RESUMEN
BACKGROUND AND OBJECTIVES: The risk of haemophiliacs contracting variant Creutzfeldt-Jakob disease (vCJD) via treatment with factor VIII concentrates is not known. Therefore, in order to determine the extent to which the vCJD agent might be removed during the preparation of factor VIII concentrate, the partitioning of a bovine spongiform encephalopathy (BSE)-derived agent was measured over the main purification step used to prepare the Scottish National Blood Transfusion Service high-purity factor VIII concentrate (Liberate). MATERIALS AND METHODS: Murine-passaged BSE (strain 301V), in the form of a microsomal fraction prepared from infected brain, was used to 'spike' a solution of factor VIII of intermediate purity. The 'spiked' starting material was subjected to solvent-detergent treatment and then to anion-exchange chromatography with Toyopearl DEAE-650M. All fractions were tested for 301V infectivity using a murine bioassay, including the procedures used to clean the ion-exchange media after use. RESULTS: BSE 301V infectivity was reduced by 2.9 log(10) in the fibrinogen fraction and by 2.7 log(10) in the factor VIII fraction. Over 99% of the added 301V infectivity remained bound to the ion-exchange column after elution of factor VIII. A large quantity of infectivity was subsequently removed by washing the ion-exchange media with 2 m NaCl. No further BSE 301V infectivity was detected in column eluates after treatment with 0.1 m NaOH or a second wash with 2 m NaCl. CONCLUSIONS: Results using a BSE-derived agent suggest that vCJD infectivity would be substantially removed by the ion-exchange process used in the preparation of fibrinogen and factor VIII concentrate. Although 301V infectivity remained bound to the ion-exchange matrix following elution of factor VIII, this appeared to be eliminated by the procedure used for cleaning the ion-exchange media after each use.
Asunto(s)
Cromatografía por Intercambio Iónico , Síndrome de Creutzfeldt-Jakob/transmisión , Encefalopatía Espongiforme Bovina/transmisión , Etanolaminas/química , Factor VIII/aislamiento & purificación , Fibrinógeno/aislamiento & purificación , Polímeros/química , Proteínas PrPSc/aislamiento & purificación , Adsorción , Animales , Bioensayo , Química Encefálica , Bovinos , Síndrome de Creutzfeldt-Jakob/sangre , Humanos , Ratones , Ratones Endogámicos , Microsomas/química , Proteínas PrPSc/efectos de los fármacos , Proteínas PrPSc/patogenicidad , Sensibilidad y Especificidad , Cloruro de Sodio/farmacología , Hidróxido de Sodio/farmacología , Solventes , VirulenciaRESUMEN
The levels of hepatitis A antibody were studied in factor VIII products manufactured by the Scottish National Blood Transfusion Service. It was found that the current high-purity factor VIII product has no detectable hepatitis A antibody, whereas the superseded intermediate-purity product had significant antibody levels (6,700 mIU/ml). Although the finished high-potency factor VIII product has no detectable hepatitis A antibody, significant levels of antibody are found in the early stages of product manufacture. This antibody may offer some protection against hepatitis A contamination present in very occasional plasma donations. Antibody to parvovirus B19 is also present at intermediate stages in the manufacture of high-potency factor VIII, but its significance is not known. Hepatitis A antibody levels were also measured in normal immunoglobulin. These data indicate that antibody levels in the plasma pools used to manufacture factor VIII are falling.
Asunto(s)
Seguridad de Productos para el Consumidor , Factor VIII , Anticuerpos Antihepatitis/sangre , Hepatovirus/inmunología , Factor VIII/aislamiento & purificación , Humanos , Plasma/microbiología , EscociaRESUMEN
During product development of a factor VIII concentrate (Dutch blood banks) the conversion from unsterilized to autoclaved freeze-drying buffer caused impaired product characteristics after severe dry heat treatment (80 degrees C for 72 h). Analysis of the freeze-drying buffers showed the presence of fructose and glucose in heated buffers, resulting from hydrolysis of sucrose. The detrimental effect of glucose and fructose on solubility, yield of factor VIII and color of the heat-treated product was confirmed by freeze-drying and heating products spiked with increasing levels of these monosaccharides. The effect of the use of freeze-drying buffers autoclaved with and without sucrose was examined in two other factors VIII concentrates, S(8) and Z8 (Protein Fractionation Centre, Edinburgh, UK). If sucrose was present during autoclaving of the buffer, a slightly lower yield over freeze-drying and 80 degrees C heat treatment was observed. Since glucose is present as a substrate in the medium for the host cells during cultivation of viruses, its potential effect on the 80 degrees C heated product (Dutch blood banks) was examined during the validation study of the inactivation of HIV-1 and Sindbis. The cultivation cycles for the virus inocula were simulated and residual glucose levels measured. In the supernatant medium of the host cell culture used for the propagation of Sindbis no residual glucose was found. Glucose however was found in the supernatant medium of the host cell culture for propagation of HIV-1, and therefore small molecular weight substances were removed from the actual HIV-1 inoculum by ultrafiltration.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Factor VIII/aislamiento & purificación , Fructosa , Glucosa , Calor , Tampones (Química) , Liofilización , VIH-1/fisiología , Humanos , Presión , Desnaturalización Proteica , Virus Sindbis/fisiología , Solubilidad , Ultrafiltración , Replicación ViralRESUMEN
Different manufacturers use several different processes for the production of intravenous immunoglobulin. Several manufacturers include a production step where the immunoglobulin is treated with low levels of pepsin at pH 4. A series of experiments were undertaken to assess whether or not pH 4/pepsin treatment could inactivate a range of test viruses. Acid-labile viruses such as vaccinia, herpes simplex, mumps and Semliki Forest virus were found to be susceptible to pH 4/pepsin treatment whereas poliovirus type 2, an acid-stable virus, was completely resistant to this treatment. In immunoglobulin preparations, viral contaminants are likely to be present as antibody/virus complexes and such complexing was found to help protect the test viruses from inactivation by pH 4/pepsin treatment. Despite this protection, at least 99% of the test inoculum of two susceptible viruses (vaccinia and herpes simplex) was found to be inactivated after treatment and the subsequent dissociation of virus/antibody complexes. It is concluded that pH 4/pepsin treatment may contribute to the safety of intravenous IgG by inactivating potential viral contaminants.
Asunto(s)
Contaminación de Medicamentos/prevención & control , Inmunoglobulinas/normas , Pepsina A/farmacología , Virus/efectos de los fármacos , Anticuerpos Antivirales/inmunología , Humanos , Concentración de Iones de Hidrógeno , Inmunoglobulina G/inmunología , Inmunoglobulinas/inmunología , Virus/inmunologíaRESUMEN
The biosynthetically radiolabelled secreted proteins of human lymphoblastoid cell lines have been analysed by sucrose gradient velocity sedimentation. The addition of detergent to the gradient buffer dramatically improves the recovery of immunoglobulins from cell line supernatants, allowing the gradient fractions to be analysed further both serologically and biochemically. The results of these analyses show that IgM is synthesised in the 19S form and can be clearly separated from other components which include free light chains. Immunoglobulin appears to form a much larger portion of the proteins secreted by human lymphoblastoid lines than has formerly been recognised, a finding which may be of practical importance for the development and exploitation of human monoclonal antibodies.