Note: Descriptions are shown in the official language in which they were submitted.
~ ~t~Z~66
1 This invention relates to a process for pre-
paring immunoglobulin suitable for intravenous injection.
Since the immunoglobulin possesses antibody
activity to many pathogens, it is administred to patients
deficient in ~rarious antibodies to protect them from
injection diseases or for the therepeutic purposes. The
immunoglobulin preparations have been developed in two
types, the one being for the intravenous in~ection and
the other being for the intramuscular in~ection, and
both are widely in clinical use.
The known methods for the preparation of
intravenous administration type include that ~n which
crude immunoglobulin is treated with proteases such as
pepsin and plasmin, that which employs chemical modifica--
; 15 tion of immunoglobulln by acylation and other chemicalmeans, and that utilizing fractionation of the plasma or
of Cohn's plasma~fractions~using polyethylene glycol,
Pluronics (polyoxyethylene-polyoxypropylene copolymer),
or the llke.
In the lmmunoglobulin for intravenous admini-
stration obtalned by the method of pepsin treatment,
7S IgG has been transformed into F(ab')2 by the digestive
action of pepsin, which has a disadvantage of very short
half-life in vivo. In the method of plasmin treatment,
although 60 to 70% of the ~mmunoglobulin remain in the
:~i72~
1 form of normal immunoglobulin, the remainder has been
converted into low molecular substances which decrease
the half-life in vivo of the product as in the case of
pepsin treatment. In the case of chemical modification,
there is a possibility of the development of fresh
antigenicity depending upon the type of modifying radical
and other conditions, bringing about the problem of safety
when the modified immunoglobulin is to be repeatedly
administered.
The fractionation with polyethylene glycol or
Pluronics~is generally belleved to be the most desirable
means to recover the immunoglobulin in hhe form existing
in the living body, that is, unmodified and undecomposed
immunaglobulin. The procedure has already been dis-
closed in detail by Polson and Coval [Japanese Patent
Application "Kokai" (Laid-open) Nos. 46,8I4/1975,
91,321/1976 and 20,415/1978]. However) if the poly-
ethylene glycol or Pluronic fractionation is carried
out under known conditions, it is possible to obtain an
immunoglobulin preparation containing no aggregate-
type i~munoglobulin and suitable for intravenous in~ection,
but the yield is alwaDs low. Thereforeg an improvement
in the yield has been eagerly awaited.
The present inventors conducted st~dies to
2~ elucidate the cause for the decrease in yield and as a
result found that in the step of removing the aggregate-
type immunoglobulin at a low concentration of polyethylene
glycol or a Pluronic added to the raw immunoglobulin
~ 7~a ~ 7 c~r~ - 2
.
~ 7 ~
1 material, the non-aggregate-type immunoglobulin is also
removed at the same time. Further, it was also found
that an immunoglobulin material contâining a comparatively
large amount of aggregate-type immunoglobulin is ~ractio-
nated with polyethylene glycol or a Pluronic, the lossin non-aggregate-type immunoglobulin becomes also large.
The present inventors further made an effort to solve
the novel technical problem associated with the a~oresaid
difficulty inherent in the prior art and, as a result,
found that the yeild of non-aggregate-type immunoglobulin
is remarkably improved by treating the raw immunoglobulin
material with an acid to dissociate the aggregate-type
immunoblobulin and then subjecting the material to the
fractionation with polyethylene glycol or a polyoxyethylene-
polyoxypropylene copolymer. Based on this finding, thepresent invention has been accomplished.
An object of this invention is to provide a
process for preparing in a hlgh yield an immunoglobulin
suitable for use in intravenous injection from a plasma or
Cohn's fraction I+II+III, fraction II+III, fraction II, or
fraction III obtained by subjecting a plasma to the Cohn's
cold alcohol fractionation.
Other objects and advantages of this invention
will become apparent from the following description.
According to this invention there is provided
a process for preparing an immunoglobulin suitable for
intravenous injection, which comprises treating a plasma
or the Cohn's fraction I+II~III, fraction II~III, fraction
_ 3 _
~ ~ 7'~
1 II, or fraction III obtained by sub~ecting a plasma
to Cohn's cold alcohol fractionation with an acid at pH
3.2 to 5.0 and at 4 to 15C for 30 to 180 minutes,
adding to the resulting material at pH 4.6 to 5.4 an
alkylene oxide polymer or copolymer having a molecular
weight of 2,000 to 20,000 to a concentration of 4.5 to
5.5% (W/V), removing an aggregate-type immunoglobulin
as a precipitate, and adding again said polymer or
copolymer at pH 8.o to 9.0 to a concentratiQn of 6 to
13% (W/V) to recover as a precipitate a non-aggregate-type
immunoglobulin containing substantially no aggregate-type
immunoglobulin.
The plasma used as starting material according
to this invention is preferably that originated from
human blood in view of the problem of antigenicity. The
Cohn's plasma fractions I+II~ II+III, II, and III are
substantially ~ and ~-globuIin (IgG, IgA, IgM).
The plasma ~-globulin ~raction is obtained by the conti-
nuous precipitation with cold ethanol as described in
2a detall in Journal of Clinical Investigation, 23, 417
(1944) and Journal of the American Chem~cal Society, 68,
479 (1946).
The acid treatment is carried out by keeping
the starting material under acidic conditions. By this
treatment, the aggregate-type immunoglobulin in the raw
material is dissociated first into a non-aggregate type.
The acid conditions are pH 3.2 to 5.0, preferably 3.8
to 4.2 and an ionic strength of 0.002 to 0.30, pre~erably
_ I+ _
117~6
1 0.10 to 0.20. Other pH conditions are undesirable because
if pH is below 3.2, denaturation of the protein will
take place, while if it i5 above 5.0, the said dis-
sociation of the aggregate-type immonoglobulin becomes
insufficient. The concentration of protein is not criti-
cal, but is preferably 2 to 10% (W/V)~ because of the
ease of operation. The temperature of acid treatment is
4 to 15C. Other temperatures are not desirable,
because if the temperature is below 4C, the aforesaid
dissociation will be insufficient, while if it exceeds
15C, decomposition of the protein will take place. The
duration of the acid treatment is 30 to 180 minutes.
If the duration of treatment is shorter than 30 minutes,
the aforesaid dissociation becomes insufficient, while
too long a duration of treatment is the waste of time
and even sometimes glves unfavorable results. The acids
used for the treatment include inorganic acids such as
hydrochloric acid and phosphoric acid and organic acids
such as acetic acid and citric acid.
After such acid treatment as described above,
a fractionation is performed by uslng an alkylene oxide
polymer or copolymer having a molecular weight of 2,000
to 20,300, to produce a high-purity non-aggregate-type
immunoglobulin in a high yield. The alkylene group of
the alkylene oxide polymer used in the fractionation
is that having 1 to 4 carbon atoms such as methylene,
ethylene, propylene or butylene group. The alkylene
o~ide copolymers include copolymers of two or more alkylene
~ 7Z 1 ~ ~
1 oxides such as polyoxyethylene-polyoxypropylene copolymer.
The present inventors found that in the frac-
tionation using an alkylene oxide polymer or copolymer
having a molecular weight of 2,000 to 20,000, the pH
conditions are a very important factor and the yield and
purlty of the immunoglobulin are markedly improved only
in a very limited pH range of from 4.6 to 5.4, preferably
from 4.8 to 5.2. This is clearly shown also by the experi-
mental results (Table l) obtained by dissolving a fraction
II+III paste in o.6% aqueous sodium chloride solution
to a protein concentration of 5%, treating the resulting
solution with an acid at pH 3.5 and 10C for 60 minutes,
fractionating the solution with polyethylene glycol
(5% in concentration) at varied pH in the range of 4.0
to 6.3, and determining the yield and purity of the
non-aggregate-type immunoglobulin.
Table 1 pH conditions in polyethylene
glycol fractionation.
. _ . ,
pHIgG recovery, % IgG purity, %
4.1 60 35
4.3 58 60
5.0 70 98
5.5 45 95
6.o 10 93
~ 2 ~ ~
1 When a polyoxyethylene-polyoxypropylene copolymer
of an average molecular weight of 15,000 is used in place
of the polyethylene glycol, a higher yield and higher puri-
ty of the non-aggregate-type immunoglobulin are obtained
also under the above pH conditions than those obtained
under other pH conditions.
The above polymer or copolymer is added to
the starting material to a concentration of 4.5 to 5.5% ,
preferably 5% (W/V) and the precipitated impurities, e.g.
aggregate-type immunoglobulin, are removed by a cu~tomary
means, e.g. centrifuging (1,000-5,000 rpm). To the
supernatant thus fractionated, is further added the
above polymer or copolymer to a concentration of 6 to
13%. By adjusting pH to 8.o to 9.0, the intended non-
aggre~ate-type immunoglobulin is precipitated and can be
recovered by a customary means, e.g. centrifuging
(1,000 to 5,000 rpm). The recovery of the intended
product under the above conditions is 60% or more. The
precipitate which was formed is again dissolved, for
example, in a physlological saline or~a 0.02 M acetate
buffer solution admixed with o . 6% of sodium chloride,
2% of mannit and l% of albumin, and the~resulting solution
is passed through a bacterial filter to obtain an
intravenous immunoglobulin solution~suitable for clinical
use. The immunoglobulin in this solution shows no change
in its properties upon dispensing the solution in small
portions into vials and lyophilizing. Accordingly, ~hen
the product is intended for long-term storage, it can be
-
216~
1 made into the ~orm of lyophilized preparation.
The immunoglobulin prepared by the present
process contains substantially no aggregate-type immuno-
globulin and the anticomplementary activity is less
than 20 units, as assayed on a solution of 5% in concent-
ration, the purity as Ig~ being 90% or above.
The invention is illustrated below in detail
with reference to Examples J but the invention is not
limited thereto.
In Examples, the recovery of immunoglobulin
was determined by the single immunodiffusion and the purity
by the electrophoresis using a cellulose acetate membrane.
The anticomplementary activity was assayed by the method
of Kobat and Mayer [Experimental Immunochemistry, p 225
(1961)] and the method o~ Nishioka and Okada ["Biochemistry
of Immunity", p 103 (1971), Kyoritsu Publishing Company].
'
Example l
The-Cohn's fraction II+III paste (l kg) obtained
by the cold alcohol fractionat~on was dissolved in 10
liters of a o.6% aqueous sodium chloride solution. The
solution was ad~usted to pH 3.8 with l_ hydrochloric
acid and stirred at 4C for 60 mlnutes to effect acid
treatment. To the solution was added 500 g of poly-
ethylene glycol (average molecular weight 47000). While
allowing the polyethylene glycol bo dissolve, pH of the
solution was gradually increased with lN aqueous sodium
hydroxide solution. As soon as the pH reached 5.0, the
-- 8 --
~ ~ '7Z~66
1 precipitate was remvoed by centrifugation (4,000 rpm)
to obtain a clear supernatant. The recovery of IgG in
the supernatant was 85% based on the fraction II+III
and the purity of IgG was 97%. To the supernatant was
added another 500 g of polyethylene glycol (molecular
weight 4,000). While being stirred mildly, the super-
natant was adjusted to pH 8.5 wlth lN aqueous sodium
hydroxide solution. The immunoglobulin precipitated
under the above condltions was recovered by centrifuga-
tion (4~000 rpm). The whole of the recovered precipitatewas dissolved in a 0.02 M acetate buffer solution (pH 6.6)
containing 1% of human albumin and using the same solvent
the concentration was adjusted to 5%. The solution was
sterilized by passing through a Millipore filter
(Millipore Co.) and asepticalLy dispensed in small
containers. One half of the dispensed liquor was
immediately lyophilized to yield a dry preparation.
The ultimate yleld of IgG from the starting
material was 84%, as contrasted with 63% when the acid
treatment at pH 3.8 to the starting material was omitted.
The Furity was 95%, excluding the albumin which was
added afterwards. The anticomplementary activities
(at a protein concentration of 5%) of the liquid prepara-
tion and the solution of lyophilized preparation were
found to be 14 and 16, respectively.
A 5% solution was administered~to 5 mice,
about 20 g in body weight, at a dose of 1 ml per mouse.
Neither decrease in body weight nor any anomaly in Pilo~
_ 9 _
~17~6~
1 erection was noted during an observation period of one
week. The lyophilized preparation was tested after one
year of storage at 4C, but no change in solubility and
anticomplementary activity was observed as compared with
the initial preparation.
Example 2
A Cchn's fraction II paste (500 g) obtained by
the cold ethanol fractionation was dissolved in 10 liters
of a 0.1% sodium chloride solution. Immunoglobulin
containing no a~gregate type was recovered from the
solution in a manner similar to that in Example 1. The
yield in terms of IgG was 80%, as contrasted with 62%
when the acid treatment (pH 3.8) was omitted. The
purity was 97%.
In a manner similar to that in Example 1, the
immunoglobulin obtalned above was dissolved in a 0.5%
sodium chloride solution to a protein concentration of
5%. After addition of 1% of mannit, the solution was
passed through a bacterial filter and lyophilized.
20 ~The lyophilized preparat~on was dlssolved in distilled
water for lnjection to a protein concentration of 5%.
The anticomplementary activity at this concentration was
found to be 13.
Example 3
The proeedure of Example 1 was repeated~ except
that the polyethylene glycol (average molecular weight
.
- 10 _
.
1172166
1 4,000) was replaced by the same amount of a polyoxy-
ethylene-polyoxypropylene copolymer (average molecular
weight 15,000). The immunoglobulin was recovered with
the same results as in Example 1.
:
:
.
-- 11 -
