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Background and Objectives: Complement factor H (CFH) acts as major regulator of the alternative pathway of complement and its mutations and polymorphisms predispose to various human diseases. We aimed to develop a scalable purification process of CFH from human plasma fractions to supply a pathogen-safe and functional CFH concentrate., Materials and Methods: Starting with intermediates of cold ethanol fractionation of plasma, CFH purification was performed with three chromatographic steps including solvent detergent treatment and nanofiltration. CFH functionality was tested by a haemolysis assay using sheep erythrocytes, by determining decay acceleration activity on C3 convertases and cofactor activity of C3b cleavage. CFH identity was confirmed by Western blot and mass spectrometry., Results: Three scalable chromatographic steps highly purified full-length and native CFH from human plasma fractions. The purification process enabled the removal of truncated and dysfunctional CFH species, yielding a native CFH concentrate as demonstrated in sensitive functional in vitro assays., Conclusion: This novel process provides a pathogen-safe and functional CFH concentrate that can be produced on an industrial scale and is suitable for pre-/clinical studies., (© 2012 The Author(s). Vox Sanguinis © 2012 International Society of Blood Transfusion.)

Background and Objectives: A high concentration of large size polymers in intravenous immunoglobulin preparations was always correlated with high anticomplementary activity (ACA). In former days, high ACA was also linked to adverse reactions in patients. The goal of this study was to scrutinize critical parameters of the ACA assay and the influence of different polymer variants of IgG on the complement consumption., Materials and Methods: Critical reagents as the complement and the preparation of erythrocytes were investigated. The influence of molecular integrity of IgG on the ACA was tested by subjecting IgG solutions ranging from pH 4.5 to 7.0 to heat treatment at 60 degrees C., Results: The different complement batches had a significant impact on the test result of the ACA assay. It was demonstrated that polymers, provoked by heat treatment at pH values above 5.5, consumed complement almost completely whereas a polymer content up to 12% formed under acidic conditions did not lead to an increase in ACA., Conclusion: It was shown that suitable complement batches have to be identified in a screening procedure. Furthermore, it could be demonstrated that IgG polymers formed in the neutral pH range during heat treatment were potential ACA inducing compounds. Manufacturing the IVIG preparations under acidic conditions may help to avoid the formation of those ACA active polymers. Thus, intensive analysis of ACA during process development and validation is recommended.

Background and Objectives: The pathomechanism of thrombotic thrombocytopenic purpura (TTP) and atypical haemolytic uraemic syndrome (aHUS) is associated with a severe deficiency of ADAMTS13 and factor H. The aim of this study was to quantify the levels of ADAMTS13 and factor H in the pharmaceutically licensed plasma for transfusion, Octaplas, and the universally applicable plasma, Uniplas (development product, working title). Furthermore, Octaplas batches of blood groups A, B, O, AB, and plasmas derived from different sources were compared., Materials and Methods: Twenty-four Octaplas and three Uniplas batches were selected for the study. ADAMTS13 activities were measured by fluorescence resonance energy transfer assay, ADAMTS13 antigen levels were quantified using enzyme-linked immunosorbent assay test kit, while factor H antigen levels were detected using radial immunodiffusion (RID) methods. In addition, von Willebrand factor (vWF) multimeric analyses were performed., Results: Both Octaplas, produced from US and European plasma of different blood groups, and Uniplas contain ADAMTS13 antigen and activity levels as well as factor H concentrations at normal levels without significant differences. In addition, Octaplas and Uniplas show a vWF multimeric pattern comparable to normal plasma., Conclusion: The study revealed that Octaplas and Uniplas contain normal levels of ADAMTS13 at low batch-to-batch variations. Therefore, both products can substitute the missing or neutralized protease activity in TTP patients and thereby limit vWF-dependent (platelet-related) thrombosis. In addition, both plasma products contain factor H at a physiological level, and, thus can be used efficiently in the treatment of aHUS patients, which have been shown to benefit from plasma administration.

Highly purified, plasma-derived factor IX (FIX) concentrates are produced in large part by a combination of anion exchange and heparin affinity chromatography. However, the concentrates still contain some accompanying proteins. The main impurity has turned out to be the adhesive glycoprotein, vitronectin. It occurs in concentrates exclusively in its multimeric form, in contrast to the situation in plasma. The multimeric vitronectin can be removed either by nanofiltration with a crossflow system or by size-exclusion chromatography. When these FIX concentrates are used as therapeutic agents, the fact has to be taken into account that considerable amounts of multimeric vitronectin are given to the patient. The physiological consequences of the dosage of this protein have not yet been investigated. Although no thrombogenicity has been reported in connection with the above-mentioned FIX concentrates, we recommend that the impurity should be removed from the preparation with the methods described here.

The biochemical and immunochemical aspects of the development of inhibitors with a plasma-derived, double-virus inactivated factor VIII (FVIII) concentrate (marketed as Octavi SDPlus in Germany and Bisinact in Belgium) are described. A total of 12 cases of inhibitor formation (predominantly type II) were reported in Germany, 8 in Belgium but none in Portugal. Initially, the only difference between the non-pasteurised, SD virus-inactivated product Octavi and the pasteurised product Octavi SDPlus appeared to be pasteurisation, though subsequently, the quality of source material for the product was found to differ in different countries. Separation studies revealed the presence of a 40 kDa peptide fragment in some batches. It was subsequently shown that there was a strong correlation between inhibitor development and batches containing the 40 kDa marker, and a relationship between elevated markers of coagulation activation (FPA in particular) and the occurrence of the 40 kDa marker. Further work revealed that analytical methods commonly used for quality control were not suitable to highlight batch-to-batch differences. It was concluded that inhibitor potential (neoantigenicity) in Octavi SDPlus arose due to two effects; degradation of FVIII already present in source material; and heating of unstable FVIII degradation products. In this case, inhibitors were not caused by the overall production process, nor by GMP failures. The problem of inhibitor potential can be avoided if appropriate preventive measures are taken. Further work is needed to prove non-neoantigenicity and to reinforce the scientific findings, and to characterise pilot batches.