New orphan disease therapies from the proteome of industrial plasma processing waste- a treatment for aceruloplasminemia.

Plasma-derived therapeutic proteins are produced through an industrial fractionation process where proteins are purified from individual intermediates, some of which remain unused and are discarded. Relatively few plasma-derived proteins are exploited clinically, with most of available plasma being directed towards the manufacture of immunoglobulin and albumin. Although the plasma proteome provides opportunities to develop novel protein replacement therapies, particularly for rare diseases, the high cost of plasma together with small patient populations impact negatively on the development of plasma-derived orphan drugs. Enabling therapeutics development from unused plasma fractionation intermediates would therefore constitute a substantial innovation. To this objective, we characterized the proteome of unused plasma fractionation intermediates and prioritized proteins for their potential as new candidate therapies for human disease. We selected ceruloplasmin, a plasma ferroxidase, as a potential therapy for aceruloplasminemia, an adult-onset ultra-rare neurological disease caused by iron accumulation as a result of ceruloplasmin mutations. Intraperitoneally administered ceruloplasmin, purified from an unused plasma fractionation intermediate, was able to prevent neurological, hepatic and hematological phenotypes in ceruloplasmin-deficient mice. These data demonstrate the feasibility of transforming industrial waste plasma fraction into a raw material for manufacturing of new candidate proteins for replacement therapies, optimizing plasma use and reducing waste generation.

(+)-syn-Benzotriborneol an enantiopure C3-symmetric receptor for water.

(+)-syn-Benzotriborneol forms stable complexes with one molecule of water. This is due to the ability of the host to form three hydrogen bonds with water, to act simultaneously as a hydrogen-bond acceptor and donor, and to a perfect geometrical match between the pair. We report experimental (X-ray and neutron diffraction, VT NMR, DSC, TGA) and stereochemical studies carried out to elucidate and quantify the molecular and thermodynamic aspects of this supramolecular complex.

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.

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.

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 assay's 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 Spearman's 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.