Claudio Farina

A new biocompatible nanoparticle delivery system for the release of fibrinolytic drugs

The preparation of novel biocompatible polymeric nanoconstructs suitable to load sensitive bioactive protein agents is reported. Nanoparticles were prepared as based on hybrid polymeric matrices consisting of synthetic bioerodible alternating copolymers of maleic anhydride and n-butylvinylether hemiesterified with 2-methoxyethanol and grafted with poly(ethylene glycol) segments and monoclonal antibody single chain fragment specific for fibrin clot. The prepared nanoparticles were loaded with proteolytic enzymes (trypsin and urokinase), encapsulating up to 2500UI of urokinase/mg of dried nanoparticles. The release of the enzyme from nanoparticles resulted time controlled and it was assessed that in case of administration of urokinase-loaded nanoparticles, the enzyme would preserve its thrombolytic properties more efficiently in respect to free drug administration. Moreover, the nanoparticles showed a good in vitro biocompatibility, suitable for biomedical applications. The stability (shelf life) of the prepared nanostructured dosage forms was evaluated. The drug-loaded nanoparticles resulted stable under stressed conditions (35 degrees C for 13 weeks) in a lyophilized form and preserved their morphological and functional characteristics when stored in suspension for 18 months at 4 degrees C.

Characterization of factor VIII pharmaceutical preparations by means of MudPIT proteomic approach.

For a good clinical outcome of Haemophilia A substitutive therapy a detailed characterization of factor VIII (FVIII) concentrates is required, in order to disclose the eventual relations between differently composed concentrates and their biological effects. This preliminary work could be a first step towards a deep structural characterization of FVIII concentrates, using the fast and simply manageable MudPIT technology, which enables the identification and characterization of protein mixtures taking advantage of both the high separation capacity of two-dimensional chromatography and the powerful peptide characterization ability of tandem mass spectrometry. The aim of this study was to evaluate the suitability of for the characterization of FVIII molecule in complex mixtures such its commercial concentrates, both plasma-derived and recombinant, and for the determination of the protein composition of different FVIII preparations. By means of Multidimensional Protein Identification Technology (MudPIT) it was possible to assess the presence of factor VIII in its preparations and to identify most of the contaminant proteins without gel separation. In particular, 125 and 42 proteins were identified in plasma-derived and recombinant concentrates, respectively. Concerning investigation of FVIII, 24 different peptides were identified in plasma-derived corresponding to 7, 29, 27, 19 and 67 of percentage coverage for A1, A2, A3, C1 and C2 domains, respectively. About its multimeric carrier von Willebrand factor (VWF), we have sequenced 42% of domain interacting with A3 and C2 domains of FVIII. Finally, it has been observed that normalized parameters, such as total peptide hits obtained by SEQUEST may be used for evaluation of the relative abundance of FVIII in different preparations.

Effectiveness of nanofiltration in removing small non-enveloped viruses from three different plasma-derived products

The objective of this study was to assess the ability of nanofiltration of albumin solution, prothrombin complex (PTC) and factor IX (FIX) to remove two small, non‐enveloped DNA viruses, parvovirus B19 (B19V) and torque teno virus (TTV). Virus removal was investigated with down‐scale experiments performed with sequential steps of 35‐nm and 15‐nm nanofiltrations of products spiked with virus DNA‐positive sera. Viral loads were determined by real‐time PCRs. The 15‐nm nanofiltration removed more than 4.0 B19V log from all the products, TTV was reduced of more than 3.0 log from albumin solution and FIX by 35‐nm and 15‐nm nanofiltrations, respectively, being viral DNA undetectable after these treatments. Traces of TTV were still found in PTC after the 15‐nm nanofiltration. In conclusion, nanofiltration can be efficacious in removing small naked viruses but, since viruses with similar features can differently respond to the treatment, a careful monitoring of large‐scale nanofiltration should be performed.

A simple method for large-scale purification of plasma-derived apo-transferrin.

We investigated and optimized a purification process, suitable for industrial scale, to obtain pharmaceutical grade apo‐Tf (apo‐transferrin), preserving its physiological properties and functions. Apo‐Tf was obtained from fraction IV subfraction 1 and IV subfraction 4 (fraction IV‐1,4), a waste product of the Cohn fractionation process, performing a single chromatographic run and two viral inactivation/removal steps. The structural integrity and the biological activity of the final product were extensively tested. The yield of apo‐Tf produced was 80% on laboratory scale and 90% in scale‐up lots, and the purity was higher than 95%. The purified protein preserves iron‐ and receptor‐binding activities and shows a normal glycosylation pattern. The single chromatographic step process presented here provides an efficient means to prepare commercial quantities of the protein. The final product is sterile and two viral inactivation/removal steps were introduced into the process.

Quality control of recovered plasma for fractionation: an extensive Italian study

BACKGROUND: This study was aimed at obtaining significant information on the quality of whole‐blood plasma (WBP) delivered to a private pharmaceutical company by the blood transfusion centers (BTCs) of 10 Italian regions.

Apotransferrin inhibits interleukin-2 expression and protects mice from experimental autoimmune encephalomyelitis

Transferrin (Tf) has a major role in T cell activation and proliferation. Here, we investigated whether Tf exerts immunomodulatory effects on T cells and in development of T-cell driven experimental autoimmune encephalomyelitis (EAE). While treatment of concanavalin A-stimulated splenocytes with apotransferrin (ApoTf) did not affect release of IL-1β, TNF, IFN-γ, IL-17, IL-4, and IL-10, it markedly and dose-dependently down-regulated synthesis of IL-2 in these cells. ApoTf also inhibited IL-2 generation in purified CD3+ T cells and the effect was accompanied with down-regulation of MAPK p44/42 and NFκB signaling. Despite impeded IL-2 release, proliferation of splenocytes was not inhibited by ApoTf. Importantly, ApoTf ameliorated EAE in mice and significantly reduced ex vivo IL-2 production in proteolipid protein-specific lymphocytes. Thus ApoTf may be a promising beneficial agent for multiple sclerosis.

Evaluation of von Willebrand factor activity in factor VIII/von Willebrand factor concentrates with the automated von Willebrand factor: activity IL test.

The von Willebrand ristocetin cofactor assay is still the main cleared measurement test used to evaluate von Willebrand factor (vWF) activity in concentrate samples containing vWF. Although the assays performance has been improved over the years, the test reliability is still affected by a high interassay and interlaboratory variability; moreover, it requires skilled technicians and significant time. An automated HemosIL vWF:activity test, already set up on plasma samples, was then applied to factor VIII/vWF concentrates to verify its suitability in routine analysis of concentrate samples. As first step precision, linearity and accuracy were assessed. Then, 40 commercial batches of a high purity factor VIII/vWF concentrate were examined with this new method. The Spearmans correlation between the results obtained with the HemosIL vWF:activity assay and vWF activities determined by established procedures was evaluated. Comparisons of the means between HemosIL vWF:activity and the other tests were calculated with the Wilcoxon and Bland–Altman tests. Validation study showed satisfying within-run and between-run precision values. Activities determined with HemosIL vWF:activity had good correlation with those determined as ristocetin cofactor, Imubind (vWF:Imubind) and collagen-binding. Wilcoxon test, used to compare the means between the HemosIL vWF:activity and the other activity assessments, proved no significant variation with vWF:Imubind, but displayed a slight variation with von Willebrand ristocetin cofactor and von Willebrand factor:collagen-binding. This automated HemosIL vWF:activity test could be included in routine determination of vWF activity in concentrate samples and supports traditional von Willebrand ristocetin cofactor because it is reliable, reproducible and sensitive.

Different behavior of erythrovirus B19 and torquetenovirus in response to a single step of albumin purification

BACKGROUND:  The safety of human serum albumin (HSA) is of special interest with respect to virus transmission because of the wide use of this blood product as a therapeutic agent and also, added to other products, as an excipient or a stabilizer. Conflicting data are reported concerning HSA contamination by small, naked viruses such as the erythrovirus B19 (B19V) and the anellovirus torquetenovirus (TTV). This study has been performed to assess the effect of the HSA purification procedures on the viral contamination.

Anti-A and anti-B hemagglutinin depletion during Cohn purification process of 5% immunoglobulin

Polyvalent immunoglobulin G (IgG) products obtained by fractionation of human plasma are widely used to treat a broad range of conditions, including immunodeficiency syndromes and autoimmune, inflammatory, and infectious diseases. For high‐quality products and to minimize adverse events related to the use of intravenous IgG (IVIG) it is very important to perform detailed analyses of their components. One of these components, that in rare cases can cause severe hemolytic conditions, is the amount of hemagglutinins, natural antibodies that bind A and/or B (anti‐A or ‐B) antigens present in red blood cells (RBCs).