CA1340737C - Process for the preparation of pasteurized immunoglobulin preparation - Google Patents

Abstract

A process for the production of a pasteurized immunoglobulin preparation is described, which comprises heating a solution of an immunoglobulin in the presence of a carboxylic acid or one of its salts and/or of a saccharide until viable pathogens, which may remain undetected in conventional tests, in particular hepatitis viruses or HTLV III ("Aids") viruses, are inactivated i.e. are rendered incapable of reproduction and/or of being intracellularly or otherwise reproduced. A preparation of this type can be used for therapy or prophylaxis or as component of a diagnostic kit.

Description

A process for the preparation of a pasteurized immunoglobulin preparation The invention :relates to a process for the production of an immunoglobu:Lin preparation, in which, for the purpose of inactivation of pathogens, a solution of an immunoglo-bulin is heated in the presence of stabilizers and is, if necessary, subsequently purified.
A process is neseded which permits inactivation of pathogens, such as viruses, by pasteurization of immuno-globulin preparations; thereby the full activity of the immunoglobulin must be retained. In general, when immunoglobulins are prepared by conventional processes there is a reduction in the potential risk of infection to such an extent that the content of viruses or viral antigens is often merely reduced to below the detection limit of the test system used. However, the detection limit is either usually inadequate to unambiguously rule out a risk of ~:nfection; or further, there are not test systems appropriate for detecting certain contamination viruses. Hence a heat treatment is expedient. It is claimed that the inactivation renders the infectious agent incapable of reproduction and/or of being intra-cellularly or otherwise reproduced.
To avoid their denaturation it is necessary to stabilize the immunoglobulins during their prolonged exposure to heat. To inactivate viruses it is indispensable to heat at the highest permissable temperature for a prolonged period.

1340'37 A process is described in EP-A-0 124 506, in which ammonium sulfas=a is added to an immunglobulin solution, and the suspension is heated at 60°C for 10 hours.
However, when an immunoglobulin solution is treated as described in Example 16 of that patent application, the formation of polymeric immunoglobulin occurs. A
maximum limit o f 10$ of polymeric immunoglobulin is permitted in the European Pharmacopoea in an immuno-globulin solution for intramuscular administration.
In EP-A-0 144 714 it is described that a Cohn fraction II+III (J. Am.Chem.Soc. (1946) 68, 459) can be pasteuriz-ed only under mild conditions, preferably at 52°C for about 30 minute's; even when there has been previous removal of the euglobulins and dialysis of the solution to remove ethanol, aggregates are nevertheless produced.
It is doubtful whether absolute virus inactivation is reliable under these conditions.
In The Lancet, of November 19, 1983, pages 1198-99, there is a description of a process in which human immunoglobulin was heated to 60°C for 10 h in solution containing 45$ (w: v) sorbitol and 15~ (w: v) glycine.
Afterwards neither a loss of activity nor an increase in the aggregate content was observed.
A process for t:he pasteurization of human plasma with the addition of sugar alcohols, amino acids or saccha-rides is described in EPatent-A-0 139 975. In the pasteurization of plasma the immunoglobulin is protected by the other plasma proteins. The stabilizing effect of albumin on IgG is known.
The present invention relates to a process for the preparation of a pasteurized immunoglobulin preparation, which comprise; heating a solution of an immunoglobulin in the presence of a carboxylic acid or one of its salts and/or of a sac:charide until pathogens in particular hepatitis viruses or HTLV III ("Aids") viruses, are inactivated (i,.e. rendered incapable of reproduction or of being intrac:ellularly or otherwise reproduced).' Conditions suitable for such inactivation are known to the expert. It is customary to heat at about 60°C for about 10 hours.
This carboxylic' acid is preferably an aliphatic carboxy-lic acid which can preferably be substituted with one or more additional. carboxyl groups or one or more amino groups or one or more hydroxyl groups. It preferably contains two to ten carbon atoms. This carboxylic acid is preferably g~lycine, glutamic acid, citric acid or tartaric acid.
A preferred salt of a carboxylic acid is a soluble metal salt, in particular an alkali metal or alkaline earth metal salt, especially the sodium or magnesium salt.
The salt of glutamic acid which is used is preferably an alkali metal salt, especially the monosodium salt (sodium glutamate).
The carboxylic acids or their salts can be added in an amount up to or exceeding the saturation limit, preferab-ly from 0.4 to 0.6 g per milliliter of the immunoglobu-lin solution which is to be stabilized.
The saccharide which is preferably used is a mono- or disaccharide, with particular preference for sucrose.
These saccharides are preferably added in an amount of 0.5 to 1.0 g per milliliter of the immunoglobulin solution which is to be stabilized.

The carboxylic acids or their salts or the saccharides can be added i:n an amount up to or exceeding the satur-ation limit of the immunoglobulin solution which is to be stabilized.
The addition of these substances may cause a precipitat-ion of the immunoglobulins. In this case, the resulting suspension is nevertheless heated without detrimental effect on the :immunoglobulin.
The pH-value i:a adjusted to 5-8.5, preferably 6.5-7.5, for the heating process.
Using the clairned process it is possible to obtain pasteurized imnnunoglobulin solutions whose polymer content is below 10$. In the preferred embodiments the polymer content: remains unchanged, within the range of experimental error variation of the method, compared with that of the unheated immunoglobulin solution.
The starting material for the process according to the present invention can be a purified immunoglobulin which, in the literature is called gamma-globulin, IgG, imunoglobulin Gr or fraction II based on J.Am.Chem.Soc.
71, 541 (1949). Immunoglobulins of this type are mainly derived from the step-wise precipitation which result from the fractionation of plasma. Immunoglobulin-con-taining precipitates are the fraction A of Vox.Sang. 7, 414 (1962), or fraction II and III of J.Am.Chem.Soc. 68, 459 (1946).
It is also possible to use modified immunogloblins as starting materials. Immunoglobulins of this type can be modified by chemical modification, for example sulfito-lysis, or enzymatic treatment, for example peptic elimination of the Fc portion. Proteolytic cleavage of the immunoglobulin molecule with pepsin at pH 4 results ,~- - 5 -mainly in F(ab)2 fragments with a molecular weight of about 100,000 and a sedimentation coefficient determined in the analytical ultracentrifuge of about 5 S (S =
Svedberg unit).
Products of this type contain uncleaved immunoglobulin of 7 S (molecular weight about 150,000) but virtually no immunoglobulin polymers. However, more extensively fragmented portions with a molecular weight below 5 S
are observed at concentrations below 10~.
It has been surprisingly found, that the claimed process is also suitab:Le for solutions of immunoglobulins which contain ethano:L .
When purified :immunoglobulins are obtained using etha-nol, often the final concentration step of the product-ion process consists of a complete precipitation of the immunoglobulins with ethanol and the subsequent removal of the precipitate by centrifugation.
Dissolution of the precipitate to an approximate 10$
solution resulta in a residual alcohol content which amounts to about 4~ by volume. The ethanol is usually removed by freeze-drying or ultrafiltration. If the heating were to be carried out in the alcohol-free solution, for example after ultrafiltration, in the presence of stabilizers it would be necessary to repeat the ultrafiltration to remove these additives subsequent-ly. Thus, to e~;pedite and economize the processing it is advantageous to perform the heating in the presence of ethanol.
It was surprisingly found, that, upon addition of carboxylic acid., no increase in aggregation was observed when the immunoglobulins were heated in the presence of ethanol at 60°C' for prolonged periods, for example 40 h.

It is known that ethanol normally denatures immunoglobu-lins at elevated temperatures. Accordingly, heating of immunoglobulins even in the presence of carboxylic acids or their salts as stabilizers did produce a higher content of polymers in the presence of alcohol, than in a procedure without alcohol.
An example of an ethanol-containing immunoglobulin solution is a fraction containing gamma-globulin called fraction II+III: by Cohn et al., J.Am.Chem.Soc. (1946), 68, pages 459 e:t seq., or fraction A by Nitschmann (Kistner and Ni.tschmann, Vox Sang. (1962), 7, 414).
Fractions of this type contain not only gamma-globulins but also lipoproteins, euglobulins, alpha- and beta-glo-bulins and minor amounts of albumin. The gamma-globulin content is about 40-80 g in 100 g total protein. When 100 g of fraction II+III are dissolved in 250 ml of distilled water, the alcohol content of the solution is 4-5 ml/100 ml. A fraction II+III of this type can there-fore be pasteurized in the manner according to the invention.
Table 1 shows the contents of polymeric immunglobulin after application of the process described, compared with the state of the art. In each case, heating at 60°C
was continued for 10 hours. The immunglobulin content was 10-11 g of protein per 100 ml of solution. The pH
was 7.

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The contents of: the higher molecular weight portions resulting from the heating can be further reduced by known processes;.
To this end it is advantageous to replace the added stabilizer, for example by ultrafiltration, by an ionic medium which is suitable for the chosen purification process.
The duration of heating can be varied within certain limits.
To test the efficacy of the process which has been described, an i:mmunoglobulin in solution containing 9.9 g of protein per 100 ml and 3.6 g of ethanol per 100 ml was mixed with 1 g of sucrose and 0.15 g of glycine per 1 ml of solution. Rous sarcoma virus (RSV) was added in a concentration of 1 x 104 infectious RSV units/ml (U/ml) and then the solution was heated at 60°C. After heating for one hour, the virus content had decreased below the detection limit.
It is evident from Table 2 that the heating which has been described lead no effect on the antibody acitivity.

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'- ~. 1340737 The examples w'.hich follow illustrate the invention:
Example 1 Heating of an :immunoglo~bulin in solution with sodium glutamate 200 ml of a virtually pure solution of immunoglobulin with a protein concentration of 90 g/1 were stirred while 120 g of sodium glutamate (monosodium salt of glutamic acid) were adeed. This resulted in the preci-pitation of the' immunoglobulin. The pH-value of the suspension was adjusted to 7; it was then heated and stirred at 60°C: for 10 hours. The mixture was cooled to room temperature and then the precipitate was removed by filtration or centrifugation. The precipitate was dissolved in distilled water. The glutamate was removed by dialysis or ultrafiltration. The solution was ad-justed to the desired protein content and made isotonic.
Herefrom 110 m7. with a protein concentration of 155 g/1 were obtained. The polymer content was 1.6% (1.2%
unheated).
Example 2 Heating of an i.mmunoglobulin solution with sucrose and glycine 89 kg of sucrose and 13.3 kg of glycine were added to 89 1 of immunoglobulin solution which had a sodium chloride concentration of 1 g/1, a protein concentration of 97 g/1 and 3.6% ethanol by volume. The pH-value was adjust-ed to 7 and then the mixture was heated and stirred at 60°C for 10 hours. The solution was diluted with 100 1 i3~~'~3~

of 0.3 g/100 ml sodium chloride solution and was steri-lized by filtration. The stabilizers were removed by ultrafiltration in a known manner. The solution was then made isotonic and adjusted to a protein concentration of 160 g/1.
Consequently 51 1 of solution with a polymer content of 2.6% (2% in the unheated solution) were obtained. The residual sucrose concentration was 0.04 g/l.
Example 3 100 g of sucrose and 15 g of glycine were added to 100 ml of sulfonatE:d immunoglobulin with a protein concen-tration of 100 g/1 and a sodium chloride concentration of 3 g/1. The pH-value was adjusted to 7.3 and then the mixture was heated and stirred at 60°C for 10 hours.
The solution was then cooled to room temperature and diluted with 1'l0 ml of 0.3 g/100 ml sodium chloride solution. The stabilizers were removed by ultrafiltrat-ion. The solvent was replaced by a 0.3 g/100 ml sodium chloride solution. The solution of the immunoglobulin was made isotonic and adjusted to a protein concentrat-ion of 50 g/1.
195 ml of solution with a polymer content of 5.6% (5.8%
in the unheated solution) were obtained by this proce-dure.
Example 4 13.7 1 of an inununoglobulin solution which had undergone peptic cleavage (with a protein concentration of 180 g/1 and a sodium chloride concentration of 3.2 g/1) were 1340'~3'~

diluted with 13.5 1 of a 0.3 g/100 ml sodium chloride solution. Then 27.2 kg of sucrose and 4.08 kg of glycine were added. At pH 7 the mixture was then heated to 60°C
and stirred fo:r 10 hours. The solution was then cooled to room temperature and diluted with 45 1 of 0.3 g/100 ml sodium chloride solution. Sterilization by filtration was followed b:y substantial removal of the added stabi-lizers by ultrafiltration. The solution was then made isotonic and adjusted to a protein concentration of 50 g/1. 48 1 of solution with a residual sucrose concen-tration of O.O:L g/1 were obtained. The content of components of <iefined molecular weights was determined in an analytical ultracentrifuge as follows:
Starting material: S less than 5 = 8.8%
S about 5 = 75.5%
S about 7 = 15.7%
S greater than 7 = 0 %
Heating final product: S less than 5 = 8.1%
S about 5 = 77.5%
S about 7 = 14.4%
S greater than 7 = 0 %
Example 5 Heating of a dissolved ethanol-containing fraction II+III in the presence of ethanol 200 g of fraction II+III were dissolved under stirring in 500 ml of distilled water. To the solution (about 700 ml) 700 g of sucrose and 0.3 to 0.2 mol/1 glycine were added. The pH-value was adjusted to about 7 and the solution was then heated and stirred at 60°C for 10 hours. The heated solution was then fractionated. 304 ml of immunoglobulin solution with a protein concentration of 66 g/1 were obtained. The polymer content was 1.1%.

1340'~3~

For comparison an unheated treatment also using 200 g of fraction II+III produced 208 ml of immunoglobulin solution with a protein concentration of 96.8 g/1. The polymer content was 2.0$.

Claims (10)

1. A process for the preparation of a pasteurized immunoglobulin preparation, which comprises heating a solution containing up to 5% ethanol and an immunoglobulin in the presence of one or both of a carboxylic acid or one of its salts and of a saccharide until viable pathogens are inactivated.

2. The process as claimed in claim 1, wherein the process is performed in the presence of a carboxylic acid or one of its salts and a saccharide.

3. The process as claimed in claim 1, wherein the carboxylic acid is an aliphatic carboxylic acid which may be substituted.

4. The process as claimed in claim 1, wherein the carboxylic acid is an aliphatic carboxylic acid which has two to ten carbon atoms and is substituted with one or more of one or two additional carboxyl groups, one or two amino groups and one or more hydroxyl groups.

5. The process as claimed in claim 1, wherein the carboxylic acid is glycine, glutamic acid, citric acid or tartaric acid.

6. The process as claimed in claim 1, wherein the saccharide is a mono- or disaccharide.

7. The process as claimed in claim 1, wherein the saccharide is glucose, fructose, galactose or sucrose.

8. The process as claimed in any one of claims 1 to 6, wherein the viable pathogens are hepatitis viruses or HIV
viruses.

9. The use of an immunoglobulin preparation as claimed in claim 1 for therapy or prophylaxis.

10. The use of an immunoglobulin preparation as claimed in claim 1 in diagnostic reagent kits.