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We report here results of in vitro comparisons of the Fc functional activity of a second-generation intravenous immunoglobulin (IGIV) preparation (Venoglobulin-I) and a third-generation IGIV product that includes a deliberate virus-inactivation step (Venoglobulin-S). Both formulations showed equivalent Fc-mediated function against viral antigens (rubella, influenza A, and influenza B) by single-radial hemolysis test, and against group B Streptococcus, Staphylococcus aureus and Escherichia coli by opsonophagocytosis assay. In addition, we showed by three different immunochemical reactions and by HPLC analysis that both preparations consisted of mostly monomeric IgG and contained very low levels of complement-fixing IgG aggregates. However, IgG aggregation induced by heating at 63 degrees C markedly enhanced fixation of Clq and C3 and binding to Raji cells, indicating that the IgG molecules retained their complement-fixing capacity. Thus, the incorporation of a virus inactivation step in the manufacture of our third-generation IGIV did not alter the Fc functional activities of the IgG, as measured by these in vitro assay systems.

We report here the results of our evaluation of virus inactivation during the manufacturing steps of two intravenous immunoglobulin (IGIV) preparations. Virus inactivation and/or removal by processing steps, such as ethanol fractionation and polyethylene glycol precipitation, and deliberate virucidal steps, such as solvent/detergent treatment and pasteurization, were tested on a variety of human pathogenic and experimental model viruses, including human immunodeficiency, Hepatitis C, Mumps, Vaccinia, Chikungunya, Vesicular Stomatitis, Sindbis, and ECHO viruses. All viruses were successfully inactivated and/or eliminated by the processing steps studied. In some cases, however, multiple steps were required. We conclude that the incorporation of steps deliberately designed to inactivate or remove viruses during the production of IGIV provides an extra measure of viral safety.

Keeping the pH constant during the incubation of sample and complement solutions was an inportant factor to obtain a reproducibla ACA value. Lyophilized complement shows a pH of 8.8-9.0 after reconstitution. The pH of frozen complement is 7.2-7.4. When the ACA of an intravenous IgG (IVIG) was tested with the reconstituted complement, the ACA value was higher than when the preparation was tested with frozen complement.Ionic strength also affected the ACA test of an IVIG which did not contain ionic substances such as NaCl or glycine. Even when the pH and ionic strength of commercial IVIG preparations were standardized by dialysis, the concentration of IgG polymer did not necessarily correlate with the ACA value.

Control sera are an essential component of in-vitro clinical diagnostic reagents. We have developed a control serum for HBsAg, anti-HCV, anti-HIV and anti- Treponema pallidum testing. Since this control serum is prepared from human plasma, we paid special attention to viral safety. We incorporated three viral inactivation methods into the manufacturing process that maintain the necessary characteristics of a stable control serum.

To compensate for the hypoprotein and hypoalbuminemia of familial amyloidotic polyneuropathy (FAP) patients, 800ml of fresh frozen plasma (FFP) was intravenously administered and change in total and variant transthyretin (TTR) levels were measured in the plasma. After injection of FFP, total plasma TTR levels were elevated and variant TTR levels decreased from 24 to 48h, accompanied by an elevation of plasma total protein, albumin levels and TTR levels. To elucidate the mechanism of this phenomenon, a large amount of purified normal TTR from normal human plasma was intravenously injected in mice and FAP patients. By intravenous injection of 3mg of the purified TTR to C57Black6, the expression of TTR mRNA decreased from 6 to 24h post injection, and gradually increased up to 48h post injection. After injecting 400mg of normal TTR in each of 3 FAP patients, total plasma TTR levels were elevated and variant TTR levels decreased significantly from 24 to 48h. These results suggested that down regulation of the harmful protein by replacement of its normal form of the protein occurred by this method. This phenomenon should be applied as the basis for one of the useful methods for decreasing the harmful proteins in the circulation.

Cholesterol derivatives, including 7 alpha-hydroxycholesterol (7 alpha-HC), 7 beta-hydroxycholesterol (7 beta-HC), and 7-ketocholesterol (7-KC), were isolated from the Cohn fraction IV-I of normal human sera and were shown to be the moieties responsible for the suppression of lymphocyte proliferation. When 5-10 micrograms/ml of 7 alpha-HC, 7 beta-HC or 7-KC were added in vitro to lymphocyte cultures, incorporation of 3H-thymidine (3H-TdR) into the lymphocyte DNA was suppressed by about 50% when compared with the control. When 10-50 mg/kg/day of 7 alpha-HC, 7 beta-HC and 7 KC were administered in vivo to skin grafts on rats, the prolongation of skin grafts was observed. The concentration of 7-KC in cancer patient sera was 2 to 3 times the amount in normal pooled human sera.

A method for the heat treatment of human IgG solution at 60 degrees C for 10 h was established. Human immunodeficiency, mumps, vaccinia and 4 other viruses were added to the IgG solution in 33% sorbitol and heated at 60 degrees C. Those viruses were inactivated within 1 h. Heat-treated intravenous IgG (IVIG-H) was prepared by heat treatment and polyethylene glycol (PEG) fractionation. Conventional nonheated intravenous IgG (IVIG-C) was prepared from the same source paste by the fractionation method. No physicochemical or biological difference was observed between the heated and control IVIG preparations.

A large-scale purification method for hepatitis B surface antigen produced in a recombinant yeast (Saccharomyces cerevisiae) was established. The resulting HBsAg was greater than 99% pure and suitable for vaccine use. The yeast-derived HBsAg was structurally and biochemically similar to plasma-derived HBsAg. The anti-HBs antibody producing potency of the yeast-derived vaccine in mice was significantly higher than that of the plasma-derived vaccine. The yeast-derived vaccine induced protective antibody against hepatitis B virus of either adr or ayw subtype in a chimpanzee efficacy study. These observations demonstrate the usefulness of the yeast-derived vaccine as a second-generation hepatitis B vaccine.

Immunoglobulin samples (HIV-Ig) were prepared by cold ethanol fractionation of human plasma containing antibody against human immunodeficiency virus (HIV). The ability to prevent viral spreading was studied using either human T-cell leukemia virus type I (HTLV-I)-carrying MT-4 cells or in a coculture system using MOLT-4 cells and virus-producing MOLT-4/HIVHTLV-IIIB cells. Treatment of HIV-infected MT-4 cells with HIV-Ig effectively blocked the appearance of antigens of HIV and the virus-induced cytopathic effect. HIV-Ig blocked multinucleated giant cell formation in the MOLT-4 and MOLT-4/HIVHTLV-IIB coculture system.

More than 10(4) plaque-forming units (pfu)/ml of HIV are inactivated during the alcohol fractionation step from plasma to fraction (Fr)-II+III, greater than 10(4) pfu/ml is inactivated from Fr-II+III to Fr-II and greater than 10(4) pfu/ml is inactivated during the polyethylene glycol (PEG) fractionation process from Fr-II+III to intravenous IgG (IVIG). The total inactivation rate from plasma to IVIG via Fr-II+III or Fr-II was calculated to be greater than 10(8) or 10(12), respectively. The PEG fractionation method produces an intact and unmodified IVIG. In addition, the PEG fractionation method at a low ionic strength was found to be effective for the elimination of greater than 10(5) units of other viruses, including hepatitis B, vesicular stomatitis and Sindbis viruses.

Conditions for obtaining a large quantity of monomeric IgG which can be used for intravenous injections in humans was successfully established with human serum IgG preparation obtained using the ethanol fractionation method. When dissociation of IgG aggregates and denaturation of IgG monomers contained in the starting material were examined at various pH values, the treatment for 60 min at 28 degrees C at pH 3.8-4.0 was found to be optimum for obtaining such monomeric IgG. At this pH range, the dissociation of IgG aggregates into monomeric IgG reached maximum, without producing polymeric IgG. When monomeric IgG was treated at this acidic pH condition, its biological and physicochemical properties such as acid difference spectrum, molecular weight, plasmic digestion ratio, and complement-, antigen- and protein A-binding activities remained unchanged. The fact that large scale production of monomeric IgG can be easily performed under such conditions is discussed.

Before HBsAg screening of plasma was instituted, the preventive effect of immunoglobulin preparations (ISG) against Hepatitis B was not clearly defined. Since HBsAg screening became a requirement, all ISG has shown Anti-HBs and it further became clear that such preparations are effective in preventing Hepatitis B.Recently, cases of Hepatitis NANB associated with administration of intravenous immunoglobulin (IVIG) were reported. Since there is no NANB screening test presently available and IVIG tends to be administered in a large volume, a virus inactivation treatment should be performed on IVIG.Now that heat treatment at 60°C for 10 hours, which had previously been considered to be impossible or unnecessary for immunoglobulin, has become possible, an intravenous immunoglobulin preparation which is more ideal in stability and safety is available.

A sensitive and specific assay system for anti-HBc, the passive hemagglutination (PHA) method, has been established. The reactivity of PHA cells prepared by conjugating purified recombinant HBcAg-particles with fixed sheep blood cells (SRBC) was highly specific to monoclonal- and polyclonal anti-HBc IgGs. The sensitivity of PHA method was higher than that of radioimmunoassay (RIA). However, uncertainty for the positivity of anti-HBc still remained in plasma with the PHA titers lower than 2(5). A relatively high ratio (19%, 37 of 196) of anti-HBc-positive plasma, which had been confirmed to be HBsAg negative, was demonstrated in blood donors whose bloods have been considered suitable for transfusion. The hazards of anti-HBc-positive bloods and the importance of anti-HBc detection in plasma are discussed in this paper.

The efficacy and safety of a recombinant yeast-derived hepatitis B vaccine were evaluated in 209 subjects after three administrations at 0, 4 and 20 weeks. Subjects were divided into four groups given 5 micrograms vaccine subcutaneously, 10 micrograms subcutaneously, 10 micrograms intramuscularly and 20 micrograms subcutaneously to define the effective dose and to compare the effect of administration. Seroconversion of the antibody to hepatitis B surface antigen after the third vaccination reached 96.6% in the group given 5 micrograms vaccine subcutaneously and 100% in the other groups. The final geometric mean antibody titres were 700 IU/l in subjects given 5 micrograms subcutaneously, 2004 IU/l in those given 10 micrograms subcutaneously, 4674 IU/l in those given 10 micrograms intramuscularly and 3342 IU/l in those given 20 micrograms subcutaneously. In the groups given 10 micrograms, the early seroconversion rate of the antibody to hepatitis B surface antigen and the geometric mean antibody titres after the third vaccination were significantly higher in subjects administered intramuscularly than subcutaneously (P less than 0.05). No major adverse effects were observed and minor reactions were the same as, or less than, those reported for the plasma-derived vaccine. Before and after administration, no significant fluctuation in the yeast antibody titre was observed. These results demonstrate the efficacy and safety of the yeast-derived vaccine, and show that 10 micrograms was the effective dose.