Katharina Pock

Targeted profiling of atherogenic phospholipids in human plasma and lipoproteins of hyperlipidemic patients using MALDI-QIT-TOF-MS/MS

OBJECTIVES Phospholipids (PLs) are increasingly recognized as key molecules with potential diagnostic value in acute inflammation, CVD and atherosclerosis. We introduce a pioneer mass spectrometry (MS)-based approach aiming to investigate the relationship of specific plasma PL-subsets with atherogenic blood parameters in young patients with familial hyperlipidemia representing high-CVD-risk groups. METHODS Plasma of carefully phenotyped FH and FCH patients as well as normolipidemic subjects (age 13 ± 5 years, n = 20) was used. Clinical parameters were assessed using standard laboratory techniques and lipids were subjected to a direct targeted monitoring using LC-ESI-SRM- and MALDI-QIT-TOF-MS/MS, respectively. Statistical analysis was performed to evaluate correlations between PL data and the clinical parameters. RESULTS Most characteristically significant differences of SM/PC and PC/LPC ratios and positive correlations between SM vs. LDL-C (r = 0.946; p = 0.004) and LPC vs. VLDL-C (r = 0.669; p = 0.218) were observed in FH in contrast to the other study groups. OxPC levels were found in the range of ∼2-20 μmol/L with predominance of short-chain aldehydic species (e.g. SOVPC). A positive correlation of OxPCs with IMT (r = 0.952; p = 0.052) and HDL-C (r = 0.893; p = 0.016) but negative correlation with OxLDL (r = -0.910; p = 0.096) was observed. CONCLUSIONS Our study was a first attempt to use a MALDI-QIT-TOF-MS/MS based clinical lipidomics approach to investigate atherogenic dyslipidemia in young patients with familial hyperlipidemia. This technique represents a promising platform for clinical screening of lipid biomarkers in the future.

Use of high-resolution techniques for the characterization of clotting factor VIII.

Dealing with the structural characterization of clotting factor VIII (FVIII) requires the application of several high-resolution analytical techniques. Besides the analytical point of view, a detailed knowledge of FVIII structure, production and therapeutic application is necessary. This review gives an overview of most of the currently applied analytical methods and how they deal with the complex analytical problem, investigating FVIII in a sample matrix containing large amounts of accompanying plasma proteins.

Characterization of clotting factor IX in plasma-derived preparations by electrophoretic techniques.

Clotting factor IX preparations from human plasma (pdFIX) have been characterized using electrophoretic methods like sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis. Factor IX prior to and after activation with factor XIa was separated by one- and two-dimensional polyacrylamide gel electrophoresis and on isoelectric focusing gels. The main differences between the band patterns of the two pdFIX preparations are due to their purity. Vitronectin was identified by immunological techniques as major accompanying plasma protein, separated from factor IX and characterized by isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis.

Electrophoretic analyses of clotting factor VIII concentrates

Abstract Preparations of clotting factor VIII (FVIII) are complex protein mixtures, regardless of whether they are made by genetically engineered cells or isolated from human plasma. Commercially available FVIII preparations contain either von Willebrand factor (vWF) or human serum albumin (HSA), both of which function as stabilizers. Apart from these, the preparations contain other plasma proteins as impurities. Such protein mixtures were analyzed with SDS–PAGE and 2D-electrophoresis. Single bands and spots were identified by immunoblot using monoclonal and polyclonal antibodies. The aim was to detect every preparation which has an increased level of FVIII cleavage products. Chromatographic methods and SDS–PAGE under reducing and non-reducing conditions usually do not show any differences between single preparations. Only by a combination of separations under non-reducing and increasingly reducing conditions cleavage products of FVIII can in some cases be detected in immunoblot. In 2D electrophoresis the detection of proteins with high molecular masses was achieved by applying a mixture of thiourea and urea. The spots in 2D-electrophoresis of plasma-derived FVIII preparations have been identified as FVIII and vWF, but also as impurities, chiefly from fibrinogen. It was shown that especially 2D-electrophoresis and immunoblot can be used for better characterization of FVIII concentrates. The 2D protein maps of FVIII concentrates will be an efficient tool for quality control of such products.

Influence of HSA and IgG on LDL oxidation studied by size-exclusion chromatography and phospholipid profiling using MALDI tandem-mass spectrometry.

In the present study a direct detection approach combining size-exclusion chromatography (SEC) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem-mass spectrometry (MALDI-QIT-TOF-MS/MS) was applied to investigate the influence of HSA and IgG on LDL oxidation in vitro. SEC analysis showed an increase of protein aggregation during LDL-oxidation that could be essentially suppressed in the presence of HSA. In parallel, lipid peroxidation measured by TBARS assay over 24h was inhibited by 95-100% in the presence of HSA but only 0-34% by IgG, respectively. MALDI phospholipid profiles showed considerable decrease of signals from PCs containing sn-2 PUFAs (18:2 or 20:4) accompanied by increase of sn-2 LPCs indicating for specific breakdown of PUFA-containing PLs during LDL-oxidation. These effects were nearly 100% inhibited in the presence of HSA but not by IgG, respectively. Among known pro-atherogenic PL species present in human plasma sphingomyelin (SM16:0) was bound in significant amounts to HSA but not IgG after incubation with oxLDL. Moreover, our investigation showed that LPCs containing SAFAs (16:0 or 18:0) were specifically bound to HSA, while those containing PUFAs (18:2 and 18:3) were preferentially associated with IgG. In summary, the presented methodology provides a promising platform for studying lipid-protein interactions in vivo.

Thawing of Pooled, Solvent/Detergent-Treated Plasma octaplasLG®: Validation Studies Using Different Thawing Devices

Background: The aim of this study was to perform validation of the thawing process for solvent/detergent-treated plasma octaplasLG® using different thawing devices. Optimized settings for temperature and thawing time should be defined based on the results of both temperature measurements and extensive biochemical characterization studies. Methods: octaplasLG units were thawed using water bath systems (i.e. MB-13A, QuickThaw® DH4), dry tempering systems (i.e. plasmatherm, SAHARA-III), and microwave oven (i.e. transfusio-therm® 2000). Optimized thawing conditions were defined. Subsequently, using the selected thawing conditions, octaplasLG units were thawed and tested on product release parameters. Results: The fastest thawing was observed for the microwave oven. All octaplasLG units thawed by different devices and optimized thawing conditions were clear and free of solid and gelatinous particles, indicating no protein denaturation or overheating. In addition, no significant differences were found in the coagulation and inhibition activity and hemostatic potency of octaplasLG when thawed by the different devices tested. All parameters after thawing were within the product release specification levels. Conclusion: Our study demonstrated that octaplasLG can be thawed using all above listed devices without any negative influence on the plasma quality, presupposed that optimized settings defined for this plasma product are used.

Biochemical characterization and stability of immune globulin intravenous 10% liquid (Panzyga ® )

Panzyga® is a new glycine-formulated immune globulin intravenous 10% liquid for the treatment of patients suffering from immunodeficiencies and autoimmune diseases. Panzyga® is a high purity, native and functional IgG product with an IgG subclass distribution equivalent to normal plasma. The levels of hemagglutinins and accompanying plasma proteins (including IgA and IgM) are low. Potential procoagulant activity is not detectable. Functional activity of the IgG was demonstrated by opsonophagocytosis and receptor binding assays. Dynamic light scattering measurements and fluorescent dye binding were used to characterize the integrity of the IgG molecule. Panzyga® is stable under refrigerated storage for at least two years regarding all assessed physicochemical and functional parameters; it can also be stored at room temperature for at least twelve months within its total shelf-life.

Biochemical characterization, stability, and pathogen safety of a new fibrinogen concentrate (fibryga®)

Fibryga® is a new lyophilized fibrinogen concentrate for intravenous use for the treatment of congenital fibrinogen deficiency. fibryga® is produced from pooled human plasma and the final product is characterized by high purity, integrity, and pathogen safety. Functional activity of fibrinogen was demonstrated by cross-linking studies and thromboelastometry; integrity of the fibrinogen molecule was demonstrated by size exclusion chromatography and the detection of only trace amounts of activation markers in the final product. Pathogen safety of fibryga® was proved by downscaling studies for the two dedicated pathogen inactivation/removal steps, i.e. solvent detergent treatment and nanofiltration. Fibryga® is stable for at least three years when stored at room temperature. In conclusion, the performed studies demonstrated that fibryga® meets the requirements for a state-of-the-art fibrinogen concentrate, such as a satisfactory activity profile combined with a favorable pathogen safety profile and stability.