AU598268B2 - A process for the preparation of a pasteurized immunoglobulin preparation - Google Patents

Abstract

The process is characterised in that a solution of an immunoglobulin is heated in the presence of a carboxylic acid or of one of its salts and/or a saccharide under such conditions that pathogens which are capable of multiplication, in particular hepatitis viruses or HTLV III ("Aids") viruses, are inactivated. Such a preparation can be used for therapy or prophylaxis.

Description

COMOWALHOF AUSTRALIA 586 PATENTS ACT 1952-69Frm1 COMPLETE

SPECIFICATION

(ORIGINAL)

Class I t. Class Application Number: Lodged: du'. ujuell, C2~rplete Specificpation Lodged: 0 t eo p P&ip~ity 0 Accepted: Published: tr qtlPtiu BSA oorr'IA

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aRelctod Art: a a Name of Applicant: 0 Address of Applicant: tOctual Inventor.

4 Address for Service BEHRINGWERKE AKTIENGESELLSCHAFT D-3550 Marburg, Federal Republic of Germany HANS MULLER and H-ELMUT GEIGER EDWD. WATERS SONS, 50 QUEEN STREET) MELBOURNE, AUSTRALIA, 3000.

Complete Specification for the Invention entitled,: A PROCESS FOR THE PREPARATION OF A PASTEURIZED IMMUNOGLOBULIN PREPARATION The following statement is a full description of this invention, including the best method of performing It known to US BEHRINGWERKE AKTILNGESELLSCHAFT 86/B 014 Ma 571 Dr. Ha/Sd.

A process for the preparation of a pasteurized immur.globulin preparation The invention relates to a process for the prcduction of an immunoglobulin preparation, in which, for the purpose of inactivation of pathogens, a solution of an immunoglobulin is heated in the presence of stabilizers and is, if necessary, subsequently purified.

A process is needed which permits inactivation of pathogens, such as viruses, by pasteurization of immunoglobulin preparations; thereby the full activity of the imnurnoglobulin must be retained. In general, when imunoglobulins 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 inadequlate to unambiguously rule out a risk of infection; 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 rendeirs the infectious agent incapable of reproduction and/or of being intracellularly 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 proldnged period.

2 -2- A process is described in EP-A-0 124 506, in which, ammonium sulfate is added to an immunglobulin solution, and the suspension is heated at 60°C for 10 hours.

However, when an immnunoglobulin solution is treated as described in Example 16 of that patent application, S the formation of polymeric immunoglobulin occurs. A Li maximum limit of 10% of polymeric immunoglobulin i> permitted in the European Pharmacopoea in an immunoglobulin solution for intramuscular adminiztration.

In EP-A-0 144 714 it is described that a Cohn fraction II+III (J.Am.Chem.Soc. (1946) 68, 459) can be pasteurized only under mild conditions, preferably at 52°C for about 30 minutes; even when there has been previous removal of the eaglobulins 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% sorbitol and 15% glycine.

Afterwards neither a loss of activity nor an increase in the aggregate content was observed.

A process for the pasteurization of human plasma with the addition of sugar alcohols, amino acids or saccharides is described in EPatent-A-0 139 975. In the pasteurization of plasma the immunoalobulin 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 comprises heating a solution of an immunc4robulin I1u 3 in the presence of a carboxylic acid or one of its salts and/or of a saccharide until pathogens in particular hepatitis viruses or tFV- TTT AIDS viruses, are inactivated rendered incapable of reproduction or of being intracellularly or otherwise reproduced).

Conditions suitable for such inactivation are known to the expert. It is customary to heat at about 60 0 C for about 10 hours.

This carboxylic acid is preferably an aliphatic carboxylic 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 glycine, glutamic acid, citric acid or et 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 preferauy 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, preferably from 0.4 to 0.6 g per milliliter of the immunoglobulin 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 to 1.0 g per milliliter of the immunoglobulin lrth solution which is to be stabilized.

4 The carboxylic acids or their salts or the saccharides can be added in an amount up to or exceeding the saturation limit of the immunoglobilin solution which is to be stabilized.

The addition of these substances may cause a precipitation of the immunoglobulins. In this case, the resulting suspension is nevertheless heated without detrimental effect on the immunoglobulin.

The pH-value is adjusted to 5-8.5, preferably 6.5-7.5, for the heating process.

Using the claimed process it is possible to obtain pasteurized immunoglobulin 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.

41 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 G 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-containing 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 sulfitolysis, or enzymatic treatment, for example peptic elimination of the Fc portion. Proteolytic cleavage of the immunoglobulin molecule with pepsin at pH 4 results t 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 It has been surprisingly found, that the claimed process is also suitable for solutions of immunoglobulins which contain ethanol.

When purified immunoglobulins are obtained using ethanol, often the final concentration step of the production 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 solution results 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 subsequently. Thus, to expedite 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 0 C for prolonged periods, for example 40 h.

t 6 It is known that ethanol normally denatures immunoglobulins 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 et seq., or fraction A by Nitschmann (Kistner and Nitschmann, Vox Saing. (1962), 7, 414) Fractions of this type contain not only gamma-globulins but also lipoproteins, euglobulins, alpha- and beta-globulins 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 ml/100 ml. A fraction II+II of this type can therefore be pasteurized in the manner according to the invention.

i 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 0

C

was continued for 10 hours. The immunglobulin content was 10-11 g of protein per 100 ml of solution. The pH was 7.

ll TABLE 1 Contents in 100 mL of the globulin solution Ethanol NaCL Glycine (ml) (g) 0 0.3 2.25 4 0.1 0 0 0.3 2.25 4 0.3 2.25 0 0.3 2.25 4 0.3 2.25 2 0.3 2.25 2 0.12 0 4 0.12 0 2 0.12 0 2 0.12 0 4 0.1 0 4 0.1 2.25 4 0.1 0 0 0.3 2.25 4 0.3 2.25 Added to 100 mL of globulin solution Polymer content before after heating* (g/100 g globulin) Substance i Ammonium sulfate Lysine, HCL Monosodium citrate Mono-Na L-glutamate 11 it 11 11 Glucose Fructose GaLactose Sucrose Mono-Na L-glutamate Sodium tartrate

I

Amount (g) 80 100 60 60 60 60 60 60 100 60 60 100 100 60 50 50 Glycine Glyci ne G ly ci ne Lysine, HCL Sucrose Substance Amount (g) 15 15 15 15 50 1.7 3.3 1.3 1.1 1.1 1.1 1.2 2.3 3.0 2.3 2.3 2.2 3.3 3.0 1.3 1.1 10.8 3.4 27.7 1.4 1.8 3.1 5.1 1.7 4.1 4.3 3.6 17.3 Analytical gel chr-matography using Ultrogel ACA 34. The transmission signals continuous flow photometer are converted into extinctions, and an area elution constructed and evaluated.

measured in a profile is 8 The table shows that, after application of the claimed process, the undesired increase in polymers of immunoglobulin is, as a rule, very low even in the presence of alcohol. This finding is, moreover, confirmed by other test methods, for example by determination of the K anticomplementary activity.

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 purificatioi process.

The duration of heating can be varied within certain limits.

To test the efficacy of the process which has been described, an immunoglobulin in solution containing 9.9 g of protein per 100 ml and 3,6 g of ethanol per 100 ml 1 was mixed with l 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 10 infectious RSV units/ml (U/ml) and then the solution was heated at 60C., 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 had, no effect on the antibody acitivity.

labLe 2 Stabilizer Amount added Ethanol mLflOO ml Heating at 600 hours Aat i -tetanus1 Antibody titer Anti -HBsAg 2 Anti rubeLLa 3 (g per- ml) Na glutamate GLucose Na gLutamate Na glutamate! gLycine Na gLutamate! suc rose Sucrosefg Lycine GLucose/gLycine 0.6 0.6 0.6 4048 4048 4048 8096 8096 8096 8096 8096 8096 0.22 0.20 0.19 0.31 0.22 0.29 0.28 0 .27 0.21 30720 30720 30720 30720 30720 30720 30720 30720 30720 0.6/0.15 0.610.5 1/0.15 1/0-15 Indirect bemaggLutination assay I.H-A. (reciprocal titer) Radioimmunoassay (in international units/mI) ELisa (enzyme-Linked immunosorbent assay; reciprocaL titer)

C-,

'4 10 ii The examples which follow illustrate the invention: Example 1 Heating of an immunoglobulin in solution with sodium glutamate 200 ml of a virtually pure solution of immunoglobulin with a protein concentration of 90 g/l were stirred while 120 g of sodium glutamate (monosodium salt of glutamic acid) were adeed. This resulted in the precipitation of the immunoglobulin. The pH-value of the Ssuspension was adjusted to 7; it was then heated and stirred at 60 0 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 j by dialysis or ultrafiltration. The solution was adjusted to the desired protein content and made isotonic.

i Herefrom 110 ml with a protein concentration of 155 g/l were obtained. The polymer content was 1.6% (1.2% unheated).

Example 2 Heating of an immunoglobulin 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/l, a protein concentration of 97 g/1 and 3.6% ethanol by volume. The pH-value was adjusted to 7 and then the mixture was heated and stirred at 0 C for 10 hours. The solution was diluted with 100 1 11 of 0.3 g/100 ml sodium chloride solution and was sterilized 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/l.

Consequently 51 1 of solution with a polymer content of 2.6% in the unheated solution) were obtained. The residual sucrose concentration was 0.04 g/1.

Example 3 i 100 g of sucrose and 15 g of glycine were added to 100 Sml of sulfonated immunoglobulin with a protein concen- Stration of 100 g/l and a sodium chloride concentration of 3 g/l. The pH-value was adjusted to 7.3 and then the mixture was heated and stirred at 60 0 C for 10 hours.

The solution was then cooled to room temperature and diluted with 170 ml of 0.3 g/100 ml sodium chloride solution. The stabilizers were removed by ultrafiltration. 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 concentration of 50 g/l.

195 ml of solution with a polymer content of 5.6% (5.8% in the unheated solution) were obtained by this procedure.

Example 4 13.7 1 of an immunoglobulin solution which had undergone peptic cleavage (with a protein concentration of 180 g/1 and a sodium chloride concentration of 3a2 g/1) were 12 12 diluted with 13.5 1 of a 0.3 g/100 ml sodium chloride bslution. Then 27.2 kg of sucrose and 4.08 kg of glycine were added. At pH 7 the mixture was then heated to and stirred for 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 by substantial removal of the added stabilizers by ultrafiltration. The solution was then made isotonic and adjusted to a protein concentration of g/l. 48 1 of solution with a residual sucrose concentration of 0.01 g/l were obtained. The content of components of defined 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 Example Heating of a dissolved ethanol-containing fraction S II+II in the presence of ethanol 200 g of fraction II+III were dissolved under 4tvrring in 500 ml of distilled water. To the solution (ab~t 700 ml) 700 g of sucrose and 0.3 to ml/ g0y e aY.e added. The pH-value was adjusted to ab0Wo 7 oh solution was then heated and etirred hours. The heated solution was the .4 ml of immunoglobulin solution with a .iton of 66 g/1 were obtained. The polymez 1.1%.

I. :i 13 For comparison an unheated treatment also using 200 g of fractioh II+III produced 208 ml of immunoglobulin solution with a protein concentration of 96.8 g/l. The polymer content was i

LI

ii i y I .a« -14 COMPARATIVE EXAMPLE 1.

inactivation of Herpes Sirnolex Virus Type 1 (HSV-i) by Heat Treatment at 60 IC Szolution inactivation of HSV-I Rate Timie (hours) loa 10

ID'

0 /m1 inunmungobuiin Solui:on 6,28 without. erthanol.

mg/im2 ~unalobulin solution 5,2 .2 with -1.3 ('ol/VoI) wiz~ zaoi a/mi glycine I /ral sucrose)

Claims (5)

1. "A method for production of a pasteurized immunoglobulin solution, characterized in that an immunoglobulin solution with an ethanol content of up to 5 vol. is mixed in the presence of 0.02 0.6 g/ml of a soluble carboxylic acid or one of its salts and/or with 0.1 1.0 g/ml of a saccharide, whereby the saccharide is glucose, fructose, galactose or sucrose, and is heated over a period of 2 40 hours at C until certain pathogens, especially hepatitis viruses or Aids viruses, are inactivated and the immunoglobulin remains essentially unaltered in its polymer content, while the total polymer content is

2. "A method according to Claim 1, characterized in that ,o °the carboxylic acids may be substituted with one or several o 'o carboxyl, amino or hydroxyl groups, whereby the carboxylic o acid is glycine, glutamic acid, citric acid or tartaric acid". 4 44

3. The process as claimed in claim 1, wherein the carboxylic acid is glycino, glutamic acid, citric acid or tartaric acid. o

4. The use of an immunoglobulin preparation as claimed in claim 1 for therapy or prophylaxis. 4o 0

5. The use of an immunoglobulin preparation as claimed in claim 1 in diagnostic reagent kits. DATED this 31st day of January 1990. BEHRINGWERKE AKTIEMGESELLSCHAFT o44444 4 4 WATERMARK PATENT S TRADEMARK ATTORNEYS TH ATRIUM, 290 BURWOOD ROAD HAWTHORN, VICTORIA 3122, AUSTRALIA