María Susana Vitali

Preparation of lyophilized and liquid intravenous immunoglobulin G: development and scale-up.

Intravenous immunoglobulin G (IVIG) preparations are used in several disorders: primary and secondary immunodeficiencies, autoimmune and systemic inflammatory diseases, and in infectious diseases as effective therapy. In this work, we report a method of processing IVIG (lyophilized and liquid) from Cohn fraction II (FII) paste.

Implementation of an 'in-house' PCR assay validated for the detection of hepatitis C virus RNA.

Dear Sir ‘The manuscript has been seen and approved by all authors, it is not under active consideration for publication, has not been accepted for publication, nor has it been published, in full or in part (except in abstract form)’. Since 1 July 1999, the European Pharmacopeiamonograph 0853 ‘human plasma fractionation’ has required that the manufacturing plasma pools are tested for hepatitis C virus (HCV) RNA by validated nucleic acid technology (NAT). For this reason, manufacturers are encouraged to adopt strategies for minipool testing (between 100 and 520 donors/plasma pool) to avoid the loss of completely manufactured pools and to facilitate donor tracing in the event of positive results (World Federation of Hemophilia, 2003). The Committee for Proprietary Medicinal Products has introduced guidelines for testing and complete validation of the chosen ‘in-house’ or commercial NAT method to guarantee a robust technique.TheEuropeanMedicinalEvaluation Agency requests a minimal sensitivity of 100 IU mL by NAT on each production of plasma pools, prior to the release of the final blood products (European Agency for Evaluation of Medical Products, 1997). Since 2001, in UNC-Hemoderivados, the plasma pools for fractionation have been systematically tested for NAT with an in-house method optimized and validated in our laboratory. Herein, we describe the implementation of an assay using polymerase chain reaction (PCR) to detect HCV RNA as well as the results obtained during the past 6 years (2001–2007) regarding the screening of minipool blood donations. To avoid contamination,RNAextraction and reverse transcription, pre-PCR reagent preparation, DNA amplification and gel electrophoresis of PCR products were performed in separate rooms (Kwok & Higuchi, 1989). Viral RNAs were extracted from plasma samples according to the modified method described previously (Chomczynski & Mackey, 1998). Briefly, 200 mL of plasma were denatured by a guanidine thiocianate solution; the viral RNA was extracted with phenol– chloroform in an acid environment and concentrated by alcoholic precipitation. The dried pellet was dissolved in 10 mL of ribonuclease-free water, containing 20 units of recombinant ribonuclease inhibitor (RNAsin; Promega, Madison, WI, USA). For cDNA synthesis, the RNA solution was added to a mixture containing 75 pmol of gene-specificprimers (Novati et al., 1992) and5 reverse transcription buffer, incubated at 65 C for 15 min and then 200 IU Moloney murine leukemia virus reverse transcriptase (MML-V) and 20 mM of dNTPs were added and incubated at 37 C during 90 min. Next, 5 mL of cDNAwere amplified in a PCRmix containing 1 9 mM of MgCl2, 0 16 mM of dNTPs, 25 pmol outerprimer and 1 IU Taq polymerase and amplified by 35 cycles (94 C for 25 s, 55 C for 25 s and 72 C for 25 s). Then, 5 mLof thePCRproductswereadded toamixture containing 2 8 mM of MgCl2, 0 22 mM of dNTPs, 25 pmol inner-pair primers and 2 5 IU Taq polymerase and amplifiedby 40 cycles (94 C for 15 s, 63 C for 15 s and 72 C for 15 s). Denaturation and extension steps were always performed at 95 and 72 C, respectively. The amplification products (210 bp) and internal control (260 bp) were resolved by electrophoresis in a 2 5% agarose gel containing ethidium bromide. RNA internal controls was synthesized by cloning techniques with insertions of a 50 bp in target sequences and was designed to have the same primers binding regions as the target sequence. In the initial experiment, also a Southern blotting hybridisation with P-labeled probe and autoradiography during the development of method was used (Novati et al., 1992). The assays were considered valid only when all the controls were positive. The characteristics considered most important for the validation of the analytical procedure were specificity, detection limit and robustness of the assay. The specificity was evaluated with 100 HCV RNAnegative plasma pools, and the ability to detect all the six HCV genotypes was probed during different proficiency studies with satisfactory results. The detection Correspondence: H. Guglielmone, Laboratorio de Hemoderivados, Universidad Nacional de Córdoba, Córdoba, Argentina, Av. Juan Filloy s/n. Ciudad Universitaria, X5000HRA, Córdoba, Argentina. Tel.: 154 351 4334122; fax: 154 351 4334124; e-mail: hgugli@bioclin.fcq.unc.edu.ar

Studies in animal model on the thrombogenicity of a new prothrombin complex concentrate from Argentina

Dear Sir Prothrombin complex concentrate (PCC) is a therapeutic agent human plasma derived that contains vitamin K-dependent coagulation factors (Roberts & Eberst, 1993). These may be manufactured as three factors (II, IXandX)or four factors (II, VII, IXandX), depending on the purification procedure (Chandra & Wickerhauser, 1978; Feldman & Winkelman, 1991). Furthermore, some concentrates have also variable quantities of protein Z and physiologic coagulation inhibitor proteins C and S (Romisch et al., 1998). PCC administration has been used in the treatment of hemophilia B, severe liver failure and consumption coagulopathies as well as in the therapy for the reversal of warfarin anticoagulation in emergency settings (Lankiewicz et al., 2006). However, the therapeutic use of PCC may be accompanied by adverse events, including allergic reactions, transmission of blood-borne viruses and thrombotic episodes (Watson & Ludlam, 1997). The thrombogenic components of these concentrates have been mainly attributed to activated coagulation factors and also to the presence of coagulant-active phospholipids, zymogen overload and the lack of inhibitors antithrombin and/or protein S (Kohler, 1999). To avoid the thrombotic complications of PCC, the International Society on Thrombosis and Haemostasis (ISTH) has recommended in the past the use of heparin in the formulation of the final product (Menache, 1976). However, strong evidences support that the quality of PCC plays a crucial role in the occurrence of thromboembolic events, whichmainly dependon the purification methods used during its manufacture (Kohler, 1999). As a consequence of these observations, we developed a PCC that meets the criteria of presumed low thrombogenicity because the content of vitamin K-dependent clotting factors and inhibitors is well balanced and the thrombin and activated factors activities were negligible by in vitro assays. This study was designed to evaluate a full-dose-related response for our PCC, using the in vivo stasis model of thrombogenicity (Wessler test), adapted to rats. Different lots (n 1⁄4 3) of PCC were studied and manufactured from human plasma at the Laboratorio de Hemoderivados, Universidad Nacional de Córdoba production facility (Córdoba, Argentina). The factors II, IX andXwere isolated from cryosupernatant using ion-exchange batch chromatography, and the coagulation factors were eluted using buffer of increased ionic strength. A first step of viral inactivation was performed with solvent–detergent and then a second chromatographic step was carried out. The proteins of interest were eluted, and the protein concentration was adjusted by ultrafiltration procedure and formulated with or without heparin. Finally, the PCC was aseptically distributed in a vial and lyophilized and a second step of viral inactivation using dryheat (100 C for 30 min) was applied (Bernardi et al., 2003). PCCs were standardized according to factor IX (F IX) activity, following the European Pharmacopoeia procedures using International Standard preparations provided by the National Institute for Biological Standard and Control (Lamb et al., 1991). The method of Wessler et al. (1959) was used to test the thrombogenicity of the concentrates. Briefly, female Wistar rats (230 12 g) were anaesthetized with chloral hydrate (400 mg kg i.p.) and a 1-cm segment of the jugular vein was exposed through a midline central incision; the PCC (0 5 mL i.v. for 10 s in all the cases) or sample control was given by the cannulated femoral vein. The venous segment was tied off exactly 15 s after the beginning of infusion and the development of thrombi in the isolated segment of jugular vein was determined 10 minutes later by visual inspection using scores as reported (Wessler et al., 1959). All procedures were performed according to the guidelines established by The Animal Models Subcommittee, Scientific and Standardization Committee of the ISTH (Levi et al., 2001). PCCs (496 42 IU F IX per vial) were reconstituted according to obtain doses of 25, 50, 100 and 200 IU F IX kg in the presence or absence of heparin were used, and some vials of PCC (50 and Correspondence: Maria Eugenia Bernardi, Area de Desarrollo de Productos y Procesos, UNC-HEMODERIVADOS, Av. Valparaiso s/n, Ciudad Universitaria X5000HRA, Córdoba, Argentina. e-mail: mebernardi@hemo.unc.edu.ar