Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1998-07-09
2003-11-11
Saunders, David (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C424S130100, C424S176100, C424S177100, C424S804000, C530S390100, C530S390500, C530S415000, C530S416000, C530S418000, C530S861000
Reexamination Certificate
active
06646108
ABSTRACT:
The invention relates to a process for the isolating IgG and IgA as well as the IgA solutions obtainable according to this method.
Immunoglobulins (Ig) are specific immune proteins in blood plasma, lymph and in other body secretions of all vertebrates. Immunoglobulins are synthesized from B-lymphocytes. Monomeric immunoglobulins each consist of two L (light) and H (heavy) chains which are bound to each other by disulfide bridges. Immunoglobulins are glycoproteins which function as antibodies and whose formation is stimulated by antigens. Quantitatively, they constitute approximately 20% of the total plasma proteins.
Up to now, five main classes of immunoglobulins have been identified in humans (IgA, IgD, IgE, IgG and IgM) which differ in their H-chains, in their serum concentration, molecular weight (approximately 146000 to 970000), carbohydrate content, electrophoretic mobility and their biological properties. The main classes IgA and IgG can be divided into sub-classes (for example IgA1, IgA2). The diversity of the immunoglobulin classes and subclasses as well as their diverse different specificity in binding antigens arises through combinations of various genetic building blocks.
Immunoglobulin A (IgA) represents the main antibody class in external secretions, such as saliva, tears and mucus of the bronchial and intestinal tract. Thus, immunoglobulin A forms one of the first defence lines against bacterial and viral pathogens.
In the pure monomeric form, IgA consists of two light (L) and two heavy (H) chains; in the dimeric secretory form, two such monomers are coupled by so-called J-chain (joining chain). In the secretions of the mucous membranes and glands, dimers with an additional secretory component (so-called SC-component) are present above all.
In solution, plasmatic IgA monomer is present in equilibrium with non-covalently bound IgA dimer. In this equilibrium, the maximal portion of dimeric IgA amounts to approximately 25% of the total IgA.
IgA consists of two sub-classes, IgA1 and IgA2 which are present in a native relationship of about 80% by weight to 20% by weight. This relationship can be altered in the course of isolation. The native ratio of kappa to lambda light chains (measured in U/dl) in an immunoglobulin preparation amounts to approximately 1:1.
IgA only represents approximately 3-4% of the total protein of normal human serum. During purification of IgA, a marked tendency to form complexes and aggregation has been observed. Therefore, isolation of monomeric IgA from serum was mostly associated with low yields and, up to now, only a few methods among the numerous methods of production are known which are also suitable for commercial production. Main impurities of IgA preparations are essentially the various sub-classes of immunoglobulin G whose separation requires additionally purification steps which further reduce the yield of IgA.
The known and common methods for purification of immunoglobulins are mostly based on differences in physical properties, such as for example solubility in aqueous systems (purification by fractionated precipitation), number of charges (purification by ion exchange chromatography) or a difference in the molecular size (purification by molecular size exclusion chromatography).
For a long time, inorganic carrier materials have also been used for carrying out chromatographic purification methods, for example, for the separation of plasma proteins. Thus, Al (OH)
3
for example is used for separating the prothrombin complex from other proteins contained in plasma and this method is also applied on a commercial scale. Other inorganic materials, such as for example aluminium oxide, calcium phosphate or various compounds of silicon, are also preferably employed in the preparation on a commercial scale.
Hydroxylapatite which is obtained according to a special production method in sintered form is known to be employed as an adsorption agent in general chromatography from EP 242 544. Hydroxylapatite is described as a chemical substance with the formula Ca
10
(PO
4
)
6
(OH)
2
, i.e. as a hydroxylated calcium phosphate, which is mostly present in particulate form. Therefore, hydroxylapatite can be considered as a particular form of calcium phosphate. Among others, Hydroxylapatite is proposed as a suitable material for the separation of biological macromolecules, for example for proteins such as immunoglobulins or enzymes or for the separation of RNA, DNA, viruses or plasmids.
A method for the production of an intravenously administerable immunoglobulin-containing pharmaceutical is known from DE 39 27 112. This pharmaceutical consists of IgA, IgG and IgM in concentrated form and is obtained by a multi-step purification process which among others also includes an absorption to calcium phosphate in the presence of caprylic acid. However, in this method, the immunoglobulins are not separated from each other, therefore, a pharmaceutical preparation is provided which contains all three groups of immunoglobulins in a certain mixed proportion.
Additionally, pharmaceutical compositions based on immunoglobulins, for example immunoglobulin A, have already been proposed for prevention and treatment of bacterial and viral infections (see JP A 478 59815).
A problem which is faced particularly using starting material of human origin for the production of immunoglobulins is the virus safety of the obtained product. Despite selection of donors and testing of the individual donor plasmas, it cannot be excluded that infectious pathogens, especially hepatitis viruses or retroviruses such as HIV are present in the pool of donations as a result of the low sensitivity of some tests for example.
Although depletion/inactivation of viruses by more than 10
15
units was described in the production of an immunoglobulin preparation by fractionated alcohol precipitation according to Cohn (see Wells et al., Transfusion 26 (1986) 120-213 for example), the danger of insufficient virus inactivation, i.e. an inadequate virus safety of the preparation exists especially when using intermediate products from the Cohn fractionation.
In common methods for the inactivation of viruses, additional aggregate formation of the immunoglobulins is expected. This is demonstrated to a particular degree with methods of heat treatment which are preferably applied because, aside from lipid-coated viruses, non-lipid coated viruses (for example hepatitis A viruses) are also effectively inactivated.
It has been shown that a substantial portion of IgA can form mulitmers and/or can be polymerized during the above mentioned heat treatment. However, as explained above, aggregated IgA of this type reduces the yield of monomeric IgA obtained with the aid of various purification methods. Aggregates, for example IgG aggregates, also cause among others an increase in anti-complement activity and lead therewith to intolerability reactions after intravenous administration. For this reason, some purification methods according to the state of the art in which a step for the inactivation of viruses is carried out, in paticular a heat treatment step in the presence of stabilizers. Such a method is described in EP 177 836 for example. However, a disadvantage of using stabilizers is that this requires a further removal step. A further disadvantage of using stabilizers is also the simultaneous stabilization of viral proteins and, therewith, the kinetics for inactivation of viruses becomes worse.
Object of the present invention is to separate the immunoglobulins IgG and IgA in an immunoglobulin-containing starting material from each other and also from their high molecular aggregates as well as from other high molecular contaminating substances which were either already present in the starting material or were formed during the purification steps by a method which is simple to carry out and easily transferred to large scale production.
The above object is solved according to the invention by a method which is characterized in that
(i) IgG and optionally IgA are adsorbed to a solid inorganic carrier material,
(i
Eibl Martha
Leibl Heinz
Mannhalter Josef
Tomasits Regine
Wolf Hermann
Baxter Aktiengesellschaft
Heller Ehrman White & McAuliffe
Saunders David
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