g
B-propiolactone per 100 ml of solution at a temperature
of 5C (pH 8.0~ and irradiated with ultraviolet light.
~, . .
The thus obtained serum has the following composition
~: (based upon S,000 milligrams contained in 100 milliliters):
4,
SubstanQe ontent Function
Albumin 3,060 On~tic activity; transport
of nutritive materials, vitam-
ins, hormones, medicaments.
IgG 820 Ant1body to viruses and
bacterla,
IgA 185 Antibodies protecting the
mucous membrane.
IgM 75 Anti~odles to bacteria and
toxines.
~; Prealbumin 17 Tyroxine bond.
. ~
5~ Oæ1-antitrypsin162
d42-macroglobulin 141 Inhi~itors to pr~teolytic
enzymes.
~aptoglobulin ~10 ~onding and transport of free
haemoglobulin.
Haemopexin 75 Bondlng and transport of free
haemin.
Transferrin 195 Transport of Fe+~
Coeruloplasmin1~ Transport of Cu~ as
peroxidase.
Cholines~erase3.0 Elml Splitting of succinylcholin.
: . ' .
;;,; , . ~ ~he above Table also reveals the functions of the variOus
constltuents of the serum protein composition.
he composition prepared in accordance with the conventional
process is commercially available. This conposition mav be
:'
' ;:,
applied without complication.
~, ' ' ' ' ~
However, it is considered to be disadvantageous with
respect to the preparation and application tha-t the
composition is very costly because of the high-quality
starting material - i.e. human blood - and cannot be
made available to a sufficient extent. Add to this that
the contents of the particularly highly active antibodies
IgG, IgA, IgM is relatively low in correspondence with
the presence of these substances in the human blood~
: ' :
Accordingly, it is the object of the present invention to
provide a process for the preparation of a serum protein
composition for intravenous administration, in which the
starting substances are less expensive and rare. This
prccess should allow to use for the preparation of
valuable serum protein compositions such starting
substances which have no, or inadequately only, been
processed or recovered heretofore. It is a further object
of the invention specifically to increase the content of
active antibodies or other proteins of the serum without
thereby increasing the cost of preparation to any
substantial degree.
In the above mentioned process, this object is sol~ed hy
usin~ as the starting material, fractions of human blood
plasma which are recovered from human serum with the use
l~OZ694
of one or more fractionating steps ~erving to enrich
(concentrate) or ~solate, respect1vely~ individual ones
of the blood protein ~onstituents with these latter
constituen~s being substantially preserved in their
native form;
and ~haracterized by the fact that the fractions of the
starting material are proviaed with a composition o-E
albumins and globulins equivalent to that of the blood
serum, by adjustment (standardization) of the mixture,
stabilization, purification ana concentration or
dilution, respectively. Especially, this i~ performed by
the steps ~ - d of claim 1.
The generalized wording o the claim for patent proteation
~tates that, although it is started with human blood as
the starting material, the preparation of serum protein
composition in accordance with the present process is no
longer performed directly on the basis of the native serum,
but that other intermediate ractionation products are
employed which result from the fractionation of blooa~ In
this connection, it is an objec~ of the present invention
to employ well~known process products which h~ve not been
utilized heretofore, to useuncomplicated process steps,
and to increase the recovery of the proportions of useful
products present in the hum~n blood.
: i :
In particular, the process according to the invention
makes use of the fact that the ~lood fractionation according
to COHN yields certain fractions which could heretoore not,
or incompletely only, be further processed.
~,
The process according to COHN is based upon the -~
fractionation of the blood plasma with the use of e~hanol
as a precipitant under precisely determined conditions
of temperature, of the pH, of the ion densities and of
the ethanol content. The COHN process is described, for
lnstanc~, in the following pu~lications:
~OHN, E.J. et al.: "JOAmer.Chem.Soc." 72 (1950),
p~ 465 - 474
COHN, E.J. et al.: "~.Amer.Chem.Soc." 68 ~1g46),
p. ~59 - 475
U.S. Patents 2,390,074 and 2~469,193.
.',~ :;
The enclosed flow diagram shows the various Process steps~
In thls diagram, the expression "~ of ethanol" always
refers to percents by volume, measured at 25C. The startin~
plasma is obtained from donor blood. 500 milliliters o~
human blood are each collected in 50 milliliters of a
4 % solution of sodium Citrate. Initially, t~e solid
constituents of the blood are separated from the plasma;
the plasma of a plurality of donors is psoled.
26~4
.
- 8
i
In step a accor~ing to the flow diagram, the crude
~fibrionogen is removed from the plasma by addin~ th~eto
'. cold 53.3 ~ ethanol up to a concentration o:E 8 ~.
The temperature is maintained at from -2t5 to -3,0VC.
The precipltate P 1 - fibrinogen - is removea ~y
centrifuglng. The supernatant S 1 is further processed.
. . :
Step b again comprises the addition of 53.3 '~ ethanol
to the supernatant liquid S 1 until a concentration of
from 18 to 25 ~ of ethanol in the liquid reachad. ~he
temperature is kept at -5~C, and the pH is adjusted to .
5.8. The precipitate P 2 is precipitated, which is
designated as fraction II~III or as y-globulin fraction~
This precip1tate includes the immune globulins and other
physiologically si~rificant protein5. The precipitate
P 2 is removed by centrifuging at -5C. The supernatant
liquid S ~ is further processed.
.,;:
Step c involves the further treatment of the supernatant
S 2. The ethanol concentration is set to 40 ~. Similarly
as ln ~tep b, the temperature and the pH value are set
to -7 C and to 5.8, respectLvely. By centrifuging, the
precipitate P 3, also termed fraction IV, is o~tained.
The latter contains the ~-globulins and B-~lobulins.
'
:i
The supernat,ant liquid - Supernatant S 3 - is thereaf ter
~' further processed. To this end, the conditions of temperature ;~
~ 7C, pH = 408)and ethanol concentration = 40 % are
'~ malntained. In this wayl precipitate P 4 is precipitated,
namely to the so-called crude al~umin, which is purified an
steriliæed in a manner known per se. The supernatant
: liquid - Supernatant S 4 - is disposed of.
;:
The ~-globulin fraction II~III is further processed hy the
following steps: ~
-.
..
Pr~cipitate P 2 is suspended in a citrate phosphate ~uffer
a~ a pH of from 7.0 to 7.4. The temperature is held at
15C. 3 % of polyethylene glycol (PEG) 4,000 or 2.5 ~ of
PEG 6,000 are added.
The PEG used in practice comprises a mixture of non-
voIatile polyethylene glycols which are solu~le both in
water and in organic liquids and which have a molecular
weight on the order o from 4,000 to 20,000. A mixture
of polyethylene glycols of this type having an average weight
of 4,000 :is termed PEG 4,000.
Upon thorough agitation and after a reaction period of
from 1/2 40 4 hours, the mixture is centrifuged, whereby
~z~
I
I - 10 -
Supernatant S 5 and fraction III-1 (precipitate P 5) are
~ , , .
formed. Supernatant S 5 is again treated under modified
conditions ~pH 4,6; PEG 4,000 up to about 5.5 %;
temperature = i5C) and fractionated. Fraction III-2
(precipitate P 6) and Supernatant S 6 are formed, the
latter containing primarily the immune globulin IgG The
globulins of fractions III-1 and III-2 are enriched or
concentrated to substantial degree with the immune
globulins IgG, IgA and IgM. Further, ~ antitripsin,
~2- haptoglobulin, coeruloplasmin, transferrin and
haemopexin are present in concentrated form.
In general, fractions III-1 and III~2 are not further
process a. These fractions are available in large
quantities, because it is in the first line the a~bumin
that is recovered from the blood, while the ~-globulins
play a secondary role only.
As the starting products for the process according to the
invention, mainly fraction IV as well as fractions III-1
and III-2 of the above-described fractionation are employed
~precipitates P 3, P5, P 6).
These fractions are mixed in predetermined rations bv
adjusting the ratio to from 50 to 70 % of albumin of from
50 to 30 % of globulins, respectively, based on the total
protein. X-t is possible in th~s way to select in the
above-~entioned ratio the same values as are present in
the native serum proteins, too.
Elowever, hy correspondingly increasing the additio:ns of
the globulin portion or of the proportions of fractions
III-1 and III-2, it is also possible to specifically
increase the proportion of globulins in the form o IgM
~nd IgA.
. . .
In a preferred mode of operation, it is contemplated
that fractions IV, III-1 and III-2 ~sediments) are
suspended in a normal saline solution or in a suitable
bu~fer solution, respectively, i.n the following
quantities:
Fraction IV fxom 30 to 80 percent ~)~ weight
Fraction III-1 fxom 0 to 70 percent by weight
Fraction III-2 from 0 to 70 percent by weight.
based upon a total protein content corresponding to
100 % each.
Likewise, it is possible to recover the serum protein
compound from fractions III-1, III-2 or IV only~ and to
prepare these fractions from separate fractions in a
z~
12
stable, dissolved form as the.preparation or composition,
for the concentration of specific plasma proteins~
Similarly, desired ratios of components may be produced
- upon separately preparing the flnal products o~tained
from fractions III~ 2 and from fraction IV.
':
~he substanc~s mixed in a fi~ed ratio and suspended in
a suitable buffer or saline solution are subjected to a
pre-purification. In such pre-puriication, the lipid
factors of the ~- and ~-glo~ul~n series are remaved
by adsorption to suitable adsor~ents (Aerosil, bentonite
or other sillcic acid containing complex compounds).
Thereupon, th~ product is purified by filtering and
dialyzed, and the solution is concentrated. Normally,
a seperate sterilizing step is not necessary. However~
such sterilizat~on may be performed in the well-known
manner. For example, two methods are used for
eliminating viruses:
(a~ Pasteurizing by heating to 60C for 1~ hours.
Thi.s method suffers from the drawback that numerou~
serum proteins are denaturaliæed under the above
conditions. ~,
- 13 - .
(b) Combination of B propiol~cthonetreatment and
ultrav}olet irradiation~method according to
Lo Grippo; Fed. Proceedings l5, page 518, 1959).
~his method allows to sterilize sera and plasmas
under mlld conditions; the storage stability is
not improved thereby, however.
In general, in accordance with the present process the
mixture of the starting components, after having been
aaiusted or standardized to a specific protein content
and to a specific ratio of the proteins, is subjec~ed
to a similar process as is conventional for the serum
~W. Stephan, loc. cit;). Surprlsin~ly~ it is in this .:
way possible to prepare a serum protein composition
which is superior to the conventional composition in ;
lts e~ectiveness or activity, due to its increased
content of immune globulines Ig~ or IgA, respectivelya
Of course, it ~s feasible to remove from the individual
fractions as such the constltuents tending to beCome
denaturalized and thereafter to mix, concentrate an~ I
stabilize the purified fractions. Also, it is feasible
to use as the startin~ material a ~ractionation product
from a ~ractionation with rivanol. To its end, fractions/
1,
6~ -
I II, III and IV according to STEINB~CH are suitable,
I which fractions are described in Vox Sanguin.(V. 23,
pages 92 to 106)~
Preferably, operating with adsorbents is effec-ted in a
concentration of from 200 to 500 milligrams of silicic `~
acid per gram of total protein and at a temperature of
up to 50C. The substances are intimately mixed,
whereupon the adsorbent loaded with the lipoProteins
is separated. `;
As experiments have shown, the most favorable results
are obtained at temperatures of between 20 and 50C or at
p~ values of between 6.5 and 8, respectivelv.
The followings examples serve to explain the process in
greater detail. :~
Example 1
To 300 liters of normal saline solutian (aqua destillata
containing 1.7 percent by weight of NaCl, adjusted to ~
pH of 4.6 by using 0j2 n of HCL) - termed solvent in the
following -, 10 kp of COHN fraction IV showing the
folloNing analysis of the protein constituents, are
.
Zfi~4L r
~1 5
added and suspended therein:
55 % of albumin
10 % of ~1-glo~ulin
8 % of ~2-glo~ulLn
15 ~ of B-glo~ulin
12 ~ of ~ globulin
Fraction IV contains about 50 g of solids CProtein. salts)
and 50 % of residual moisture (N20; residual ethanol).
Further, S kp (kiloponds) of COHN fraction III-1
(subfraction) are added, w~ich fraction shows the
following analysis of the solid constituents ~percent
by weight):
15 % of albumin
2 % of ~1-globulins
13 % of ~2-globulins
17 ~ of B-globullns
53 % of ~ globullns,
and 5 kp of COHN subfraction III-~ are suspended there~n,
whlch subfraction has the follow1ng composition:
2~
- 16
8 ~ of al~umin
~ % Of ~.1-globulins
9 % Qf ~2-globulins
25 % of ~--globulins
55 % of ~-globulins.
The ration between solids and residual moisture is
substantially identical in all of these fractions.
The 20 kp pf "moist" fractions are suspended and s~irred
~n the so~ent. ~ereby, the p~ is maintained at a constant
value of 4~6. Soluble substances present in the fractions
a~e sissolved or suspended ln the solvent. Coexisting,
insoluble components cause turbidity. The insoluble
substances comprise a portion of 12 percent by weight,
based on the total content of solid substah~es. The .
lnsoluble substances involve particularly denaturalized
globulins, including lipoproteins wh~ch are not required
in the further course of the process. These latter
substances are therefore removed in a centrifuging
s-tep.
The supernatant li~uid is slightly turbid and of
yellowish color.
.
. By adding 0.2 n of NaOH, the pH is adjusted to
7.4. Pure colloidal silicic acid is added to the solution
untiL a concentration of 5 percent by weight is reachecl, and
this silicic acid is admixed by stirring. The :Liquid is
heated to 45C (about 1C per minute), whi.le ]ceeping the p~l
constant. When the final temperature of 45C is reached,
the liquid i5 agitated for 4 hours. Duriny this process
step, the storage instable proteins are bonded to the
silicic acid to form a precipitate which is removed by
centrifuging.
The supernatent liquid from the centrifuying step
is subjected to a clarifying filtration over active carbon
filters (~,anufactured by the firrm Seitz (AKS-Filter)). The
thus obtained filtrate is a clear liquid of arnbe~ color.
The liquid is concentrated to about 25~ of the initial
vol~ne by means of a dialysis concentration method
(Millipore~ cassette syste, pore size of the membrane or
diaphragm 10,000 Daltons). Following this step~ the
solution has a total protein concentration of from about 3.4
to 4.5 percent by weight.
An analysis shows that substantially all of the
blood constituents having a moleeular weight of less than
10,000 have been removed from the concentrate by the
dialysis concentration step. Tbese constituents include
particularly those substances which are undesirable in a
protein e.g. the oligo pep-tides showing a vaso-active
effectiveness.
Following this, the concentrate is su~jected to a
dialysis with three times the volume of a 9 percent weight
NaCl solution for the final removal of the above-ment:ioned
"impurities" and for the adjustment of the electrolyte. The
` - 17
.: . .
dialysis is performed in the same devices and with the same
diaphragms as have been employed in the concentration step.
This processing is followed by another
concentration step at the termination of which step a total
concentration of the protein of from 6 to :l0~ is present.
Then, the phy~iological conditions of the isotonic
pararneters accordlrig to the human blood are standardized by
means of a NaCl solution, and the total protein
concentration is set to 5 percent by weight.
The analysis of the protein recovered according to ~
example 1 shows the following:values: ~ ;
(based on 100 milliliters and a
Protein - content of 5,000 milligrams)
Albumin 2,925 "
IgG ~ 1,500
IgA 3 ao
IgM ~ 285 "
The thus standardized;solution is additionally
filtered for clarification through a germicidal filter (EKS
~20 II; Seitz,:Kreuznach). The sterlle filtration is performed
with the aid of membrane filters.
In its finally bottlel ~tate, the composit.ion is
now ready for intravenous administratlon.
~XAMPLE 2
~ ~ In the salne manner as in example 1, the following ,~
: materials are dissolved in 300 liters o~ solvent:
7,5 kp of COHN fraction IV
4 kp of COHN fraction III-l
and 8,5 kp of COHN fraction III-2
The composition of these fractions is identical to
that of exarnple 1. The fractions are mixed by stirring with
18
. - ..
the solvent, and the pEI is maintained at 4.6.
60 grams of Aerosil 2491/380 per liter of solution
are added to the latter, and the pH i.s brought to 7.6. The
IlliXtUre i5 agitated for 4 hours at 47C and thereafter
coolecl to 18C and subjected to alluvial filtratiorl. q~his
filtration step is perfor~ed in an upright alluvial ~r
precoat filter, type CHF-S of the firm Schenk, E'ilterbau,
Schwab. Gmund. As tXe filtering aid, ~yflo~ Super Cel is
employed in a concentration of 8 percent by weight, based
upon the filter volwne. Likewise, kieselguhr Celite~ 545
in a concentration of 5% may ~e used as the filtering aid.
EXAMPL~ 3
Following the purification, the various solutions
are combined and subjected to a dialysis concentration
method equivalent to that of example 1. However, the
solutions, if desired, may be processed into the final
concen-trate in the rnanner explained in example 1, whereupon
these solutions may be selectively used individually or
cornbined i.n any desired manner.
EXAMPLE 4
lQ kp of fraction III-l and 10 kp of fraction
III-2 are di.ssolved in 200 liters of a phosphate-citrate
buffer solution (0,066 moles of~phosphate-citrate), within a
period of about 3 hours. The pN is set to 7~50, and after a
further dilution to 300 liters by using the above-mentioned
, ~,
~'` buffer solution and with the addition of 100 grams/liter of
Aerosil/bentonite ~2:1), the solution is stirred or agitated
for 4 hours at 45C. After cooling to 10C, the suspension
is bright filtered on an alluvial or precoat filter
precoated with 1,0 kg of kieselguhr-celite~ 545 per scluare
meter of filter area with a filter performance of 50
- 19
liters/square meter hour. I~is is followed by purification
filtration (using a Seitz-AKS4 filter) and by dialysis
concentration a~, explained in examples 1 to 3. The fincll
concentration of the solution ready for use is set to 5~ of
protein. This provides the following average composition:
(Protein 5,000 milligrams)
albumin about 1,800 to 2,000 rmilligranls
globulins 3,000 to 3,200 milligrams
IgG about 1,450 rnilligrams
IgA " 750 "
' ' ~!
IgM " 500
ÆXAMPLE 5
Instead of a fractionated precipitation, the COEIN
fractions I to III and IV-l may be precipitated in a single
step at a concent~ration of 40~ of ethanol and a pH of 5.8.
Fraction IV-l means a sub~raction of fraction IV (eompare
e.g. U.S. Patent 2,710,293). The precipitate is isolated.
20 kp of the precipitate are suspended in 300 liters of
solvent according to example 1 and further processed in the
manner described in example 1.
EXAMPIJE 6
It is known to effect a fractionated precipitation
of the h~nan plasma with Rivanol (2-ethoxy-6, 9 diarnino-
acidinil~acetate) (STEINBUCH; Vox Sanguin. 23, p~ 92 to
106). In accordance with the processes described in the
latter publication, precipitates II, III and IV are
obtained. These precipitates may be processed to yield the
serum protein cornposition according to the invention, too.
10kp of precipitates II, III and IV each, a total of 30 kp,
are suspended in 300 litres of the solvent accordiny to
exarnple 1 and processed in accordance with the process
explained in example 1.
- 20
