A workshop organized by the European Medicines Agency and the European Directorate for the Quality of Medicines and HealthCare was held in London, UK on November 28-29, 2013, to provide an overview of the current knowledge of the characterization of new factor VIII (FVIII) and factor IX (FIX) concentrates with respect to potency assays and testing of postinfusion material. The objective was to set the basis for regulatory authorities' discussion on the most appropriate potency assay for the individual products, and European Pharmacopoeia (Ph. Eur.) discussion on whether to propose revision of the Ph. Eur. monographs with respect to potency assays in the light of information on new FVIII and FIX concentrates. The workshop showed that for all products valid assays vs. the international concentrate standards were obtained and potency could be expressed in International Units. The Ph. Eur. chromogenic potency assay gave valid assay results which correlate with in vivo functionality of rFVIII products. For some modified rFVIII products and all modified rFIX products, one-stage clotting assay methods result in different potencies depending on the activated partial thromboplastin time reagent. As a consequence, monitoring of patients' postinfusion levels is challenging but it was pointed out that manufacturers are responsible for providing the users with appropriate information for use and laboratory testing of their product. Strategies to avoid misleading determination of patents' plasma levels, e.g. information on suitable assays, laboratory standards or correction factors were discussed.

The use of von Willebrand factor collagen-binding assay (vWF:CBA) as an alternative method for the quantification of the physiological activity of vWF in plasma from patients with von Willebrand disease (vWD) and in blood clotting factor (F) VIII concentrates (FVIII/vWF concentrates) for the therapy of vWD is currently being discussed. We compared two vWF:CBAs that are distinctive with regard to the type and structure of collagen and the coating principle used. After analyzing samples of a plasma pool from normal donors, we received results that were in very good compliance with both methods. However, significantly different results (p < or =0.005) were obtained when FVIII/vWF concentrates were tested. In an attempt to elucidate these discrepancies, vWF multimers were separated by heparin affinity chromatography and analyzed by vWF antigen enzyme-linked immunosorbent assay (ELISA), ristocetin cofactor activity test, both vWF:CBAs, and a multimer analysis. From our data we conclude that the assay with pepsin-digested collagen (human, type III) that was covalently linked to preactivated microtiter plates (vWF:CBAPDC) revealed a higher affinity for low and medium vWF multimers, whereas the assay with collagen fibrils (equine, type I) that were adsorbed to microtiter plates (vWF:CBACF) predominantly bound high vWF multimers. Based on results reported by others, we assume that the discrepancies between both vWF:CBAs were not related to the type and species of collagen used. Taken together our results imply rather that fragmentation of collagen by pepsin digestion or subsequent covalent linkage to the microtiter plate, or both, increased the affinity for low and medium vWF multimers, whereas the fibrillar structure of collagen was required for the binding of high vWF multimers, which exhibit the highest physiological activity in primary hemostasis.

Osteocalcin is one of the major noncollagenous proteins specific to mineralized connective tissues of vertebrates. A cDNA clone encoding the chicken osteocalcin gene was isolated, and the complete coding sequence for the 97-amino-acid pre-pro-osteocalcin was deduced. The 48-amino-acid pre-pro-peptide contains the expected hydrophobic leader sequence and the dibasic Lys-Arg sequence preceding the NH2-terminal His of the mature 49-amino-acid chicken osteocalcin, which is believed to be necessary for pro-peptide cleavage. The pro-peptide sequence also contains the expected motif of polar and hydrophobic residues, including Phe at -16, which targets vitamin K-dependent gamma-carboxylation of the three specific Glu residues at positions 17, 21, and 24 in the mature protein. Northern blots of total RNA were prepared from embryonic and adult chicken tissues (bone, brain, heart, intestine, kidney, muscle) and probed with chicken osteocalcin cDNA. The appearance of a single 0.5 kb mRNA species confirms that bone is the major site of osteocalcin expression in vivo. In primary osteoblasts isolated from 17-day embryonic chicken calvaria, an osteocalcin mRNA of similar size is expressed concurrently with culture mineralization in vitro. Hypertrophic chondrocytes from 12-day ventral vertebrae and from the cephalic half of 17-day caudal sternae also express osteocalcin mRNA, but nonhypertrophic chondrocytes from the caudal half of 17-day sternae do not express osteocalcin mRNA.

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