Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
  • A61L2/02—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using physical phenomena
  • A61L2/04—Heat
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39591—Stabilisation, fragmentation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
  • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
  • A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
  • A61L2/0023—Heat
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators

Definitions

• 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.
• IgG Immunoglobulin G
• 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.
• 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.
• 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.
• an immunoglobulin solution such as an IgG solution
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• Figure 1 shows: (a) Dependence of aggregate generation on pH in immunoglobulin during pasteurization at 60°C for 10 hours.
• Figure 2 shows the effect of protein concentration an sorbitol concentration on aggregate generation during pasteurization at 60 C C for 10 hours.
• Immunoglobulin Purification Immunoglobulins were purified from normal plasma pools using Cohn fractionation or chromatographic methods. Cohn fractionated product
• 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).
• 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 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.
• 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 CH 50 /mg where CH 50 refers to 50% haemolytic units of complement. This assay has been correlated with the BP assay and the limit of 10 CH 50 /mg is within the BP limit for anticomplementary activity.
• Immunoglobulins must have an Fc function of greater than 60% of this standard to meet BP requirements (BP 1994).
• 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.
• 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 shows that increasing sorbitol concentrations stabilise immunoglobulin during pasteurization thus resulting in less aggregate formation.
• 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%).
• 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.
• 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 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.
• Diphtheria antitoxin (lU/ml) 1 .9 1 .2 2.1 2.0 (1-2.9)
• 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 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
• 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
• DHBV duck hepatitis B virus
• EMC encephalomyocarditis
• Theilar's virus as a model for non-enveloped viruses e.g. hepatitis A virus (HAV);
• 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.
• Intramuscular immunoglobulin (IMIG) products are commonly derived from Cohn
• 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.
• IMIG-VI 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.
• CH50 refers to 50% haemolytic units of complement.
• 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.
• 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.

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