HRP931103A2 - Hepatitis a virus vaccine - Google Patents

Abstract

A complete, highly reproducible, commercial scale process for the production of stable hepatitis A virus comprises optimal growth, collection and purification conditions of the antigen to obtain> 95% pure protein-based HAV. Further stages of processing provide an inactivated formulation of the HAV vaccine that is immunogenic, well tolerated and effective in protecting against infection by virulent HAV. The content of proteins, carbohydrates, lipids, DNA and RNA is determined in the product.

Description

This is a continuation-in-part of pending application serial number 07/926,873, filed 08/10/92, the priority of which is claimed under 35 USC §120.

The field of this invention is the production of a highly purified vaccine for hepatitis A virus. 1973 Feinstone et al., /Science 182, p. 1026/ identified the etiological agent of infectious hepatitis, later known as hepatitis A virus, using immune electron microscopy.

In vitro culture of hepatitis A virus (HAV) was first reported by Provost et al.. /P.S.E.B.M. 160, p. 213, 1979/ according to a procedure in which the liver of infected marmosets is used as an inoculum for liver explant culture and rhesus fetal kidney cell culture (FRhk6) /U.S. Patent 4,164,566/.

In a later invention, direct inoculation of HAV that had not previously passed through a subhuman primate was successfully used to initiate in vitro propagation of HAV (Provost et al.. P.S.E.B.M. 167, p. 201 (1981): U.S. Patent 5,021,348

From this paper, the attenuation of HAV through in vitro culture is shown. In addition, it was shown that with repeated passages in vitro, HAV cultures became more productive and the rate of replication increased as the virus became more adapted to cultured cells. A further development was the demonstration in subhuman primates of the protective efficacy of both live attenuated virus (Provost, et al.. J. Med Viol. 20, p. 165 (1986)) and formalin-inactivated HAV /U.S. Patent 4,164,566; U.S. Patent 5,021,348; Provost et al., in Viral Hepatitis and Liver Diseases, pp. 83-86, 1988-Alan R. Liss. Inc/. From previous work, it has become clear that either inactivated or attenuated, immunogenic HAV are possible vaccine candidates. However , for human use a commercial-scale reproducible process is needed to produce high-purity antigen as a safe HAV vaccine that should be commercially available.

Various procedures have been described for partial purification of HAV virus (virions) for testing and initial characterization of the virus. See, for example, Hornbeck, C.L. et al., Intervirology 6; 309-314 (1975); Locarnini, S.A. et al., Intervirology 10; 300-308 (1978); Siegl, G. et al., J Virol. 26, 40-47 (1978); Siegl, G. et al., J. Gen. Virol. 57, 331-341 (1981); Siegl, G. et al., Intervirology 22, 218 (1984); Hughes, J.V. et al., J. Virol. 52, 465 (1984); and Wheeler, C.M. et al., J. Virol, 58, 307 (1986).

All these procedures use cumbersome, impractical and prohibitively expensive steps, such as sucrose gradient centrifugation or CsCl-density gradient centrifugation. In addition, these procedures were adapted to relatively small production scales, and although some workers reported that their product was highly purified, careful analysis of the work showed that insufficient parameters were examined to support a claim for any absolute level of purity. Thus, Lewis et al., (European Patent Application 0302692 published February 8, 1989) describes a process comprising a) destruction of HAV infected cells, extraction and concentration; (b) passing through an anion exchange column at a high salt concentration to remove nucleic acid contaminants; and (c) gel filtration chromatography. In later work, Lewis et al., (European Patent Application 0468702, published July 18, 1989) modified this process by including an ion exchange absorption/desorption stage to remove contaminating carbohydrates. In both of these published works, the scale at which HAV is produced is relatively small and the problems presented by this invention for large-scale, commercially scaled-up production are not considered. In addition, the purity of the product was determined using comassage or silver staining of the gel electrophoresed product. This analysis procedure provides a measure of protein purity, but is not quantitative, and does not provide any information such as the presence or absence of other contaminants such as lipids, carbohydrates, nucleic acids, or low molecular weight organic substances.

In both EPO468702A2 and EP0302692A2, it was reported that earlier procedures for the collection and purification of HAV used one or more steps that likely prevent approval by the Food and Drug Administration, due to their use of detergents and exogenous enzymes. Both detergents and exogenous enzymes are used in this invention, but the invention describes procedures for removing these useful reagents, and demonstrates that the product contains less than detectable amounts of these additional substances, while being as pure or purer than the HAV products shown in the published literature. literature.

Likewise, Moritsugu et al., (European Patent Application 0339668, published November 2, 1989), describe a process comprising:

(a) solubilization, extraction, DNase/RNase concentration, and proteinase treatment;

(b) sucrose density fractionation (which separates RNA containing empty capsids); and

(c) gel filtration chromatography.

Once again, this process is limited in scale of production, especially with regard to the degree of fractionation by sucrose density. In addition, product purity is difficult to determine and is not stated in any absolute sense in that publication.

Lino et al., /Vaccine, vol # 10, p. 5-10 (1992)/ have published improvements to Moritsugu's procedure. The product of their procedure was determined by SDS-polyacrylamide gel electrophoresis and densitometry to show that there was less than 1.9% residual exogenous protein in their purified HAV. They also concluded that there was less than 5 pg of residual host cell nucleic acids in 0.5 μg of their purified HAV. However, due to the limitations of their production process, large-scale production of HAV is impractical, and absolute purity based on multiple parameters is not specified.

In another work, Kusov et al., /Vaccine, 9, 540 (1991)/ cultivated HAV in Vero-like cells, and purified the virus by organic extraction, DNase treatment and chromatography on porous silica layers. Again, there is no indication here of the absolute purity of the product, nor is there any indication of scale.

Despite the uncertainty in the published literature regarding HAV vaccine purity, it is possible to compare vaccine products on the basis of relative antigenicity (see Table 1, Example 11) which shows the comparative amounts of HAV in different products listed by their manufacturers, compared to the product from this invention.

The product of the present invention is an immunogen with the absolute purity characteristics described in this disclosure, representing a level of HAV purity and production scale previously only aspired to. This invention is far beyond the level of semi-powered small-scale procedures published for the production of HAV vaccines, and for the first time is a reality for the commercial production of a reproducible high-quality, highly purified, safe HAV vaccine.

The procedure, which includes cultivation of HAV in a large-scale stationary surface area bioreactor, virus harvesting with detergent, nuclease treatment, concentration, organic solvent extraction, ion-exchange chromatography, and gel filtration chromatography, provides a reproducible means of producing a stable vaccine for HAV products with a HAV protein purity of > 95%. DNA, carbohydrate, RNA and lipid levels are determined for the vaccine.

Sl. 1 Complex process diagram of the main processes used to produce hepatitis A vaccine for clinical trials. The left side represents the prior art procedure used for phase I/II clinical trials using the rolling bottle procedure. The new process used for the Monroe efficacy study (New England J. of Med., 327:453-457 1992) is on the right, and the manufacturing process according to this invention is shown in the center.

Sl. 2 Analytical granulation analysis during the procedure using a HPSEC TSK PW 4000 column eluted with phosphate buffered saline, PBS. This assay is calibrated to high molecular standards and represents a linear surface response with hepatitis A virus dilution. The assay is particularly useful in later purification steps. Shown here are four chromatograms from the last four stages of the production process.

Chromatography is registered using double UV detection at 214 nm (upper trace using indicator), and respectively 260 nm (lower trace using indicator). Using this assay, the peak corresponding to hepatitis A virus is first visible after resuspending the PEC pellet (panel A). A chloroform extraction step removes much of the high-molecular-weight, UV-absorbing material (panel B). Anion exchange chromatography serves to concentrate the virus (panel C) and preparative size exclusion chromatography provides a preparation of highly purified virus (panel D). Purified hepatitis virus has poor absorption in the 260 or 280 nm region so that only a trace is present using the indicator at 214 nm. Under the conditions of this assay, the HAV product is given as a single, symmetrical peak and therefore the assay provides a measure of HAV purity, independent of SDS PAGE analysis.

SI. 3 Growth of MRC-5 cells on glass, polystyrene and 316 stainless steel. Cell surface concentration is plotted as a function of time. Cells are inoculated onto coupons of each material at a density of approximately 10,000/cm 2 and the coupon is sacrificed daily for cell counting. Cells on glass coverslip coupons (microscopic) and on 316 stainless steel coupons are cultured in 150 cm2 petri dishes containing 90 ml of medium (0.6 ml/cm2); cells grown in T-bottles of 25 cm2 are cultured with 7.5 ml medium (0.3 ml/cm2).

Sl. 4 MRC-5 cell growth and residual glucose concentration for cells grown on 316 stainless steel coupons. Cell surface concentration (cells/cm 2 ) and glucose concentration (mg/cm 2 ) are plotted as a function of time (d) for cells grown on 25 cm 2 stainless steel coupons.

Sl. 5 Light micrograph of MRC-5 cells grown on stainless steel mesh. Cells were inoculated at 5,000-10,000 cells/cm2 and cultured in excess for 2 weeks.

Magnification 40X.

Sl. 6 Staining of viable and non-viable MRC-5 cells grown on 316 stainless steel mesh. Cells were stained with fluorescein diacetate (FDA) and ethidium bromide (EB) according to the protocol described in the Materials and Methods section of Example 14, and then observed using a scanning laser confocal microscope; the material was scanned (decomposed) in 57 μm increments. Figure 6A corresponds to fluorescence from fluorescein and shows viable cells. Figure 6B is the fluorescence due to ethidium bromide staining of DNA, showing almost complete cell viability. A positive control for ethidium bromide is shown in Figure 7.

Sl. 7 Ethidium bromide staining of non-viable MRC-5 cells. Cells grown on 316 stainless steel mesh were rendered non-viable by treatment with 100% methanol. After washing well with PBS and allowing to dry, cells were stained with FDA/EB to serve as a negative control for FDA and as a positive control for ethidium bromide (pictured). No fluorescence was observed due to the production of fluorescein for the negative control.

Sl. 8 Seeding efficiency is plotted for each element in a dauze static mixer reactor for protocols using gravity sedimentation or media recirculation. The elements are numbered from 1 to 5, where 1 is the element at the top and 4 or 5 are the elements at the bottom of the element used in gravity sedimentation experiments and 3 for recirculation experiments).

Seeding efficiencies shown here are percentages of total seeding; total seeding efficiency is of the order of 90%. The data for recirculation is the average value from 3 experiments, while the value for gravity sedimentation is from one experiment. Recirculation speeds were approximately 6 cm/h.

Sl. 9 Glucose uptake rates (GUR) as a function of time for reactors containing solid titanium elements and 316 stainless steel mesh. GUR on a cm2 basis (μg/cm2 -d) versus time (d) is shown in Figure 9A, while GUR on a reactor volume basis is plotted in Figure 9B. The results in Figure 9A are dependent on the determined surface areas of the elements, while Figure 9B does not represent any determination since both reactors are of the same volume.

Sl. 10 Percentage of total protein observed in subsequent lysates for a constant mixer with a static network. Protocols for lysates 1-4 are described in the text. Microscopically, they were free of cellular debris, while the network elements contained considerable cellular debris after four lysis.

Sl. 11 Glucose uptake rates as a function of time for a 316 stainless steel static mesh mixer reactor. The reactor was infected with an MOI of 1 on day 7 and harvesting was performed on day 28.

Sl. 12 The percentage of total HAVAg protein in subsequent lysates for the static grid mixer shown in Figure 11. Protocols for lysates 1-4 are described in the text. The total protein of lysate 1 was artificially high due to inefficient PBS washing.

Sl. 13 Effect of MOI on cell growth and glucose consumption. Cells were infected at an MOI of 0.1, and 10 with HAV CR326FP28. No differences in cell density (13A) and residual glucose (13B) were observed until 8 days after infection of this system. A complete reloading of the media was carried out on day 8.

Sl. 14 Cellular protein measured according to BSA standard according to the number of cells. The most suitable interpolation line is described by the equation: Y(104) = -0.09 + 0.42X, r=0.99. This figure shows that a single curve provides a means to measure the number of MRC-5 cells using total protein, regardless of the "state" of the cells.

Sl. 15 Figure 14 is plotted again, showing the time for each score point.

Sl. 16 The conversion of total protein to cell number closely resembles the cell density obtained using a Coulter Counter. Discrepancies are believed to be due to small volumes of lysis buffer used in T-25a collection (2X 0.08 ml/cm2 was used for consistency).

Sl. 17 Purification via solvent extraction step, HPSEC profile (production batch consistency).

Sl. 18 Purification via solvent extraction step, HPSEC profiles (test series).

Sl. 19 Effect of mixing time on Hep A/impurity ratio.

SI. 20 Effect of mixing time on the surfaces of Hep A and impurities (shaker, 50 ml bottles, 6 minutes).

Sl. 21 Effect of mixing time on Hep A surfaces and impurities (Shaker, 500 ml bottles, 2 minutes).

Sl. 22 The effect of mixing time and the volume of the test tube on the surfaces of Hep A and impurities (Shaker).

Sl. 23 The effect of the solvent/water ratio on the Hep A/ impurity ratio.

Sl. 24 The effect of the solvent/water ratio on the ratio of Hep A surfaces and impurities (Mućkalica).

Sl. 25 Effect of mixing time and type on the Hep A/impurity ratio.

Sl. 26 Hepatitis A sample stability determined by HPSEC.

Sl. 27 Effect of salt on the solubility of hepatitis A (at 4 degrees).

Sl. 28 Effect of salt on the solubility of hepatitis A (at 4 degrees).

Sl. 29 Calibration curve of molecular weights, PW4000xl.

Sl. 30 Log peak area of hepatitis A according to log concentration of hepatitis A.

Sl. 31 Hepatitis A calibration curves.

Sl. 32 Comparison of samples of filtered lysate and nuclease-treated lysate, UV at 214 nm.

Sl. 33 Comparison of samples of filtered lysate and nuclease-treated lysate. UV at 260 nm.

Sl. 34 Sample of resuspended PEG pellets. UV at 214 nm.

Sl. 35 Sample of the chloroform-extracted aqueous phase from Rx2011008. UV at 214 nm.

Sl. 36 Sample anion exchange product from Rx2009854. UV at 214 nm.

Sl. 37 Product of size exclusion chromatography. UV at 214 nm.

Sl. 38 Sample size exclusion chromatography (SEC) product from Rx2011008. UV at 214 nm.

Retained peak visible at about 49 minutes.

Sl. 39 Correlation of EIA and HPSEC results from samples from 11 batches.

Sl. 40 Area percentage values from 4 samples from 13 hepatitis A series.

A commercially adaptable purification method of the present invention encompasses the purification of any hepatitis A virus (HAV), whether derived from attenuated or virulent HAV, produced in any cell strain, line or culture suspected of HAV infection. In addition, all HAV capsids, whether empty (without viral nucleic acids) or complete (containing viral nucleic acids) are encompassed within the methods of the present invention.

HAV at passage twenty-eight (P28CR326F'; for the classification of variants arising from CR326, such as strain CR326F and CR326F', and the relationship of this nomenclature according to absolute passage level, see Provost et al., J. Med Virol., 20: 165-175, 1986) is used to infect MRC-5 cells in this invention, for illustrative purposes only, and the production material is cultured at P29. P28CR326F' is an attenuated HAV strain. Other HAV strains are encompassed by the present invention, including HAV strains that can be attenuated by conventional techniques: Other suitable cell lines for HAV propagation include Vero, FL, WI-38, BSCl, and FRhK6 cells. These and other HAV propagation and cell culture systems are discussed in Gerety, R.J. "Active Immunization Against Hepatitis in Gerety, R.J. (ed.) Hepatitis A Academic Press 1984, pp. 263-276; and Ticehurst. J.R.. Seminars in Liver Disease 6, 46-55 (1986). In principle, any cell line as which is any human diploid fibroblast cell line, can serve as a host cell for HAV provided it is susceptible to HAV infection.The primary cell line for vaccine production is MRC-5.

The commercial-scale process according to this invention includes the following steps:

(a) Cultivation of a large amount of hepatitis A virus (HAV) in a suitable culture reactor and medium

Preferred ways of accomplishing this include, but are not limited to, the use of HAV adapted for growth in human diploid lung cells suitable for vaccine production. WI3B cells or MRC-5 are suitable: These cells are grown on micro-carriers or cell layers in flasks or spinners. The process according to this invention uses large surface area, multilamellar bioreactors such as "NUNC CELL FACTORY, COSTAR CUBE Static Surface Reactor (SSR) static mixer reactor" NUNC cell Factory, COSTAR cubic static surface reactor (SSR), or static mixer reactor as which is described in the U.S. Patents 4,296,204, 4,415,670, or as further described herein in a stainless steel mesh static surface mixer, depending on the desired scale of production. Accordingly, in a preferred embodiment of the present invention, MRC-5 cells are grown in cell layers in an 85,000 cm2 COSTAR cubic SSR, or SSR with an even larger surface area, infected at a multiplicity of infection (MOI) of HAV sufficient to achieve efficient infection of the culture cell. An MOI of about 0.01 - 10 is suitable. The seed batch is conveniently obtained using HAV from the supernatant fraction of SSR incubated for about 28 days.

In a preferred embodiment, HAV is cultured on MRC-5 cells on a stainless steel grid. (See Example 14). According to this embodiment, a bioreactor is used in which the elements of the mixture are made of any suitable, non-toxic material on which cells can grow. Such elements can be made of stainless steel, titanium, plastic or similar materials that can be prepared in a regular three-dimensional surface. 316 stainless steel compound elements such as those available from Sulzer Biotech Systems (especially SMV, SMX, EPack EX, DX, BX, CY) are primarily used. Each element consists of a uniform surface area of layers of mixtures on which cells can be grown. Each element of the regular network of the mixture is oriented at angles of 0-90° to each other in a sterile reactor system. The configuration can be a linear column or any other geometric shape adjusted to accommodate the elements of the mixture and which makes good use of the volume. The cells are then seeded and allowed to attach (bind) to the large surface provided with the mixture. Once cells are well established in culture, they can be fed at a spray rate sufficient to maintain nutrient turnover and remove waste products. At any suitable cell density, cells are infected with HAV as described later.

The advantage of mesh elements is that the fibrous layer provides more surface area per volume unit. The geometrically precise mixture enables the uniform growth of fibroblasts over the surface, to known and constant depths, which makes it possible to control the delivery of nutrients. The uniformity of the mixtures also allows for predictable points of contact between the fibers, which allows rapid migration of cells to cover the fiber mesh. The static mixer configuration controls the proximity of the growth surfaces to each other so that natural biological growth and "fouling" of the reactor is prevented. This is a serious problem that occurs in randomly packed bed reactors, where the non-uniformity of the packing allows for localized regions of cell growth that then cause a decline in nutrient flux and subsequent cell starvation.

It is understood that the scope of the present invention includes, in addition to passage 18, P18, p28, p29, p72 of the CR326F Hav strain, and any HAV variant strain, whether attenuated or virulent. Attenuated variant strains can be isolated by serial passage in cells, animals or other procedures. See, for example, Provost, P.J. et al., Proc. Soc. Exp. Biol. Honey. 170.8 (1982); Provost, P.J. et al., J. Med. Virol. 20, 165 (1986): U.S. Patent 4,164,566 and 5,021,348 for attenuation details. Purification procedures are quickly and easily adaptable for attenuated or virulent HAV strains.

HAV is allowed to replicate in the SSR until peak virus production. For attenuated cell culture, adapted HAV such as CR326FP28 is taken from 7-35 days, depending on the initial MOI and cell density, during which time the nutrient medium is constantly circulated through an opening that enables gas exchange. The cell culture medium can be any medium that allows active growth of MRC-5 cells and HAV replication. Primarily, the culture medium is replenished and removed at a rate between about 0.010 and 0.3 ml/cm2/day. This rate is flexible since the rate of cell mass gain is complete exposure to the medium.

Accordingly, in a preferred embodiment of the present invention, MRC-5 cells are grown on cell layers in a 340,000 cm 2 SSR (for example, using four 85,000 cm 2 elements) or an even larger area SSR grid, infected at a multiplicity of infection (MOI) of HAV of about 0.1 - 1.0, and HAV is allowed to replicate until viral antigen production peaks. This takes about 28 days, during which time the nutrient medium is re-added and circulated through the culture reactor and gas exchange port. The culture medium can be any medium that supports the growth or maintenance of MRC-5 cells and HAV replication. We have determined that the most suitable medium is one containing a basic medium, such as Williams' Medium E, supplemented with suitable growth factors. As a source of growth factors, we have determined that calf serum supplemented with iron is the most suitable.

(b) Collection of cultured HAV

The procedure for collecting cultured HAV will be dictated, to some extent, by the geometry of the culture reactor. Generally, after culturing for about 28 days with replacement of the nutrient medium, the culture medium is drained and the HAV is collected. Previously, for small-scale propagation of virus, this was achieved by scraping, then freezing to a semifluid state and optionally sonicating, to optimize the release of cell-bound virus. In the process of the present invention, cell-bound HAV is released into the minimal volume of the collection solution. Primarily, the collection solution contains a component effective to render the cells permeable to HAV. Such components are known in the art. Primarily, a detergent such as Triton X-100, NP-40, or an equivalent is added at the lowest possible effective concentration to aid subsequent removal. A supply of about 0.05-0.5%, and preferably 0.1% Triton X-100 has been found to be adequate to achieve efficient extraction of HAV from cells cultured in NUNC cell factories, COSTAR cubic, or any layered cell culture. The advantage of using detergent extraction, especially from an SSR such as COSTAR cubic, is that the geometry of such a culture reactor is such that it does not allow collection by mechanical means, except to the extent that HAV is in the culture supernatant which can be separated and the HAV concentrated and regenerated. However, the amount of HAV found in the upper layer of the culture fluid is usually only a small fraction of the total HAV that can be regenerated from the cells. Collection of HAV using detergent from cultured cells is highly efficient.

(c) Treatment of collected HAV

Once HAV has been collected in essentially cell-free form from the culture supernatant, from the cells, or both, it is desirable to concentrate the HAV to allow downstream processing and purification. In a prior art HAV purification process, where HAV was collected by mechanical means and only small amounts of material were purified, HAV was extracted with an organic reagent such as methylene chloride or methylene chloride: isoamyl alcohol (24:1, v/v) , and then by concentrating the aqueous phase with the addition of a water-dissolved synthetic polymer, such as polyethylene glycol. It has now been discovered that detergents and exogenous enzymes can be used in a commercial scale process to produce whole HAV vaccine with the removal of additional materials to the point where traces are undetectable in the purified product.

In one embodiment of the present invention, the detergent, such as Triton X-100, that was used to collect the HAV is removed by concentration/diafiltration, and then the remaining detergent is removed, for example with an AMBERLITE nonionic polymer adsorbent such as XAD. A useful procedure showing removal of Triton X-100 below about 10 μg/ml is published by Hurni, W.M., and Miller, W.J. /Journal of Chromatography, 559, 337-343 (1991)/. The detergent used to collect HAV cannot be detected by this analysis in the product according to the implementation of this procedure. In addition, many other contaminants are removed, yielding a partially purified HAV product.

In another embodiment of the invention, after detergent collection, nuclease treatment is used for concentration, followed by an anion exchange binding step, prior to concentration/diafiltration, followed by XAD treatment.

In this embodiment, a non-specific nuclease of high specific activity, and highly homogeneous, is primarily used. The non-specificity ensures cleavage of both DNA and RNA, and the homogeneity of the enzyme ensures that only one enzyme is added, without the addition of other protein contaminants. In addition, it is convenient that, in the process of this invention, the nuclease is detergent-resistant, so that it can be added directly to the detergent-collected HAV. Any number of nucleases can fit this criterion. It was found that BENZONASA, obtained as a recombinant protein by the company Nycomed Pharma A/S (Denmark), is particularly suitable for this purpose. A supplement of between 0.1 μg - 100 μg/l of collected HAV, preferably 10 μg/l, was found to be acceptable for the removal of contaminating free nucleic acid.

The addition of a minimal amount of enzyme is important to facilitate subsequent enzyme removal in downstream processing steps, yielding a product with undetectable residual enzyme levels (less than 10 pg/ml).

Since the nuclease treatment occurs in a relatively dilute solvent, we have found it convenient to bind HAV by anion exchange chromatography in a dilute (90 to 150 mM) salt solvent, and then elute the HAV by adding about 1.5 column volumes of the higher ionic strength solvent. (≥ about 0.3 M). HAV is substantially concentrated by this level of binding and, in addition, most detergent, BENZONASE and many other contaminants flow through the column. Therefore, the HAV eluted from the binding column is partially purified. Any of a number of ion exchange resins known in the art can be used for this step, including those listed in EPO468702A2. We have found that a particularly suitable resin for this purpose is the use of DEAE TOYOPEARL 650M (Tosohaas).

(d) Concentration of HAV

After harvesting the HAV and concentrating /diafiltration/ XAD or treating with nuclease/binding, the HAV is concentrated with a water-dissolved synthetic polymer effective to concentrate the proteins. Polyethylene glycol (PEG), which has a molecular weight of between approximately 2,000 daltons and 12,000 daltons, is effective for this purpose. Typically, NaCl is added to partially purified HAV to a final concentration of between approximately 150 mM and 500 mM. PEG is then added to a final concentration of between approximately 2% (w/v) and 10% (w/v), preferably about 4%. The resulting 4% PEG settled partially purified HAV is concentrated and centrifuged, the upper liquid layer is separated and the pellets are resuspended for further processing. We have found that the main contaminant is removed in a separate upper liquid layer from this stage. Removal of the protease increases the yield, stability, and purity of the HAV product during further purification. The resuspended PEG pellets are optionally sonicated to aid dissolution prior to organic extraction, as described below.

(e) Organic extraction

Resuspended PEG pellets from the above are extracted by adding a mixture of halogenated lower alkanes containing 1-6 carbons, such as methylene chloride, chloroform, tetrachloroethane, or the like, and antifoams, such as isoamyl, alcohol. The volume to volume ratio of halogenated lower alkane to antifoam is between about 15:1 and 50:1 and is preferably between about 20:1 and 30:1. Applicants have found that organic extraction significantly increases the purity of the final HVA product. Chloroform is better than methylene chloride for organic extraction at this stage.

Such extraction removes, in addition to other contaminants, lipid and lipid-like substances. Therefore, the resuspended PEG pellets are diluted with any of the different buffers, including phosphate buffered saline. TRIS, carbonate, bicarbonate, or acetate buffers. Since HAV capsids are stable in slightly acidic conditions, buffers in the slightly acidic pH range are acceptable. One preferred buffer is TNE (10 mM TRIS-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA). Phosphate can be used instead of TRIS-HCl.

Primarily, the resuspended PEG pellets are organically extracted by addition of chloroform/isoamyl alcohol (24:1, w/v) with an organic to water ratio between about 0.5:1 and 3:1. with strong swirling. This extract is then clarified by centrifugation at 4,000 g for 10 minutes at 20°C. The aqueous phase is left, and the interface and organic phase are re-extracted with a volume of TNE or buffer equivalent equal to between 0.3 and 0.6 of the original sample volume, and both aqueous phases are combined, yielding the extracted PEG pellets.

The degree of solvent extraction plays a key role in the Hepatitis A purification process by depositing protein impurities on the separation surface as well as extracting lipids into the solvent phase. The considerable purification achieved by this extraction during production stable runs can be seen from the HPSEC chromatogram in Figure 17, where the impurity peak at 23 min is a useful marker for studying impurity removal during this stage. In contrast to this, in the course of smaller half-run series, a significant proportion of this peak was retained in the extracted product (Figure 18). Several variables have been examined in order to understand and control this degree of increase in production. The following were studied: ratio of volume of solvent to water, type of shaking, time of mixing and intensity of mixing in several test tubes and size of bottles (See Example 15).

(f) Ion exchange chromatography

The extract from above is subjected to anion exchange chromatography on resin, gel or matrix. Typical anion exchange matrices include, but are not limited to,

DEAE cellulose;

DEAE agarose;

DEAE Biogel;

DEAE dextran;

DEAE Sefadex;

DEAE Sepharose;

Aminohexyl Sepharose;

Ecteola cellulose;

TEAE cellulose;

QAE cellulose;

mono-Q; or

Benzoylated diethylaminoethyl cellulose;

One primary anion exchange matrix is DEE TOYOPEARL 650M (Tosohaas). General background information on ion exchange chromatography can be found, for example, in E.A. Peterson, "Cellulosic Ion Exchangers" in Work, T.S. and cooperation. Laboratory Techniques in Biochemistry and Molecular Biology Vol. 2, part II, pages 223 et seq. North-Holland 1970.

The organically extracted resuspended PEG pellets are diluted, if necessary, with 10 mM TRIS, 1 mM EDTA, pH 7.5, or equivalent buffer, and NaCl is added to obtain a NaCl concentration between approximately 0.3 M and 0.35 M if necessary. contaminating free nucleic acid is removed. Under these conditions, HAV passes through the column in a filtration manner. However, if the HAV preparation has previously been treated with nuclease, the organic extract is adjusted to between 0.1 M and 0.15 M NaCl, preferably with 0.12 M NaCl, before the next step.

At the NaCl molarity described above, HAV capsids bind to the anion exchange matrix while certain residual contaminants such as carbohydrates pass through the matrix. The column is washed with several column volumes of 0.12 M NaCl to further remove remaining contaminants. The HAV capsids are then eluted from the anion exchange column using one column volume plus the original sample volume of 0.35 M NaCl. Alternatively and more conveniently, HAV is eluted by gradient elution up to about 1 M NaCl or equivalent salt, with HAV elution at about 0.3 M NaCl.

(g) Gel filtration

The final stage of gel filtration chromatography comes after anion exchange chromatography. Sepharose CL-4B (Pharmacia) is typically used, but many other types of gel filtration matrices can be substituted. See, for example, Fischer, L., "Gel Filtration Chromatography" in Work, T.S. et al., Laboratory Techniques in Biochemistry and Molecular Biology Elsevier (1980). We have discovered a particularly convenient procedure for applying size exclusion chromatography to the purification of HAV at this level. This involves elution of as little sample volume as possible by anion exchange purified HAV on TOYOPEARL HW55S and HW65S tandem columns.

While specific sequences of anion exchange chromatographic steps followed by gel filtration are typical protocols for the purification of HAV capsids in the present invention, it is understood that the sequences can be varied. For example, gel filtration may precede an anion exchange step. We found the previously mentioned sequence to be more convenient.

(h) Deposition of HAV

A completely unexpected discovery in the course of large-scale production of HAV, relating to the solubility of the virus, arose as a result of the large quantities of HAV produced according to the present invention. We found that at a salt concentration of 0.25-0.30 M at which HAV is eluted from the ion exchange column, virus deposition occurs if the HAV concentration is above about 10-20 μg/ml.

This was revealed by HPSEC analysis of representative samples, which showed that the AQX product (from Choroform Extracted Aqueous Phase) and the size-exclusion product (SEC) (from Size-Exclusion Product) were highly stable upon deposition at 4 °C; but the anion exchange product (IEX) (from Anion Exchange Product) showed a marked loss of hepatitis A after storage at 4°C.

To confirm this finding, a stability study was started with another set of samples. AQX, IEX product, and SEC product were each analyzed (in triplicate) by HPSEC immediately after genesis. The concentration of hepatitis A was then determined by comparing the areas of the obtained peaks according to the SEC hepatitis A calibration curve. In addition, an aliquot of the IEX product was diluted 1 in 5 with phosphate buffered saline (0.12 M NaCl, 6 mM sodium phosphate, pH 7.2) and quantitative determination was immediately performed. The stability of these 4 samples at 4°C was recorded by HPSEC over several days.

The initial hepatitis A concentration and the value obtained 4 to 5 days later for several of these purified hepatitis A flow samples are given below.

[image]

The full results, including intermediate time points, are shown in Figure 26.

As seen with the first batch, the IEX product shows a marked loss of hepatitis A, with a loss of about 25% occurring on overnight storage (hepatitis A concentration drops from 59 to 45 μg/ml).

The diluted IEX product and the AQX product show much greater stability, with hepatitis A losses of only about 10% during the first 100 hours of storage. The SEC product shows no loss of hepatitis A, essentially the same result was obtained for the first batch.

One difference between these two series is the formation of a visible precipitate that occurs with the second series. The presence of sediment requires centrifugation of the entire IEX product prior to the SEC stage. This centrifugation leads to large losses of settled hepatitis a; the concentration after centrifugation was 29 μg/ml. A table of yields calculated from HPSEC data is shown below.

[image]

An unexpectedly low SEC yield indicates that aggregation or precipitation on the SEC column may be occurring. In fact, some aggregates were observed using HPSEC. A 7 ml sample of the IEX product that is chromatographed on a smaller set of tandem SEC columns (immediately after the IEX recovery is complete) shows a 71% recovery of hepatitis A, which is much more typical for this stage. Since this sample did not lose hepatitis A during centrifugation (as it had not settled yet) the yield of 71% is about 3 times that obtained on a production scale going from IEX to SEC products. The smaller-scale tandem SEC gave a much higher dilution factor, which may explain the higher yield.

A precipitate observed during the purification of these batches coinciding with the loss of virus, as determined by HPSEC, was surprisingly found to be dissolved in aqueous solvents of low ionic strength (eg, 6 mM sodium phosphate), so that the precipitation was reversible. The precipitate is redissolved by adding 6 mM sodium phosphate, which leads to rapid dissolution. In contrast, resuspending the precipitate in 120 mM sodium chloride with 6 mM sodium phosphate or in solvents of higher ionic strength leads to little change. Based on HPSEC retention times, the precipitate solubilized in 6 mM sodium phosphate was the same size as monomeric hepatitis A, while silver-stained SDS-Page showed that the solubilized precipitate had the same protein bands seen in the final SEC product. Based on these data, the hepatitis A purification was modified to include dilution of the ion exchange product to prevent precipitate losses (See Example 16), such that a 1:1 dilution yields a salt concentration of about 0.15 M or lower. It is also desirable to achieve a HAV concentration lower than 20 μg/ml.

(i) HPSEC consistency analysis

High Performance Size Exclusion Chromatography (HPSEC) analysis was developed for the quantitative determination of hepatitis A concentrations and impurity levels in samples from the hepatitis A purification process.

Hepatitis A samples from the procedure from a total of 14 batches obtained according to this invention were analyzed; the resulting database was used to characterize hepatitis A levels and contaminant profiles for each of the purification steps. The conditions of HPSEC analysis, the security of the data showing the accuracy and precision of the analysis, the comparison of HPSEC and EIA data, and the results from the HPSEC analysis of 10 series, are shown in Example 17 and figures

29-46.

(j) Inactivation and formulation of vaccines

Additional procedural steps of a conventional and well-known nature are or may be necessary to obtain purified HAV capsids for vaccine use. For example, treatment with formalin, sterile filtration, and absorption onto carriers or adjuvants are typical basic steps for obtaining a formalin-inactivated vaccine.

See, for example, Provost, P.J. et al., Proc. Soc. Exp. Biol. Honey. 160, 213 (1979); Provost, P. J., et al., J. Med. Virol. 19, 23 (1986). HAV can be inactivated by heat, pH changes, irradiation, treatment with organic solvents such as formalin or paraformaldehyde: Typically, HAV inactivation is performed at a ratio of 1/4000 formalin. We found that formalin inactivation proceeds much faster and more efficiently at four times the standard concentration, and a formalin concentration of 1/1000 reduces the inactivation time by a factor of 4 without adversely affecting immunogenicity. Inactivated HAV is then absorbed or co-precipitated with aluminum hydroxide to provide an adjuvant and carrier effect.

Since the dose required for the immunological efficiency of this product is very low, it is convenient to complex or absorb the inactivated HAV on a carrier. Aluminum hydroxide was found to be completely acceptable for this purpose as it forms a solid complex with HAV and prevents virus loss to the walls of the container.

Accordingly, the present invention provides a process for obtaining on a commercial scale hepatitis A virus of greater than 95% purity of the HAV protein, comprising the steps of:

(a) culturing hepatitis A virus in large quantities in cell layers in large surface areas of a static surface reactor,

(b) removing the hepatitis A virus from the cell culture, by permeating the detergent into the cell layers so that the hepatitis A virus is released from the cell layers without actually removing, lysing or destroying the cell culture in the layers,

(c) optional removal at this stage of non-hepatitis A virus specific nucleic acids from the collected HAV, either by nuclease treatment or ion exchange nucleic acid absorption,

(d) concentrating the hepatitis A virus removed from the cell culture, using membranes and diafiltration, or binding using an ion exchanger,

(e) removing residual non-hepatitis A virus specific proteins from the concentrated hepatitis A virus of step (d) using a combination of organic extraction, PEG precipitation, ion exchange, including removal of contaminating nucleic acid, if not previously completed in step (c), and then gel permeation chromatography as the final step,

(f) regenerating the purified hepatitis A virus of step (e).

In a preferred embodiment of the present invention, the process for the production on a commercial scale of an inactivated hepatitis A vaccine comprises the steps of:

(a) Cultivation of large quantities of hepatitis A virus (HAV) in NUNC cell factories, (NUNC, CELL FACTORIES) COSTAR CUBES, or in static surface bioreactors of even larger surface areas where the cell layer is MPC-5 cells mounted on a regular solid grid of a mixture of solid stainless steel, titanium or titanium stainless steel;

(b) Collecting the cultured HAV by removing the culture medium and adding a collection solution containing about 0.05 to 0.5% Triton X-100, preferably 0.1%;

(c) Treatment of harvested HAV with nuclease to ferment contaminating cellular nucleic acids:

(d) Concentration of nuclease-treated HAV by binding to an ion exchange column and eluting the bound HAV with about 0.35 M NaCl, followed by precipitation with polyethylene glycol;

(e) Extraction of concentrated HAV with chloroform/isoamyl alcohol (24:1, v/v) with vigorous stirring, and separation and retention of the aqueous phase;

(f) Chromatography of the aqueous phase from the organic extraction on an ion exchange matrix at about 0.12 M NaCl, and elution of HAV with a gradient to a higher concentration of NaCl (1 M is adequate) and immediately diluting the collected solution from the HAV elution to a salt concentration of about 0.15 M or lower, and primarily up to a HAV concentration of about 20 μg/ml or lower;

(g) Chromatography of HAV eluted from ion exchange on a size-exclusion chromatography resin, primarily on a TOYOPEARL HW55S and HW65S tandem device in which the HAV enters the matrix volume;

(h) Inactivation of gel filtered HAV by treatment with formalin at 1/1000 followed by sterile filtration, co-precipitation with aluminum hydroxide and fractionation into unit dose equivalents of up to about 25 ng - 100 ng of inactivated purified HAV per dose.

The HAV obtained in this invention is highly purified and highly antigenic. The product is a purified hepatitis A virus preparation where more than 95% of hepatitis A virus protein is specific based on SDS PAGE and silver staining analysis and less than 0.1% of the mass of the preparation is non-HAV nucleic acid and less than 4% is lipid. The level of carbohydrate detected as glucose in the hydrolyzed product sample is between about 0% and 200% by weight of protein present, with non-glucose carbohydrate present at less than 15% by weight of protein. In preferred embodiments, glucose and non-glucose carbohydrates, such as ribose, are present at less than about 20% by weight of the protein. The preparation retains its antigenicity when inactivated with formalin.

Small amounts of inactivated virus are adequate to induce protective immune responses. Primarily, a dose of 25-100 ng is administered once, twice or three times over a period of several months, if necessary to induce high titers of anti-HAV immune responses.

The purified hepatitis A virus preparation has the following characteristics, where each characteristic is presented on the basis of the Eig protein. The procedure given in parentheses was used to determine these characteristics:

a) lipid content (gas chromatography): < 0.1 μg

b) carbohydrate detected as < 0.1 μg

glucose 2.5 μg;

(TFA hydrolysis, Dioney)

c) non-glucose carbohydrate: < 0.2 μg;

(TFA hydrolysis, Dionet)

d) specific protein of hepatitis A vaccine < 0.95 μg;

(amino acid analysis and "western blot"),

e) specific RNA of hepatitis A virus 0.1-0.2 μg

f) contaminating DNA < 0.0004 μg

The presence in the product of a hydrocarbon component consisting primarily of glucose is considered insignificant in terms of tolerance, immunogenicity and protective efficiency of the inactivated adjuvanted vaccine. However, it is important that the levels of non-glucose carbohydrate (probably ribose from the degradation of HAV-specific RNA) are very low in the product. It is also important to emphasize that it has only become possible to define the characteristics listed here since the discovery of a process capable of producing sufficiently large quantities of product, so as to allow the relevant analyzes to be performed.

In one specific formulation of the present invention, the hepatitis A virus vaccine has the following nominal composition per 25-100 units/0.1-1 ml dose:

[image]

In another specific formulation, the hepatitis A virus vaccine has the following nominal composition per 25 units/0.5 ml dose:

[image]

Purified inactivated hepatitis A virus according to the present invention has been found to be effective in preventing infection when a 25 ng sample (equivalent to 25 unit doses of one of the above formulations) is administered based on EIA analysis against a reference standard characterized by amino acid analysis. No serious adverse experiences have been observed in healthy adults, adolescents or children that can be considered related to the vaccine. Doses ranging from 6 to 50 units have been administered to adults, adolescents and children. Faster, higher seroconversion rates were observed in adults as the doses administered increased. This phenomenon is less pronounced in children.

One month after a single dose of 25 units, 83% of adults (≥ 18 years) and 97% of children and adolescents (2 to 17 years) were seroconverted. Two doses of 25 units given two weeks apart increase the seroconversion rate in adults to 93%. Seroconversion seven months after the second or third dose of 25 units leads to greater than 99% seroconversion in both age groups. Persistence in seropositivity is about 100% up to 12 months and 36 months in both age groups. Therefore, it is clear that a single dose of 25 units is sufficient to achieve seroconversion in at least 80% of any recipient population within one month of immunization. Immunization with higher doses shows seroconversion in a higher percentage of recipients.

On a 25 ng dose basis, one "COSTAR CUBE" can be assembled according to the method of this invention, yielding between about 5,000 and 10,000 doses or more. The process is also suitable for collecting and processing several "COSTAR" cubes at the same time. One SSR with or without a net, in order to increase the area, gives a significantly larger number of doses that are collected per production flow.

The following examples are provided to illustrate specific embodiments of the present invention, but the examples should not be construed as the only way to carry out various aspects of the present invention.

EXAMPLE 1

PRODUCTION OF HEPATITIS A VIRUS BASIC SEED

The procedure for large-scale production of virus seeds involves the infection of MRC-5 cell monolayers in NUNC CELL FACTORIES (NFC) of 6000 cm2. MRC-5 cells are grown in factories to confluent monolayers, and then infected with virus at an MOI of 0.1. After infection, the cells are incubated for 28 days with weekly replacement of medium containing 10% zap/zap fetal calf serum. It was found that high serum concentrations, 2-10% w/w, enable greater virus production than low levels, from 0.5 to 2% w/w. At the end of this cycle, the supernatant contains large amounts of infectious virus, in this example 107.3 TCID50 per milliliter, which is collected directly from the NFC, without cell lysis, and is used as a source of virus seed. In this way, the large quantities of infectious virus required for large-scale production are obtained, which is more reproducible and easier than using rolling bottles or balloons, - or mechanical collection of cells.

EXAMPLE 2

INCREASE IN VACCINE PRODUCTION

To support phase III clinical trials, laboratory scale-up has become an important consideration for hepatitis A vaccine production. To support scale-up of the manufacturing process, modifications to previously known HAV propagation and purification procedures are required. Two basic cell propagation procedures have been used, NUNC CELL FACTORIES, and static surface reactors, such as the COSTAR CUBE. This example describes the NUNC CELL FACTORY process. Example 3 describes the COSTAR cubic SSR procedure.

NUNC cell factories used for the Monroe efficacy study

A revised process has been developed that allows for improved scale-up of the process to propagate larger quantities of attenuated hepatitis A vaccine than previously possible. This was initially achieved by replacing standard rolling bottles with NUNC cell factories (NCF). The basic unit of the NUNC CELL FACTORY (NCF) is a vessel with a culture area of 600 cm2. NCF forming dishes are manufactured from polystyrene, treated for optimal cell attachment and spreading (Maroudas, 1976, J. Cell Physiol. Vol. 90, pp. 511-520. The 10 unit dishes described here have 10 identical culture units (dishes) with with a total surface area (area) of 6,000 cm2. In two corners there are specially constructed openings that form vertical tubes that connect the containers in multiple layers. These vertical tubes terminate in two adapters that receive air filters and a specially designed Teflon connector. All units are sterilized by irradiation and are received from the manufacturer already ready for use.

The medium in the NCF for MRC-5 cells is very similar to that obtained in the rolling flasks. Cells are grown in static culture with the cells attached to a solid substrate covered with a liquid medium and the supply of nutrients and oxygen to the cells is via diffusion. The use of NCF plants increases the reproducibility of operations, while mimicking the conditions used in the production of rolling bottles. However, the downstream collection and processing process is different, as further described below.

EXAMPLE 3

COSTAR CUBE static surface reactor used for commercial scale production process

The further increase was a challenge to obtain as large an area as possible per reactor, while enabling good control of the environment through the bioreactor. NUNC CELL FACTORIES provided a partial solution to this challenge. Better solutions are provided by COSTAR cubic (COSTAR CUBE) and STATIC MIXER reactor with static surface (SSR). The former consists of densely packed polystyrene layers in a sterilizable plastic cube (COSTAR CS2000). A total of 85,000 cm2 of usable growth area is available in a single cube (compared to 6,000 cm2 for 10 vessels in a NUNC cell factory. In the latter reactor, described in U.S. Patents 4,296,204 and 4,415,670, metal, ceramic, glass, or plastic static mixing elements form a dense packed growth surface; these elements can be fabricated and arranged to provide larger surface areas than the (COSTAR CUBE) COSTAR cube in a single reactor, for large production processes. HAV is successfully cultivated in both types of reactor. SSR has a circulation hole that allows media recirculation and refeeding nutrients through the saturator, in order to control the pH and dissolved gas levels of the medium, to achieve the oxygen demand of the culture, and to achieve nebulization of the medium. This optimization is not possible in roller bottles or in NCF. In addition moreover, the medium is changed in order to improve the availability of large quantities of the key raw material. serum (Cat. A-2151, Hyclone Laboratories, Inc., Logan, UT or equivalent) has been used successfully to replace fetal bovine serum.

EXAMPLE 4

Harvesting hepatitis A virus from NUNC cell factories or static surface reactors by Triton lysis

Neither the NUNC cell factory nor the static surface reactor is suitable for mechanical scraping to release layers of MRC-5 infected cells. Cell-bound hepatitis A virus is efficiently regenerated using cell lysis with Triton, and purified using a new procedure. The solution obtained by the Triton lysis process was more diluted than the preparation obtained by mechanical scraping of rolling bottles. The solution obtained from the Triton collection is concentrated by diafiltration and then treated with an absorbent, XAD, to remove the remaining Triton.

Triton removal down to less than 10 μg/ml was demonstrated using in-process capillary zone electrophoresis analysis. HAV is then precipitated with polyethylene glycol (PEG), extracted with chloroform: isoamyl alcohol. Contaminating DNA is removed using a DNA filter column followed by anion exchange chromatography and size exclusion chromatography. The procedure involving both NCF growth and Triton lysis did not significantly affect the immunogenicity of the purified formalin-inactivated hepatitis A vaccine.

EXAMPLE 5

Nuclease treatment and binding chromatography

The scale-up, productivity, and consistency of the procedure were increased by Triton treatment of the harvested material with an endonuclease, followed by anion exchange chromatography binding to concentrate the virus (see Figure 1). This modification eliminates the need for a concentration/diafiltration step, XAD treatment, and a DNA filter column.

A non-specific endonuclease active against both RNA and DNA was selected. This endonuclease, BENZONASE, was isolated from Serratia marcescens and cloned into Escherichiacoli. It is produced as a highly purified, well-characterized recombinant protein by Nycomed Pharma A/S (Denmark). This particular endonuclease was chosen, partly because it has a very high specific activity and can be added directly to the crude material collected by Triton in very small amounts (10 mcg BENZONASE/1 collected material). Assay procedures have been developed, using a "WESTERN Blot" for specific BENZONASE protein and a kinetic assay for enzyme activity, to measure enzyme removal by the purification process. Most (> 90%) of the added endonuclease activity can be considered to be in the wash fractions and the fractions that flow through the binding step of anion exchange chromatography (described below). The level of BENZONASE is reduced by a factor of more than 100 in the product of the binding column.

EXAMPLE 6

ADDITIONAL STEPS OF THE PROCEDURE

a) Degree of binding

Hepatitis A virus is concentrated from nuclease-treated Triton-collected material on an anion-exchange column by application at low ionic strength and elution step in 0.35-0.5 M sodium chloride in phosphate buffer. Hepatitis A virus is concentrated 30-fold by this degree of binding and Triton is reduced to undetectable levels, as determined by in-process CZE analysis.

b) Precipitation with PEG and extraction with chloroform

Hepatitis A virus from concentration/diafiltration/XAD or that eluted from the binding column is precipitated with polyethylene glycol (PEG). Under favorable conditions, this step is highly selective and the main contaminant, which is otherwise co-purified with the virus, remains dissolved and is discarded in the fraction of the upper liquid layer. The PEG precipitate containing the virus is resuspended and extracted with chloroform: isoamyl alcohol (24:1) to remove dissolved lipids and proteins denatured with chloroform that remain at the interface between the aqueous and organic phases.

c) Anion exchange chromatography and size exclusion chromatography

The aqueous extract from the chloroform extraction step is applied to an anion exchange column at low salt content and then eluted with a NaCl gradient up to 1.0 M salt. The final stage of size exclusion chromatography yields a highly purified virus preparation (see Figure 2).

d) Inactivation

Formalin inactivation of hepatitis A virus was previously performed using 93 μg/ml formaldehyde/ml (1:4000 formalin and at a temperature of 37°C for antigen concentrations close to dose levels. Under these conditions, virus inactivation follows first-order decay kinetics with a half-life of approx. 2 hours, corresponding to a loss of about 3.5 log10 TCID50 (50% tissue culture infective dose) per day A reduction of 7 logs of virus infectivity is achieved in about 2 days, but during inactivation it extends up to twenty days to ensure a large safety factor that the virus is completely inactive.

Small-scale experiments have confirmed that as the concentration of formaldehyde increases, the rate of inactivation increases, as measured by the loss of TCID50. The rate of inactivation varies proportionally with formaldehyde concentration, as expected for pseudo-first-order inactivation kinetics. A tissue culture assay of two hundred dose equivalents confirms complete inactivation when the virus is incubated with 370 μg/ml formaldehyde (1:1000 formalin) virus at 37°C for 5 days. Therefore, these modified inactivation conditions provide a safety factor similar to that of the previously used conditions. This was confirmed by three large-scale half-run inactivations performed under identical conditions, followed by absorption using aluminum hydroxide, yielding a vaccine for clinical trials. The rate of inactivation was about four times higher than the rate previously observed at lower concentrations of formaldehyde, which is in excellent agreement with small-scale tests. Samples of these two aluminum hydroxide-absorbed vaccines were tested for immunogenicity in mice, with an ED50 for each similar to that of earlier vaccines (less than 2 ng).

e) Absorption of aluminum hydroxide

Absorption on aluminum hydroxide is performed by co-precipitation of formalin-inactivated virus with aluminum hydroxide. The procedure used for all clinical preparations was essentially the same, with the only variable being the concentration of virus in the formalin-inactivated crude solvent. Direct deposition of the inactivated virus on aluminum hydroxide was performed first by adding potassium aluminum sulfate to the formalin-inactivated crude solution. Finally, a precipitate is formed by the addition of sodium hydroxide. Formaldehyde and residual salts are eliminated by removing the upper layer of liquid from the suspension and replacing it with physiological saline.

EXAMPLE 7

PURITY OF HAV PRODUCTS

Analytical size exclusion chromatography (HPSEC), EIA and protein as well as SDS-PAGE were used to document the purification process to ensure consistent yields of high purity vaccines. HPSEC provides a way to record nucleic acid losses during nuclease treatment as well as to determine purity in later stages of the process. The chromatograms in Figure 2 show that the peak corresponding to hepatitis A is clear in the PEG precipitate. Hepatitis A virus is enriched in a solvent extraction step and then concentrated in an ion exchange chromatography step. Final gel filtration yields a highly pure hepatitis A preparation (> 95% by SDS-PAGE and silver staining). As an independent measure of the purity of the preparation, HAV is a uniquely included peak in the HPSEC analysis (see Figures 2, 37 and 38).

II. Removal of impurities

CZE analysis of in-process samples indicates that Triton is removed by the diafiltration concentration stage in the Monroe process and the binding column in the production process. In addition, BENZONASA flows through the binding column. The amount of BENZONASE present in the anion exchange step of the product is below the detection level (< 18 femtograms of activity and less than 13 pg BENZONASE mass/25 unit doses of hepatitis A; the limit of detection in this analysis is 12.5 pg).

EXAMPLE 8

Purification of hepatitis A virus harvested from NUNC cell factories or static surface bioreactors by Triton lysis: the Monroe study

HAV was propagated essentially as described in Example 2 using NCFs. MRC-5 cells were grown in batches of sixty 6000 cm 2 NCFs at 37°C for days using medium consisting of Williams Medium E supplemented with 10% v/v fetal calf serum and neomycin sulfate. On day 7, cells are refed (supplied) with fresh medium containing enough virus to infect the cultures at about 0.1 MOI, and switched to medium at 32°C. Cultures are further incubated at 32°C for 21 days with re-feeding once a week. HAV is harvested by treating cell layers with lysis buffer containing 0.1% Triton X-100, 10 mM TRIS, 1 mM MgCl2 pH 7.5. The obtained lysate from NCF is collected and frozen until further processing.

Four liters of frozen hepatitis A virus collected from a NUNC cell factory culture by treatment with TRITON X-100 are detected in a water bath at 20-30°C for 4 hours and then filtered. The filtered lysate is concentrated tenfold on a Pellicon apparatus using a membrane cut of 300,000 molecular weight. The concentrated lysate, approximately 400 ml, is then diafiltered against 10 mM Tris-HCl buffer, pH 7.5 containing 150 mM NaCl and 1 mM EDTA. Triton is removed by batch treatment with AMBERLIT XAD-4 (Rohm and Haas Company) polymer resin at 30 mg/ml lysate. After filtration to remove XAD-4, the lysate was adjusted to 510 mM NaCl and then precipitated by adding polyethylene glycol (PEG 8000, Sigma) to a 5% (v/v) final concentration. The mixture is mixed vigorously, incubated at 4°C for 1 hour and then centrifuged at 1000 x g at 4°C for 10 minutes. After centrifugation, the upper liquid layer is removed and discarded. The precipitate is then dissolved in 6.2 mM sodium phosphate buffer, pH 7.2 containing 120 mM NaCl and 1 mM EDTA. The resuspended pellets are then extracted with an equal volume of chloroform: isoamyl alcohol (24:1 v/v). The mixture is shaken vigorously and then centrifuged at 3000 x g for 10 minutes at 20°C. The separation surface containing the undissolved material is re-extracted in the same mixture of organic solvents using 30% of the initial volume. After centrifugation, the two aqueous phases are combined. The chloroform extract is then adjusted to 350 mM NaCl and chromatographed on DEAE-Sepharose CL6B (Pharmacia) by filtration to absorb DNA. The fraction flowing through the column containing HAV is collected and diluted by a factor of three with 6.2 mM sodium phosphate buffer, pH 7.5 and chromatographed on a TOYOPEARL DEAE 650M (Toso Haas) with gradient elution up to 1 M NaCl. Hepatitis A eluted at 15-30% of 1 M buffer, corresponding to 15-30 mS. Fractions separated from this region were then chromatographed on Sepharose CL4B (Pharmacia) in 6.2 mM sodium phosphate buffer with 120 mM NaCl. The loading of this column is adjusted to be greater than 1% but less than 5% of the column volume. The product was eluted between 46 and 64% of the total column volume.

The final purified batch was sterile filtered to 0.22 micron and inactivated with 1:4000 formalin reagent (formalin is 37-40% formaldehyde by weight) at 37°C for 20 days. These conditions show first-order degradation kinetics with a half-life of 2 hours and a loss of approximately 3.5 log10 TCID50/24 hours. The inactivation period has been extended to 20 days to ensure a high degree of safety. Potassium aluminum sulfate is added to the inactivated hepatitis A and then the solution is precipitated with the addition of sodium hydroxide to pH 6.6-6.8. Fromaldehyde and residual salts are removed by centrifugation, whereby the upper liquid layer is removed and replaced with physiological saline.

EXAMPLE 9

Purification of hepatitis A harvested from COSTAR cubes with Triton X-100,

using nuclease treatment and binding chromatography: A production-scale process

Production-scale material is obtained using the SSR process for virus propagation. In this procedure, the COSTAR cube is used as a cell growth surface; the cube is bound into a COSTAR CS2000 saturator using an external port and a pump to drive the media flow, as described in Example 3. MRC-5 cells are inoculated into COSTAR CUBE SSR, and grown at 37°C for 7 days. Spraying was started two days after the introduction of cells. The reactor was continuously sprayed with Williams' Medium E supplemented with 10% zap/zap iron-supplemented calf serum and neomycin sulfate. The reactor pH is controlled between 6.9 and 7.6, and the dissolved oxygen in the reactor reaches between 15 and 100% air saturation in the medium. Cell growth was allowed to proceed for a total of 7 days, and virus was introduced into the reactor at approximately 0.1 MOI, using a batch of virus obtained according to the procedure of Example 1. The SSR temperature was reduced to 32°C, and feeding was continued for 21 days. The SSR is then drained and the medium is replenished with 0.1% w/v TRITONAR, 10 mM TRISR, pH 7.5 buffer to release HAV. For four batches of virus produced by this process in COSTAR cube systems as shown in Example 10, HAVAg production averaged 2.8 times the historical average value in NCF reactors using the Monroe surface-based propagation process. Increased SSR productivity allowed for easy scale-up of the breeding process to production scale.

The crude lysate is treated with BENZONAS to release the virus from the nucleic acid adducts and the virus is then bound by anion exchange chromatography. Treatment with BENZONAS eliminates the need for a concentration/diafiltration step, treatment with XAD-4 and a DNA filter column (See Example 8).

Twenty-five liters of COSTAR cube Triton X-100 collected material is treated with 5-25μg BENZONASE/1 lysate in the presence of 1 mM MgCl2 and then filtered through a 0.22 micron filter. The mixture is incubated for 18 hours at 25°C. The enzyme-treated lysate is applied to a TOYOPEARL DEAE 650 M anion exchange column equilibrated with 4.7 mM sodium phosphate buffer, pH 7.2 containing 90 mM NaCl.

Hepatitis A is eluted (average 800 ml) with 6.2 M sodium phosphate buffer containing 0.35 M NaCl and 1 mM EDTA. Test procedures using kinetic analysis for enzyme activity have been developed to record enzyme removal during the procedure. The results show that >90% of the endonuclease activity can be attributed to the wash fractions and the fractions flowing through the binding column. In addition, the total nuclease activity, including that from added BENZONASE, in the product from the binding column is reduced by a factor of >100 compared to the amount initially present in the lysate.

The concentration of NaCl hepatitis A product from the binding column is adjusted to 420 mM and the virus is precipitated with polyethylene glycol (PEG 8000, Sigma) at a final concentration of 4.5%. The mixture is mixed vigorously and then incubated for 1 hour at 4°C. After incubation, the pellet is collected by centrifugation at 1000 x g for 10 minutes at 4°C. The upper liquid layer is removed and discarded. The PEG pellet is resuspended in 6.2 mM sodium phosphate buffer, pH 7.2 containing 120 mM NaCl and 1 mM EDTA. Organic extraction of the resuspended PEG precipitate is performed by adding 1.5 volumes of chloroform: isoamyl alcohol (24:1, zap/zap). Phase separation is assisted by centrifugation at 3000 x g for 10 minutes at 20°C and the upper, aqueous phase is retained. The separation surface is extracted again with 30% of the original volume of the organic mixture, centrifuged and the second aqueous phase is combined with the first.

The aqueous extract is chromatographed on a TOYOPEARL DEAE 650 M (Toso Haas) anion exchange matrix and eluted with a gradient of 1 M NaCl. Hepatitis A virus is eluted at 15-30%c of a 1 M gradient which corresponds to 15-30 mS. Fractions from this region were separated and subjected to size exclusion chromatography. Size exclusion chromatography used two columns, TOYOPEARL HW55S and HW65S. Under these conditions, hepatitis A virus was in the included volume for both matrices and a tandem column device was used to separate both higher and lower molecular weight impurities from hepatitis A virus. The columns were equilibrated with 6.2 mM sodium phosphate buffer, pH 7.2, which contains 120 mM NaCl. The eluted hepatitis A in the symmetric peak and product fractions were collected.

The sterile 0.22 micron filtered product from size exclusion chromatography was inactivated at approximately 500 units/ml (one unit corresponds to about 1 ng of viral protein) with 370 μg/ml (1:1000 dilution of formalin which is 37-40% formaldehyde by weight) 5 days at 37°C. Inactivated hepatitis A virus was sterile filtered to 0.22 micron. Co-precipitation with aluminum hydroxide is done by initially adding potassium aluminum sulfate and then titrating to pH 6.6-6.8 with sodium hydroxide.

Residual salts and formaldehyde are removed by a sedimentation/decantation process in which the top liquid layer is removed and replaced with physiological saline.

EXAMPLE 10

The product obtained by the process, as described above, is analyzed for: protein by amino acid analysis (by gas-phase hydrolysis followed by PITC labeling and reverse-phase high-resolution liquid chromatography); HAV antigen by HAV antigen solid-phase enzyme immunoassay (antigen bound using human polyclonal antibody preparation followed by detection with mouse monoclonal antibody and goat-anti-mouse antibody/peroxidase conjugate) carbohydrate (including detection of glucose and non-glucose carbohydrate; non-glucose carbohydrate was present in the product at a level of less than 0.1 per μg of product protein) by high pH anion exchange chromatography and pulsed amperometric detection (HPAEC-PAD, acid hydrolysis using 2 N TFA followed by HPLC analysis). DNA of the host cell by hybridization ("dot blot" procedure using an Alu DNA fragment as a hybridization probe); fatty acid by gas chromatography (methyl esterification of fatty acids, followed by capillary gas chromatography); RNA hydrolysis and reverse-phase HPLC analysis of ribonucleotides.

In addition, the results shown below, obtained according to the previous procedures, SDS-PAGE analysis and protein detection using silver staining or using "western blot" with anti-HAV antibodies revealed only the presence of HAV specific proteins by applying 50-300 ng of protein. In addition, the presence of full capsids was demonstrated by analysis of HAV-specific RNA by hybridization with HAV-specific DNA probes, sucrose density gradients, and reverse-phase HPLC. HAV specific RNA was found to be present in the product at about 0.1-0.2 μg per μg of protein. This level of HAV-specific RNA is consistent with a ratio of about 1/3 full capsids to about 2/3 empty HAV capsids. Finally, an independent measure of HAV purity is provided by the analysis shown in Figure 2, using HPLC analysis, which shows a single peak of the HAV product.

Absolute concentrations of protein, carbohydrate, lipid, and DNA are presented below in section A. Data from section A were transformed below in section B by normalization to micrograms of protein, and in section C, as a weight percent of protein (which indicates the ratio of mass of carbohydrate present, to example, and the mass of the protein present, multiplied by 100).

The results of these analyzes are presented below for eight preparations of HAV obtained by the commercial-scale process of the present invention.

A. Composition analysis

[image]

* The limit of detection (LOD) for the first sample was 0.0045 μg/ml.

For subsequent samples, the sensitivity was increased so that the LOD was 0.003 μg/ml.

Protein % by weight

[image]

Preliminary data characterize the product of this invention, prior to inactivation and co-precipitation with aluminum hydroxide, as an HAV preparation having the following characteristics, on a microgram protein basis (with protein that is greater than 95% pure HAV protein by SDS PAGE and silver staining) :

Carbohydrate consisting of deafness 0.1 - 2.5 μg

Non-glucose carbohydrate 0.2 μg

DNA 0.004 μg

Lipid 0.1 μg

HAV RNA 0.1-0.2 μg

Primarily, this analysis is performed on the purified HAV product of this invention, when the HAV product is most concentrated, prior to inactivation and formulation. However, provided with sufficient amounts of inactivated or final formulated vaccine, this assay is sure to yield substantially similar results. In one particularly suitable embodiment, the vaccine according to the present invention has the following nominal composition per 25 units/0.5 ml dose:

[image]

* Extrapolated from analyzes of a purified batch of virus

# From the analysis of demonstration samples of the series.

& Based on activity analysis.

EXAMPLE 11

Comparison of different HAV preparations

The purified HAV product of this invention was compared with the description of other HAV products as described by their manufacturers. The results of this comparison are shown in Table 1:

TABLE 1

COMPARISON OF INACTIVATED VACCINES

FOR HEPATITIS A

[image]

a) Joint effort of Chemo-Sero-Therapeutic Research Institute Chiba Serum Institute, and Denka Bio-Research Institute.

b) Based on the data of amino acid analysis (AAA) (amino acid analysis).

c) Defined by enzyme immunoassay of HAV antigen; the calibration standard was purified virus, 90% pure by SDS-PAGE/silver staining, protein defined by AAA.

d) Defined by enzyme immunoassay of HAV antigen; calibration standard undefined.

e) Defined by SDS-PAGE analysis.

f) Defined by HAV antigen to total protein ratio.

From these data and the information provided above in Example 10, it is apparent that the product of this invention is much purer (based on HAV antigen to total protein ratio and amino acid analysis), more completely characterized, and contains the lowest amount of virus protein effective to achieve seroconversion and protection than any product previously described for which analytical data is available.

EXAMPLE 12

Vaccine efficacy - Monroe's study

The vaccine was obtained by infecting layers of MRC-5 cells cultured in NUNC cell factories with CR326F HAV. The culture was maintained for several weeks using regular feeding. At appropriate times, infected MRC-5 cell monolayers were harvested by detergent treatment and HAV was purified according to the method of the present invention, as shown in Figure 1 for NCFs. Aqueous virus is absorbed on aluminum hydroxide and stored at 2-8 C. Doses of 0.6 ml each contain 25 units of hepatitis A virus antigen, based on radioimmunoassay against a purified standard reference virus, and 300 μg of aluminum hydroxide. The placebo contained 300 μg of aluminum hydroxide and thimerosal at a 1:20,000 dilution. Healthy, seronegative vaccine recipients at high risk of HAV infection received either a single dose of 0.6 ml of placebo vaccine. Blood samples were taken on the day of vaccination and one month later. Almost 100% of vaccine recipients were free of HAV infection since a waiting time of 50 days was taken into account to search for individuals who had acquired HAV infection before vaccination. On the other hand, a significant number of placebo recipients acquired HAV infection after the 50-day search period.

The population studied was large enough so that the protection of vaccine recipients is statistically significant.

EXAMPLE 13

Virus production in a static mixer reactor

Hepatitis A virus was propagated in an SSR consisting of a static mixer column connected to a saturator using an external port and pump system (Charles River Biological Systems CRBS 5300). The static mixer reactor is manufactured from titanium metal elements having a total surface area of about 260,000 cm2, in an 8" diameter 40" long column. MRC-5 cells are introduced into the reactor and grown for 7 days at 37°C in Williams Medium E supplemented with 10% v/v fetal calf serum and Neomycin sulfate. Cell growth is allowed to proceed for 7 days, and part of the medium is changed three times weekly, obtaining a feed rate equal to the NCF Monroe method. The virus is introduced into the reactor at approximately 0.1 MOI. The SSR temperature is reduced to 32°C and feeding continues as above for 22 days. The medium is then drained from the SSR and refilled with 0.1% w/v TritonR buffer to release HAV. This reactor produces approximately 50% more HAVAg on a surface area basis than a control T-flask inoculated using the same reagents, feed (feed), propagation and harvesting in a similar manner. The HAV reactor production is also compared favorably with the NUNC cell factories of Example 2.

EXAMPLE 14

HAV PRODUCTION ON STAINLESS STEEL MESH STATIC MIXER ELEMENTS

14.1 MATERIALS AND PROCEDURES

Cell culture

All studies used MRC-5 cells at PDL ≤ 40. Cells were cultured in Williams E medium containing 10% iron-fortified calf serum, 2 mM glutamine, and 50 ml/l neomycin sulfate at 37°C. In studies using a spray and stirred tank reactor, Pluronic F-68 is added to the medium. A cell density of 10,000 cells/cm 2 was used to inoculate new culture surfaces. Cell counts were performed using a Coulter counter and/or hemacytometer with Trypan blue staining to determine viability.

Cell culture on metal coupons

A solid metal or metal mesh coupon about 5 cm x 5 cm is placed in a 150 cm2 glass petri dish and sterilized. After allowing to cool, 90 ml of medium is placed in the dish and then the inoculum of cells is added. The amount of media is sufficient to completely cover the metal surfaces. After 1 day at 37°C, the metal part is removed using sterile forceps, and placed in a new sterile petri dish (to prevent cell growth on the glass surface) containing 90 ml of medium. Solid layers are treated with trypsin, washing twice with phosphate-buffered saline (PBS) and adding 2 ml of trypsin to the metal. After observing cell release, trypsin is carefully pipetted several times above the surface to ensure complete removal.

Raw material for virus and infection

All studies used the raw material for the hepatitis A virus, CR326F P28, with a titer of 108 TCID50/ml. MOI values shown here are calculated as TCID50/cell, not PFU/cell. Cultures were infected by adding virus stock to fresh medium and adding this to the culture. After infection, the cultures were incubated at 32°C.

Liveability coloring on network coupons

Cell viability was registered using fluorescein diacetate (FDA) and ethidium bromide (EB) staining. In viable cells, FDA is cleaved by an esterase in the cytoplasm, yielding free fluorescein that fluoresces green. EB rapidly intercalates into the nuclear DNA of non-viable cells and fluoresces red. Cells are labeled with both colors simultaneously. The staining solution consisted of 100 μl of FDA stock (5 mg/ml in acetone) and 200 ul of EB stock (10 mg/ml in PBS) in 50 ml of PBS. The mesh material was then incubated in 5 ml of this solution for 5-6 minutes. After labeling, the mount was washed twice in PBS (20 ml per wash) and placed between the lower plate and the upper cover plate of the microscope slides. The samples were then observed using a BioRad MRC-600 confocal microscope.

Static mixer reactor

Mesh (known as "E-pak") and solid plate 316 stainless steel static mixer elements (SMV configuration) were obtained from Koch Engineering (Wichita, KS). All elements were approximately 2" x 2", constructed of type 316 stainless steel or titanium. The area of solid elements is thus determined to be 1300 cm2, and the mesh elements are thus determined to be 2300 cm2. The surface to liquid volume ratio of these last elements was approximately 26 cm2/ml (about 24 cm2/ml based on the total volume of the reactor). The five elements are nominally placed in a thread-like glass column of 5 cm by 26.5 cm with PTFE parts for connection at the end. The elements are placed in a configuration with mixing directions perpendicular to successive elements. A column with elements constitutes a reactor for cell growth. Stirring tank satellite vessels, connected to electronic controllers, were used to control the pH of the medium at 7.2-7.3 and up to 80-90%. The medium is recirculated from the mixing tank into the reactor using a peristaltic pump.

Seeding of the reactor was performed by providing a low circulation rate to allow the cells to attach. The inoculum was placed in the system and typically recirculated at 100 ml/min for 5 minutes to mix the cells and medium. Very little cell attachment occurred during this rapid recirculation period. The flow rate was then reduced to about 2 ml/min, which provided a linear rate through the reactor close to the cell sedimentation rate. Velocities around the cell sedimentation rate were found to be optimal for attachment rates and even distribution of cells throughout the reactor.

Cell lysis procedure

Cells were lysed in Triton lysis buffer containing 0.1% Triton X-100, 10 mM Tris, and 1 mM MgCl2. T-bottles were washed twice with 0.3 ml/cm2 PBS, and lysed twice with 0.1 m1/cm2 lysis buffer for 1 hour. Protocols for the lysis reactors are described in the text.

Analytical procedures

Cell metabolism was measured using a Kodak Ektachem DT60 analyzer. Hepatitis A viral antigen (HAVAg) was analyzed using a monoclonal antibody enzyme-linked immunoassay. Total protein of cell lysates was quantified using the Coomassie blue Bio-Rad protein assay (Cat #500-0001) against the bovine serum albumin standard.

RESULTS AND DISCUSSION

Growth of MRC-5 cells on solid metal coupons

Prior to this study, work on the static mixer was performed on titanium elements. It was desirable to test cell growth on surfaces made of different alloys, which, in addition to being cheaper than titanium, provide greater plasticity (extensibility) for ease of static mixer production. Two grades each of stainless steel and Hastelloy grades B and C were tested. Metal coupons were inoculated with approximately 10,000 cells/cm2. After 4 days, the cells were trypsinized (treated with trypsin) and the final cell density was recorded:

Material Cell/cm2

Titan 1.4x105

316 stainless steel 1.6x105

316L stainless steel 1.2x105

Hastelloy B 2.2x103

Hastelloy C 1.3x104

Growth on both types of stainless steel was similar to that on titanium; cells did not grow on Hastelloy B and grew poorly on Hastelloy C. Differences in cell densities for stainless steel and titanium should not be considered as superior; the differences are probably due to different analysis.

Most data for hepatitis A virus procedures have been obtained for cell growth on plastic holders, eg, culture bottles made of polystyrene material. For comparison, growth curves of MRC-5 cells on polystyrene, glass, and 316 stainless steel are shown in Figure 3. Cells grew similarly on all surfaces. The maximum specific growth rates are similar (0.034 h-1 for plastic, 0.032 h-1 for glass, and 0.039 h-1 for 316 stainless steel). The lower final cell density obtained for plastic is likely due to nutrient limitation. Growth on plastic was performed in T-bottles, while growth on glass and stainless steel was performed in petri dishes. Due to the difference in the volume of media per cm2 in each system, there is an imbalance in the results of available nutrients. Cell growth and glucose consumption for stainless steel coupons are shown in Figure 4 .

Growth of MRC-5 cells on metal mesh coupons

In an attempt to maximize surface area per unit volume, the metallic mesh material was tested in petri dishes for the growth of MRC-5 cells. For certain combinations of wire diameter and spacing, a piece with the same design surface area as a solid coupon may present a larger total area for cell growth. Layers of meshes can then be formed or arranged in the reactor in such a way as to maximize the available surface area per unit volume of the reactor. To examine cell growth, a single grid surface, MRC-5 culture was grown using the techniques described in the Materials and Methods section.

Growth of MRC-5 cells was illustrated on mesh constructed from either 316 stainless steel or titanium. Mesh coupons with wire diameters ranging from 100 μm to 400 μm, weaves of 14 x 100, 120 x 110, 40 x 200, and 107 x 59 wires per inch were tested. Cells grew on all samples, to larger or smaller extents. In the early stages, cells grew on the surface of the wire. Soon after, the cells begin to bridge the intercavity spaces, especially in the corners. Over time, the cells completely fill the intercavity spaces and form multi-layered structures. By visual inspection, the mesh bears very high cell densities, which are more than scaled up by surface area. Direct cell surface concentrations are not available since multilayer growth on a grid is not amenable to trypsinization. Preliminary results show that intercavity size is an important factor, as cells are not able to bridge large intercavities, and a network with small intercavities reduces the area to the limit of the same area as the surface of a smooth plate. The construct material did not affect cell growth, and it was concluded that any biocompatible material that could be woven into the mesh layers was suitable for use. Woven metal meshes have the advantage of being easily cleaned and reused for many cycles of cell growth, as described below.

From here on, only stainless steel mesh with a weave of 107 wires x 59 wires per inch, and a wire diameter of 160 μm (giving a wire spacing of 77 μm x 270 μm) will be quoted. The grid static mixer elements discussed below are constructed of this material. In addition to providing an increased surface area, the mesh allows cells to grow in multiple layers. A micrograph of the multi-layer growth of cells on this network is shown in Figure 5. It was interesting to determine whether the cells throughout the layers are all viable, or whether the cells in the center of the multiple layers are necrotic. For this purpose, fluoroscein diacetate and ethidium bromide were used, as detailed in the Materials and Methods section. Examining effective cross-sections of the network using a laser confocal microscope, it is evident that the vast majority of cells were viable based on the fact that ethidium bromide did not intercalate the DNA core and that fluorescein diacetate was cleaved, yielding free fluorescein. Figure 6A shows the fluorescein signal for a series of efficient sections through the cell layers. Figure 6B shows the ethidium bromide signal for the same sections. Separate coupons of cells rendered non-viable by treatment with methanol served as a positive control for ethidium bromide and as a negative control for fluorescein diacetate in these experiments. This control is shown in Figure 7.

Cell seeding on static mixer grid elements

Uniform seeding is important for optimal productivity and for maintaining a low, uniform doubling rate for the production of biologicals. Static reactor network elements were obtained, consisting of the same material examined above. The cylindrical reactor is oriented with a vertical axis. Although earlier studies were conducted with a reactor with a horizontal axis to increase sedimentation of cells onto the growth surface, attachment to a vertical surface was found to be just as effective and is the preferred mode of operation. In the course of this work, cells were seeded at approximately 10,000 cm2. Cells are seeded in multi-element reactors, where the mixing axes of the individual elements are anywhere between parallel or ninety degrees to each other. For the purposes of liquid mixing and optimal reactor operation, a vertical configuration is preferred.

Cells were not seeded in earlier trials where non-circulating velocities were used which gave a surface velocity of 5 cm/min. Seeding efficiency was increased by filling the reactor and allowing cells to seed by gravity sedimentation for 2 hours. This routinely provided a seeding efficiency greater than 90% (seeding efficiency - number of cells seeded/total number of cells in inoculum x 100%), however seeding was non-uniform.

As shown in Figure 8, the element at the bottom contains the most cells, while the element at the top contains the fewest. From these data, it was calculated that the average settling speed of the MRC-5 cell preparation is 6 cm/h. In a further experiment using four elements, the medium is recirculated for seeding at a rate approximately equal to the deposition rate. Uniform seeding is obtained with seeding efficiency greater than 90% for these conditions (Figure 8). The above results demonstrate that the surface velocity of the medium through the reactor affects both the rate of cell attachment and the final distribution of cells in the reactor.

Comparison of cell growth on a solid bed and on network elements of a static mixer reactor

Two reactors were installed and operated simultaneously to describe the differences between the solid layer and the mesh elements. The solid elements are constructed of titanium, and the mesh elements are constructed of stainless steel. It was determined that the area is 1300 cm2 for the solid elements and 2300 cm2 for the mesh, which shows that the ratio of the mesh to the solid surface is 1.8. Solid layer elements work in conjunction with grid elements to provide control for all experiments. The reactors were seeded with approximately 10,000 - 20,000 cells/cm2. The reactors were operated in a batch mode until the glucose concentration fell below 120 mg/dl, when the medium sparging was initiated. Both reactors were inoculated with the same harvested MRC-5 cells, and fed from a common medium reservoir to eliminate any differences in these variables. The glucose uptake rate (GUR) for each reactor is shown in Figure 9. Figure 9A compares the reactors by cm2 using the surface areas listed above; these surfaces are considered approximate. Figure 9B compares the systems based on reactor media volume since each reactor was the same size, i.e., five 2" elements with a 1 L satellite media vessel. Figure 9B does not include surface area approximations. Glucose uptake rate is used as a measure of growth cells and is used to determine the onset of the stationary phase. The last point in Figure 9 shows the time when the reactors were collected by triton lysis. The cells were visually confluent with the solid matrix and the interstitial spaces on the mesh were nearly filled on the mesh elements, indicating that more growth. Then the medium was sprayed at a rate of 0.19 l/h for the solid reactor, and the glucose concentration in the medium at this point was 1.16 g/l. The spraying rate was 0.26 l/h for the mesh reactor, and the glucose concentration in the medium was 0.69 g/l These results demonstrate the better metabolic activity obtained for the mesh reactor compared to the solid reactor for the cell growth phase.

These reactors were drained and cells were lysed using detergent buffer to extract cellular protein, a measure of cell mass in the reactor. For these experiments, the lysis procedure for harvesting cell-bound protein from each reactor was identical: 2 L PBS at room temperature was circulated through the reactors at 100 ml/min for 5 min and then drained. This washing process to remove protein is repeated twice. Then 1 L of lysis buffer, from the Materials and Methods section, is recirculated through the reactor at 400-500 ml/min, 37°C, for 1 hour. This is repeated once more (lysates 1 and 2) and subsequent lysates are cycled for a further 2 h for lysate 3, and 4 h for lysate 4. The percentage of protein removed in each lysate is presented in Figure 10. When protein calculations are converted to cell density using correlations presented in Appendix 1, solid static mixer elements contained 3x105 cells/cm2, while mesh elements contained 6x105 cells/cm2. The former number is believed to be representative of a solid-bed reactor, as all cells were removed during lysis as demonstrated by wiping elements and observing the wiped material under a microscope. However, not all cellular debris was removed from the mesh elements as observed using a microscope, so the final count is low as predicted. At the base of the reactor, gauze elements carry a concentration greater than 3 times that of solid elements. Provided the area calculations are correct, this difference in total protein is greater than the area ratio and is evidence for multilayered cell growth.

Production of HAVAg on static mixer grid elements

HAVAg production is demonstrated for a reactor using static mixer grid elements. The object of this experiment was to determine the viral propagation occurring on stainless steel mesh elements bearing multilayer growth: this is not an optimized system for HAVAg production. For this experiment, complete media replacements were used, rather than media scattering. The GUR profile for this experiment is shown in Fig. 11; The GURs calculated from this figure are based on glucose values obtained on the day after media refeeding. Cells were infected at an MOI of about 1 on day 7. Cell density for MOI calculations was determined based on the rate of glucose consumption at the time of infection. As observed with procedures such as Example 9, the rate of glucose uptake levels off shortly after infection and then declines, consistent with infection of the cells.

The observed decline in Figure 10 is more vertical than that observed with the procedure of Example 9, and is likely due to a more uniform degree of infection (Example 9 used an MOI of 0.1). Viral antigen is collected 21 days after infection by lysis with Triton detergent. The cell lysis protocol is outlined below.

Lysate 1: recirculation of lysis buffer through a static mixer for 1 hour at 37°C

Lysate 2: recirculation of lysis buffer through a static mixer for 1 hour at 37°C

Lysate 3: sonication for 1 hour in the Branson bath for sonication 0.7 l of lysis buffer

Lysate 4: freezing of elements in 1 l of lysis buffer for 12 hours, thawing and treatment with ultrasound for 1 hour.

The percentage of total protein and HAVAg released in these washes is shown in Figure 12. The first two lysates using recirculation were very efficient in removing HAVAg from the cells. This experiment used only 2 PBS washes before lysis, and was not completely efficient, therefore the percentage of total protein of lysate 1 is artificially high, and that of 2, 3 and 4 are low. One collected sample was analyzed for HAVAg by EIA before freezing. Based on the percentages of each lysate determined later, this corresponded to a yield of antigen on the surface twice that of Example 9. The goal of this experiment was realized by viral growth as demonstrated by HAVAg. This experiment demonstrates the usefulness of a grid static mixer reactor for HAV production.

Although the reactor was probably harvested late based on the GUR profile, the antigen yields were relatively high, indicating susceptibility of multiple layers to infection. The data also show the importance of MOI in HAV production; by infection at MOI 1, it appears that the process duration should be reduced by more than one week and collected on day 20. Based on various data, infection time and MOI are important variables for the production process; these parameters are important in reducing the culture length. Cell growth experiments show that infected cells continue to grow at the same rate similar to uninfected cells, even for cultures infected at high MOI with CR326F P28 (Figure 13). This is despite infection and temperatures up to 32°C, the temperature that is allowed to grow for this strain of the virus. The maximum specific growth rate calculated from these curves is 0.015 h-1, approximately half the growth rate of uninfected cells at 37°C.

The fact that the cells behave similarly during the first week of HAV infection is important in the operation of the reactor, since it is desired to achieve the highest possible concentration of cells regardless of the infection time and MOI. This experiment demonstrates that optimization of cell growth duration, infection duration, and MOI are intertwined.

Cleaning of static mixer elements

It is desirable to have a growth surface that can be reused for biological production, to eliminate waste disposal, enable automation of the reactor process, and increase culture reproducibility. Typically, the mesh elements are washed with distilled water to remove media salts (500 ml/min for 1 hour), placed in 1 L of 5% (w/v) NaOH solution and autoclaved for 45 minutes. After cooling, the elements are washed with distilled with water until they are free of NaOH (as determined by the pH of the wash solution). This procedure is extremely effective in removing cell debris by microscopic examination. It is believed that far fewer unpleasant treatments will be required to achieve equivalent cleaning, and that much more suitable for self-cleaning of the production reactor. The mesh elements treated with this procedure were reused for the last ten cycles with no change in cell growth characteristics. The elements in the virus growth study above were obtained in this way, demonstrating the utility of the used, cleaned elements for HAV propagation.

FINDINGS

These studies demonstrated that the static mixer elements constructed of stainless steel mesh were capable of supporting multilayer growth of MRC-5 cells. Multilayered elements were shown to be viable, with no indication of necrosis. Infection tests show that these multilayers are susceptible to hepatitis A infection and in a non-optimized system produce antigen in excess of what is normally achieved using solid media such as those in Example 9. With the culture microcarrier, clumping of cells can occur in poor defined multilayers; static mixer with grid provides well-defined multi-layer elements due to masterful geometry and because of this, limitations in nutrients and accumulation of waste are less of a problem. Cell growth within the interstices of the mixture closes the interstices and creates a plugged pattern similar to that of solid bed elements. This is why the system uses the benefits of static mixer technology and leads to predictable and uniform cell nutrition. This is in contrast to other types of packed bed reactors such as randomly packed fiber beds, packed glass bead beds, hollow wave reactors, or ceramic monolithic reactors. In these types of reactors, cell growth causes deterioration of the media flow pattern and creates poorly nourished pockets of cells. Overall, the reusable reactor containing static mixer elements with mesh combines greater potential for culturing MRC-5 cells and hepatitis A antigen production.

EXAMPLE 14 - APPENDIX 1

EXCERPT

Total protein from MRC-5 cell lysates was determined quantitatively and compared with direct cell counting (using a hemacytometer and using a Coulter Counter). Healthy cells in early exponential, late exponential, and stationary growth phases, as well as cells at 1, 2, and 3 weeks post-infection show that measurements of total protein from each culture lysate can be directly compared to cell density using a single plot. This simple correlation provides ways to determine cell density in reactors that are not applicable to direct counting techniques.

DISCUSSION

Protein analysis and sample collection

The Bio-Rad Protein Assay (Cat.# 500-0001) is a rapid, sensitive assay for the quantitative determination of total protein. Early experiments showed that the current lysis buffer (0.1% Triton X-100, 10 mM Tris-HCl, 0.1 mM MgCl2) did not interfere with the Coomassie blue-based assay. For consistency, all dilutions, including those of the standard protein (bovine serum albumin) were made in Triton lysis buffer. The fact that triton cell lysates are often flocculent in nature indicates that samples may require further treatment before being analyzed. A large flocculated precipitate leads to high protein titers. However, after two cycles of rapid freezing (in ethanol at 70°C) and thawing, the size of the flocculated precipitate is reduced to the point where a homogeneous suspension is obtained. In addition, by analyzing several dilutions of a single sample, easily detectable non-significant points originating from a poorly prepared sample can be revealed. The samples were also centrifuged and the clarified upper liquid layer was used; this treatment provided similar results but was considered unnecessary. The results presented here are for cell lysates frozen a minimum of twice and mixed well before they were analyzed.

Protein concentration as a function of cell density

For a standard curve that allows the conversion of total protein to a specific number of cells, the cells throughout the process must consist of approximately equal amounts of protein. To determine this, T-bottles were inoculated with equal numbers of cells. At certain intervals in the procedure, two bottles are sacrificed. One bottle was treated with trypsin (trypsinized) and cells were counted using a hemacytometer and Coulter counter, while the other bottle was subjected to detergent lysis, i.e., it was washed twice with 0.3 ml/cm2 PBS, lysed twice with 0.08 ml/cm2 buffer for lysis. This was done for cultures in early exponential growth phase, late exponential growth phase, stationary growth phase as well as for cultures at 1, 2 and 3 weeks post-infection. The cumulative results for these experiments are shown in Figure 14. The best interpolation line is described by the equation: Y(104) =

-0.09 + 0.42X. This calculation shows that a single curve provides a means to determine the number of cells by measuring total protein, regardless of the "state" of the cells.

Cells in this experiment were infected with HAV CR326F P28 at an MOI of 1. At 3 weeks post-infection, trypsinization confirmed inefficiency in cell harvesting. However, since cell densities at 1 and 2 weeks post-infection were approximately equal, and apparent cell lysis at 3 appeared to be absent, the cell densities used for 3 weeks were based on data obtained at 2 weeks. Figure 14 is plotted again, showing the time for each specific data point. By determining the data at 3 weeks after infection, there was no great influence on the slope and break of the curve (see Figure 15).

Using the above correlation, total protein data for several culture lysates as in Example 9 gave an average of 490 μg/ml protein. This translates to approximately 3.3 x 10 5 cells/cm 2 , and is generally in agreement with cell densities determined using a hemacytometer in control T flasks. Taken together, total protein from Triton cell lysates is believed to provide a useful approximation of cell density at the time of virus collection.

EXAMPLE 15

Examination of the mixing of the extraction stage with a solvent

Excerpt

During hepatitis A purification, the solvent extraction step was critical in removing impurities still present at the PEG pellet stage. In order to fully understand and control this step, a series of experiments were performed to determine the important parameters for solvent extraction. Two key variables were identified: duration of mixing and intensity of shaking, which was determined by the size of the bottles used. The solvent to water volume ratio was not a significant factor in the course of the test, nor was the use of a mechanical shaker as opposed to manual shaking or vortexing.

Materials and Procedures

Samples of the resuspended PEG precipitate were mixed with a solvent of chloroform and iso-amyl alcohol (24:1 CHCl3 : IAA) in different proportions from 0.5:1 to 3:1 (phase ratios of solvent to water). Mixing was done by hand by vigorous shaking, vortexing (for 15 ml tubes) or on a 3D mechanical shaker (Glas-Col, Terre Haute Ind) at a motor speed of 100 (with tubes placed horizontally for 15 ml and 50 ml tubes). Mixing times ranged from 20 seconds to 1 hour.

After mixing, the samples were centrifuged to separate the phases. A Beckman J6-MI centrifuged with a JS 4.2 oscillating rotor at 3800 rpm for 10 minutes was used for 50 ml test tubes and 500 ml bottles. For small tubes, initially a Dynac II centrifuge was used at 1000 rpm for 5 minutes, then for subsequent experiments, samples were transferred to 50 ml tubes for centrifugation as above.

Aqueous phases were removed with a glass pipette and analyzed by HFLC for size under the following conditions: two TSK P4000x1 columns (TosoHaas) in series, the mobile phase was RCM 8 (also listed as CM563, which is 150 mM NaCl in 6.2 mM of phosphate buffer, pH 7.2) at 0.32 ml/min, registered at 214 and 260 nm (80 minute flow time). Results are expressed as peak areas or peak area ratios based on 214 nm UV absorption. For initial experiments in 15 ml test tubes, one TSK column (flow time 40 minutes) was used. Two columns were then used to obtain better resolution between the peaks for HAP A and impurities.

Experiments were initially performed in 15 ml test tubes on a 2-6 ml scale and the samples were mixed by hand by shaking or vortexing. When scattered results were obtained from the first few batches, mixing was performed with larger volumes (in 50 ml test tubes, filled to a maximum of 24 ml) on a mechanical shaker. Samples were also taken from 500 ml bottles during processing and batch production. The bottles were shaken for a short time (for example 20 seconds). The phases were allowed to separate by standing for about 1 minute and 600 μl samples were removed from the upper phase. The bottle was returned to the shaker for another short time and then sampled again. These samples were later rotated in a microcentrifuge for 2 minutes at maximum speed.

THE RESULTS

1. Mixing time

From preliminary small-scale experiments in 15 ml tubes, mixing time appeared to be an important variable, with increased mixing time resulting in better impurity removal. From Figure 6, it is clear that the ratio of HepA to impurities increased with longer mixing times. In addition, in Figure 20, it can be seen that the area of the impurity peak decreases significantly with the mixing time.

With 50 ml tubes, it was shown that mixing time was extremely important, with 3 minutes required in this example for maximum impurity removal (Figure 20). In this case, the analysis is performed with tandem columns for better resolution and only a small decrease in Hep A surface area was observed.

The effect of mixing time was also studied in the 540 ml bottles used in production. Samples were taken every 20 seconds during the 2 minutes of total mixing time. This experiment was performed with two different solvent ratios: 2:1 and 3:1, which also corresponds to two different volumes of the aqueous phase in the bottles (about 60 ml and 90 ml, the solvent volume remained constant at 180 ml).

Figure 21 shows that the level of impurities decreases with mixing time, and that they are completely removed after 1 minute and 40 seconds. The time used in the production was 2 minutes, which ensures a significant removal of impurities. For even more complete removal, it is extended to 3 minutes. Differences in solvent volumes and ratios appear to have little effect in the field used.

This experiment was extended to a longer mixing time in both 50 ml and 15 ml tubes. Figure 22 shows that possibly the same degree of purity is achieved in all test tubes used, but that it takes 6-8 minutes in smaller test tubes and less than 2 minutes in 500 ml centrifuge bottles. These differences will be discussed further in section 3. In order to determine whether the virus is affected by prolonged shaking, a 50 ml tube is mixed for 1 hour on a mechanical shaker. The HPSEC results did not show any further decrease in the Hep A peak area and the EIA results (Table 1) showed that the antigenicity was the same at 1 hour as at 2 minutes of mixing. A process flow from the same batch shows a similar EIA value. Therefore, to ensure complete removal of impurities in the product, the minimum mixing time, as seen in the diagram, can be extended without reducing the virus yield.

TABLE 1

Effect of prolonged shaking on Hep A by HPSEC and EIA (50 ml tubes)

[image]

2. Volume ratio

The volume ratio of the solvent to the aqueous phase for one extraction was 1.5 for the semi-powered batches (mixed in one flask with a magnetic stirrer), and between 2 and 3 for the demonstration batches (shaking in 500 ml flasks). Therefore, the effect of the volume ratio between 0.5 and 3 on purity was examined. Initial experiments on a small scale (in 15 ml tubes, mixing for 1 minute) were performed with four different production batches of the initial PEG precipitate, Figure 23 shows that no clear correlation was observed between the solvent to aqueous phase ratio and impurity removal. The scatter in the results is likely due to variables in the starting material and inconsistency of shaking.

The sample size was scaled up to 50 ml tubes and a mechanical shaker was used for 2 minutes with the tubes placed horizontally. The results are shown in Figure 24. Again, although there is some scatter in the data, there is no clear trend based on the solvent to water ratio. This was confirmed by mixing time experiments in 500 ml bottles with 2:1 and 3:1 solvent ratios (Figure 21) showing very little difference in impurity removal. Therefore, it appears that the solvent ratio is not an important variable, and that an increase in the solvent ratio did not contribute to the increased purity seen in the stable series.

3. Mixing type and container size

No trend could be observed for the effect of manual shaking towards vortexing in small (15 ml) test tubes, as the results were scattered for experiments from different production batches (Figure 23). One data point in Figure 19 also shows good agreement between manual shaking and vortexing. In larger test tubes, manual shaking and mechanical shaking were compared. In Figure 25, it can be seen that manual shaking gives only slightly different results than a shaker for the same size and filling of tubes. Table 2 shows the results from experiments in 500 ml bottles. Out of a batch of six bottles, one is mixed by hand while the others are shaken mechanically. A sample was taken for each type of mixing and HPLC analysis showed that they were very similar, both giving high purity, confirming that the type of shaking is not critical, but that the size of the bottle used is very important, and that 500 ml bottles are better than 50 ml tubes. ml for the same shaker mixing time, this is also illustrated by the data in Figure 22.

TABLE 2

Effect of different types of mixing on the Hep A/impurity ratio (2 minutes)

[image]

The filling level of the test tubes was also taken into consideration. 50 ml tubes filled to the level of 10.5 ml (solvent + water) give very similar results to a typical filling of 21 ml. similarly, 500 ml bottles filled with 240 or 275 ml show equivalent amounts of impurity removal. (Figure 21). During all mixing experiments, 15 ml tubes are filled to either 0.13 or 0.4 of their full capacity. For 50 ml tubes, most were filled to 0.53 capacity (with one experiment at 0.26), and 500 ml bottles were filled between 0.48 and 0.57 of full capacity. Since similar levels were used for the three types of test tubes and the results obtained could not be related to the filling level, it probably does not have a strong effect in this area. It is likely, however, that if the tubes are completely filled, there will be no headspace and it will be difficult to mix them. As a result, this will reduce the efficiency of removing impurities.

Another variable factor that can be important is the width to height ratio of the test tubes: 0.13 for 15 ml test tubes, 0.26 for 50 ml test tubes and 0.58 for 500 ml bottles. It is likely that a larger width-to-height ratio is beneficial for mixing since the resulting shaking model results in a larger interfacial area.

Data from earlier semi-propellant series with the same impurity present in the PEG pellets were also examined. Four batches were extracted with the solvent for 1 minute in one bottle equipped with a magnetic stirrer or a rotary stirrer. The solution was then distributed into centrifuge bottles for phase separation by centrifugation. The ratio of solvent to water was 1.5. The obtained ratios of Hep A to impurities ranged from 0.48 to 2.7. An additional half-run batch was shaken in 250 ml conical centrifuge bottles for 1 minute with a solvent to water ratio of 1. The purity ratio (Hep A/impurities) obtained was 0.87. All of these results are comparable to those obtained in 15 or 50 ml tubes under similar conditions, but are much lower than the typical areas of 4 to 21 (for 8 lots) obtained in full-scale demonstration runs. This is consistent with the longer mixing times and larger bottles used on a production scale.

Conclusion

We can conclude from this data that the difference in purity between the full-scale demonstration batches and the earlier semi-powered batches is that the shaking is done in larger (500 ml) bottles and for a longer time (2 minutes vs. 1 minute). The volume effect is probably due to the larger interfacial area created by shaking in these bottles. 500 ml bottles are typically filled to a level of less than 300 ml, resulting in a large head area that is filled during mixing, resulting in a significant solvent/water/air interfacial area. It is likely that the reduced interfacial area may be responsible for the lower purity of the solvent-extracted product obtained in small tubes. However, since the mixing time is an important variable, the purity in the 50 ml tubes eventually reaches the same level as in the 500 ml bottles.

Other variables such as test, solvent to water ratio and type of shaking were not found to be important in the study area.

EXAMPLE 16

Solubility of hepatitis A virus

As described earlier, significant loss of hepatitis A was observed based on HPSEC analysis of process streams from productive batches. During purification, it was determined using HPSEC quantification that more than 50% of the virus was lost during the night between the purification steps by ion-exchange chromatography and size-exclusion chromatography. Simultaneously with this loss was the appearance of a sediment that is removed by centrifugation.

It was hypothesized that the precipitate was hepatitis A and that the precipitation was caused by the high ionic strength of the buffer used to elute the virus from the ion-exchange column. Attempts were made to dissolve the precipitate with an aqueous solution of sodium chloride and sodium phosphate. Based on the loss of virus using HPSEC, the quantitative determination of the concentration of the precipitate (actually sludge sludge) was determined to be several mg/ml, aliquots of the sludge sludge were mixed bolo with 6 mM sodium phosphate, 0.15 N sodium chloride in 6 mM sodium phosphate, 0.3 N sodium chloride in 6 mM sodium phosphate, 0.5 N sodium chloride in 6 mM sodium phosphate, or 1 N sodium chloride in 6 mM sodium phosphate.

When mixed with even a small amount of 6 mM sodium phosphate, the precipitate dissolves within seconds. However, when mixed with a solution containing sodium chloride no major difference in appearance (apart from obvious dilution) was observed. The raw material solution for obtaining the raw material solution for hepatitis A is obtained by dissolving 0.35 ml of the suspension with 5.65 ml of 6 mM sodium phosphate. It was observed that the complete dissolution of the visible particles occurred after the addition of only about 0.5 ml of sodium phosphate solvent. Raw solvent samples were mixed with varying amounts of 1 N sodium chloride in 6 mM sodium phosphate to achieve target concentrations of 0.15 N, 0.3 N, or 0.5 N sodium chloride (Table I).

TABLE 16-I

[image]

The concentrations of these solvents were then quantified using HPSEC and then quantified two weeks later using HPSEC. These data are presented in Table II and plotted in Figures 27 and 28.

Figure 27 shows the initial hepatitis A concentrations measured in the solvent approximately one day after salt addition. Using HPSEC, it was determined that the raw material solvent has a concentration of about 100 mg/ml, which corresponds to about 2 mg/ml in the settled sludge. (The amount of hepatitis A in this sludge then roughly corresponds to that loss observed during production. Since the precipitate was observed to dissolve with only a small amount of phosphate solvent added, the solubility in low-salt solvents was determined to be greater than or equal to about 1 mg /ml). The concentration of antigen in the solvents containing sodium chloride was much lower than that suitable for diluting the stock solution with saline. In addition, HPSEC analysis of the solvents containing the salt solvent shows that the aggregated material is present in large proportions in those solvents containing the salt (Table II).

TABLE 16-II

HPSEC RESULTS FOR HEPATITIS A IN SALINE SOLVENTS

(DAY 1)

[image]

This shows that hepatitis A aggregates and then precipitates out of solution even at concentrations below 50 mog/ml when in saline. Figure 27 also shows the approximate composition of the product eluted from the ion-exchange column (represented as the IEP point). It is significantly above the level of antigen that can be maintained in the solvent for even one day, and without significant changes in the purification process, it is necessary to dilute the product immediately after it is eluted from the ion-exchange column. To minimize dilution, and to minimize the volume that will then be applied to the preparative size exclusion column, dilution should be made with 6 mM sodium phosphate to maintain nominal pH control while reducing ionic strength. and antigen concentration. A one to one dilution was determined to reduce the antigen concentration to a point that was below the curve in Figure 27 represented by IEP* at about 25 mg/ml and 150 mM sodium chloride in phosphate. Based on this data, dilution is included in the purification process.

Figure 28 shows the concentrations measured in the solvents described on day 1 and day 14. It is apparent that the antigen concentrations fell over time between day 1 and day 14, and that the one-to-one dilutions do not dilute the antigen below the solubility curve, but merely stabilize the virus solution. and serves to slow down the attack of aggregation and deposition. It is possible that further optimization of the dilution will lead to further improvement in product yield.

Finally, as a check that the stock solution contains hepatitis A and not some other material that will co-elute on HPSEC with the solvent, the stock is subjected to SDS-PAGE and silver staining. When compared to the SEC product, the same protein bands are clear.

EXAMPLE 17

HIGH RESOLUTION SIZE EXCLUSION CHROMATOGRAPHY FOR REGISTRATION OF HEPATITIS A PURIFICATION SAMPLE

Introduction

A procedure was developed to characterize and record production performance during hepatitis A purification. Analytical High Resolution Size Exclusion Chromatography (HPSEC). (High Performance Size Exclusion Chromatography) with UV detection at 214 nm and 260 nm provides analysis of relative amounts of hepatitis A virus and key impurities (aggregates, nucleic acid adducts, hepatitis A and 660 kDa impurities) in production samples. In addition, HPSEC was used to quantitatively determine the hepatitis A concentration in samples from the last 3 purification steps (extraction, ion exchange and preparative SEC). This analysis was used to record the hydrolysis performance of BENZONASE and to provide a footprint of the Hep A impurity profile at each production step for post-process references.

Hepatitis A process samples from 10 production runs were analyzed using HPSEC to provide process performance data for all purification steps. Data from these assays together with additional stability studies provide both confirmation of the HPSEC assay and a clear indication of the consistency of the hepatitis A procedure.

This Example aggregates both the confirmation data of the HPSEC analysis and the results of the analysis of 10 production batches. The report is organized into the following sections:

SECTION I Instrumentation and chromatographic conditions

Description of the HPSEC column and HPLC instrumentation i

work conditions

SECTION II Analysis and calibration procedures

Description of obtaining calibration standards and analysis procedures

SECTION III Linearity, accuracy and reproducibility

statistical determination

SECTION IV Analysis of results

A set of chromatograms from one batch is shown, along with

by describing the type of data collected for each production sample

and recommended caution limit

SECTION V Sample storage conditions

Storage studies for each type of production sample are

performed to determine stability at 4-6 C

SECTION VI Comparison of EIA and HPSEC results

SECTION VII Process performance consistency

APPENDIX A Results of HPSEC analysis of process samples from 10 batches

SECTION I Instrumentation and chromatographic conditions

Materials and reagents

CM 80 (MMD) phosphate buffered saline containing 6 mM sodium phosphate 120 mM sodium chloride; pH 7.2

Instrumentation

(An equivalent replacement can be made except with a column)

HPLC System; RAININ Rabbit pump equipped with a 10 ml salt washing head

Auto sampling device:

RAININ AI-1 is equipped with a recirculation cooler to maintain the sample at 2-6 C

Detector: RAININ UV-M two-channel UV detector

Data acquisition system: Dynamax HPLC Method Manager,

Version 1.1 runs on the Apple Macintosh SE

Column: TosoHaas TSK PW4000x1, 7.8 x 300 mm L

HPLC microampules - glass or polypropylene.

Chromatographic conditions

Column: TosoHaas TSK PW4000x1, 7.8 x 300 mm L

Mobile phase: CM 80 (6 mM sodium phosphate 120 mM sodium chloride, pH 7.2)

Flow rate: 0.32 ml/min

Detection: UV 214 nm and 260 nm S IV detector output

Injection volume: 50 ul

Time between injections: 55 minutes

TSK PW4000x1 column

The column used is TSK PW4000x1, which contains a substrate based on 5 um methacrylate. A calibration curve obtained from polyethylene oxide (PEO) standards is shown below. Dotted lines show the retention times of the 3 dissolved materials of interest; hepatitis A aggregates, Hepatitis A, and 660 kDa contaminant. The dotted lines also show the retention times of 2 low molecular weight hydrophobic compounds, vitamin B12 and acetone. Both compounds are on the right side of the calibration curve; the retention of these compounds shows the hydrophobic nature of the polymer substrate. Retained peaks are seen in several process samples (see Figure 29). They mostly elute near the salt peak and are assumed to be low molecular weight compounds.

SECTION II Analysis and calibration procedures

Obtaining calibration standards

A six-level calibration curve was used for the quantitative determination of hepatitis A. Calibration standards are obtained by diluting the purified hepatitis A product. The SEC product with a concentration of 12 µg/ml was used as a standard. Six calibration standards covering the concentration range of 0.75 to 12 ug/ml are obtained by diluting the SEC product with CM80 as shown below.

[image]

Standards are obtained by adding hepatitis A solution and CMBO directly to HPLC microampules. The calibration curve for hepatitis A is obtained by plotting log hepatitis A peak area against log concentration. A typical calibration curve is shown in Figure 30.

Log-log transformation of the calibration curve data was performed to reduce the dependence of the calibration curve on high concentrations of the calibration standard. For chromatography of biological macromolecules, data variability tends to increase as concentrations increase. For HPLC calibration curves, the slope of the regression line is therefore primarily determined using the highest concentration standard, which is also the most variable.

Plots of 13 hepatitis A calibration curves (obtained as described above over a 2-month period) are shown in Figure 31. On the left is a linear plot of the 13 curves, and on the right is a log-log transformation of the same 13 curves. The linear plot shows a marked "flaring" of the curves from the common origin, clearly demonstrating the increase in variant hepatitis A peak areas as the concentration increases. The result is a variant of the intercurve in slope values.

log-log transformation of these calibration curves makes the variability (RSD) constant over the entire concentration range. The result is a reduced intercurve of slope variation, as shown in Figure 31B.

Procedures for obtaining a sample

A. Production samples of hepatitis A:

Production samples from the following stages were analyzed:

1. Filtered lysate

2. Nuclease-treated filtered lysate

3. PEG precipitate resuspended

4. Chloroform extracted aqueous phase

5. Anion exchange product

6. Exclusive product by size

Filtered lysate, nuclease-treated lysate, and size-exclusion product samples do not need to be diluted prior to injection. A sample of the resuspended PEG pellet should be centrifuged (10,000 x g for 5 minutes) to remove any remaining particles, prior to injection. A sample of the resuspended PEG pellet is diluted 1:4 (v/v) with CM80 prior to injection. The chloroform-extracted aqueous phase and the anion exchange product are analyzed undiluted and analyzed diluted 1:1 (zap/zap) with CM80 before injection to obtain a concentration within the calibration curve. For all samples, the analysis is performed in triplicate and the results from the three analyzes are taken as an average. Samples are stored in an auto sampler at 2-6°C while awaiting injection.

Triton is poorly retained on the TSK PW4000 x 1 column, and additional eluate is required to remove it. Therefore, samples containing Triton (filtered lysate and nuclease-treated filtered lysate) should be injected with a CM80 blank to ensure removal prior to injection of other samples. Samples can be run in reverse order starting with the SEC product to prevent late elution of interfering peaks.

Analysis procedure

1. Inject 50 ul of each of the six calibration standards. The retention time of hepatitis A should be 19.0 +/- 1 minute.

2. Prepare the samples as described above. 50 μl is injected. Each sample is analyzed in triplicate.

SECTION III Linearity, accuracy and reproducibility

Thirteen calibration curves covering the range from 0.75 to 12.0 μg/ml were obtained on 13 different days over a period of 5 weeks. The regression equation obtained for each curve was used to calculate the concentration of each of the calibration standards comprising that curve. The results calculated from the 13 curves were compared with the nominal concentrations for these 6 calibration standards as shown below in Table 1.

TABLE 17-1

Comparison of measured according to nominal concentrations of pure

of hepatitis A. Calculation based on the regression line

NOMINAL CONCENTRATIONS OF HEPATITIS A

CALIBRATION STANDARDS

calibration

curve

[image]

Between-day reproducibility is very good for all six calibration levels, with RSD values less than 3% over the entire concentration range. Accuracy is also very good; for 12, 9, 6, 3, 1.5 ug/ml and 0.75 ug/ml standards, all measured concentrations were within 5% of the nominal concentration.

A peak labeling study

To further determine the specificity and accuracy of the HPSEC analysis, a peak labeling experiment was performed.

The anion exchange product and the chloroform-extracted aqueous phase are labeled with an equal volume of SEC product from the same batch. The results are shown in Table 2 below. All samples were analyzed in triplicate.

TABLE 17-2

Regeneration of hepatitis A labeled in production samples

[image]

For both samples, complete recovery was obtained, demonstrating specificity and accuracy by HPSEC concentration determination.

SECTION IV Analysis of results

Excerpt

As a series, HPSEC chromatograms provide a snapshot of the hepatitis A purification process. Comparison of filtered lysate to nuclease-treated lysate provides confirmation of BENZONASE activity. For these samples, chromatograms were obtained at both 214 nm and 260 nm and integrated.

Comparing the last 4 production samples, PEG pellets resuspended, chloroform extracted aqueous phase, anion exchange product and SEC product show release of 2 major production contaminants; aggregated material 660 kDa protein contaminant 660kDa. Calculating the percent surface area of hepatitis A for these samples provides confirmation that each stage of the procedure is working as intended to remove these contaminants. Finally, HPSEC analysis is used to calculate the concentration of hepatitis A in the final 3 stages of the procedure.

Samples of filtered lysate, nuclease-treated lysate, resuspended PEG pellets, chloroform-extracted aqueous phase, anion exchange product and SEC product from 10 batches of hepatitis A produced at MMD were analyzed using HPSEC. The chromatographic profile for each procedure sample was highly reproducible throughout these 10 runs.

In this section, a chromatogram for each of the 6 production samples is presented, along with a description of the information obtained for each sample, and typical triplicate analysis results.

Filtered lysate and nuclease-treated lysate

Chromatograms of samples of filtered lysate and nuclease-treated lysate were obtained at wavelengths of 214 nm and 260 nm. For each of these samples, the chromatographic profile obtained at 214 nm is completely different from the profile obtained at 260 nm. Comparative analysis of these chromatograms reflects the purpose of the HPSEC analysis of these samples: to find a quantifiable difference between the filtered lysate and the nuclease-treated lysate that will indicate the completeness of BENZONASE hydrolysis. Therefore, in this section, the profiles of filtered lysate and nuclease-treated lysate are compared; first at 214 nm, and then the same comparison was made at 260 nm. Characteristic data obtained for each sample are presented, as well as a recommendation for the quantitative determination of the completeness of BENZONASE activity.

Filtered lysate and nuclease-treated lysate at 214 nm

Chromatograms of the filtered lysate and the nuclease-treated lysate at 214 nm are shown in Figure 32. At 214 nm, both samples show a series of partially resolved peaks. Due to the low resolution obtained for these samples, integration of individual peaks in the hepatitis A region is impossible; the only information obtained from these samples at 214 nm is the total UV area for all peaks. The results of the triplicate analysis of the samples are also shown in Figure 32. This total UV214 nm area does not include the retained peak at 36 minutes. The total UV214 nm area of the nuclease-treated lysate sample is 15% lower than that of the filtered lysate sample as a result of BENZONAS hydrolysis; as a visual comparison of the 2 chromatograms (Figure 32) shows a loss of absorption in the hepatitis A region. However, a defined measure of this hydrolysis is not possible since the low resolution of the peaks involved. The total UV214 nm area was obtained for 9 batches, these values are given in Appendix A. These total UV214 nm areas do not provide any direct or unique process performance information, their use was only to register the stability of the sample, therefore the calculation of the total UV214 area is not necessary for routine publication.

(A) FILTERED LYSATE

Filtered lysate Total UV surface 214 nm (uV)

1 27,248,238

2 26,404,744

3 26,782,947

Average 26,811,976

Apartment. Dev. 344,966

(B) NUCLEASE TREATED LYSATE

Nuclease-treated lysate Total UV surface 214 nm (uV)

1 22,728,263

2 22,841,564

3 22,784,419

Average 22,784,749

Apartment. Dev. 46,256

Filtered lysate and nuclease-treated lysate at 260 nm

Chromatograms of the filtered lysate and the nuclease-treated lysate at 260 nm are shown in Figure 33. At 260 nm, the filtered lysate shows a more specific profile, with up to 6 separated peaks. The first two peaks, with retention times of 17.2 and 19.1 minutes, comprise the hepatitis A-nucleic acid adduct and hepatitis A. Since both peaks have some degree of nucleic acid associated with them, they dominate the 260 nm HPSEC profile.

Comparing the filtered lysate sample with the nuclease-treated lysate at 260 nm (Figures 33A and 33B) provided a clear and unequivocal confirmation of BENZONASE activity. The prominent peaks evident in the filtered lysate sample were completely removed by hydrolysis with BENZONASON, Figure 33B shows no discernible peaks at 17 and 19 minutes.

Confirmation of hydrolysis with BENZONAS

Analysis of filtered lysate and nuclease-treated lysate samples from 9 batches is shown below in Table 17-3. The UV260 nm area for the 17 and 19 minute peaks for these samples shows that the addition of BENZONASE leads to the complete removal of these peaks for all tested batches. Therefore, for this sample the recommended criterion for determining BENZONASE activity will be a reduction of the 17+19 minute UV260 nm peak in the nuclease-treated lysate to less than 10% of that in the filtered lysate.

TABLE 17-3

Reduction of UV260nm surface using BENZONASE for 9 series.

FILTERED NUCLEASE TREATED

LYSATE (A) LYSATE (B) REMAINS

SURFACE. 17+19 min Pikovi SURFACE 17-19 min Pikovi A260

Series # (260 nm) (260 nm) (B/A X 100)

984 1,240,987 0 0

985 947,988 0 0

1047 1,085,719 0 0

102 708,663 0 0

101 831,179 0 0

108 1,060,441 0 0

100 895.005 0 0

139 1,377,974 0 0

236 1,281,608 0 0

Average = 1,018,495 0 0

Stan Dev = 205,034

FIGURE 33 (A) FILTER LYZATE

Filtered Area 17+ 19 min peaks (UV) Total UV Area i(UV) 260 nm lysate

1 1,192,597 3,193,055

2 1,259,216 3,034,891

3 1,271,147 3,094,820

Average 1,240,987 Average 3,107,589

StanDev 34,562 StanDev 65,198

FIGURE 33 (B) LYZATE TREATED WITH NUCLEASE

Nuclease-treated lysate Surface 17+ 19 min peaks (UV)

1 0

2 0

3 0

______________

Average and StanDev =0

Resuspended PEG pellet sample

Below in Figure 34 is shown the chromatogram of the resuspended PEG pellet sample at 214 nm. The characteristic profile of this sample consists of several partially separated peaks: aggregated material; hepatitis A peak, 660 kDa contaminant, other low molecular weight contaminants, and salt peak.

The percent area of hepatitis A is calculated for this sample (Hep A peak area) total peak area with 100) total area does not include the salt peak. The results for the triplicate analysis of the resuspended PEG pellet sample are shown below.

Suspend again. PEG pellets: Hep A surface area percentage

1 25.9

2 27.0

3 27.4

_________

Average 26.8

Flat. Dev. 0.7

A typical profile for this sample has the 660 kDa contaminant as the major component, with the amount of aggregates being the most variable of the components.

The amount of aggregate in a typical batch is lower than what is seen. The results of HPSEC analysis of resuspended PEG pellet samples from 10 batches are presented in Table A2 of Appendix A. The values of the hepatitis A surface percentage range from 18.5% to 32.0%; all of which were successfully purified by the following production steps.

Therefore, a strict precautionary limit is not required for this sample; certain area percentage values greater than 18% are acceptable, however the effect of having lower area percentage values will not be known until they are encountered in the hepatitis A procedure.

A sample of the aqueous phase extracted with chloroform

The chromatogram for a sample of the chloroform-extracted aqueous phase is shown below in Example 35. The hepatitis A peak is the major peak in this sample (excluding the salt peak) and is well separated from the 660 kDa contaminant in small aggregates.

For this sample, the concentration of hepatitis A was determined and the area percentage of hepatitis A was calculated. The calculation of the area percentage does not include the salt peak. The results of a triplicate analysis of a sample of the chloroform-extracted aqueous phase are shown below.

Chloroform extr. Concentration of hepatitis A Percentage of surv. hepa-

water phase ug/ml titis A

1 28.64 61.0

2 29.76 62.8

3 30.65 61.2

_______________ __________________

Average 29.68 Average 61.7

Flat. Dev. 0.82 Stan.Dev. 0.8

The results of the HPSEC analysis of samples of the chloroform-extracted aqueous phase from 10 batches are shown in Table A2 of Appendix A. The values of the hepatitis A surface percentage for the full batches ranged from 60.0 to 72.8%; the two half-series were significantly higher, with surface percentage values of 79.4 and 85.2, respectively. Hepatitis concentrations were more variable, ranging from 14.4 to 29.7 ug/ml. Variability in hepatitis A concentrations may be related to variation in the volumes of samples that flow up through consistent batches.

Comparing the HPSEC profile of the PEG pellet (Figure 34) to the chloroform-extracted aqueous phase (Figure 35) provides a clear indication of the importance of this step to the hepatitis A process; this step selectively removes large amounts of the 660 kDa contaminant without significant loss of hepatitis a yield. Although the 660 kDa contaminant can be removed in two subsequent chromatography steps, in both cases if the 660 kDa is a major component of the nutrient solvents, a peak reduction strategy is required which will lead to to a decrease in the yield of hepatitis a. In addition, extensive optimization experiments have shown that the extraction efficiency is quite sensitive to changes in mixing times (see Example 15). The value of the hepatitis A surface percentage for the chloroform-extracted phase provides quantitative verification during the procedure of successful extraction and removal of impurities.

Therefore, a cautious limit of >60% hepatitis A surface area percentage value for the chloroform-extracted aqueous phase sample is recommended. Area percentage values below 60% should be considered potentially indicative of process problems (such as those with process mixing apparatus), especially if this occurs regularly.

Anion exchange product

Figure 36 shows a sample of the anion exchange product. The hepatitis A peak is the major component of this sample with small amounts of aggregates, the 660 kDa contaminant, and low molecular weight contaminants also present.

For this sample, the hepatitis A concentration was determined, as well as the surface area percentage of hepatitis A, excluding the salt peak and the retained peak. The results of the triplicate analysis for the anion exchange product sample from Rx2009854 are shown below.

Anionic product Concentration Percentage of area

changes in hepatitis A hepatitis

(ug/ml)

___________________ ______________ __________________

1 31.98 82.8

2 32.63 81.4

3 32.37 81.6

____________ _____________

Average 32.32 Average 81.9

Stan.Dev 0.27 Stan.Dev 0.6

The results of the HPSEC analysis of a sample of anion exchange products from 10 batches are shown in Table A2 of Appendix A. The values of percentage area obtained range from 78.6 to 87.4% for full batches, with values for half batches greater than 90%. The increased area percentage values found for the half-batch are not surprising given the increased purity of the feedstock.

Concentration values increased significantly (over 2 times) for these series; this variation, as well as the one observed in the extraction degree, is probably due to the variation in sampling for these batches.

In the hepatitis A procedure, anion exchange chromatography functions as the final finishing step; it removes some 660 kDa contaminants and other low molecular weight compounds, but the purity of the anion exchange product depends on the purity of the feed to this stage, this stage cannot remove large amounts of 660 kDa without loss of yield due to peak reduction. That is why this stage cannot compensate for errors in the previous processing. Typically the surface area percentage of hepatitis A is increased by 10-20% through the anion exchange chromatography step, the higher the purity of the feed, the higher the purity of the resulting product. For this sample, it is recommended that the cautious limit be area percentage values of >75% with an increase in area percentage of > 10% through this stage. When both criteria are met, the anion exchange process and stage both function as disclosed. Failure to achieve a 75% surface area value is in itself possible for an anion exchange stage if previous stages fail, failure to achieve an area percentage increase of > 12% is likely to indicate a problem in the process very specific to the anion exchange stage, such as is the formation of ridges, dysfunction or formation of channels in the column.

Product of size exclusion chromatography (SEC)

The profile of the SEC product shows highly purified hepatitis A as shown in Figure 37 for the SEC product.

For this sample, the concentration of hepatitis A is determined. The results of the double analysis of the SEC product are shown below.

Sample analysis Hepatitis A concentration

(ug/ml)

________________ ______________________________

1 17.34

2 16.34

______

Average 16.84

Flat. Dev. 0.50

The results of the HPSEC analysis of SEC products from 10 batches are shown in Table A2 of Appendix A. For this sample, no area percentage values were calculated. The profile shown in Figure 37 clearly shows a product of high purity, with no detectable amounts of aggregates and 660 kDa contaminants.

However, SEC analysis of the product over a 60 minute period showed the presence of a small retained peak at about 49 minutes as shown in Figure 38. The size of this peak varies from batch to batch, with the batch shown (Figure 38) having the largest amount of observed data. Identification of this retained peak should be performed before calculating the percent area value for this sample, which is indicative of its purity. While this identification is being made, the percent area values for this sample will only include peaks between the excluded and included volumes (retention times of 16-32 minutes). With these restrictions, the area percentage values for all tested series were 100%.

SECTION V Sample stability

Process samples were shown to be stable for up to several months as measured by HPSEC and EIA. Small losses can be observed in the degree of ion exchange, generally less than 10% over a period of two weeks. However, after this initial decline, even these samples remained stable. In addition, the final SEC product is stable for periods of one year.

SECTION VI Comparison of EIA and HPSEC results

EIA and HPSEC results obtained for 3 samples (chloroform-extracted aqueous phase, anion exchange product and SEC product) from 11 batches (batch 2 to 12) were compared.

A plot of log HPSEC concentration plotted against log EIA concentration is shown for these 33 samples below in Figure 45. The interpolated regression line for these data has a slope of 0.88 and a correlation coefficient (r) of 0.94. The value of the slope approaches 1, and the r value of 0.94 indicates that a very good correlation exists between these two analyses, especially considering the inherent variability of each measurement.

SECTION VII Maintenance of Procedure Consistency

HPSEC analysis of batches 2 to 14 reveals a very stable and reproducible process. Based on extensive HPSEC analyzes of in-process samples taken from production batches, the purification process provides excellent reproducibility for the removal of major contaminants. Figure 46 provides one measure of this, where the area % of monomeric virus is applied for batches. This measure of stream composition for the final stages of purification shows great consistency across these batches. Series 10 and 11, which show higher purity at the extraction and anion exchange stages, are both half-size series.

EXAMPLE 17; APPENDIX A

TABLE 17-A1

Data overview for lysate and nuclease-treated lysate samples. All samples were analyzed in triplicate, and the results of the three analyzes were averaged.

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APPENDIX A (CONTINUED)

TABLE 17-A2

Data overview for samples of resuspended PEG pellets of chloroform-extracted aqueous phase, anion exchange product, and SEC product. All samples were analyzed in triplicate, and the results of the three analyzes were averaged.

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EXAMPLE 18

RESULTS OF THE KLIMC EXAMINATION

The purified vaccine according to this invention was applied to healthy people. No serious adverse effects attributable to the vaccine have been identified.

The efficacy of a single dose of 25 units of vaccine was demonstrated in children in the Monroe study, the results of which were published in the August 13, 1992 issue of the New England Journal of Medicine, /Werzberger et al., NEJM 327 (no. 7 ); 453-457, (August 13, 1992)/. The differences between the Monroe study material and the production material, both of which form part of this invention, are highlighted in Figure I, and both are contrasted with the Phase I/II studies, material produced in rolling bottles and collected by mechanical means, with the present limitations in the scale of production contained in this procedure. One dose of 25 units (25 ng of HAV protein) of the purified hepatitis A virus preparation according to the present invention is sufficient to achieve over 95% seroconversion in children and adolescents 2-16 years of age within 4 weeks of immunization. This same dose provides 100% protection in children and adolescents starting 21 days after administration of one dose of the vaccine.

Weight and age have now been identified as responsible for variability in adults. One month after a single dose of 25 units, 83% of adults (≥ 18 years) and 97% of children and adolescents (2 to 17 years) seroconverted. Two doses of 25 units given two weeks apart increased the seroconversion rate in adults to 93%. Seroconversion seven months after the second or third dose of 25 units is greater than 99% seroconversion in both age groups. Persistence in seropositivity is about 100% up to 12 months and 36 months in both age groups. Therefore, it is clear that a single dose of 25 units is sufficient to achieve seroconversion in at least 80% of any recipient population within one month of immunization. These data are given in Table 18-1 (treatment regimen indicates a dose of 25 units given at 0 and 24 weeks or 0, 2, and 24 weeks: GMT = geometric mean anti-HAV antibody titer). Immunization with higher doses induces seroconversion in a higher percentage of recipients.

Table 18-1

Seroconversion at the indicated week after

initial immunizations

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In addition to the above data, one-tenth of the patients (cohort) found to be seronegative at 4 weeks (23 cases in total) were retested at 24 weeks and elicited an anamnestic response (> 10-fold in antibody titer) at 28 weeks. Two individuals showed an approximately six-fold increase in titer after six months of dosing. The remaining 21 per unit, 91% showed seroconversion and anamnestic response. Therefore, these individuals are permanent seroconverters and are likely to be protected by immunization with the vaccine of this invention, even when they are clearly seronegative for an extended period after immunization.

Claims (23)

1. A process for producing hepatitis A virus on a commercial scale with greater than 95% purity with respect to protein by SDS polyacrylamide gel electrophoresis and silver staining analysis, comprising the steps of: a) culturing hepatitis A virus in large quantities in cell layers in a static surface reactor with a large surface area (SSR); b) removing the hepatitis A virus from the cell culture layers using detergent permentation of the infected cell layers, so that the hepatitis A virus is released from the cell culture; c) optimal removal of non-hepatitis A virus specific nucleic acids at this stage from the collected hepatitis A virus, using nuclease treatment; d) concentrating the hepatitis A virus removed from the cell culture, using membranes and diafiltration, or binding using ion exchange; e) removal of non-hepatitis A virus specific proteins from the concentrated hepatitis A virus of step (b) by a combination of organic extraction, PEG precipitation, ion exchange, including removal of contaminating free nucleic acid if not previously completed in step c), and a gel permeation chromatography step ; and f) regeneration of the purified hepatitis A virus from step (e). 2. Purified hepatitis A virus, indicated by the fact that it was obtained by the process according to claim 1. 3. The method according to claim 1, characterized in that step a) comprises the inoculation of virus A obtained from the upper liquid layer of HAV infected with SSR into a reactor with a static surface in which the cells are cultivated in layers of cells, and the culture medium is constantly circulated through the reactor with a static surface and an opening in which control of the reactor, saturation and replenishment of culture components is achieved. 4. The method of claim 1, wherein step b) comprises harvesting the hepatitis A virus by treating the hepatitis A virus-infected cell layers with between about 0.05% to 1% Triton-X 100 solution to release the hepatitis A virus. 5. The process according to claim 1, characterized in that step c) comprises hydrolysis with BENZONAS. 6. A process for the production on a commercial scale of an inactivated vaccine for the hepatitis A virus, which is more than 95% pure HAV, on a protein basis, characterized by the fact that it comprises a) culturing large amounts of hepatitis A virus (HAV) in NUNC CELL FACTORIES, COSTAR CUBES or STATIC SURFACE REACTORS (SSRa), including SSR comprising regular elements on which cells are capable of growing at high densities per unit volume, in which it has been established cell layer of MRC-5 cells; b) collecting cultured HAV by removing the culture medium and adding a collection solution containing 0.1% Triton X-100; c) treating the collected HAV with BENZONAS to hydrolyze contaminating nucleic acids; d) concentrating BENZONAS treated HAV by binding to an ion exchange column and elution of the bound HAV with about 0.35 M NaCl, followed by precipitation with polyethylene glycol; e) resuspending the PEG-precipitated HAV and extracting the concentrated HAV with chloroform/isoamyl alcohol (24:1 v/v) with vigorous stirring, and separating the remaining aqueous phase; f) chromatographing the aqueous phase from the organic extraction on a DEAE TOYOPEARL 650 M anion exchange matrix at about 90 to 150 mM NaCl, and eluting HAV with a gradient up to about 1 M NaCl, and diluting the collected HAV fractions to a salt concentration below about 150 mM and a HAV concentration below about 20 μg/ml so that HAV deposition is minimized or eliminated; g) collection of HAV peak fractions from step f) and HAV chromatography on tandem TOYOPEARL HW55S and HW65S size exclusion columns; h) inactivation of gel-filtered HAV by treatment with formalin at a concentration of 1/1000 for five days, followed by sterile filtration, absorption with or co-precipitation with aluminum hydroxide, and distribution into unit doses, equivalent to 25-100 ng/dose of inactivated purified HAV . 7. A method for preventing HAV infection, indicated by the fact that it includes immunization with an immunologically effective amount of the product according to the method from claim 6. 8. HAV vaccine, characterized by the fact that it is produced according to the process of claim 6. 9. A process for cultivating HAV, comprising growing HAV in a COSTAR CUBE or STATIC SURFACE REACTOR, optionally including regular titanium or stainless steel mesh elements. 10. The method of claim 9, comprising producing between about 5,000 and 50,000 doses of 25 ng inactivated HAV greater than 95% pure HAV per vaccine dose, from a single COSTAR cube culture vessel. 11. Purified preparation of hepatitis A virus, characterized in that more than 95% of the protein is specific for hepatitis A virus, and less than 7% of the protein mass of the preparation is non-HAV nucleic acid or lipid and between about 0-180% of the preparation is a carbohydrate consisting of glucose. 12. The preparation according to claim 11, characterized in that the hepatitis A virus is inactivated by formalin. 13. The preparation according to claim A, characterized in that it is formulated as a 25-100 ng dose of protein-based HAV with about 100-1000 ug aluminum hydroxide. 14. Purified preparation of hepatitis A virus, indicated where each characteristic is listed by ug of protein base, and the procedure in parentheses was used to determine the characteristics: a) lipid content (gas chromatography): < 0.1 μg; b) hydrocarbon detected as glucose: 0.1-2.5 μg; non-glucose carbohydrate < 0.1 μg; (TFA hydrolysis, Dionex) c) specific protein for hepatitis A virus > 0.95 μg; (amino acid analysis and "western blot") d) specific RNA for hepatitis A virus 0.1-0.2 μg; e) contaminating DNA < 0.001 μg. 15. The preparation according to claim 14, characterized in that the hepatitis A virus is activated by formalin. 16. The preparation according to claim 15, characterized in that it is absorbed on aluminum hydroxide. 17. Purified preparation of inactivated hepatitis A virus, characterized in that one dose of 25 units, equivalent to about 25 ng of hepatitis A virus protein based on amino acid analysis, is sufficient to achieve 95% or greater seroconversion in children and adolescents 2- 16 years of age within 4 weeks of immunization. 18. A purified preparation of inactivated hepatitis A virus, characterized in that one or three doses of 25 to 100 units, equivalent to about 25 ng to 100 ng of hepatitis A virus based on ammonium acid analysis, are sufficient to achieve 80-95% seroconversion in recipient population within 4-28 weeks of immunization. 19. The method of claim 1, comprising culturing hepatitis A virus in a static surface reactor, and suitable culture medium is replaced and removed at a rate of about 0.010 and 0.3 ml/cm2/day. 20. The method according to claim 1, characterized in that it comprises the culture of the hepatitis A virus in a reactor with a stationary surface, and the culture medium is changed and removed at a constant rate. 21. Hepatitis A virus vaccine, characterized by having the following nominal composition per dose of 25-200 units/0.1-1 ml: [image] 22. Hepatitis A virus vaccine, characterized by having the following nominal composition per dose of 25 units/0.5 ml: [image] 23. A purified preparation of inactivated hepatitis A virus, characterized in that an iodine dose of 25 units, equivalent to 25 ng of hepatitis A virus protein based on amino acid analysis, is sufficient to achieve 95% or greater protection in children and adolescents 2-16 years of age already after 21 days after immunization.