KR950703054A - Hepatitis A Virus Vaccine (HEPATITIS A VIRUS VACCINE) - Google Patents

Abstract

The present invention is perfect for producing a stable hepatitis A virus (HAV) including optimized growth, harvesting and antigen propagation conditions for obtaining hepatitis A virus (HAV) having a purity of 75% or more based on protein. A highly reproducible method on a commercial scale. An additional processing step produces an inactivated HAV vaccine formulation that is immunogenic, resistant and effective when preventing infection with strongly toxic HAV. The production of proteins, carbohydrates, lipids, DNA and RNA is characterized by content.

Description

Hepatitis A Virus Vaccine (HEPATITIS A VIRUS VACCINE)

Since this is an open matter, no full text was included.

Figure 1 is a synthetic flow diagram of the major methods used in the production of hepatitis A virus vaccine for clinical trials. The left description shows prior art methods used in Phase I / II clinical trials using the Rotarian method. The novel method used for Monroe efficacy studies (New England J. of Med., 327: 453-457 1992) is the right branch and the manufacturing method of the present invention is central.

Figure 2 is an analytical sizing intermediate assay using a phosphate buffered saline, HPSEC TSK PW 4000 column eluted with PBS. This analysis plots high molecular weight standards and shows hepatitis A virus dilution and linear area response. This analysis is particularly amenable to later stages of purification. Four chromatograms are shown here from the last four steps of the preparation method. Chromatography is monitored at 214 nm (top trace) and 260 nm (bottom trace) by double UV detection, respectively. Using this assay, peaks corresponding to hepatitis A virus can be seen after re-establishment of PEG pellets (panel A). The chloroform extraction step removes much of the high molecular weight UV absorbing material (panel B). Anion exchange chromatography concentrates the virus (panel C) and preparative size exclusion chromatography yields highly purified virus (panel D). Purified hepatitis A virus shows poor absorbance in the 260 or 280 nm range and therefore only a 214 nm trace exists. Under the conditions of this assay, the HAV product is a single, symmetric peak, so this assay provides a measure of HAV purity independent of the analysis by SDS PAGE. 3 shows MRC-5 cell growth on glass, polystyrene, and 316 stainless steel. Cell surface enrichment is plotted as a function of time. Cells are seeded onto coupons of each material at a density of approximately 10,000 / cm ² and one coupon per day for cell counting. A medium glass coverslips and 316 stainless steel incubation cells on the plate in pupon stylish PET 150cm ² containing medium (0.6m1 / cm ²⁾ 90m1 and the cells grown in 25cm ² flask T- (0.3ml / cm ²⁾ 7.5 Incubate with ml.

Claims (23)

a) incubating a large amount of hepatitis A virus in a cell sheet in a static surface reactor (SSR) having a large surface area, and b) removing the hepatitis A virus from the cell sheet culture by infiltrating a detergent into the infected cell sheet. Thereby freeing the hepatitis A virus from the cell culture, c) optionally removing the non-hepatitis A viruses, particularly nucleic acids at this stage, from the harvested hepatitis A virus by nuclease treatment, d) membranes And concentrating the hepatitis A virus removed from the cell culture by diafiltration, or by capture by ion exchange, and e) removing the contaminating nucleic acid if not previously completed in step c). , Non-hepatitis A virus specific protein from the concentrated hepatitis A virus of step (d) by PEG precipitation, combination of ion exchange, and gel permeation chromatography, f) step (e) Hepatitis A method of producing a commercial scale biruseu having at least 95% pure, based on the protein by, Bolivar SDS acrylamide gel electrophoresis and the stain analysis comprising the step of recovering the purified hepatitis A biruseu. Purified hepatitis A virus obtained by the method of claim 1. The method according to claim 1, wherein step a) is inoculated with hepatitis A virus derived from the supernatant of HAV infected SSR into a fixed surface reactor in which MRC-5 cells are incubated in a cell sheet, and the culture medium is fixed surface reactor and reactor. Constant circulation in a loop to achieve conditioning, aeration and supplementation of culture components. The method of claim 2, wherein step b) comprises harvesting the hepatitis A virus by releasing the hepatitis A virus by treating the hepatitis A virus infected cell sheet with a Triton-X 100 solution of about 0.05% to 1%. How to. The method of claim 1, wherein step c) comprises digesting with BENZONASE. (a) Large amounts of A in NUNC CELL FACTORIES, COSTAR CUBES, or STATIC SURFACE REACTORS (SSRs) containing SSRs containing regular mesh elements capable of growing cells at high density per unit volume in which cell sheets of MRC-5 cells are installed. Hepatitis virus (HAV0) was cultured, (b) cultured HAV was harvested by removal of the culture medium and addition of harvesting solution containing 0.1% Triton X-100, and (c) treatment of HAV harvested with BENZONASE Contaminating free nucleic acid was digested, (d) concentrated BENZONASE treated HAV by capture on an ion exchange column, eluted HAV captured with about 0.35 M NaCl, followed by precipitation with polyethylene glycol, and (e) PEG precipitated. HAV is resuspended, HAV concentrated with chloroform / isoamyl alcohol (24: 1, v / v) with vigorous stirring is extracted and the aqueous layer is separated and maintained, (f) DEAE TOYOPEARL 650M anion at about 90-150 mM NaCl. On the exchange matrix Chromatography of the aqueous layer from the organic extract and eluting HAV with a gradient up to about 1M NaCl concentration and diluting the pooled peak HAV fraction below salt concentration below 150 mM and HAV concentration below about 20 μg / ml to minimize or eliminate HAV precipitation. , (g) collecting the peak HAV fraction from step f) and chromatography HAV on a tandem size exclusion column of TOYOPEARL HW55S and HW65S, (h) treatment with formalin at 1/1000 concentration for 5 days and then sterile filtration Dissolving the gel filtered HAV by adsorption to aluminum hydroxide or co-precipitation using the same and dispensing the protein in unit doses ranging from 25 to 100 ng per dose of inactivated purified HAV. Commercial scale preparation of inactivated hepatitis A virus vaccine with 95% or more pure HAV as a reference. A method for preventing HAV infection, characterized by immunization using an immunologically effective amount of the product according to the method of claim 6. HAV vaccine prepared according to the method of claim 6. A method of culturing HAV, comprising growing HAV in a COSTAR CUBE or STATIC SURFACE REACTOR, optionally comprising a regular mesh element of titanium or stainless steel gauze. 10. The method of claim 9, wherein about 5,000 to 50,000 doses are produced from a single COSTAR CUBE culture vessel at 25 ng per vaccine dose of inactivated HAV of HAV with a purity of at least 95%. Purified formulation of hepatitis A virus, wherein at least 95% of the protein is specific for hepatitis A virus, less than 7% of the protein mass of the preparation is a non-HAV nucleic acid or lipid and about 0 to 180% of the preparation is a carbohydrate consisting of glucose. . 12. The formulation of claim 11, wherein the hepatitis A virus is formalin inactivated. The formulation of claim 12 formulated as a HAV at a dose of 25-100 ng based on protein with about 100-1000 μg aluminum hydroxide. Purified preparations of hepatitis A virus having the following characteristics (each feature is for 1 μg on a protein basis and the method in parentheses is used to measure each feature). a) lipid content (gas chromatography) <0.1 μg; b) carbohydrates detected as glucose <0.1-2.5 μg; Non-glucose carbohydrates <0.1 μg; (TFA hydrolysis, Dionex) c) Hepatitis A virus-specific protein <0.95 μg; (Amino acid analysis and Western blot) d) 0.1 to 0.2 μg of hepatitis A virus, in particular RNA; e) contaminating DNA <0.001 μg. 15. The formulation of claim 14, wherein the hepatitis A virus is formalin inactivated. The formulation of claim 15 adsorbed onto aluminum hydroxide. Based on the amino acid analysis, a single 25 unit dose equivalent to about 25 ng of hepatitis A virus protein was sufficient to achieve a seroconversion rate of 95% or higher in children and young adults aged 2 to 16 within 4 weeks of immunization. Purified Sanctions of Virus. One to three 25-100 unit doses equivalent to about 25-100 ng of hepatitis A virus, based on amino acid analysis, are insufficient to achieve 80-95% seroconversion of the recipient population within 4-28 weeks of immunization. Purified Formulation of Activated Hepatitis A Virus. The method of claim 1, comprising a hepatitis A virus culture in a fixed surface reactor, wherein a suitable culture medium is supplemented and removed at a rate of about 0.010 to 0.3 ml / cm ² / day. The method of claim 1 comprising a hepatitis A virus culture in a fixed surface reactor, wherein the culture medium is supplemented and removed at a constant rate. Hepatitis A virus vaccine having the following standard composition per 25-100 units / 0.1-1 ml dose. A type A infectious virus vaccine having the following standard composition per 25 unit / 0.5 ml dose. Based on amino acid analysis, a single 25 unit dose equivalent to about 25 ng of the Type A chlorinated viral protein is sufficient to achieve a serum conversion rate of 95% or higher in children and young adults aged 2 to 16 as fast as 21 days post-immunization. Purified formulation of hepatitis virus. ※ Note: The disclosure is based on the initial application.