Biochemical characterization of LR769, a new recombinant factor VIIa bypassing agent produced in the milk of transgenic rabbits.

BACKGROUND: The bypassing agent factor VII (FVIIa) is a first-line therapy for the treatment of acute bleeding episodes in patients with haemophilia and high-titre inhibitors. FVIIa is a highly post-translationally modified protein that requires eukaryotic expression systems to produce a fully active molecule. A recombinant FVIIa was produced in the milk of transgenic rabbits to increase expression and provide an efficient, safe and affordable product after purification to homogeneity (LR769). AIM: To present the biochemical and functional in vitro characteristics of LR769. RESULTS: Mass spectrometric analyses of the intact protein and of heavy and light chains revealed a fully activated, mature and properly post-translationally modified protein notably regarding N/O-glycosylations and γ-carboxylation. Primary structure analysis, performed by peptide mapping, confirmed 100% of the sequence and the low level or absence of product-derived impurities such as oxidized, deamidated and glycated forms. Low levels of aggregates and fragments were observed by different chromatographic methods. Higher order structure investigated by circular dichroism showed appropriate secondary/tertiary structures and conformational change in the presence of Ca2+ ions. Finally, activated partial thromboplastin time and thrombin generation assays showed the ability of LR769 to decrease coagulation time and to generate thrombin in haemophiliac-A-plasmas, even in the presence of inhibitors. CONCLUSION: The innovative expression system used to produce LR769 yields a new safe and effective rhFVIIa for the treatment of haemophilia A or B patients with inhibitors.

Investigation of monoclonal antibody dimers in a final formulated drug by separation techniques coupled to native mass spectrometry.

Therapeutic monoclonal antibodies (mAbs) are highly complex proteins that must be exhaustively characterized according to the regulatory authorities' recommendations. MAbs display micro-heterogeneity mainly due to their post-translational modifications, but also to their susceptibility to chemical and physical degradations. Among these degradations, aggregation is quite frequent, initiated by protein denaturation and then dimer formation. Here, we investigated the nature and structure of the high molecular weight species (HMW) present at less than 1% in an unstressed formulated roledumab biopharmaceutical, as a model of high purity mAb. HMW species were first purified through preparative size-exclusion chromatography (SEC) and then analyzed by a combination of chromatographic methods (ion-exchange chromatography (IEX), SEC) coupled to native mass spectrometry (MS), as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and capillary gel electrophoresis under non-reducing conditions. Both covalently and non-covalently bound dimers were identified at a proportion of 50/50. In-depth characterization of the HMW fraction by SEC and IEX hyphenated to native MS revealed the presence of three mAb dimer forms having the same mass, but differing by their charge and size. They were attributed to different compact and elongated dimers. Finally, high-resolution middle-up approaches using different enzymes (IdeS and IgdE) were performed to determine the mAb domains implicated in the dimerization. Our results revealed that the roledumab dimers were associated mainly by a single Fab-to-Fab arm-bound association.

Planetary radio astronomy observations from voyager 2 near saturn.

Planetary radio astronomy measurements obtained by Voyager 2 near Saturn have added further evidence that Saturnian kilometric radiation is emitted by a strong dayside source at auroral latitudes in the northern hemisphere and by a weaker source at complementary latitudes in the southern hemisphere. These emissions are variable because of Saturn's rotation and, on longer time scales, probably because of influences of the solar wind and Dione. The electrostatic discharge bursts first discovered by Voyager 1 and attributed to emissions from the B ring were again observed with the same broadband spectral properties and an episodic recurrence period of about 10 hours, but their occurrence frequency was only about 30 percent of that detected by Voyager 1. While crossing the ring plane at a distance of 2.88 Saturn radii, the spacecraft detected an intense noise event extending to above 1 megahertz and lasting about 150 seconds. The event is interpreted to be a consequence of the impact, vaporization, and ionization of charged, micrometer-size G ring particles distributed over a vertical thickness of about 1500 kilometers.

Planetary radio astronomy observations from voyager 2 near jupiter.

The Voyager 2 Planetary Radio Astronomy experiment to Jupiter has confirmed and extended to higher zenomagnetic latitudes results from the identical experiment carried by Voyager 1. The kilometric emissions discovered by Voyager 1 often extended to 1 megahertz or higher on Voyager 2 and often consisted of negatively or, less frequently, positively drifting narrowband bursts. On the basis of tentative identification of plasma wave emissions similar to those detected by Voyager 1, the plasma torus associated with Io appeared somewhat denser to Voyager 2 than it did to Voyager 1. We report here on quasiperiodic sinusoidal or impulsive bursts in the broadcast band range of wavelengths (800 to 1800 kilohertz). A Faraday effect appears at decametric frequencies, which probably results from propagation of the radiation near its sources on Jupiter. Finally, we discuss the occurrence of decametric emission in homologous arc families.

Voyager 2 radio observations of uranus.

Within distances to Uranus of about 6 x 10(6) kilometers (inbound) and 35 x 10(6) kilometers (outbound), the planetary radio astronomy experiment aboard Voyager 2 detected a wide variety of radio emissions. The emission was modulated in a period of 17.24 +/- 0.01 hours, which is identified as the rotation period of Uranus' magnetic field. Of the two poles where the axis of the off-center magnetic dipole (measured by the magnetometer experiment aboard Voyager 2) meets the planetary surface, the one closer to dipole center is now located on the nightside of the planet. The radio emission generally had maximum power and bandwidth when this pole was tipped toward the spacecraft. When the spacecraft entered the nightside hemisphere, which contains the stronger surface magnetic pole, the bandwidth increased dramatically and thereafter remained large. Dynamically evolving radio events of various kinds embedded in these emissions suggest a Uranian magnetosphere rich in magnetohydrodynamic phenomena.

Outburst of Jupiter's synchrotron radiation after the impact of comet Shoemaker-Levy 9.

Jupiter's nonthermal microwave emission, as measured by a global network of 11 radio telescopes, increased dramatically during the Shoemaker-Levy 9 impacts. The increase was wavelength-dependent, varying from approximately 10 percent at 70 to 90 centimeters to approximately 45 percent at 6 and 36 centimeters. The radio spectrum hardened (flattened toward shorter wavelengths) considerably during the week of impacts and continued to harden afterward. After the week of cometary impacts, the flux density began to subside at all wavelengths and was still declining 3 months later. Very Large Array and Australia Telescope images of the brightness distribution showed the enhancement to be localized in longitude and concentrated near the magnetic equator. The evidence therefore suggests that the increase in flux density was caused by a change in the resident particle population, for example, through an energization or spatial redistribution of the emitting particles.

Voyager planetary radio astronomy at neptune.

Detection of very intense short radio bursts from Neptune was possible as early as 30 days before closest approach and at least 22 days after closest approach. The bursts lay at frequencies in the range 100 to 1300 kilohertz, were narrowband and strongly polarized, and presumably originated in southern polar regions ofthe planet. Episodes of smooth emissions in the frequency range from 20 to 865 kilohertz were detected during an interval of at least 10 days around closest approach. The bursts and the smooth emissions can be described in terms of rotation in a period of 16.11 +/- 0.05 hours. The bursts came at regular intervals throughout the encounter, including episodes both before and after closest approach. The smooth emissions showed a half-cycle phase shift between the five episodes before and after closest approach. This experiment detected the foreshock of Neptune's magnetosphere and the impacts of dust at the times of ring-plane crossings and also near the time of closest approach. Finally, there is no evidence for Neptunian electrostatic discharges.

A generic method for intact and subunit level characterization of mAb charge variants by native mass spectrometry.

Monoclonal antibodies (mAbs) are heterogeneous macromolecules that display a complex isoform profile as a result of the large series of modifications they can undergo. Product-related charge variants that are associated with a loss of biological activity or affected half-life and immunogenicity are especially important. Consequently, they are often considered critical quality attributes such that acceptance criteria and controls should be established. The characterization of mAbs charge variants has long been a time and resource consuming task. Recent successes in the use of salt mediated pH gradient ion exchange chromatography with volatile mobile phases have shown there to be significant promise in using online mass spectrometric (MS) detection to facilitate peak detection. In this study, a newly developed 3 µm non-porous cation exchange column technology was investigated for its capability to be hyphenated to MS for the purpose of characterizing mAb charge variants. A 2 mm ID format was selected for the ease of configuring it to classical MS ESI ion sources. A monoclonal antibody reference material from NIST (RM 8671; NISTmAb) was used in its intact and IdeS/IgdE-digested forms to test for column performance and MS sensitivity. Furthermore, three different mAbs with highly basic isoelectric points (pI) were analyzed in their native and proteolyzed forms to demonstrate the straightforward application of the developed technique even with mAbs having strong retention on cation exchange media. The MS detection of low-abundance charge variant species (<0.1%) demonstrated there to be acceptable sensitivity and dynamic range even from routine analyses. The capability of the column to separate different mAbs having high basic pI was demonstrated, and it was found that slight adjustment of ammonium acetate concentration in the eluent can be a convenient way to rapidly optimize a separation if necessary. Linearity was shown to exist between protein mass loads of 2.5 and 50 µg while an optimal balance between chromatographic resolution and MS sensitivity was observed between 5 and 10 µg. Excellent run-to-run and column-to-column repeatability was achieved in terms of retention times, resolution and recovery.

Development and validation of the mental health professional culture inventory.

AIMS: No instrument has been developed to explicitly assess the professional culture of mental health workers interacting with severely mentally ill people in publicly or privately run mental health care services. Because of theoretical and methodological concerns, we designed a self-administered questionnaire to assess the professional culture of mental health services workers. The study aims to validate this tool, named the Mental Health Professional Culture Inventory (MHPCI). The MHPCI adopts the notion of 'professional culture' as a hybrid construct between the individual and the organisational level that could be directly associated with the professional practices of mental health workers. METHODS: The MHPCI takes into consideration a multidimensional definition of professional culture and a discrete number of psychometrically derived dimensions related to meaningful professional behaviour. The questionnaire was created and developed by a conjoint Italian-Canadian research team with the purpose of obtaining a fully cross-cultural questionnaire and was pretested in a pilot study. Subsequently, a validation survey was conducted in northern Italy and in Canada (Montreal area, Quebec). Data analysis was conducted in different steps designed to maximise the cross-cultural adaptation of the questionnaire through a recursive procedure consisting of performing a principal component analysis (PCA) on the Italian sample (N = 221) and then testing the resulting factorial model on the Canadian sample (N = 237). Reliability was also assessed with a test-retest design. RESULTS: Four dimensions emerged in the PCA and were verified in the confirmatory factor analysis: family involvement, users' sexuality, therapeutic framework and management of aggression risk. All the scales displayed good internal consistency and reliability. CONCLUSIONS: This study suggests the MHPCI could be a valid and reliable instrument to measure the professional behaviour of mental health services workers. The content of the four scales is consistent with the literature on psychosocial rehabilitation, suggesting that the instrument could be used to evaluate staff behaviour regarding four crucial dimensions of mental health care.

Characterization of Human Serum Albumin isoforms by ion exchange chromatography coupled on-line to native mass spectrometry.

Human Serum Albumin is the most abundant protein of the plasma and displays a wide range of non-oncotic properties such as antioxidant activity, distribution in tissues and organs of binding molecules and clearance of toxic compounds. Albumin is susceptible to numerous post-translational modifications and particularly related to its free thiol group at Cys34 which is the main circulating scavenger of reactive oxygen species. The characterization of these modifications is of high interest for the diagnosis and treatment of patients with liver diseases and for the structural integrity assessment of albumin as a therapeutic protein. In this study, an ion exchange chromatographic method coupled on-line to native mass spectrometry was developed in order to bridge an effective charge variants separation method with a powerful identification technique for a detailed characterization of albumin isoforms. The chromatographic performance of the method allows the separation of 9 different isoforms that were on-line characterized by MS as oxidized, glycated, deamidated and N/C-terminal truncated forms. The method is also able to detect Cu(II) ions binding to the N-terminal site of the protein which is an important antioxidant feature of albumin. Finally, the method showed preliminary good performance parameters in term of linearity, precision and sensitivity for characterization of purified albumin as well as albumin from raw plasma for clinical and pharmaceutical purposes.

Charge variants characterization of a monoclonal antibody by ion exchange chromatography coupled on-line to native mass spectrometry: Case study after a long-term storage at +5°C.

Numerous putative post-translational modifications may induce variations of monoclonal antibodies charge distribution that can potentially affect their biological activity. The characterization and the monitoring of these charge variants are critical quality requirements to ensure stability and process consistency. Charge variants are usually characterized by preparative ion exchange chromatography, collection of fractions and subsequent reverse-phase liquid chromatography with mass spectrometry analysis. While this process can be automatized by on-line two-dimensional chromatography, it remains often complex and time consuming. For this reason, a straightforward on-line charge variant analysis method is highly desirable and analytical laboratories are actively pursuing efforts to overcome this challenge. In this study, a mixed mode ion exchange chromatographic method using volatile salts and coupled on-line to native mass spectrometry was developed in association with a middle-up approach for a detailed characterization of monoclonal antibodies charge variants. An aged monoclonal antibody, presenting a complex charge variant profile was successfully investigated by this methodology as a case study. Results demonstrate that deamidation of the heavy chain was the major degradation pathway after long-term storage at 5°C while oxidation was rather low. The method was also very useful to identify all the clipped forms of the antibody.

Glycation of polyclonal IgGs: Effect of sugar excipients during stability studies.

A number of intravenous immunoglobulin preparations are stabilized with sugar additives that may lead over time to undesirable glycation reactions especially in liquid formulation. This study aimed to evaluate the reactivity of sugar excipients on such preparations in condition of temperature, formulation and concentration commonly used for pharmaceutical products. Through an innovative LC-MS method reported to characterize post-translational modifications of IgGs Fc/2 fragments, a stability study of IVIg formulated with reducing and non-reducing sugars has been undertaken. The rate of polyclonal IgGs glycation was investigated during 6months at 5, 25, 30 and 40°C. High levels of glycation were observed with reducing sugars such as glucose and maltose in the first months of the stability study from 25°C. Non-reducing sugars presented a low reactivity even at the highest tested temperature (40°C). Furthermore, a site by site analysis was performed by MS/MS to determine the glycation sites which were mainly identified at Lys246, Lys248 and Lys324. This work points out the high probability of glycation reactions in some commercialized products and describes a useful method to characterize IVIg glycated products issued from reducing sugar excipients.

Activation of leukocyte movement and displacement of [3H]leukotriene B4 from leukocyte membrane preparations by (12R)- and (12S)-hydroxyeicosatetraenoic acid.

Both (12R)- and (12S)-hydroxyeicosatetraenoic acid were demonstrated to produce aggregation of rat leukocytes and enhance human leukocyte chemokinesis. (12R)-Hydroxyeicosatetraenoic acid was 10-20-fold more potent than (12S)-hydroxyeicosatetraenoic acid but at least 500-fold less potent than leukotriene B4 in these assays. These relative potencies are correlated with the potencies of (12R)- and (12S)-hydroxyeicosatetraenoic acid for competition of [3H]leukotriene B4 binding to rat and human leukocyte membrane preparations.

Calcium mobilization and right-angle light scatter responses to 12-oxo-derivatives of arachidonic acid in neutrophils: evidence for the involvement of the leukotriene B4 receptor.

The biological activities of two carbonyl compounds derived from arachidonic acid, (5Z,8Z,10E,14Z)-12-keto-5,8,10,14-eicosatetraeno ic acid (12-OxoETE) and (5Z,8Z,10E)-12-oxo-5,8,10-dodecatrienoic acid (12-OxoDTrE) were investigated. The ability of these compounds to induce a mobilization of calcium and to trigger a right-angle scatter response in isolated peripheral blood human neutrophils was determined. The two compounds induced a rapid and dose-dependent increase in the concentration of cytoplasmic free calcium; these effects were clearly detectable at concentrations greater than or equal to 10(-8) M. Pre-exposure of neutrophils to leukotriene B4 completely abolished the calcium mobilization induced by 12-OxoDTre and 12-OxoETE, while pre-exposure of the cells to the carbonyl compounds only slightly reduced the response to subsequent stimulation of neutrophils by leukotriene B4. The carbonyl compounds also induced a decrease in right-angle light scatter and these effects were abolished by pretreatment of neutrophils with leukotriene B4. These data demonstrate that 12-OxoETE and 12-OxoDTrE show significant agonist activities towards human neutrophils and strongly suggest that their mechanisms of action involve the leukotriene B4 binding sites or a common activation sequence.

Stereoselective carbonyl reductases from rat skin and leukocyte microsomes converting 12-ketoeicosatetraenoic acid to 12(S)-HETE.

Cell-free preparations from rat polymorphonuclear leukocytes and skin were found to catalyze the reduction of 12-keto-5,8,10,14-eicosatetraenoic acid (12-KETE) to 12-hydroxyeicosatetraenoic acid (12-HETE). The reductase activity was associated with the microsomal fraction and showed a marked preference for NADH over NADPH as reducing cofactor. Characterization of the reaction product by chiral phase HPLC of the methyl ester derivative indicated that 12-KETE reduction generated almost exclusively 12(S)-HETE. The results demonstrate that rat skin and leukocyte microsomes possess an NADH-dependent 12-KETE reductase activity that forms 12(S)-HETE as a major product. The identification of stereoselective 12-KETE reductases provides a basis for further defining the role these enzymes may play in the regulation of 12-KETE levels and in the protection against degradation of 12-KETE to the pro-inflammatory 12(R)-HETE by selectively generating 12-HETE of the S configuration.

2,3-Diarylcyclopentenones as orally active, highly selective cyclooxygenase-2 inhibitors.

Cyclopentenones containing a 4-(methylsulfonyl)phenyl group in the 3-position and a phenyl ring in the 2-position are selective inhibitors of cyclooxygenase-2 (COX-2). The selectivity for COX-2 over COX-1 is dramatically improved by substituting the 2-phenyl group with halogens in the meta position or by replacing the phenyl ring with a 2- or 3-pyridyl ring. Thus the 3,5-difluorophenyl derivative 7 (L-776,967) and the 3-pyridyl derivative 13 (L-784,506) are particularly interesting as potential antiinflammatory agents with reduced side-effect profiles. Both exhibit good oral bioavailability and are potent in standard models of pain, fever, and inflammation yet have a much reduced effect on the GI integrity of rats compared to standard nonsteroidal antiflammatory drugs.