Virus preparations and methods

Details Virus preparations and methods Virus preparations and methods

1999-03-05

2001-07-31

Mosher, Mary E. (Department: 1648)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Virus or component thereof

C424S231100, C435S239000

Reexamination Certificate

active

06267967

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the production and purification of viruses and to the harvesting and purification of virus preparations from virus-infected cell cultures, for example for experimental and therapeutic purposes, e.g. for the production of pharmaceutical formulations such as virus vaccines. In particular aspects the invention relates to methods and arrangements for the production of preparations of herpesviruses. Other aspects of the invention will be apparent from the description given below.
BACKGROUND OF THE INVENTION AND PRIOR ART
Several methods are known for producing live virus preparations, e.g. herpesvirus preparations, for vaccine and other purposes.
For example, U.S. Pat. No. 3,985,615 (Osaka Res Foundation: T Kubo et al) shows production of live attenuated varicella virus for vaccine use by culture comprising passage in guinea pig primary embryonic tissue cells. U.S. Pat. No. 5,024,836 (Merck: W J McAleer et al) relates to production of lyophilized vaccine preparations based thereon.
DD-209738 (Cent Cerc Bioprep: IV Patrascu) illustrates production of another type of herpesvirus, for use as vaccine against Marek's disease is produced by (a) culturing specific-pathogen-free chicken embryo cells on dextran microspheres; (b) inoculating the culture at 80% confluence with turkey herpes virus strain FC-126 (clone 1, IIIb); (c) collecting the infected cells in SPGA medium (sucrose, phosphate, glutamate, bovine albumin fraction V) when the cytopathic effect is 80%; (d) subjecting the suspension to three ultrasonic pulses of 1 minute duration at 2 minute intervals and centrifuging it to recover a first crop of vaccine; (e) resuspending the sediment in SPCA medium and repeating step (d) to obtain a second crop of vaccine (to increase the vaccine yield by almost 2%); (f) freezing the combined vaccines at −100 deg.C. prior to determining the virus titre; and (g) diluting the SPCA medium and freeze drying.
JP06234659-A (Z H Handai Biseibutsubyo Kenkyukai) describes, in an example, production of herpesviral vaccine on human diploid fibroblast MRC-5 cells cultured in MEM medium at 37 deg.C.: comprising inoculation of varicella virus Oka strain seed virus at a MOI of 0.03 to MRC-5 cells and culture at 37 deg.C. for 2 days. Virus is then suspended in a solution containing 6.4 g NaCl, 0.16 g KCl, 2.3 g Na2HPO4, 12H2O, 0.16 g KH2PO4, 50.0 g sucrose, 1.0 g Na L-glutamate, 2.0 g gelatin, 25.0 g gelatin hydrolysate and 0.1 g EDA-3Na per l.
EP 0 573 107, U.S. Pat. No. 5,360,736 and U.S. Pat. No. 5,607,852 (Merck: P A Friedman et al) describe processes for production of attenuated varicella zoster virus vaccine, including a process for preparing live, attenuated, cell-free varicella-zoster virus (VZV) vaccine that comprises: (a) Culturing VZV infection-susceptible cells, selected from human diploid cells, to confluency in monolayer culture, under conditions of sufficiently high nutrition to achieve a high degree of cell replication, and supplying a non-metabolizable disaccharide; (b) infecting the cells cultured according to step (a) at as close to the point of confluency as possible with as high a multiplicity of infection of VZV-infected cells as practical; (c) maintaining the VZV-infected culture in a state of high nutrition for about 22-96 hours and harvesting at the point of peak infectious VZV production; (d) washing the V/V-infected culture with a physiologic solution, optionally containing a lysosomotropic agent, such as ammonium chloride or chloroquine, prior to harvesting the VZV infected cells; (e) Harvesting the VZV infected cells into a minimal volume of a stablizing solution and either disrupting the cells immediately or freezing the cells for later disruption, (l) Disrupting the VZV-infected cells to optimally release cell-associated VZV, and removing cellular debris, to provide a cell-free VZV preparation. The process discloses use of cell densities of up to ca. 500,000 cells/cm2 in conventional culture vessles. The process is proposed for mass production of live vaccine. Appropriate nutrient medium for growing cells in monolayer culture in that connection is described as consisting essentially of SFRF-2 medium supplemented with between 0.2 mg/mL an d0.4 mg/mL soybean lipid, the cells being selected from MRC-5 cells, WI-38 cells and Vero cells.
WO 92/05263 (Immunology Ltd: S C Inglis et al) and WO 94/21807 (Cantab Pharmaceuticals Research: Inglis et al) are illustrative of the provision of recombinant cells and culture methods for producing genetically disabled herpesvirus such as herpes simplex virus for vaccine purposes.
It is known that herpes simplex virus can bind to cellular surface heparan sulfate (E Lycke et al, J gen Virol (1991) 72: 1131-1137).
Viruses more widely have been shown to bind to sulfonated polysaccharides such as dextran sulfate, heparin and heparan sulfate (M Baba et al, PNAS 1988 85:6132-6136; E Lycke et al, cited above; and H Mitsuya et al Science 1988 240:646-649). It is also known to carry out affinity binding and purification of feline herpesvirus on a sulfonated derivative of beaded, regenerated cellulose with particle diameter of 80 micron and pore structure claimed to reject virus particles (P F O'Neil and E S Balkovic (1993) Bio-Technology 11(2):173-178).
It remains desirable to provide methods for treatment of herpesvirus-containing preparations, especially further purification processes capable of contributing to the manufacture of infectious virus preparations in good yield and purity, e.g. those that are to be used in vaccines.
SUMMARY AND DESCRIPTION OF THE INVENTION
According to an aspect of the invention, preparations of herpesviruses can be usefully purified by affinity purification on a solid phase (affinity binding reagent) that can competitively bind materials with affinity for heparin. The invention is for example especially applicable to infectious preparations of human herpesviruses such as herpes simplex virus (HSV), e.g. HSV type 2, which can tend to remain strongly cell-associated when grown in culture. The affinity reagent carrying the virus, which can be applied from a carrier liquid containing salt (e.g. sodium chloride or other pharmaceutically acceptable salt over about 0.4M) or containing heparin or another sulfated or sulfonated polysaccharide (e.g. in the order of about 10-250, such as about 50, micro-g/ml), can then suitably be washed and the virus recovered in actively infectious form by elution, e.g. with high-concentration salt solution or with sulfated or sulfonated polysaccharide.
Examples of suitable solid phases for use in this connection include a heparin-carrying solid phase, and solid phases with similar binding functionality, e.g. preferbly a sulfated (or sulfonated) polysaccharide binding functionality. Suitable affinity binding reagents can carry binding groups containing sulfate or sulfonate together with nonionic polar groups. For example the sulfated polysaccharides contain sulfate groups and hydroxy groups. Examples of solid phases carrying sulfated polysaccharide include dextran sulfate or heparin sulfate. Preferably, the sulfate or sulfonate groups can be carried on side chains, e.g. polymeric side-chains, relative to the material of the solid phase, and thus can be other than resins and crosslinked polymer beads that have been directly derivatised with such acid groups. Solid phases carrying other sulfate-comprising or sulfonate-comprising binding agents than those already mentioned, such as biphenyl disulfonic acid urea copolymers, or protamine sulfate, can be used.
In a preferred aspect of the invention, the affinity purification can form part of a process for producing purified preparations of herpesviruses, which comprises the steps of (i) culturing host cells infected with the virus, e.g. suitable mammalian host cells such as Vero cells of MRC5 cells, or recombinant cells derived from Vero cells, preferably cultured on microcarriers, and infected with HSV-2 (or in further embodiments, cells infected with other viruses such as VZV), (ii) harvest

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