EP0954534A1

EP0954534A1 - Pasteurization of immunoglobulin solutions

Info

Publication number: EP0954534A1
Application number: EP97930259A
Authority: EP
Inventors: Joseph Bertolini; Germano Coppola; Jeffrey Raymond Davies
Original assignee: CSL Ltd
Current assignee: CSL Ltd
Priority date: 1996-07-18
Filing date: 1997-07-18
Publication date: 1999-11-10
Also published as: KR20000067911A; WO1998003550A1; JP2000515857A; CA2261291A1

Abstract

A process for the pasteurization of an immunoglobulin solution, particularly an IgG solution, comprises heating the solution at a temperature of 50 °C to 70 °C for a period of from 1 to 20 hours, at a pH of 4.0 to 6.0 in the presence of 20 to 40 % w/v sorbitol or 25 to 45 % w/v sucrose, and wherein the protein concentration of the solution is approximately 3 % w/v or less.

Description

PASTEURIZATION OF IMMUNOGLOBULIN SOLUTIONS

FIELD OF THE INVENTION

This invention relates to the heat treatment, more particularly the pasteurization of immunoglobulin solutions, and in particular it relates to the pasteurization of Immunoglobulin G (IgG) solutions.

Pasteurization can be used as a method to inactivate viruses in the manufacture of IgG solutions for therapeutic use. It is preferable, however, that the process be carried out under conditions which maintain the physiological function of the immunoglobulin and that it produces a stable product without the need for additional purification.

BACKGROUND OF THE INVENTION

Denaturation of immunoglobulins during the manufacturing process can lead to aggregation of immunoglobulin molecules and loss of functional properties. Aggregates of immunoglobulin, when infused into patients intravenously, can result in adverse reactions due to activation of the complement cascade. In the past, manufacturers have dealt with this issue either by modification of the Fc region of the immunoglobulin molecule to prevent aggregation and reduce complement binding, or by using manufacturing processes which result in low immunoglobulin aggregate content in the final product.

Due to the diverse functions of immunoglobulins, it is desirable to produce a chemically unmodified immunoglobulin and maintain Fc mediated functions including complement activation. Thus, in manufacturing unmodified IgG for intravenous use, it is important that the aggregate content is kept to a minimum and that it remains below 3% during the shelf life of the product (British Pharmacopoeia (BP), 1994). A major challenge for manufacturers of plasma protein products has been in designing virus inactivation steps which inactivate viruses but do not denature immunoglobulin and other protein. The principal methods which have been used in the manufacture of unmodified immunoglobulin products are incubation at low pH and solvent/detergent treatment. Both these methods are effective against lipid enveloped viruses but are less so against non-enveloped viruses. Pasteurization (at 60°C for 10 hours) has been applied widely in the preparation of albumin and is effective against both enveloped and non-enveloped viruses, however the application of the pasteurization approach to immunoglobulins has been more challenging. Immunoglobulins are more susceptible to heat denaturation than albumin and it is difficult to prevent aggregation.

Processes for pasteurizing immunoglobulin solutions have however been described. Magnin et al. (1989) describe a process for pasteurizing IgG under conditions of low protein concentration, low ionic strength and acidic pH. The propensity of IgG to aggregate on heat treatment was shown to increase with increasing ionic strength, protein concentration, temperature and time of exposure to high temperatures. Pasteurization in the absence of stabilizer as described by Magnin et al. produces a product which rapidly forms aggregates on storage in solution.

Hirao etal. (1989) describe a process for pasteurizing IgG under similar conditions to those of Magnin et al. with inclusion of sorbitol to stabilise IgG during pasteurization. The preferred method involves pasteurization (preferably at 60°C for 10 hours) under conditions of low ionic strength (0.001 or less), a pH range of 4.5 to 6.5, and a sorbitol concentration of 10% to 70% w/v. Protein concentration according to the method is not restricted and the process is deemed to work over a concentration range of 0.1 to 30% w/v.

Nowak et al. (1992) describes a process for pasteurizing IgG (60°C, 10 hours) using high concentrations of sucrose and glycine to stabilise IgG during pasteurization. For intravenous preparations, the stabilisers are removed and the IgG is then treated either by low pH incubation in the presence of pepsin or by S sulphonation resulting in a modified immunoglobulin. The modification to the immunoglobulin molecule reduces complement activating IgG aggregates and allows compliance with pharmacopoeial requirements for anticomplementary activity.

It is an object of the present invention to provide a process for the pasteurization of immunoglobulin solutions, particularly IgG solutions, which allows the production of an immunoglobulin preparation with low aggregate levels, low anticomplementary activity, and retained functional activities which meet pharmacopoeial requirements for an intravenous product. Consequently, no additional purification or modification of IgG is required following the pasteurization process.

SUMMARY OF THE INVENTION

According to the present invention there is provided a process for the pasteurization of an immunoglobulin solution, such as an IgG solution, which comprises heating the solution at a temperature of 50°C to 70°C for a period of from 1 to 20 hours, at a pH of 4.0 to 6.0 in the presence of 20 to 40% w/v sorbitol or 25 to 45% w/v sucrose, and wherein the protein concentration of the solution is approximately 3% w/v or less.

Preferably, the pasteurization process is carried out at low ionic strength (conductivity of 1.OmS/cm or less; more preferably, conductivity of less than 0.3mS/cm at 25°C).

The present invention also extends to a pasteurized immunoglobulin preparation prepared by the process broadly described above.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a process for the production of a pasteurized immunoglobulin preparation which retains its therapeutic effectiveness, and in particular a preparation in which the level of aggregate and anti-complementary activity is within therapeutically acceptable limits. In accordance with this process, purified immunoglobulin preparations are pasteurized, for example at 60°C for 10h, and then formulated and dispensed without the need for additional purification to remove aggregate or modification to reduce anti-complementary activity.

The process of the present invention where sorbitol is used as a stabilizer during pasteurization utilises similar conditions to those broadly described by Hirao et al. (1989) except protein concentration is recognised as a critical parameter in this process. In preferred embodiments of this process, the immunoglobulin solution is pasteurized at 50°C to 70°C (preferably at 60°C), for a period of 1 to 20 hours (preferably 10 hours), at a pH of 4.0 to 6.0 (preferably 4.0 to 5.0), in the presence of 20 to 40% w/v (preferably 20- 33% w/v), sorbitol and at protein concentration of approximately 3% w/v or less. In preferred embodiments of the process of this invention where sucrose is used as the stabilizer, the immunoglobulin solution is pasteurized at 50°C to 70°C (preferably at 60 °C), for a period of 1 to 20 hours (preferably 10 hours), at a pH of 4.0 to 6.0 (preferably 4.4 to 5.4), in the presence of 25 to 45% w/v (preferably 30 to 40% w/v) sucrose, and at a protein concentration of approximately 3% w/v or less. Studies leading to the establishment of this process have shown that higher protein concentrations e.g. 5% w/v, result in unacceptable aggregate generation and a tendency for product to further aggregate during storage if formulated as a liquid preparation. Consequently, if pasteurization is conducted under such conditions, further purification steps are required to produce a product with acceptable aggregate content and stability (see Uemura et al, 1989). This increases the possibility of recontaminating batches with virus, results in loss of some product and complicates the manufacturing process.

In one particularly preferred embodiment of this invention, if the immunoglobulin solution is derived by a chromatographic process, it is pasteurized at a protein concentration of less than 3% w/v in the presence of 20-33% w/v sorbitol at a pH of 4.8 and at a conductivity of less than 0.3mS/cm.

In another particularly preferred embodiment, if the immunoglobulin solution is derived by Cohn fractionation, it is pasteurized at a concentration of less than 2% w/w in the presence of 20-30% w/v sorbitol at a pH of 4.8 and a conductivity of less than 0.3mS/cm.

In yet another preferred embodiment, chromatographically purified immunoglobulin is pasteurized at a protein concentration of less than 2% w/v in the presence of 30-45% sucrose at a pH of 4.4 to 5.2.

Preferably, for therapeutic use the sorbitol or sucrose is removed from the pasteurized immunoglobulin by diafiltration, and the immunoglobulin is formulated and is dispensed at 6% w/v protein concentration in 10% w/v maltose at pH 4.0-6.0, preferably pH 4.25-5.5.

Further features of the present invention are more fully described in the following Examples. It is to be understood however, that this detailed description is included solely for the purposes of exemplifying the present invention, and should not be understood in any way as a restriction on the broad description of the invention as set out above.

In the accompany drawings:

Figure 1 shows: (a) Dependence of aggregate generation on pH in immunoglobulin during pasteurization at 60°C for 10 hours.

(b) Effect of protein concentration on aggregate formation during pasteurization at 60°C for 10 hours, and the stability of the preparations.

Figure 2 shows the effect of protein concentration an sorbitol concentration on aggregate generation during pasteurization at 60^CC for 10 hours.

The following general methods are used in the Examples:

Immunoglobulin Purification. Immunoglobulins were purified from normal plasma pools using Cohn fractionation or chromatographic methods. Cohn fractionated product

(Supernatant III) was purified by a method based on Cohn-Oncley Method 6.

Chromatographically purified immunoglobulin was purified using Cohn fractionation to remove fibrinogen (Fraction I) followed by ion exchange chromatography.

Immunoglobulin Bulk Concentrate. Following purification by either method, preparations were concentrated to appropriately 10% w/v and diafiltered at pH 4.8, followed by dilution to achieve a conductivity of < 0.3mS/cm (unless stated otherwise in the Examples).

Formulation. The pasteurized solution was diafiltered to remove sorbitol while maintaining pH at 4.25 to 4.8 and then formulated as a 6% w/v solution in 10% w/v maltose at pH 4.25 to pH 5.5.

Aggregate content. Aggregate content was measured by size exclusion chromatography using a TSK3000 column (60cm X 0.5cm) with 0.1 M phosphate pH 7.0 as running buffer with samples diluted in 0.1 phosphate (BP 1988) or 0.5% NaCl (BP 1994) and a flow rate of 0.5ml/min. Elution of proteins was monitored at 280nm. Anticomplementary Activity (ACA) assay. According to the BP (1994) method, ACA activity is assayed by mixing the immunoglobulin preparation under assay with guinea pig complement. Any aggregated IgG will bind to complement resulting in neutralisation of complement activity. The remaining activity is determined by monitoring lysis of sheep red blood cells. Activity is expressed as a percentage of complement consumed against a complement control solution not exposed to immunoglobulin. In the present Examples, an analogous but modified method was used. Serial dilutions of the immunoglobulin preparation were mixed with human complement and the amount of complement remaining determined by monitoring lysis of sheep red blood cells. Results were expressed as CH₅₀/mg where CH₅₀ refers to 50% haemolytic units of complement. This assay has been correlated with the BP assay and the limit of 10 CH₅₀/mg is within the BP limit for anticomplementary activity.

Fc Function Test. This test was conducted as described in the BP (1994). Sheep red blood cells were tanned and coated with rubella antigen. Hence rubella antibodies present in the IgG preparation will bind to the antigen and specifically interact with the complement system via the Fc domain prompting red blood cell lysis. The degree of lysis correlates with Fc function. Results are expressed as a percentage of the European Pharmacopoeia Biological Reference preparation,

Batch 1 (EPBRP, Batch 1). Immunoglobulins must have an Fc function of greater than 60% of this standard to meet BP requirements (BP 1994).

EXAMPLES Example 1 Pasteurization of immunoglobulin in absence of stabilizer.

Immunoglobulin bulk concentrate prepared by Cohn fractionation was diluted to 1 % w/v adjusted to pH 4.0, 4.2, 4.8 or 5.0 and pasteurized at 60°C for 10 hours in the absence of any stabilizer.

Figure 1 (a) shows the aggregate content obtained at each condition. The data illustrates that under these conditions pasteurization of IgG results in an inordinate level of aggregation for IV infusion, rendering the product clinically unacceptable as it exceeds the pharmacopoeial limit for an intravenous immunoglobulin preparation of ≤3% aggregate.

Figure 1 (b) shows that the material pasteurized without sorbitol at 0.5% or 1 % w/v IgG and formulated at 6% w/v IgG in 10% maltose at pH 5.5 exhibits a high initial aggregate content and further increases over 13 days at both 8°C and 25°C.

The data shows that pasteurization in the absence of stabilizer generally results in aggregate content which would render in immunoglobulin product unsuitable for therapeutic use and highlights the poor reproducibility of this process.

Example 2 Pasteurization of immunoglobulin in the presence of sorbitol.

Immunoglobulin bulk concentrate derived by the chromatographic process was formulated in various concentrations of sorbitol ranging from 0% to 30% w/v, at various protein concentrations ranging from 1 % to 4% w/v. The pH was adjusted to 4.8 and the solutions pasteurized at 60°C for 10 hours.

Figure 2 (a and b) shows that increasing sorbitol concentrations stabilise immunoglobulin during pasteurization thus resulting in less aggregate formation. In fact, in Figure 2b, it can be seen that at a protein concentration of 1 % w/v in the presence of 30% w/v sorbitol, no significant aggregation occurs. Even at 2% in 20% sorbitol, aggregate content remains less than 2.0%, which is within specifications for intravenous IgG (≤3%).

It has been found that pasteurization of immunoglobulin under these conditions can result in a clinically acceptable immunoglobulin preparation with respect to aggregate content. No further fractionation is required and the process is reproducible.

Example 3 Comparison of aggregate formation following pasteurization of Cohn and chromatographically purified immunoglobulin preparations.

Immunoglobulin bulk concentrates from Cohn and chromatographically purified immunoglobulin preparations were formulated at 1 to 30% w/v IgG concentration in 15 to 30% w/v sorbitol at pH 4.8 and pasteurized at 60°C for 10 hours. Aggregate formation was monitored in the pasteurized product by size exclusion chromatography.

The results presented in Table 1 show that material derived from both processes can be pasteurized under conditions which minimize aggregation. Chromatographically purified immunoglobulin can be pasteurized up to a concentration of 3% w/v in the presence of 30% w/v sorbitol resulting in a progressive increase in aggregation with increasing protein concentration or decreasing sorbitol concentration. Cohn fractionated material was more labile with a greater tendency to form aggregates. However, at a protein concentration of 1 % w/v and at a sorbitol concentration of 30% w/v, aggregation was minimised.

These results indicate that the tendency for immunoglobulin products to aggregate reflects the purification process used to derive the product. Thus, material produced by

Cohn fractionation, which involves differential ethanol precipitation, is more labile during the pasteurization process and must be pasteurized at a lower protein concentration or higher sorbitol concentration. This may reflect perturbation of the immunoglobulin molecule by the relatively harsh fractionation conditions, resulting in increased sensitivity of the molecule to heat denaturation. The chromatographically purified material is more stable during the pasteurization process, and higher protein concentration and lower sorbitol concentrations can be used during the pasteurization process.

TABLE 1 Aggregate formation during pasteurization of chromatographically purified (PC-10, PC-11, PC-12) and Cohn fractionated (IP004, IP005, IP006) immunoglobulin at various concentrations of protein and sorbitol.

PC-10 PC-11 PC-12 IP004 pH 4.2 IP004 pH 4.8 IP005 IP006

[Protein] % Sorbitol % % Aggreg % Aggreg % Aggreg % Aggreg % Aggreg % Aggreg % Aggreg

1 15 0.08 0.24 1 .4 0.81 1.48

20 0.07 0.3 1 .73 0.76 1 .03 0.6

30 0.06 0.04 0.27 0.87 0.48 0.41 0.41

1.5 15 0.27 0.31 1.18 4.07 3.03 3 1.59

20 0.13 0.23 0.92 3.61 1.06 0.85 1.67

30 0.06 0.06 0.32 2.08 0.84 0.91 0.85

2 27 0.07 0.06 0.59 3.83 2.09 2.86 1 .87

30 0.08 0.07 0.4 3.32 2.1 3 1.42 2.41

33 0.08 0.1 1 0.45 1.6 1.54 1 .84 1 .32

3 27 1 .46 2.57 8.53 4.18 7.69

30 0.63 1.1 1 6.39 5.16 5 66

33 0.71 6.1 1 4.7 4.3

Example 4 Functional characterisation of pasteurized immunoglobulin.

Table 2 presents the antibody titre, Fc function, ACA and aggregate content of pasteurized intravenous immunoglobulin. Chromatographically purified intravenous immunoglobulin was pasteurized at a protein concentration of ≤3% w/v in the presence of 30% w/v sorbitol at pH 4.8. Following pasteurization, sorbitol was removed by diafiltration and the immunoglobulin formulated at 6% w/v protein, 10% w/v maltose, pH 4.25. Data for a non-pasteurized intravenous immunoglobulin product purified by Cohn fractionation and formulated as described above is presented for comparison.

Table 2 Characterisation of pasteurized chromatographically purified immunoglobulin.

PC-11 PC-12 PC-13 Non-pasteurised immunoglobulin

Hepatitis A Antibody (U/ml) 51 25 48 48 (27-62)

Hepatitis B Antibody (lU/ml) 3.24 1 .70 2.54 3.06 (1.6-4.0)

CMV Antibody (μ/ml) 14456 8455 8521 9815 (6439-14434)

Diphtheria antitoxin (lU/ml) 1 .9 1 .2 2.1 2.0 (1-2.9)

Tetanus Antitoxin Human 1 7 1 1 23 20(1 1-24) (lU/ml)

Fc Function (% mt.std.) 132 105 1 12 106 (90-1 13)

ACA (CH₅Jmg) 3 3 3 < 2-4

Aggregates (% aggreg) 0.4 0.1 0.8 0.1 (0-0.4)

Table 3 presents characterisation data (antibody titre, Fc function, aggregate content) of pasteurized Cohn fractionated immunoglobulin, using the same conditions of pasteurization and subsequent formulation.

Table 3 Characterisation of pasteurized immunoglobulin.

Batch Formulation % Aggregate pH

0 weeks 13 weeks 26 weeks 52 weeks

PC1 5.87 0.61 - 1 .5 0.8

PC2 5.59 0.64 - 1 .0 1.0

PC5 4.63 0.41 0.4 0.7 -

PC6 4.29 0.51 2.0 1 .2 -

PC 7 4.68 1 .8 2.3 1 .8 -

PC8 4.72 0.2 0.6 0.1 -

PC9 4.71 0.9 1.0 0.0 -

PC10 4.68 0.9 1.0 0.0 -

PC1 1 4.19 0.0 - -

PC12 4.25 0.1 0.1 - -

PC13 4.25 0.8 - - -

It can be seen that the properties of pasteurized immunoglobulin - whether derived by chromatographic processes or Cohn fractionation - are comparable to those found in the unpasteurized product. Aggregate content is elevated in pasteurized product derived by Cohn fractionation but well within specifications for an intravenous product.

Example 5 Stability of pasteurized immunoglobulin.

The prime concern with liquid immunoglobulin preparations is potential instability especially with respect to aggregate formation. Chromatographically purified immunoglobulin solution was pasteurized as a 2% w/v protein solution with 30% w/v sorbitol at pH 4.8 as described in Example 4. The pasteurized material was formulated at 6% w/v protein in 10% maltose at the pH values stated. Aggregate formation on product stored at 4°C was monitored by size exclusion chromatography.

Table 4 presents the aggregate content of chromatographically purified immunoglobulin following storage at 4°C for 13 and 52 weeks.

Table 4 Aggregate content in pasteurized chromatographically purified immunoglobulin during storage at 4°C.

IP004 IP-005 IP006 Non-pasteurised immunoglobulin

Hepatitis A Antibody (U/ml) 60 40 65 48 (27-62)

Hepatitis B Antibody (U/ml) 3.61 3.53 3.92 3.06 (1.6-4.0)

CMV Antibody (μ/mL) 18308 12568 9906 9815 (6439-14434)

Diphtheria Antitoxin (lU/mL) 2.1 2 4 2.0 2.0 (1 -2.9)

Tetanus Antitoxin Human (lU/mL) 20 26 16 20 (1 1 -24)

Fc Function (% mt.std.) - 125% 1 14% 106 (90-1 13)

ACA (CH₅₀/mg) 2 3 4 < 2-4

Aggregates (% aggreg) 1.2 2.0 0.9 0.1 (0-0.4)

It can be seen that the product is stable with respect to aggregate formation over a range of formulation pH values. Other parameters such as antibody titre (data not shown) are also stable.

This data confirms the stability of immunoglobulin preparations, pasteurized at

60°C for 10 hours if protein concentration is less than 3% and preferably less than 2% w/v, sorbitol is present at a concentration of approximately 30% w/v, conductivity is less than 0.3mS/cm and pH is approximately 4.8. Example 6 Viral inactivation studies.

The potential of the pasteurization step to inactivate viruses was evaluated using the following model viruses:

sindbis virus as a model for the enveloped viruses HIV and HCV; bovine viral diarrhoea virus (BVDV) as a model for the enveloped virus HCV; duck hepatitis B virus (DHBV) as a model for the enveloped virus HBV; encephalomyocarditis (EMC) and Theilar's virus as a model for non-enveloped viruses e.g. hepatitis A virus (HAV);

HIV.

Immunoglobulin bulk solution derived from the chromatographic or Cohn process was diluted to 2% w/v protein made 30% w/v with sorbitol and adjusted to pH 4.8. Samples prepared as described above were spiked with virus and pasteurized at 60°C for 10 hours. The results of the inactivation studies are presented in Table 5, and demonstrate the substantial inactivation of both enveloped and non-enveloped viruses achieved during pasteurization.

Table 5

Process Step Enveloped Virus Non-enveloped Virus (log₁₀ reduction factor) (log₁₀ reduction factor)

Sindbis BVDV HIV EMC Theilar's

Pasteurization > 5.2* >5.5 >4.7 > 5.4 > 5.9

Example 7 Pasteurised Immunoglobulin: Intramuscular formulation

Intramuscular immunoglobulin (IMIG) products are commonly derived from Cohn

Fraction II. Following freeze drying the material is reconstituted to a protein concentration of 16% w/v together with the addition of glycine to 22.5mg/ml and adjustment to pH6.5. It has been shown that pasteurisation to achieve viral inactivation can be applied to these intramuscular immunoglobulin (IMIG-VI) products.

IMIG-VI is manufactured from diafiltered Supernatant III, the pure immunoglobulin preparation resulting from Cohn fractionation. Viral inactivation is effected by pasteurisation at a protein concentration of 0.5-2.0% w/v IgG in the presence of 30% w/v sorbitol at pH 4.8. The pasteurised IgG is concentrated and diafiltered to remove sorbitol, and then formulated to a protein concentration of 16% w/v with glycine added to 22.5mg/ml and pH adjusted to 6.5.

The characteristics of three batches of normal IMIG-VI incorporating pasteurisation, and of batches produced by the alternative process are shown in Tables 6 and 7. IMIG-VI and IMIG were similar in most parameters measured. Antibody titers were similar for both products. IMIG-VI contains a higher aggregate content than IMIG, yet lower ACA compared to the IMIG product. Protease activity demonstrated by PKA and kallikrein was lower in IMIG-VI, whilst plasminogen was not observed in either. The Fc function activity trended higher in IMIG-VI and the IgG subclass distribution was similar for the two products.

These results show that pasteurisation is applicable to the intramuscular product as it does not adversely alter the characteristics of the immunoglobulin product.

Table 6 Preclinical Batch analysis for IMIG-VI and IMIG Final Products

Values in parentheses indicate range

Table 7 Characterisation Results for IMIG-VI and IMIG Final Products

Values in parentheses indicate range

* CH50 refers to 50% haemolytic units of complement.

** European Pharmacopoeia Biological Reference Preparation (Batch 1 ) Example 8 Pasteurised imunoglobulin stabilised with sucrose

Chromatographically purified immunoglobulin was subjected to pasteurisation at 60°C for 10 hours in the presence of sucrose. Three studies were conducted. For Studies 1 and 2 chromatographically purified immunoglobulin that had been stored for 5 months as an 8% solution at 4°C and pH 4.8 was used as the source material. For Study 3 freshly prepared chromatographically purified immunoglobulin was used.

Study 1

Studies assessing the effect of pH and sucrose concentration on IgG polymerisation following pasteurisation are shown in Table 8. Protein concentration was 1.5% w/v. The data shows that the optimum pH for minimising immunoglobulin aggregation during pasteurisation is pH4.8. It also demonstrates that increasing the sucrose concentration from 20% to 30% reduces the amount of aggregate formed. Results compliant with British Pharmacopoeia (BP) requirements (aggregate < 3% and monomer+dimer J>_ 90%) were achieved for pH 4.4 to 5.2 when 30% sucrose was used.

Table 8 Effect of pH and sucrose concentration on IgG polymerisation following Pasteurisation of Pure Immunoglobulin solutions

(1.5%w/v) .

pH* Protein Sucrose Composition Concentration

20% 25% 30%

4.0 Aggregate 9.32 7.59 7.24

Dimer 13.95 13.43 12.47

Monomer 75.31 77.57 79

4.4 Aggregate 3.82 3.21 2.67

Dimer 9.67 8.51 7.04

Monomer 85.65 87.47 89.54

4.8 Aggregate 2.31 1.57 1.21

Dimer 6.81 5.02 3.63

Monomer 90.41 92.87 94.85

5.2 Aggregate 4.79 2.01 1.43

Dimer 6.36 4.91 3.63

Monomer 88.85 93.08 94.94

Study 2

Studies on the effect of protein and sucrose concentrations on IgG polymerisation following pasteurisation of an immunoglobulin preparation are shown in Table 9. The pH was held constant at pH 4.8. The data shows that protein aggregation increases as the protein concentration increases. However, provided the protein concentration does not exceed 2% (w/v) and the sucrose concentration is _>.35% product remains in compliance with BP specifications

Table 9 Effect of protein and sucrose concentration on IgG polymerisation following pasteurisation of Pure Immunoglobulin solutions (pH4.8)

Protein Sucrose Concentration (%) Concentration %(w/v) Protein composition

30 35 40

0.5 Aggregate 0.18 0.22 0.21

Dimer 1.5 1.33 1.25

Monomer 98.32 98.45 98.54

1 .0 Aggregate 0.68 0.65 0.5

Dimer 3.3 2.71 2.27

Monomer 96.01 96.64 97.23

1.5 Aggregate 1.84 1.37 1.07

Dimer 4.92 3.85 2.73

Monomer 93.23 94.78 96.2

2.0 Aggregate 4.55 2.71 1.99 Dimer 5.57 4.28 3.38

Monomer 89.89 93.01 94.63

Study 3

The efficacy of the process was further tested by pasteurization freshly prepared pure immunoglobulin (Table 10). It is evident from the data that IgG polymerization occuring following pasteuristion of freshly prepared immunoglobulin solutions is very low and well below the levels observed in preparations which have been stored at 4°C for 5 months.

Table 10 Effect of Protein and Sucrose Concentration on IgG Polymerisation during Pasteurisation of Freshly Prepared Immunoglobulin Solutions (pH 4.8)

Protein Sucrose Concentration (%)

Concentration Protein

%(w/v) composition

30 35 40 45

0.5 Aggregate 0.02 0.02 0.03 0.04

Dimer 0.2 0.1 7 0.23 0.18

Monomer 99.77 99.81 99.74 99.78

1 .0 Aggregate 0.02 0.02 0.03 0.04

Dimer 0.26 0.31 0.32 0.33

Monomer 99.72 99.67 99.65 99.63

1 .4 Aggregate 0.01 0.04 0.03 0.04

Dimer 0.27 0.33 0.2 0.13

Monomer 99.71 99.64 99.77 99.83

1.6 Aggregate 0.01 0.03 0.04 0.05

Dimer 0.7 0.42 0.29 0.18

Monomer 99.29 99.56 99.67 99.77

2.0 Aggregate 0.01 0.01 0.02 0.06

Dimer 0.67 0.46 0.57 0.33

Monomer 99.33 99.53 99.4 99.61

The above examples demonstrate that pasteurized immunoglobulin solutions which meet BP requirements can be prepared without the need for furhter fractionation to remove polymeric or denatured molecules.

Acceptable conditions for pasterization are: sucrose at a concentration of 30%-45% (w/v), pure immunoglobulin solutions at a concentrations up to at least 2% w/v and a pH range of at least pH4.4 to 5.2. REFERENCES:

1 . Hirao, Y.K., (1989). "Process for heat treating chemically unmodified gammaglobulin". US Patent 5845199: 1-5.

2. Magnin, A.A. (1989). "Pasteurization of immunoglobulin solutions". US Patent 4849508:

3. Nowak, T.J. (1992). Virus safety of human immunoglobulins: Efficient inactivation of hepatitis C and other human pathogenic viruses by the manufacturing procedure. /. Med. Virol. 36:209-216.

4. Uemura, Y.K. (1989). Inactivation and elimination of viruses during the fractionation of an intravenous immunoglobulin preparation: Liquid heat treatment and polyethylene glycol fractionation. Vox Sang. 56:1 55-161 .

Claims

CLAIMS:

1. A process for the pasteurization of an immunoglobulin solution, which comprises heating the solution at a temperature of 50°C to 70°C for a period of from 1 to 20 hours, at a pH of 4.0 to 6.0 in the presence of 20 to 40% w/v sorbitol or 25 to 45% w/v sucrose, and wherein the protein concentration of the solution is approximately 3% w/v or less.

2. A process according to claim 1, wherein the immunoglobulin solution is an IgG solution.

3. A process according to claim 2, wherein the immunoglobulin solution is an IgG concentrate prepared by Cohn fractionation.

4. A process according to claim 2, wherein the immunoglobulin solution is a chromatographically purified IgG solution.

5. A process according to any one of claims 1 to 4, wherein said heating is carried out at low ionic strength, conductivity of 1 .OmS/cm or less.

6. A process according to any one of claims 1 to 5, wherein the protein concentration of the immunoglobulin solution is approximately 2% w/v or less.

7. A process according to any one of claims 1 to 6 wherein said heating is carried out at a temperature of 60°C for a period of 10 hours.

8. A process according to any one of claims 1 to 7, further comprising the step of diafiltration of the pasteurized immunoglobulin solution to remove sorbitol or sucrose.

9. A pasteurized immunoglobulin preparation, prepared by the process of any one of claims 1 to 8.