Virus safety of plasma products using 20 nm instead of 15 nm filtration as virus removing step.

During the manufacture of human plasma derivatives, a series of complementary measures are undertaken to prevent transmission of blood-borne viruses. Virus filtration using 15 nm (Planova15N) filters has successfully been implemented in manufacturing processes for various plasma derivatives primarily because virus filtration is a technique, mild for proteins, that can effectively remove even small non-lipid-enveloped viruses, such as HAV and parvovirus B19. However, the use of 15 nm filters has limitations with regard to protein capacity of the filters and the process flow, resulting in an expensive manufacturing step. Therefore, studies were performed to test whether the use of 20 nm (Planova20N) filters, having different characteristics compared to 15 nm filters, can be an alternative for the use of 15 nm filters. It is shown that 20 nm filtration can be an alternative for 15 nm filtration. However, the virus removal capacity of the 20 nm filters depends on the plasma product that is filtered. Therefore, an optimisation study must be performed with regard to process parameters such as pressure, pH and protein concentration for each plasma product. In this study, using optimised conditions, the virus removal capacity of 20 nm filters appears to be comparable or even better when compared to that of 15 nm filters.

Viral safety of C1-inhibitor NF.

We studied the efficacy of virus reduction by three process steps (polyethylene glycol 4000 (PEG) precipitation, pasteurization, and 15nm virus filtration) in the manufacturing of C1-inhibitor NF. The potential prion removing capacity in this process was estimated based on data from the literature. Virus studies were performed using hepatitis A virus (HAV) and human immunodeficiency virus (HIV) as relevant viruses and bovine viral diarrhea virus (BVDV), canine parvovirus (CPV) and pseudorabies virus (PRV) as model viruses, respectively. In the PEG precipitation step, an average reduction in infectious titer of 4.5log(10) was obtained for all five viruses tested. Pasteurization resulted in reduction of infectious virus of >6log(10) for BVDV, HIV, and PRV; for HAV the reduction factor was limited to 2.8log(10) and for CPV it was zero. Virus filtration (15nm) reduced the infectious titer of all viruses by more than 4.5log(10). The overall virus reducing capacity was >16log(10) for the LE viruses. For the NLE viruses CPV and HAV, the overall virus reducing capacities were >8.7 and >10.5log(10), respectively. Based on literature and theoretical assumptions, the prion reducing capacity of the C1-inhibitor NF process was estimated to be >9log(10).

Virus validation of pH 4-treated human immunoglobulin products produced by the Cohn fractionation process.

To assess the virus reducing capacity of Cohn's cold ethanol fractionation process for the production of intravenous (IVIg) and intramuscular (IMIg) immunoglobulin products, and treatment of these products at pH 4, a validation study of virus removal and/or inactivation was performed using both lipid-enveloped viruses [human immunodeficiency virus (HIV), bovine viral diarrhoea virus (BVDV) and pseudorabies virus (PSR)], and non-lipid-enveloped viruses [(simian virus 40 (SV40) and encephalomyocarditis virus (EMC)]. For the cold ethanol fractionation process, overall reduction factors of 3.0 logs, > or = 2.6 (< 5.5) logs, 4.6 logs, 5.8 logs and > or = 2.6 (< 6.2) logs were found for HIV, BVDV, PSR, SV40 and EMC, respectively. For all tested viruses the precipitation of fraction III from fraction II + III was the most effective step. From the overall reduction factors it appears that cold ethanol fractionation, although capable of reducing viral infectivity to a significant extent, is not sufficient to meet the requirements of regulatory bodies for viral safety of immunoglobulin products. However, pH 4 treatment contributes effectively to the viral safety of the final products. Treatment at pH 4.05 and 37 degrees C for 16 h, as is applied to IVIg, yields reduction factors of > or = 8.4 logs, > or = 4.0 logs, > or = 7.1 logs, 4.8 logs and 1.4 logs for HIV, BVDV, PSR, SV40 and EMC, respectively. The effectiveness of this process step could be enhanced by extending incubation to 40 h at pH 4.25 compared to 16 h at pH 4.05. The extended incubation, as applied in the production of IMIg, yields a reduction of infectivity of SV40 by > or = 5.5 (< 8.0) logs and of EMC by > or = 4.1 (< 7.1) logs. Storage of IMIg, which is formulated as a solution, at 2-8 degrees C also contributes to virus safety. For storage periods of 8 weeks or longer, reduction factors of 2 to 6 logs were found for all viruses, except for BVDV which remained unaffected. These data indicate that the production processes for IVIg and IMIg as described here have sufficient virus reducing capacity to achieve a high margin of virus safety.

Inactivation of human immunodeficiency virus by gamma radiation and its effect on plasma and coagulation factors.

The inactivation of HIV by gamma-radiation was studied in frozen and liquid plasma; a reduction of the virus titer of 5 to 6 logs was achieved at doses of 5 to 10 Mrad at -80 degrees C and 2.5 Mrad at 15 degrees C. The effect of irradiation on the biologic activity of a number of coagulation factors in plasma and in lyophilized concentrates of factor VIII (FVIII) and prothrombin complex was examined. A recovery of 85 percent of the biologic activity of therapeutic components present in frozen plasma and in lyophilized coagulation factor concentrates was reached at radiation doses as low as 1.5 and 0.5 Mrad, respectively. As derived from the first-order radiation inactivation curves, the radiosensitive target size of HIV was estimated to be 1 to 3 MDa; the target size of FVIII was estimated to be 130 to 160 kDa. Gamma radiation must be disregarded as a method for the sterilization of plasma and plasma-derived products, because of the low reduction of virus infectivity at radiation doses that still give acceptable recovery of biologic activity of plasma components.

Large-scale purification of factor VIII by affinity chromatography: optimization of process parameters.

The optimization of a new process for the extraction of human coagulation factor VIII (FVIII) from plasma with the tailor-made affinity matrix dimethylamino-propylcarbamylpentyl-Sepharose CL-4B (C3-C5 matrix) is described. First, plasma is applied to DEAE-Sephadex A-50 anion exchanger in order to separate a number of proteins, including coagulation factors II, IX and X (prothrombin complex), from FVIII. Subsequently, the unbound fraction of the ion exchanger, containing FVIII, is contacted with the C3-C5 affinity matrix. Optimization of the FVIII affinity chromatographic procedure is accomplished in terms of the ligand density of the matrix, adsorption mode (batch-wise versus column-wise adsorption and matrix to plasma ratio), and conditions of pH and conductivity to be applied on washing and desorption. In scale-up experiments, by processing 20 l of plasma, the recovery (340 U VIII:C/kg plasma) and the specific activity (s.a.) (1.2 U VIII:C/mg protein) are better than those obtained by cryoprecipitation (recovery 300 U VIII:C/kg plasma, s.a. 0.3 U VIII:C/mg protein). The newly developed process using the specially designed C3-C5 affinity matrix has potential application in the process-scale purification of FVIII.

Evaluation of wet pasteurization of a factor VIII concentrate produced by controlled-pore silica adsorption.

In the routine production of a factor VIII concentrate (produced by adsorption of contaminating proteins in cryoprecipitate to controlled-pore silica and concentration of the factor VIII effluent by ultrafiltration) the terminal dry-heat treatment has been replaced by pasteurization in the liquid state. High effectivity of this procedure with respect to virus inactivation was demonstrated using a variety of both lipid- and protein-enveloped model viruses, including HIV. Pair-wise quality control of dry-heated and pasteurized product revealed no significant differences, except in the composition of the formulation buffer. In a clinical study in which 17 patients with haemophilia A participated the pasteurized product was well tolerated and in vivo recovery and half-life of factor VIII were in the same (normal) range as found for the dry-heated counterpart.

Affinity purification of plasma proteins: characterization of six affinity matrices and their application for the isolation of human factor VIII.

For the purification of coagulation factor VIII, (1,1'-carbonyl-diimidazole [CDI]-activated) Sepharose CL-4B was functionalized with two aminoalkyl and four aminoalkyl-carbamylalkyl ligand-spacer combinations. The affinity matrices were contacted with human plasma. All affinity matrices showed complete adsorption of factor VIII (greater than 90%) and three aminoalkyl-carbamylalkyl Sepharoses gave factor-VIII recoveries of 50-65% and a factor-VIII preparation with a specific activity of 1-2 U factor VIII/mg of protein. Furthermore, no fibrinogen, immunoglobulin G and albumin could be detected in the isolated factor VIII. Optimal results were obtained using the di-methyl-aminopropyl-carbamyl-pentyl-Sepharose affinity matrix.

Virus inactivation by addition of neutralizing antibodies.

This study has shown that the principle of virus neutralization by specific antibodies is feasible at least in the case of hepatitis B. Virus neutralization is our preferred method since it entails no loss of functional activity and no risk of induction of neo-antigens in the plasma derivatives. Although double-blind clinical trials have not been performed, this study--in combination with some other studies - brought the conclusive evidence that virus neutralization in the case of hepatitis B transmission is efficacious. The suggested occurrence of side effects related to the formation of immune complexes during or after administration is not borne out by more than 7 years of experience with neutralized plasma derivatives with no reported side effects. Whether this method is useful for other viruses will depend on the availability of specific neutralizing antibodies.

Thermal inactivation of human immunodeficiency virus in lyophilised blood products evaluated by ID50 titrations.

Inactivation of human immunodeficiency virus (HIV) in lyophilised small pool cryoprecipitate, factor VIII concentrate, prothrombin complex and C1-esterase inhibitor concentrate by prolonged heat treatment (72 h, 60 degrees C) was studied. Plasma products, inoculated prior to lyophilisation, had infectious titres ranging from 10(7) to 10(10.5). Residual infectivity (TCID50) was assessed by multiple titrations on H9 cells in a macro system and subsequent detection of virus replication by determining reverse transcriptase activity. Kinetics of inactivation showed a biphasic pattern: during the first 8 h a variable TCID50 reduction up to 10(4.3) was observed, followed by an additional loss of 10(1)-10(2.7) during the next 64 h. Heat treatment for 72 h resulted in a mean TCID50 reduction of 10(5). It is concluded that prolonged heat treatment may lead to the adequate prevention of HIV transmission by lyophilised plasma products.

Contributions to the optimal use of human blood. IX. Elimination of hepatitis B transmission by (potentially) infectious plasma derivatives.

Investigations were performed concerning the elimination of the risk of hepatitis B transmission of potentially infectious plasma derivatives by the addition of a low dose of hepatitis B immunoglobulin (HBIg). To this end, clotting factor VIII concentrate, prothrombin complex, C1 esterase inhibitor concentrate, plasminogen and antithrombin III were prepared from plasma strongly positive for hepatitis B surface antigen (HBsAg). To one half of every preparation, HBIg was added up to a final concentration of 0.4 IU anti-HBs/ml (test preparations), the other half was not treated (control preparations). Furthermore, to 10(-3) diluted infectious reference plasma (Bureau of Biologics, FDA, USA), an overdose HBIg was added to a final concentration of about 0.4 IU anti-HBs/ml. 6 chimpanzees, injected either with the control plasma derivatives or with the untreated infectious reference plasma, were infected with hepatitis B virus, whereas 5 chimpanzees, injected either with the test plasma derivatives or the infectious reference plasma to which the HBIg had been added, did not show any evidence of hepatitis B infection during the follow-up of 1 year. Addition of a low dose of HBIg to potentially infectious plasma derivatives appears to be a reliable measure to eliminate the hepatitis B transmission and is preferred to other methods for labile plasma derivatives.

The effect of storage of whole blood on the association of factor VIII-related antigen and factor VIII-coagulant antigen.

To evaluate the extent of denaturation of factor VIII-coagulant activity (VIII: C) during production of factor VIII concentrates, the factor VIII-coagulant antigen (VIII: CAg)/VIII: C ratio was measured in plasma, cryoprecipitate and cryosupernatant from fresh and stored blood. This ratio was close to unity for both cryoprecipitate and other concentrates, suggesting that VIII: CAg is lost concurrently with VIII: C during cryoprecipitation and further fractionation. Storage of blood (18 h, 22 degrees C) before processing resulted in a 30% loss of VIII: C from the separated plasma; however, VIII: CAg was not affected. In cryoprecipitate prepared from this plasma, VIII: C and VIII: CAg both were 30% lower than when prepared from fresh plasma. In the corresponding cryosupernatant, however, more VIII: CAg but less VIII: C was present compared with fresh material. Gel chromatography revealed that the rise of VIII: CAg in cryosupernatant prepared from stored blood, was due to an increased amount of VIII: CAg of low molecular weight, not being associated with factor VIII-related antigen. Such an increase in dissociated VIII: CAg was not detected in the plasma prior to cryoprecipitation. It is concluded that during storage of blood, molecular changes are induced in the factor VIII-VWF complex, possibly by limited proteolysis, which make the complex more liable to dissociation during subsequent cryoprecipitation.

Heterogeneity of human factor VIII. III. Transitions between forms of factor VIII present in cryoprecipitate and in cryosupernatant plasma.

Human factor VIII in plasma is a disperse protein consisting of a series of aggregates wih different molecular weight. VIIR:Ag and VIII:C are present in all forms, but VIIR:WF is confined to the highest molecular weight forms only. After cryoprecipitation of plasma the latter are recovered in the precipitate, and the lowest molecular weight forms remain in the supernatant. Disaggregation of high molecular weight forms of factor VIII was found in vitro upon repeated cryoprecipitations. The disaggregation was detected only when the original low molecular weight forms were removed. The additional low molecular weight forms possessed VIII:C and VIIR:WF was lacking. The reverse process of aggregation of low molecular weight factor VIII to more highly aggregated forms was not observed. Exchange of VIII:C between high and low molecular weight fractions was demonstrated by gel chromatography of mixtures of hemophilic cryoprecipitate and normal concentrated cryosupernatant, and vice versa, at physiologic ionic strength. This suggests that VIII:C and VIIR:Ag are weakly and noncovalently linked in normal conditions. This was further supported by the dissociation of VIII:C from VIIIR:Ag and VIIIR:WF upon gel chromatography and cryoprecipitation at pH 6.2 The dissociation could be reversed by readjustment of the pH.

Survival of 125iodine-labeled Factor VIII in normals and patients with classic hemophilia. Observations on the heterogeneity of human Factor VIII.

Radiolabeled human Factor VIII was used to study its survival in normals and patients with classic hemophilia, and to study the heterogeneity of Factor VIII; Purified Factor VIII was radiolabeled with 125iodine (125I-VIII) without loss of its structural integrity. The survival of 125I-VIII was studied in six normals and six hemophiliacs of whom four of the hemophiliacs had received transfusions with normal cryoprecipitate before the 125I-VIII infusion. No significant difference was observed between the disappearance of Factor VIII coagulant activity and radioactivity in these hemophiliacs. 125I-VIII in plasma showed a biphasic disappearance with an average t1/2 of 2.9 +/- 0.4 h (SEM) for the first phase and 18.6 +/- 0.7 h (SEM) for the second phase, respectively. The survival of 125I-VIII was similar comparing normals and hemophiliacs. The highest molecular weight forms of Factor VIII disappear more rapidly than the lower molecular weight ones. This was established by analysis of the fractions obtained by gel chromatography of plasma collected at several times after infusion and by analysis of the in vivo disappearance of three subfractions of Factor VIII. The fraction of 125I-VIII binding to platelets in the presence of ristocetin (containing the highest molecular weight forms of Factor VIII including the ristocetin cofactor) represented about 50% of the radioactivity present in plasma after infusion and showed a t 1/2 of 11.7 +/- 0.9 h (SEM) for the second phase. The fraction, which was recovered in cryoprecipitate of the recipient's plasma, represented about 90% of the initial radioactivity and showed a t 1/2 of 16.3 +/- 0.8 h (SEM) for the second phase. The fraction of 125I-VIII remaining in the cryosupernatant plasma (containing low molecular weight forms of Factor (VIII) showed a t 1/2 of 27.2 +/- 1.1 h (SEM). The first phase of the disappearance of 125I-VIII is caused in part by the disappearance of the highest molecular weight forms, which are possibly removed by the reticuloendothelial system.