Functional in vitro assessment of modified antibodies: Impact of label on protein properties.

Labelling of therapeutic antibodies with radionuclides or fluorophores is routinely used to study their pharmacokinetic properties. A critical assumption in utilizing labelled therapeutic antibodies is that the label has no unfavourable effects on antibody charge, hydrophobicity, or receptor affinity. Ideally, the labelled protein should not have any significant deviations from the physiological properties of the original molecule. This article describes an established quality in vitro assessment workflow for labelled antibodies that ensures better prediction of changes in antibody pharmacokinetic (PK) properties after modifications. This analysis package considers degradation and aggregation analysis by size-exclusion chromatography, changes in neonatal-Fc-receptor (FcRn) affinity, and heparin interaction. FcRn binding is important for antibody recycling and half-life extension, whereas heparin affinity provides estimates on the rate of endocytosis through unspecific cell surface binding. Additionally, mass spectrometric analysis to determine the degree of labelling (DoL) completes the package and the combined analysis data allow to predict the label contribution to the PK properties of the modified antibody. This analytical strategy for labelling 11 IgGs has been investigated using 2 different IgG1 constructs and applying 7 different types of labels. Each labelling resulted in a change in the physicochemical properties of the protein. Not only can the DoL of modified IgGs lead to a change in protein properties, but the type of label also can. Furthermore, it was demonstrated that the labelling process can also influence the behaviour of labelled mAbs. An identical label on different constructs of IgG1 can cause different affinities for FcRn and heparin. Considering the assessment data, only 6 of the 11 modified antibodies from this study can be recommended for subsequent experiments. In conclusion, a suitability assessment of labelled antibodies prior to any pharmacokinetic studies is essential to reduce cost, allocate resources and reduce the number of animal experiments during pre-clinical drug development.

Assessment of Pre-Analytical Sample Handling Conditions for Comprehensive Liquid Biopsy Analysis.

Liquid biopsies as a minimally invasive approach have the potential to revolutionize molecular diagnostics. Yet, although protocols for sample handling and the isolation of circulating tumor DNA (ctDNA) are numerous, comprehensive guidelines for diagnostics and research considering all aspects of real-life multicenter clinical studies are currently not available. These include limitations in sample volume, transport, and blood collection tubes. We tested the impact of commonly used (EDTA and heparin) and specialized blood collection tubes and storage conditions on the yield and purity of cell-free DNA for the application in down-stream analysis. Moreover, we evaluated the feasibility of a combined workflow for ctDNA and tumor cell genomic testing and parallel flow cytometric analysis of leukocytes. For genomic analyses, EDTA tubes showed good results if stored for a maximum of 4 hours at room temperature or for up to 24 hours when stored at 4°C. Spike-in experiments revealed that EDTA tubes in combination with density gradient centrifugation allowed the parallel isolation of ctDNA, leukocytes, and low amounts of tumor cells (0.1%) and their immunophenotyping by flow cytometry and down-stream genomic analysis by whole genome sequencing. In conclusion, adhering to time and temperature limits allows the use of routine EDTA blood samples for liquid biopsy analyses. We further provide a workflow enabling the parallel analysis of cell-free and cellular features for disease monitoring and for clonal evolution studies.

Glycosaminoglycan / gold nanocluster hybrid nanoparticles as a new sensing platform: Metastatic potential assessment of cancer cells.

A novel fluorescent heparanase assay based on hybrid nano-assembly of gold nanocluster and glycosaminoglycan is developed. The nanoparticle probes are fabricated through the co-assembly of positively charged gold nanoclusters with negatively charged heparin molecules, which is accompanied by a dramatic size change and a 2.5-fold fluorescence enhancement. It is demonstrated that the fluorescence enhancement is due to denser aggregation of Au-thiolate complexes in the hybrid nanoparticle and the fluctuation of the fluorescence intensity is an indicator of the variation in assembly efficiency. Experiments in solution and in cell lysis media showed that the heparanase could turn-off the fluorescence with a high selectivity, which could be utilized for the assessment of heparanase activity and the metastatic potentials of different tumour cells. This assay technique is low cost, easy to prepare, and showing good performance. The co-assembly strategy has potential to be transferable to construct other functional nanomaterial.

Thromboinflammation as bioactivity assessment of H2O2-alkali modified titanium surfaces.

The release of growth factors from platelets, mediated by the coagulation and the complement system, plays an important role in the bone formation around implants. This study aimed at exploring the thromboinflammatory response of H2O2-alkali soaked commercially pure titanium grade 2 discs exposed to whole human blood, as a way to assess the bioactivity of the discs. Commercially pure titanium grade 2 discs were modified by soaking in H2O2, NaOH and Ca(OH)2. The platelet aggregation, coagulation activation and complement activation was assessed by exposing the discs to fresh whole blood from human donors. The platelet aggregation was examined by a cell counter and the coagulation and complement activation were assessed by ELISA-measurements of the concentration of thrombin-antithrombin complex, C3a and terminal complement complex. The modified surface showed a statistically significant increased platelet aggregation, coagulation activation and complement activation compared to unexposed blood. The surface also showed a statistically significant increase of coagulation activation compared to PVC. The results of this study showed that the H2O2-alkali soaked surfaces induced a thromboinflammatory response that indicates that the surfaces are bioactive.

Reference values assessment in a Mediterranean population for small dense low-density lipoprotein concentration isolated by an optimized precipitation method.

BACKGROUND: High serum concentrations of small dense low-density lipoprotein cholesterol (sd-LDL-c) particles are associated with risk of cardiovascular disease (CVD). Their clinical application has been hindered as a consequence of the laborious current method used for their quantification. OBJECTIVE: Optimize a simple and fast precipitation method to isolate sd-LDL particles and establish a reference interval in a Mediterranean population. MATERIALS AND METHODS: Forty-five serum samples were collected, and sd-LDL particles were isolated using a modified heparin-Mg2+ precipitation method. sd-LDL-c concentration was calculated by subtracting high-density lipoprotein cholesterol (HDL-c) from the total cholesterol measured in the supernatant. This method was compared with the reference method (ultracentrifugation). Reference values were estimated according to the Clinical and Laboratory Standards Institute and The International Federation of Clinical Chemistry and Laboratory Medicine recommendations. sd-LDL-c concentration was measured in serums from 79 subjects with no lipid metabolism abnormalities. RESULTS: The Passing-Bablok regression equation is y = 1.52 (0.72 to 1.73) + 0.07x (-0.1 to 0.13), demonstrating no significant statistical differences between the modified precipitation method and the ultracentrifugation reference method. Similarly, no differences were detected when considering only sd-LDL-c from dyslipidemic patients, since the modifications added to the precipitation method facilitated the proper sedimentation of triglycerides and other lipoproteins. The reference interval for sd-LDL-c concentration estimated in a Mediterranean population was 0.04-0.47 mmol/L. CONCLUSION: An optimization of the heparin-Mg2+ precipitation method for sd-LDL particle isolation was performed, and reference intervals were established in a Spanish Mediterranean population. Measured values were equivalent to those obtained with the reference method, assuring its clinical application when tested in both normolipidemic and dyslipidemic subjects.

Effectiveness and Safety Assessment of Citrate Anticoagulation During Albumin Dialysis in Comparison to Other Methods of Anticoagulation.

Liver failure is a serious and often deadly disease often requiring MARS (Molecular Adsorbent Recirculating System) therapy. Choosing the safe and effective method of anticoagulation during artificial liver support systems seems to be very difficult and extremely important. The aim of this study was to assess effectiveness and safety of regional anticoagulation with citrate in liver failure patients during MARS. We used a single center observational study. We analyzed 158 MARS sessions performed in 65 patients: 105 (66.5%) sessions in 41 patients with heparin anticoagulation, 40 (25.3%) sessions in 19 patients with citrate, and 13 (8%) sessions in only five patients without anticoagulation, that were excluded from part of the analysis. To determine the effectiveness of regional anticoagulation with citrate, probability of filter survival and changes in laboratory parameters were analyzed according to the applied method of anticoagulation. The safety of citrate was determined by Ca/Ca2+ ratio, acid-base balance, bleeding complications, and the need for blood product transfusions. The probability of filter survival in the citrate group was 94% and in the heparin group 82% (P = 0.204). There was no relationship between the method of anticoagulation and effectiveness of MARS therapy in lowering the levels of the analyzed parameters. Only one patient had a Ca/Ca2+ ratio higher than he safety margin. There were no statistically significant changes in pH and lactate level irrespective of anticoagulation; bicarbonate dropped significantly only in the heparin group (P = 0.03). The frequency of bleeding complications and the need for transfusions did not differ significantly between groups. Regional anticoagulation with citrate can be an effective and safe method of anticoagulation during MARS therapy, but requires attentive monitoring and further studies in liver failure patients.

Anti-coagulation assessment with prothrombin time and anti-Xa assays in real-world patients on treatment with rivaroxaban.

Monitoring of anti-coagulation with the direct factor Xa inhibitor rivaroxaban is considered unnecessary in a routine clinical setting. However, assessment of its anti-coagulant effect may be desirable in certain clinical situations. We assessed prothrombin time (PT) reagents and commercially available anti-Xa assays (Biophen) calibrated for rivaroxaban and heparin in comparison to liquid chromatography-mass spectrometry (LC-MS/MS) measurements of rivaroxaban concentration in samples from patients on treatment with rivaroxaban for stroke prevention in atrial fibrillation. Citrate plasma samples were obtained from 30 randomly selected patients on uninterrupted treatment with rivaroxaban for a minimum of 1 month. The anti-Xa assays, direct Xa inhibitor (DiXa-I®), and Heparin LRT® were conducted for both wide and low calibrations for rivaroxaban. Measurements were compared to LC-MS/MS using correlation, linear regression, intra-class correlation, and Bland-Altman analysis. In 30 patients (9 female) of median age 71.5 years and BMI 26.5 kg/m(2), rivaroxaban concentrations between 2.4 and 625 ng/ml (median 82 ng/ml) were measured by LC-MS/MS. PT reagents were poorly correlated with rivaroxaban concentrations (r (2) = 0.52 and 0.09). Anti-Xa assays DiXa-I (r (2) = 0.95) and Heparin LRT (r (2) = 0.97) were correlated with rivaroxaban in all concentrations, but especially in low concentrations with low calibrations (r (2) = 0.97 and 0.98, respectively). The highest agreement occurred between Heparin LRT and low rivaroxaban concentrations with a mean difference of -5.3 ng/ml (limits of agreement, 12.9 to 2.4 ng/ml). Anti-Xa assays can indirectly determine the concentration of rivaroxaban for a wide range of concentrations in real-world patients. An interpretation of anti-Xa and PT measurements in treatment with rivaroxaban requires knowledge of the local reagents.

An economic evaluation of rt-PA locking solution in dialysis catheters.

In a recent randomized trial, weekly recombinant tissue plasminogen activator (rt-PA), 1 mg per lumen, once per week, and twice-weekly heparin as a locking solution (rt-PA/heparin) resulted in lower risks of hemodialysis catheter malfunction and catheter-related bacteremia compared with thrice-weekly heparin (heparin alone). We collected detailed costs within this trial to determine how choice of locking solution would affect overall health care costs, including the cost of locking solutions and all other relevant medical costs over the course of the 6-month trial. Nonparametric bootstrap estimates were used to derive 95% confidence intervals (CIs) and mean cost differences between the treatment groups. The cost of the locking solution was higher in patients receiving rt-PA/heparin, but this was partially offset by lower costs for managing complications. Overall, the difference in unadjusted mean cost for managing patients with rt-PA/heparin versus heparin alone was Can$323 (95% CI, -$935 to $1581; P=0.62). When the costs were extrapolated over a 1-year time horizon using decision analysis, assuming ongoing rt-PA effectiveness, the overall costs of the strategies were similar. This finding was sensitive to plausible variation in the frequency and cost of managing patients with catheter-related bacteremia, and whether the benefit of rt-PA on catheter-related bacteremia was maintained in the long term. In summary, we noted no significant difference in the mean overall cost of an rt-PA/heparin strategy as a locking solution for catheters compared with thrice-weekly heparin. Cost savings due to a lower risk of hospitalization for catheter-related bacteremia partially offset the increased cost of rt-PA.

Post-heparin LPL activity measurement using VLDL as a substrate: a new robust method for routine assessment of plasma triglyceride lipolysis defects.

BACKGROUND: Determination of lipoprotein lipase (LPL) activity is important for hyperchylomicronemia diagnosis, but remains both unreliable and cumbersome with current methods. Consequently by using human VLDL as substrate we developed a new LPL assay which does not require sonication, radioactive or fluorescent particles. METHODS: Post-heparin plasma was added to the VLDL substrate prepared by ultracentrifugation of heat inactivated normolipidemic human serums, diluted in buffer, pH 8.15. Following incubation at 37°c, the NEFA (non esterified fatty acids) produced were assayed hourly for 4 hours. LPL activity was expressed as µmol/l/min after subtraction of hepatic lipase (HL) activity, obtained following LPL inhibition with NaCl 1.5 mmol/l. Molecular analysis of LPL, GPIHBP1, APOA5, APOC2, APOE genes was available for 62 patients. RESULTS: Our method was reproducible (coefficient of variation (CV): intra-assay 5.6%, inter-assay 7.1%), and tightly correlated with the conventional radiolabelled triolein emulsion method (n = 26, r = 0.88). Normal values were established at 34.8 ± 12.8 µmol/l/min (mean ± SD) from 20 control subjects. LPL activities obtained from 71 patients with documented history of major hypertriglyceridemia showed a trimodal distribution. Among the 11 patients with a very low LPL activity (< 10 µmol/l/min), 5 were homozygous or compound heterozygous for LPL or GPIHBP1 deleterious mutations, 3 were compound heterozygous for APOA5 deleterious mutations and the p.S19W APOA5 susceptibility variant, and 2 were free of any mutations in the usual candidate genes. No homozygous gene alteration in LPL, GPIHBP1 and APOC2 genes was found in any of the patients with LPL activity > 10 µmol/l/min. CONCLUSION: This new reproducible method is a valuable tool for routine diagnosis and reliably identifies LPL activity defects.

Optimizing biliary stent patency by coating with hydrophobin alone or hydrophobin and antibiotics or heparin: an in vitro proof of principle study.

PURPOSE: Although a wide range of biliary plastic and metal stents is on offer nowadays, the ideal cost-effective stent that functions permanently and that is easy to handle regarding its exchange is still not available. Therefore we tested in an in vitro model if the coating of plastic stents with hydrophobin alone or with hydrophobin and antibiotics or heparin in combination leads to an inhibition of the clogging process. METHODS: We coated commercially available biliary plastic stents with hydrophobin alone, as well as with hydrophobin and antibiotics or heparin in combination. After an incubation period of 28 days in human bile, we examined the stents by scanning electron microscopy to see whether the clogging material on its surface was reduced. RESULTS: Coating of plastic stents with hydrophobin led to a reduction in the amount of adherent material on the surface of the stents. Coupling of ampicillin/sulbactam or levofloxacin did not lead to a further reduction of the clogging material, whereas coupling with highly concentrated heparin did reduce the adherent material. CONCLUSIONS: The coating of biliary plastic stents with hydrophobin or with hydrophobin and heparin in combination seems to be a promising option to delay the clogging process.

Detection of possible economically motivated adulterants in heparin sodium and low molecular weight heparins with a colorimetric microplate based assay.

Recently, we described a 96-well plate format assay for visual detection of oversulfated chondroitin sulfate A (OSCS) contamination in heparin samples based on a water-soluble cationic polythiophene polymer (3-(2-(N-(N'-methylimidazole))ethoxy)-4-methylthiophene (LPTP)) and heparinase digestion of heparin. Here, we establish the specificity of the LPTP/heparinase test with a unique set of reagents that define the structural requirements for significant LPTP chemosensor color change. For example, we observed a biphasic behavior of larger shifts to the red in the UV absorbance spectra with decreasing average molecular weight of heparin chains with a break below 12-mer chain lengths. In addition, the oversulfation of chondroitin sulfate A (CSA) to a partially (PSCS) or fully (OSCS) sulfated form caused progressively less red shift of LPTP solutions. Furthermore, glycosaminoglycans (GAGs) containing glucuronic acid caused distinct spectral patterns compared to iduronic acid containing GAGs. We applied the LPTP/heparinase test to detection of OSCS (≥0.03% (w/w) visually or 0.01% using a plate reader) in 10 µg amounts of low molecular weight heparins (LMWHs; i.e. dalteparin, tinzaparin, or enoxaparin). Furthermore, because other oversulfated GAGs are possible economically motivated adulterants (EMAs) in heparin sodium, we tested the capacity of the LPTP/heparinase assay to detect oversulfated dermatan sulfate (OSDS), heparin (OSH), and heparan sulfate (OSHS). These potential EMAs were visually detectable at a level of ∼0.1% when spiked into heparin sodium. We conclude that the LPTP/heparinase test visually detects oversulfated GAGs in heparin sodium and LMWHs in a format potentially amenable to high-throughput screening.

Efficient and inexpensive method for purification of heparin binding proteins.

Heparin binding (HB) proteins mediate a wide range of important cellular processes, which makes this class of proteins biopharmaceutically important. Engineering HB proteins may bring many advantages, but it necessitates cost effective and efficient purification methodologies compared to currently available methods. One of the most important classes of HB proteins are fibroblast growth factors (FGFs) and their receptors (FGFRs). In this study, we report an efficient off-column purification of FGF-1 from soluble fractions and purification of the D2 domain of FGFR from insoluble inclusion bodies, using a weak Amberlite cation (IRC) exchanger. FGF-1 and the D2 domain have been expressed in Escherichia coli and purified to homogeneity using IRC resin. This approach is an alternative to conventional affinity column chromatography, which exhibits several disadvantages, including time-consuming experimental procedures for purification and regeneration and results in the expensive production of recombinant proteins. Results of the heparin binding chromatography and steady state fluorescence experiments show that the FGF-1 and the D2 are in a native conformation. The findings of this study will not only aid an in-depth investigation of this class of proteins but will also provide avenues for inexpensive and efficient purification of other important biological macromolecules.

Determination of galactosamine impurities in heparin samples by multivariate regression analysis of their (1)H NMR spectra.

Heparin, a widely used anticoagulant primarily extracted from animal sources, contains varying amounts of galactosamine impurities. Currently, the United States Pharmacopeia (USP) monograph for heparin purity specifies that the weight percent of galactosamine (%Gal) may not exceed 1%. In the present study, multivariate regression (MVR) analysis of (1)H NMR spectral data obtained from heparin samples was employed to build quantitative models for the prediction of %Gal. MVR analysis was conducted using four separate methods: multiple linear regression, ridge regression, partial least squares regression, and support vector regression (SVR). Genetic algorithms and stepwise selection methods were applied for variable selection. In each case, two separate prediction models were constructed: a global model based on dataset A which contained the full range (0-10%) of galactosamine in the samples and a local model based on the subset dataset B for which the galactosamine level (0-2%) spanned the 1% USP limit. All four regression methods performed equally well for dataset A with low prediction errors under optimal conditions, whereas SVR was clearly superior among the four methods for dataset B. The results from this study show that (1)H NMR spectroscopy, already a USP requirement for the screening of contaminants in heparin, may offer utility as a rapid method for quantitative determination of %Gal in heparin samples when used in conjunction with MVR approaches.

Combination of a two-step fluorescence assay and a two-step anti-Factor Xa assay for detection of heparin falsifications and protein in heparins.

There are several methods for sensitive detection of oversulfated chondroitin sulfate (OSCS) in heparin. Although contamination with OSCS is unlikely to be repeated, use of other compounds to counterfeit heparin must be considered. We have previously developed a two-step fluorescence microplate assay (two-step FI assay) for detection of OSCS. First, the heparin sample is incubated with heparinase I, then its increasing effect on the fluorescence intensity (FI) of the sensor molecule Polymer-H is measured (PolyH assay). The high sensitivity of the assay is shown to be based on heparinase I inhibition by OSCS. The objective of this study was to evaluate another assay option - indirect quantification of OSCS after heparinase I incubation by means of the anti-Factor Xa (aXa) activity of the remaining undegraded heparin (two-step aXa assay). We also examined, whether other heparin mimetics (HepM), direct Factor Xa inhibitors (DXI), and protein impurities are detectable by use of these assays. Heparin was spiked with different amounts of HepM including OSCS, pentosan polysulfate, dextran sulfate, curdlan sulfate, the natural contaminant dermatan sulfate, the DXI rivaroxaban, and BSA as a protein. These samples were compared with pure heparin in the two-step FI assay, the two-step aXa assay, and in the PolyH assay and the aXa assay without heparinase I incubation. Both two-step assays sensitively measured contamination with all the HepM (LOD ≤ 0.5%, LOQ ≤ 0.7%). The two-step aXa assay also detected rivaroxaban (LOD 0.3%, LOQ 0.4%), whereas the two-step FI assay was shown to be suited to determination of protein impurities (LOD 0.11%, LOQ 0.13%). Use of two different heparinase I inactivation procedures enabled clear differentiation between protein, HepM, and both contaminants. Finally, with the aXa assay the heparin potency can be determined in the same assay run, whereas the FI increase in the PolyH assay was shown to be useful for identification. In conclusion, both the two-step FI assay and the two-step aXa assay are sensitive, rapid, and simple tests for the detection of counterfeit heparin. Comprehensive information about heparin quality can be obtained by their combined use and the parallel measurement of non-incubated heparin samples.

Simple fluorescence assay for quantification of OSCS in heparin.

In 2008, heparin contaminated with oversulfated chondroitin sulfate (OSCS) penetrated the worldwide market and was associated with severe adverse effects. Feasible and reliable methods to test heparin for adulteration are needed. The objective was to develop a simple approach based on a microplate assay for quantification of heparin and sulfated glycans using the fluorescent heparin sensor polymer-H (polymer-H assay). However, both heparin and OSCS concentration-dependently increase the fluorescence intensity (FI) of polymer-H, so that OSCS in heparin cannot be detected. The idea was a two-step procedure including, first, separation of heparin by degradation with heparinase I, and then measurement of the remaining OSCS. To achieve complete heparin (unfractionated heparin (UFH), enoxaparin) degradation, several conditions (e.g. incubation time and heparinase I concentration) were optimized by using the aXa assay for monitoring. Defined UFH/OSCS mixtures incubated in this way showed a concentration-dependent FI increase in the polymer-H assay (λ ((em)) 330 nm, λ ((ex)) 510 nm). The sensitivity was unexpectedly high with an LOD/LOQ of 0.5%/0.6% OSCS content in heparin. Further experiments testing UFH/OSCS mixtures in the aXa assay confirmed our hypothesis: OSCS inhibits heparinase I resulting in incomplete heparin degradation and thus an additional FI increase of polymer-H by intact heparin. This two-step microplate fluorescence assay is a sensitive, rapid, and simple method for quantification of OSCS in heparin. In contrast with (1)H NMR and CE, neither expensive equipment nor much experience are required. It could be applied not only in the quality control of heparin, but also in clinical practice, to check the applied heparin preparation when a patient suffers any adverse effect.

Assay of possible economically motivated additives or native impurities levels in heparin by 1H NMR, SAX-HPLC, and anticoagulation time approaches.

New assays have been developed to detect the presence of economically motivated additives (EMAs) and poor manufacturing processes in heparin. Here, selected oversulfated glycosaminoglycans that are possible EMAs to heparin were synthesized and the utility of current (1)H NMR, SAX-HPLC or anticoagulation time protocols were evaluated for the detection of native impurities (chondroitin sulfate A or B (CSA or CSB), or heparan sulfate (HS)), or synthetic contaminants (oversulfated-(OS)-CSA, OS-CSB, OS-HS or OS-Heparin) spiked into heparin sodium active pharmaceutical ingredients (APIs). The range of w/w percent LOD values from the SAX-HPLC analysis for heparin spiked with CSA, CSB, HS, OS-CSA, OS-CSB, OS-HS, OS-Heparin and two partially oversulfated CSA analogs was 0.05-0.12%. The 500 MHz 1D-(1)H NMR spectra of heparin spiked with 1.0-10% CSA, CSB, OS-CSA, or OS-CSB showed unique signal pattern changes while the samples spiked with HS, OS-HS, OS-Heparin or partially sulfated CSA were more difficult to identify using NMR data. The ratio of anticoagulation time values obtained with factor Xa and factor IIa were found to remain within USP specifications in the presence of 10% amounts of these impurities or contaminants. In a separate test, using OS-CSA spiked API heparin samples, the factor Xa or factor IIa to USP standard ratio were found to fall below the USP 0.9 specification value in samples spiked at ca. weight percent of 15% or greater of OSCS. We conclude that the SAX-HPLC assay is the most sensitive and robust assay to identify and quantitate possible GAG-based EMAs in heparin.

The potential benefits of low-molecular-weight heparins in cancer patients.

Cancer patients are at increased risk of venous thromboembolism due to a range of factors directly related to their disease and its treatment. Given the high incidence of post-surgical venous thromboembolism in cancer patients and the poor outcomes associated with its development, thromboprophylaxis is warranted. A number of evidence-based guidelines delineate anticoagulation regimens for venous thromboembolism treatment, primary and secondary prophylaxis, and long-term anticoagulation in cancer patients. However, many give equal weight to several different drugs and do not make specific recommendations regarding duration of therapy. In terms of their efficacy and safety profiles, practicality of use, and cost-effectiveness the low-molecular-weight heparins are at least comparable to, and offer several advantages over, other available antithrombotics in cancer patients. In addition, data are emerging that the antithrombotics, and particularly low-molecular-weight heparins, may exert an antitumor effect which could contribute to improved survival in cancer patients when given for long-term prophylaxis. Such findings reinforce the importance of thromboprophylaxis with low-molecular-weight heparin in cancer patients.

UV-MALDI-TOF mass spectrometry analysis of heparin oligosaccharides obtained by nitrous acid controlled degradation and high performance anion exchange chromatography.

Nitrous acid degradation of heparin followed by high-performance anion-exchange chromatography (HPAEC) separation and ultraviolet matrix assisted laser desorption/ionization time-of-flight (UV-MALDI-TOF) analysis led to the structural determination of six sulfated oligosaccharides. Three different matrices (alpha-cyano-4-hydroxycinnamic acid (CHCA), nor-harmane, and dihydroxybenzoic acid (DHB)) have been used, and the complementary results obtained allowed in most cases to assign the position of sulfate groups. Based on the different cleavages produced on the purified oligosaccharides in source during the MS analysis by the use of the different matrices, this approach provides a new tool for structural analysis.