WHO Expert Committee on Biological Standardization.

This report presents the recommendations of a WHO Expert Committee commissioned to coordinate activities leading to the adoption of international requirements for the production and control of vaccines and other biologicals and the establishment of international biological reference materials. The report starts with a discussion of general issues brought to the Committee's attention and provides information on the status and development of reference materials for various antibodies, antigens, blood products and related substances, cytokines, growth factors, and endocrinological substances. The second part of the report, of particular relevance to manufacturers and national regulatory authorities, contains recommendations for the production and quality control of meningococcal group C conjugate vaccines, guidelines for regulatory expectations for clinical evaluation of vaccines, guidelines for the production and quality control of inactivated oral cholera vaccines and guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products.

Securing viral safety for plasma derivatives.

The various mechanisms that contribute to the viral safety of therapeutics prepared from human blood plasma are examined. The basic principles behind the 2 most important mechanisms for viral safety, inactivation, and removal are discussed, together with the main issues linked to manufacturing. Validation of virus safety and protein integrity is considered. Currently available methods for inactivation and removal of viruses are reviewed, followed by an overview of new and experimental methods that may have some promise for the future. This review ends with a brief discussion of the impact fractionation pool size may have on product safety and the potential new threat presented by contamination with prions.

New developments in plasma fractionation and virus inactivation.

Ethanol fractionation has been used for fifty years to prepare therapeutic proteins from human plasma. Although the method has been very successful, there are now newer techniques available, which might at least in part replace ethanol fractionation. Whether or not this will occur depends on the economic benefits the new methods offer. Although transmission of viruses through blood products is nowadays fortunately a rare event, the search for alternative inactivation and/or removal methods for viruses continues. The small, non-enveloped viruses are particularly difficult to deal with, and any new method should address this problem with a high priority. Transmission of spongiform encephalopathies (Creutzfeld-Jakob and similar diseases) by blood and blood products has not been observed so far. Preliminary experimental evidence indicates that the putative causative agent(s) are removed from the final products during fractionation.

Virus inactivation by pepsin treatment at pH 4 of IgG solutions: factors affecting the rate of virus inactivation.

BACKGROUND: IgG preparations have rarely transmitted infectious diseases; however, because such transmission has occurred a few times, manufacturers are required to present experimental proof that their specific production process removes and/or inactivates viruses that may be present in the starting material. STUDY DESIGN AND METHODS: The kinetics of virus inactivation mediated by pepsin treatment at pH 4 during the production of intravenous immunoglobulin was assessed with spiking experiments using human immunodeficiency virus, bovine viral diarrhea virus, Semliki Forest virus, and pseudorabies virus. The influence of various factors on the rate of virus inactivation also was studied by modifying the composition of the IgG solutions with respect to IgG, sucrose, and NaCl content. RESULTS: Virus inactivation at 37 degrees C was extremely rapid and resulted in a complete loss of infectivity within 5 minutes to 1 hour. Inactivation was much slower at lower temperatures. Furthermore, inactivation was dependent on the solute composition. Increasing the sucrose content from 0 to 15 percent reduced the rate of inactivation of pseudorabies virus but did not affect the rate of inactivation of Semliki Forest virus. In contrast, increasing the NaCl content from 0 to 150 mM resulted in a reduction in the rate of inactivation of Semliki Forest virus, whereas the rate of inactivation of pseudorabies virus remained unaffected. Moreover, increasing the IgG concentration from 0 to 10 percent resulted in an increased rate of inactivation of pseudorabies virus but a decreased rate of inactivation of Semliki Forest virus. CONCLUSION: Inactivation of viruses by pepsin treatment at pH 4 essentially is temperature-dependent, and the reaction rate is selectively influenced by the solute composition of the IgG solution. This has to be taken into account when safety data for different products are compared.

Effects of intravenous infusion of lipid-free apo A-I in humans.

Apolipoprotein (apo) A-I is the principal protein component of the plasma high density lipoproteins (HDLs). Tissue culture studies have suggested that lipid-free apo A-I may, by recruiting phospholipids (PLs) and unesterified cholesterol from cell membranes, initiate reverse cholesterol transport and provide a nidus for the formation, via lipid-poor, pre-beta-migrating HDLs, of spheroidal alpha-migrating HDLs. Apo A-I has also been shown to inhibit hepatic lipase (HL) and lipoprotein lipase (LPL) in vitro. To further study its functions and fate in vivo, we gave lipid-free apo A-I intravenously on a total of 32 occasions to six men with low HDL cholesterol (30 to 38 mg/dL) by bolus injection (25 mg/kg) and/or by infusion over 5 hours (1.25, 2.5, 5.0, and 10.0 mg.kg-1.h-1). The procedure was well tolerated: there were no clinical, biochemical, or hematologic changes, and there was no evidence of allergic, immunologic, or acute-phase responses. The 5-hour infusions increased plasma total apo A-I concentration in a dose-related manner by 10 to 50 mg/dL after which it decreased, with a half-life of 15 to 54 hours. Coinfusion of Intralipid reduced the clearance rate. The apparent volume of distribution exceeded the known extracellular space in humans, suggesting extensive first-pass clearance by one or more organs. No apo A-I appeared in the urine. Increases in apo A-I mass were confined to the pre-beta region on crossed immunoelectrophoresis of plasma and to HDL-size particles on size exclusion chromatography. Increases were recorded in HDL PL, but not in HDL unesterified or esterified cholesterol. Increases also occurred in LDL PL and in very low density lipoprotein cholesterol, triglycerides, and PL but not in plasma total apo B concentration. These results can all be explained by combined inhibition of HL and LPL activities. Owing to the effects that this would have had on HDL metabolism, no conclusions can be drawn from these data about the role of lipid-free apo A-I in the removal of PL and cholesterol from peripheral tissues in humans. The kinetic data suggest that the fractional catabolic rate of lipid-free apo A-I exceeds that of spheroidal HDLs and is reduced in the presence of surplus PL.

Cellular processing of copper-67-labeled monoclonal antibody chCE7 by human neuroblastoma cells.

Monoclonal antibody chCE7, an internalizing neuroblastoma-specific chimeric antibody, was derivatized with the macrocyclic amine ligand 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid tetrahydrochloride and labeled with the potential therapeutic nuclide 67Cu. Using pulse labeling and an acid elution endocytosis assay, 67Cu-chCE7 was found to be internalized into human neuroblastoma (SKN-AS) cells at a similar rate and to a similar extent as 125I-labeled chCE7. Uptake of 67Cu-chCE7 and 125I-chCE7 into the acid stable (intracellular) pool proceeded with similar kinetics during the first 2 h of internalization. However, in contrast to 125I-chCE7-loaded cells, at later times intracellular radioactivity kept increasing in the case of 67Cu-chCE7-loaded cells. It was shown that this effect is due to the intracellular accumulation of a low M(r) degradation product consisting of the 67Cu-4[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid complex, possibly with a short peptide attached to it. Degradation of both 125I-chCE7 and 67Cu-chCE7 was inhibited by chloroquine, indicating endosomal or lysosomal degradation, and a 43,000 M(r) fragment was found to be the major high M(r) degradation product in both cases. Although at times between 4 and 6 h of internalization intracellular breakdown of 67Cu-chCE7 was found to proceed more slowly, the major difference between the two immunoconjugates resides in the prolonged cellular retention of the 67Cu-chCE7 metabolite.

Internalization and degradation of monoclonal antibody chCE7 by human neuroblastoma cells.

Internalization and cellular degradation of a chimeric monoclonal anti-neuroblastoma antibody (MAb chCE7) is described. Immunofluorescence localization showed temperature-dependent redistribution of MAb chCE7 from the cell surface at 0 degrees C to vesicular structures after heating to 37 degrees C. SKN-AS cells which were pulse-labelled at 0 degrees C with radioiodinated chCE7 released 50% of the initial cell-bound radioactivity into the medium after 20 hr at 37 degrees C. Low-molecular-weight radioactivity in the medium was identified as iodotyrosine and the major intracellular degradation product was found to be an antibody fragment of 43 kDa. Degradation was blocked by chloroquine, an inhibitor of lysosomal function. MAb chCE7 binds to a carbohydrate epitope, as treatment with tunicamycin abolishes cell binding. In adherent cells in culture, inhibition of protein synthesis by cycloheximide affected cell-surface binding sites for chCE7 in a biphasic manner, with an initial increase followed by long-term decrease. Expression of binding sites was also found to be inhibited by sodium azide, by the lysosomotropic drug chloroquine and by Brefeldin A, a drug which prevents export of newly synthetized proteins to the cell surface. When cells were treated with high doses of chCE7 up to 24 hr and cell-surface binding sites were then measured after an acidic buffer wash, no loss of surface binding sites was found, indicating that pretreatment with MAb chCE7 does not induce down-regulation of its binding sites.

No evidence for protein modifications in fresh frozen plasma after photochemical treatment: an analysis by high-resolution two-dimensional electrophoresis.

To study if photochemical treatment of human plasma by methylene blue in combination with visible light induces protein alterations, we analysed by high-resolution two-dimensional gel electrophoresis (2-D PAGE) untreated and photochemically treated fresh frozen plasma collected by apheresis from the same donor (n = 8). Polypeptides were separated according to their charges by isoelectric focusing and to their sizes in polyacrylamide gels in presence of sodium and to their sizes in polyacrylamide gels in presence of sodium dodecyl sulphate, and visualized by silver staining. Despite their apparent complexity, protein maps of untreated plasma and photochemically treated fresh frozen plasma revealed identical spot patterns. Thus, photochemical treatment did not induce apparent protein pattern modifications.

Fibronectin is more active than fibrin or fibrinogen in promoting Staphylococcus aureus adherence to inserted intravascular catheters.

To further define the role of fibrin(ogen) and fibronectin in Staphylococcus aureus adherence to central venous catheters, the amount, chemical integrity, and biologic activity of these proteins adsorbed on lines inserted in hospitalized patients were prospectively studied. Polyurethane cannulas promoted a significantly lower adherence of S. aureus than polyvinyl chloride (P < .01) or Hickman (P < .001) cannulas and contained the lowest amount of immunologically assayed fibronectin but not of fibrin(ogen). Fibrinogen showed an extensive loss of adherence-promoting activity on inserted cannulas, which was related to its proteolytic breakdown, as detected by SDS-PAGE and immunoblots with antifibrinogen antibodies and confirmed by in vitro studies with purified protein fragments. In contrast, either intact or fragmented fibronectin, although present in much lower amounts than fibrin(ogen), could actively promote S. aureus adherence onto intravenous catheters.

Partitioning and inactivation of viruses during isolation of albumin and immunoglobulins by cold ethanol fractionation.

Albumin solutions invariably transmitted infectious hepatitis viruses before the introduction of pasteurisation in the final container. Immunoglobulin solutions (the older intramuscular as well as the current intravenous ones), on the other hand, only rarely transmitted hepatitis. The apparent safety of the latter was usually attributed to the presence of neutralizing antibodies and to the fractionation process. It was shown that viruses tend to concentrate in those fractions of the cold ethanol precipitation procedure which are used neither for albumin nor for immunoglobulin preparations. Additionally, ethanol alone inactivates some viruses, albeit much less at low temperatures than at room temperature. According to EC-directives, all manufacturers of stable blood products must introduce production steps which inactivate viruses or they have to prove that certain production steps, which are already being used, do inactivate viruses. In either case, the inactivation has to be validated with appropriate experiments. Procedures that are now recognized as virucidal are, e.g., pasteurisation (i.e., heating of the liquid product at 60 degrees C for 10 hours), solvent/detergent (S/D) treatment, photodynamic treatment, or incubation at pH4 with pepsin.

Production and characterization of a mouse/human chimeric antibody directed against human neuroblastoma.

Hybridoma CE7 produces a murine antibody (gamma 1/kappa) which binds to a 190-kDa cell-surface glycoprotein of human neuroblastoma. Because of its tumor specificity, it has been used routinely in clinical pathology to confirm diagnosis of neuroblastoma. We have isolated the gene segments coding for the variable regions of the immunoglobulin H and L chain of this hybridoma. These V genes were used to construct mouse/human chimeric H and L chain genes (gamma 1/kappa) which were then expressed in SP2/0 cells. A cell-binding inhibition assay showed that the specificity of the chimeric CE7 antibody (chCE7) is identical to that of the original CE7. Radioiodinated chCE7 binds to approximately 43,000 sites per neuroblastoma cell with an affinity of 10(10) M-1. In neuroblastoma-bearing nude mice, biodistribution studies with [125I]chCE7 were performed and tumor accumulations of up to 32% of injected dose/g tissue together with low blood and organ uptake were found.

Radioimmunolocalization of neuroblastoma xenografts with chimeric antibody chCE7.

This study was performed to evaluate the tumor targeting ability of chCE7 with a view to clinical applications in neuroblastoma imaging and therapy. A chimeric (mouse/human) monoclonal antibody (chCE7) of gamma 1/kappa isotype directed against a neuroblastoma-associated cell-surface glycoprotein is described. In vitro chCE7 binds with high affinity (KD approximately 1 x 10(-10) M) to SKN-AS human neuroblastoma cells. Binding studies with 125I-labeled chCE7 show temperature-dependent modulation of antigen binding and indicate that a proportion of the bound antibody is internalized due to rapid antigen turnover. In vivo biodistribution of radioiodinated chCE7 in nude mice bearing SKN-AS tumors shows optimal tumor uptake after 24 hr with about 30% of the injected dose per g. Optimal tumor/blood ratios (3.4:1) are reached after 4-5 days. Uptake in other organs including the reticuloendothelial system is low with tumor/organ ratios of 10 and more. Tumor uptake of chCE7 and the parent murine CE7 are found to be similar. Stability of chCE7 during and after radiolabeling is good with no loss of immunoreactivity in preparations labeled with 123I up to 100 mCi/mg and 80% immunoreactivity after labeling with 13 mCi/mg of 131I. Neuroblastoma xenografts can be imaged by radioimmunoscintigraphy with 123I- and and 131I-labeled chCE7.

Virus inactivation during production of intravenous immunoglobulin.

The effect of pepsin treatment at pH 4 on the infectivity of several enveloped viruses was assessed under the conditions used during the production of intravenous immunoglobulins. It was shown that the prototypes of four virus families--human immunodeficiency virus (Lentivirinae), herpes simplex virus type 1 and human cytomegalovirus (Herpesviridae), Semliki Forest virus (Togaviridae), and vesicular stomatitis virus (Rhabdoviridae)--were inactivated by this procedure. With vesicular stomatitis virus as a model, the contributions of both low pH and pepsin were demonstrated, and pepsin had a synergistic or additive action.

A purification method for apolipoprotein A-I and A-II.

Apolipoproteins A-I and A-II were isolated from precipitates obtained by cold ethanol fractionation of human plasma. The starting material used in this report was precipitate B of the Kistler and Nitschmann method which corresponds approximately to fraction III of the Cohn and Oncley procedure. Through the use of urea, chloroform, and ethanol in appropriate concentrations, apolipoproteins A-I and A-II were isolated by a simple extraction technique avoiding time-consuming ultracentrifugation. Starting from 10 g of centrifuged precipitate B, approximately 100 mg of apolipoprotein A-I and 10 mg of apolipoprotein A-II were obtained. When incubated with normal human or rabbit plasma, both apolipoproteins were readily incorporated into high-density lipoproteins. Apolipoprotein A-I obtained by the cold ethanol method activated lecithin-cholesterol acyltransferase to the same extent as apolipoprotein A-I prepared by the classical flotation method. Apolipoprotein A-II had no such properties by itself, but was capable of potentiating lecithin-cholesterol acyltransferase activity of apolipoprotein A-I.

Inactivation of viruses and safety of stable plasma products.

The safety of blood or plasma derived products with respect to the transmission of viruses has various aspects, one of them being the methods that may be used for inactivating viruses during processing of plasma products. In industry, the most important methods are heating in solution (pasteurization) or in the lyophilized state and treatment with solvents and detergents (S/D treatment). Both S/D treatment and pasteurization produce safe products. However, ethanol fractionation by itself may result in virus-safe preparations, since this procedure chemically inactivates viruses and also partitions viruses away from fractions that are subsequently used therapeutically.

Inhibition by immunoglobulins of Staphylococcus aureus adherence to fibronectin-coated foreign surfaces.

Recent data suggest that fibronectin may favor Staphylococcus aureus infection by promoting attachment to either injured tissues or implanted foreign bodies. Using a previously described in vitro assay, we show that promotion of S. aureus adherence by surface-bound fibronectin, adsorbed on polymethylmethacrylate (PMMA) coverslips, is antagonized by antistaphylococcal antibodies present in immunoglobulin G (IgG) purified from human plasma. Among the different organisms tested, the protein A-deficient strain Wood 46 of S. aureus was the most strongly inhibited by purified IgG or whole serum dose-dependently. Bacterial adherence was not influenced by preincubating fibronectin-coated PMMA with either purified IgG or whole serum. However, inhibition of bacterial adherence was directly related to the extent of IgG binding to S. aureus Wood 46. When F(ab')2 fragments of purified IgG were tested in the adherence assay, they could also reduce the interaction between S. aureus Wood 46 and fibronectin-coated PMMA. Two other staphylococcal strains were also tested in the adherence inhibition assay: Whereas the protein A-rich strain Cowan I of S. aureus was moderately inhibited by purified IgG or whole serum, S. epidermidis KH 11 was not at all inhibited by IgG which bound poorly to the bacterial cells. This study has demonstrated that bacterial coating by humoral factors, and specifically IgG, may influence significantly subsequent adherence of S. aureus to surface-bound fibronectin.