FORMULATION POUR STABILISER LES GAMMAGLOBULINES HUMAINES

FORMULATION FOR STABILIZING HUMAN IMMUNOGLOBULINS

Abrégé anglais

The invention relates to a formulation for
stabilizing human immunoglobulins such as human anti-Rho
[D] immunoglobulins. The solution is provided in an
injectable solution at 25°C and comprises human serum
albumin, sodium phosphate dihydrogen in a sufficient
quantity to maintain a pH between 5 and 7.2 and a
saccharide selected from the group consisting of glucose,
saccharose, maltose, lactose and mixtures thereof. It can
be applied either intravenously or intramuscularly. The
invention is likewise related to the use of the
formulation.

Revendications

The embodiments of the invention in which an exclusive
property or privilege is claimed are defined as follows:
1. Formulation for stabilizing human immunoglobulins
characterized in that said formulation comprises human
serum albumin, sodium phosphate dihydrogen in a
sufficient quantity to maintain a pH between 5 and 7.2
and a saccharide selected from the group consisting of
glucose, saccharose, maltose, lactose and a mixture
thereof.
2. The formulation of claim 1, wherein said human
immunoglobulins are human anti-Rho[D]immunoglobulins.
3. The formulation of claim 2, wherein the
formulation is dissolved in a solution which is
injectable at 25°C.
4. The formulation of claim 1, 2 or 3, wherein said
formulation is an aqueous solution comprising per 100 ml
of the solution:
- 0.5 - 2 g of human serum albumin;
- 4 - 6 g of the saccharide: and
- sodium phosphate dihydrogen in a sufficient
quantity to maintain a pH between 5 and 7.2.
5. The formulation of claim 1, 2, 3 or 4, wherein
said aqueous solution is formulated in an aqueous
vehicle, said aqueous solution comprising per 100 ml of
the solution:
- 1.0 g of human serum albumin;
- 4 - 6 g of the saccharide; and
- 0.28 g of sodium phosphate dihydrogen,
wherein said aqueous vehicle is formulated in
intravenous or intramuscular injectable units.

6. Use of a formulation according to claim 1, 2, 3,
4 or 5 for the stabilization of human immunoglobulins.

Description

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Background of the Invention
Rho heteroimmunization is an autoimmune response which takes place
in non sensibilized Rho (D) negative persons, when they receive or have been
exposed to blood from olio (D) positive persons. This happens for example in
s the parturition of Rho (D) positive children; in this case, if the necessary
precautions are not taken an immunological response takes place in Rho (D)
negative mothers, which leads to the formation of anti-Rho (D) antibodies so
that in the next pregnancy, haemolytic disorders take place in the case of Rho
(D) positive pregnancy or foetus, leading to foetus-maternal haemorrhages,
during normal parh~rition, spontaneous or indirect abortion, with symptoms and
signs of amniocentesis, abdominal trauma, etc.
Rho heteroimmunization also takes place in other situations for
example, in Rho (I7) negative persons subjected to transfusions with Rho (D)
positive blood or blood components, such as red corpuscules of Rho (D)
~s positive blood.
Conventional treatment in these situations is the prevention of Rho
heteroimmunization of mothers or would-be mothers as well as persons at risk
of heteroimmuniza.tion or in conditions of immunology with a reduction in
blood platelets. Said preventive treatment is based on the administration of
2o gamma immunoglobulin (IgG), according to a predetermined preventive
program according; to each case. In the case of non sensibilized Rho (D)
negative women, tree preventive treatment requires the administration of human
anti-Rho IgG as from the 28th week of gestation, when the progenitor is Rho
(D) positive or unknown. With this type of treatment, the risk of Rho
2s heteroimmunization is reduced to 0. 1 %. If said preventive treatment is
carried out within ?2 hours of birth, the risk of Rho heteroimmunization is 1
to
2% (compared to 12-13% in ordinary situations), ascribed to Rho
heteroimmunization which occurs naturally in the last three months of
pregnancy.

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The responses of Rho heteroimmunization present delicate clinical
symptoms and signs, mainly due to haemophylic (haemorrhages) and
haemolytic disorders.
In the above mentioned preventive treatment of Rho
s heteroimmunization, a fraction of gamma immunoglobulin from human plasma
formulated in injectables is administered intravenously or intramuscularly.
There are various commercial products used in prenatal and obstetric
clinic; for example Partogamma T* and the Win Rho~R~ line, which includes the
latest product known as Win Rho SD~R~, manufactured by Rh Pharmaceutical
Inc., mentioned here as a point of reference. The suppressive agent of Rho
heteroimmunization in these products, is a fraction of human IgG, obtained by
chromatographic fractionation in columns of anionic exchange of IgG of
human plasma, and. then treating the obtained fraction with solvent/detergent
so
as to inactivate the lipidic membrane of certain virus, such as the Hepatitis
C
is virus and HIV.
These products and other similar ones are formulated as lyophylized injectable
compositions, which contain glycine and sodium chloride as stabilizing system
in sufficient quantity to form a 0.10 and 0.15 M solution respectively, in
doses
of, for example, 250 to 330 mcg/ml of injectable solution.
Given the general tendency of immunoglobulins to form in solution,
* Trade-mark

215140
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additions or undesirable molecular complexes (dimers, trimers,
tetramers) conditional upon more antigenic properties, and anti-
complementary activity particularly in the case of intravenous
preparations, other stabilizing systems have been proposed such as:
polyethylene glycol human-albumin-manitol; glycine-histidine,
inorganic salts-proteins-saccharides, human albumin-glycine-manitol,
etc. of variable stability according to the stabilizer. In the case of Rho
[D] Immunoglobulin stabilized with glycine, the US Pharmacopia
(abbreviation in Spanish: USP) XXIII acknowledges a stability of not
more than 6 months, for products of this type stored at temperatures
between 2° and 8°C. (page 708).
It has been found, and this constitutes the aim of this
invention, that it is possible to prepare and have available new
compositions for the prevention of IRho heteroimmunization, formulated
with human gamma immunoglobulin, in aqueous solution of reduced
tendency to molecular addition, verified for periods of at least 24
months at room temperature.
Concise Description of the invention
One the of aims of this invention is a formulation for
stabilizing human immunoglobulins. The formulation comprises human
serum albumin, sodium phosphate dihydrogen in a sufficient quantity to
maintain a pH between 5 and 7.2 and a saccharide selected from the
group consisting of

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2151409
glucose, saccharose, maltose, lactose and mixtures
thereof .
The immunoglobulins suitable according to the
present invention may be for example human anti-Rho [D]
immunoglobulins. These human anti-Rho [D] immunoglobulins
may be dissolved in a solution which is injectable at
25°C.
Another aim of this invention is to provide a
formulation for stabilizing human immunoglobulins
characterized in that the formulation is an aqueous
solution comprising per 100 ml of solution:
- 0.5 to 2 g of human serum albumin;
- 4 to 6 g of the saccharide; and
- sodium phosphate dihydrogen in a sufficient
quantity to maintain a pH between 5 and 7.2.
Preferably, the formulation may contain 1.0 g of
human serum albumin, 4 to 6 g of the saccharide and 0.28 g
of sodium phosphate dihydrogen. The formulation is
preferably an aqueous solution which is formulated in an
aqueous vehicle. This aqueous vehicle may in turn be
formulated in intravenous or intramuscular injectable
units.
Another aim of this invention is to provide a use
of the formulation in accordance with the present
invention for the stabilization of human immunoglobulins.
A

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Detailed description of the invention
The compositions of the present invention are the
result of lengthy investigations and trials carried out
with the aim of finding preventive pharmaceutical
compositions for Rho Heteroimmunization and other clinical
symptoms and signs such as Immuno Purpura
thrombocytopaenia, cited before,capable of withholding
during prolonged periodsthe suppressing capacity of
immuno-response in affected persons.
In practice, and according to the preceding text,
the usual treatment is based on the administration of
compositions formulated with Rho [D] human IgG in vials of
intravenous or intramuscular application. As IgG has a
tendency to form antigenic molecular additions,
conventional stabilized formulations come in lyophilized
vials, being restorative in situ. Therefore, the cost of
this type of products rises, conditioned upon overloading
due to breaking up and transport. This has led to the
development of stabilized formulations of anti-Rho [D] IgG
of this invention, capable of withholding its
immunocapacity during prolonged periods and without signs
of side effects.
A,.

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Therefore, the compositions of this invention fit in within this line
and these aims, formulated by resorting to an unpublished combination of
stabilizing components capable of ensuring a prolonged permanence of
extended anti-Ro (D) activity, according to tests carried out during at least
s 24 months.
In the previous texts as well as in the text which follows it will be
understood that the term human Rho (D) immunoglobulin refers to
globulin originating from human plasma which contains antibodies for the
Rho (D) factor, as stated by the US Pharmacopia XXIII Ed (page 708)
io and equivalent products.
It should likewise be understood that the term serum albumin refers
to the albumin projections taken from the blood of healthy donors as
defined in the US Pharmacopeia XXIII Ed. page 38 and equivalent
products.
The stabilizing system of the compositions of this invention includes:
- saccharides such as glucose, saccharose, lactose, or maltose,
mixtures of same, in concentrations of 4 to 6% the weight to volume,
preferably 4.5 to 5.8% weight to volume, more preferably 4.9-5.1
weight to volume;
- serum albumin in concentrations of 0.5 to 2% weight to volume,
preferably 0.8 to 1.5% weight to volume, particularly 0.9 to 1.1.%
weight to volume, free of alpha-globulin and bilirubin;
- sodium phosphate dihydrogen in sufficient quantity to form a pH
:~ :~
.~._t ~'

-' - ~ 2 1 5 1 4 0 9
between 5 and 7.2, preferably between 5.0 and 6, depending on the
concentration of IgG and albumin, added as NaH2P04 or formed from,
for example, mixtures of Na2HP04 or Na3P04 and orthophosphoric acid.
For. example, for a formulation in accordance with the invention, which
s contains 1.0 g of human serum albumin, 5.0 g of saccharose and 2500
micrograms of anti-Ro (D) IgG taken up to a volume of 100 ml with water
reaches an optimum pH of 5.1-5.4 with 0.28 g of monohydrate sodium
phosphate dihydrogen.
The pharmaceutical compositions of this invention can easily be
to stored at room temperature in dosed units (vials) for immediate use, or in
bigger containers for transport, later use, or to be lyophylized if necessary.
In all cases in the previous paragraph, the term human plasma and
products extracted from human plasma such as Ro (D) positive IgG, refer
to plasma and/or serum albumin and IgG tested in order to verify the
is presence of HIV, HCV ~ and HbsAg antibodies (surface antigen for
Hepatitis B virus).
The following example describes and summarizes the tests and
results obtained in the study and verification of the stability and biological
activity of the composition of this invention.
2 o Batches studied
2 batches of products in two different concentrations were prepared:
(abbrev.in sp.:PS) 250 mcg (abbrev.in sp.:LP1)
PS 330 mcg LP1

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_g_
Tests carried out
Variations with time of the following parameters were assessed:
i) Appearance of solution
ii) pH of solution
a iii) Molecular distribution (abbrev. in sp.:H.P.L.C.)
iv) Anti-Rho activity
Conclusions
The results obtained show the good stability of the stabilized solution in
to accordance with this invention.
No significant changes in product activity were observed, nor the appearance
of dimerous, additions :nor proteins of low molecular weight.
Results obtained
l a The following Tables 1 and 2 summarize the results of tests carried out.
Table 1 illustrates results of tests carried out on the following composition:
Composition of this invention
PS 250 mcg LP I
Table 1
Natural stability trial
Time Molecular
Distribution
(months)Appearance pH Dimers MonomersOthers Activity
0 Colourless 5.2 0.2% 99.8% - 300 mcg
solution
6 without change5.2 0.2% 99.8% - 306 mcg
12 without change5.1 0.5% 99.5% - 290 mcg
24 without change5.1 1.2% 98.8% - 303 mcg

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Table 2 illustrates results of tests carned out on the following
composition:
Composition of this invention
PS 330 mcg LP1
Table 2
Natural stability trial
Time Molecular ion
Distribut
(months)A earance H Dimers Monomers Others Activi
0 Colourless 5.2 0.4% 99.6% - 363 mcg
solution
6 without change5.2 0.6% 99.4% - 333 mcg
12 without change5.1 1.0% 99.0% - 363 mcg
24 without change5.1 1.5% 98.5% - 333 mcg
The term "effective quantity" as used here, represents a quantity of
y

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Rh (D) positive human IgG, capable of blocking Rho heteroimmunization
in Rho (D) negative persons. The particular doses administered in
accordance with this invention, are determined by particular circumstances
of each case.
The term "treatment", as used here describes the mode and care of
patients with the aim to prevent the onset, ease symptoms and/or follow-up
the evolution and development of Rh heteroimminization and/or other
situations which might requiere the application of Rh (D) positive human
IgG.
The compositions of this invention are prepared in accordance with
known techniques using human Rh (D) IgG, human serum albumin and
remaining components, all known and easily available.