RESUMO
In the production of biological therapeutics such as monoclonal antibodies (mAbs), ultrafiltration and diafiltration (UF/DF) are widely regarded as effective downstream processing steps capable of removing process equipment related leachables (PERLs) introduced upstream of the UF/DF step. However, clearance data available in the literature are limited to species with low partition coefficients (log P) such as buffer ions, hydrophilic organic compounds, and some metal ions. Additional data for a wide range of PERLs including hydrophobic compounds and elemental impurities are needed to establish meaningful, comprehensive safety risk assessments. Herein, we report the results from studies investigating the clearance of seven different organic PERLs representing a wide range of characteristics (i.e., log P (-0.3 to 18)), and four model elements with different chemical properties spiked into a mAb formulation at 10 ppm and analyzed during clearance using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-photodiode-array-mass spectrometry (LC-PDA-MS), and inductively coupled plasma mass spectrometry (ICP-MS). The clearance data showed ideal clearance and sieving of spiked organic PERLs with log P < 4, partial clearance of PERLs with 4 < log P < 9, and poor clearance of highly hydrophobic PERLs (log P > 9) after nine diafiltration volumes (DVs). Supplemental clearance studies on seven additional PERLs present at much lower concentration levels (0.1-1.5 ppm) in the mAb formulation upstream of UF/DF and three PERLs associated with the tangential flow filtration (TFF) equipment also demonstrated the similar correlations between log P and % clearance. For model elements, the findings suggest that UF/DF in general provides ideal clearance for elements. Evidence showed that the UF/DF process does not only help mitigate leachables risk from PERLs introduced upstream of UF/DF, but also from the TFF operation itself as all three TFF-related PERLs were effectively cleared. Overall, the UF/DF clearance presented in this work demonstrated whereas highly hydrophobic PERLs and elements that exist as charged species, particularly transition metal ions, may not be as effectively cleared and thus warrant further risk assessment; hydrophilic and some hydrophobic PERLs (log P < 4) are indeed well-cleared and thus present a lower overall safety risk.
Assuntos
Filtração , Ultrafiltração , Ultrafiltração/métodos , Filtração/métodos , Compostos Orgânicos , ÍonsRESUMO
Irradiation sterilization of polymeric pharmaceutical processing systems and medical devices, an essential healthcare technology, is facing critical business continuity challenges, driving the need to qualify equivalent alternative irradiation technologies, such as X-ray. Whereas the underlying there is a paucity of cross-industry published data evaluating X-ray irradiation effects on plastics as compared to gamma irradiation. That leads to regulatory uncertainty in the levels of costly validation data regulators will require and overall apprehension in the rate of X-ray irradiation adoption. The present study evaluates the impact of X-ray versus gamma irradiation on a wide range of polymers with more than 36 single-use (SU) components, using a comprehensive set of industry aligned methods for characterization of bioprocess polymers. Whereas many of these techniques readily demonstrate changes in polymer properties following irradiation, all of the polymers evaluated demonstrated that the impact of X-ray irradiation was to the same degree or less as compared to gamma. Increased publication of studies evaluating the impact to polymers of X-ray versus gamma irradiation is critical to leveraging extensive, existing validation packages on bioprocess systems and medical devices obtained following gamma irradiation, and essential in qualifying X-ray irradiation as an equivalent technology (i.e., materials are impacted to the same extent or less than gamma) that can overcome business continuity challenges to ensure continued availability of critical patient therapies.
Assuntos
Polímeros , Esterilização , Humanos , Raios X , Raios gama , Esterilização/métodosRESUMO
Annexin A5 (AnxA5) is a potent anticoagulant protein that crystallizes over phospholipid bilayers (PLBs), blocking their availability for coagulation reactions. Antiphospholipid antibodies disrupt AnxA5 binding, thereby accelerating coagulation reactions. This disruption may contribute to thrombosis and miscarriages in the antiphospholipid syndrome (APS). We investigated whether the antimalarial drug, hydroxychloroquine (HCQ), might affect this prothrombotic mechanism. Binding of AnxA5 to PLBs was measured with labeled AnxA5 and also imaged with atomic force microscopy. Immunoglobulin G levels, AnxA5, and plasma coagulation times were measured on cultured human umbilical vein endothelial cells and a syncytialized trophoblast cell line. AnxA5 anticoagulant activities of APS patient plasmas were also determined. HCQ reversed the effect of antiphospholipid antibodies on AnxA5 and restored AnxA5 binding to PLBs, an effect corroborated by atomic force microscopy. Similar reversals of antiphospholipid-induced abnormalities were measured on the surfaces of human umbilical vein endothelial cells and syncytialized trophoblast cell lines, wherein HCQ reduced the binding of antiphospholipid antibodies, increased cell-surface AnxA5 concentrations, and prolonged plasma coagulation to control levels. In addition, HCQ increased the AnxA5 anticoagulant activities of APS patient plasmas. In conclusion, HCQ reversed antiphospholipid-mediated disruptions of AnxA5 on PLBs and cultured cells, and in APS patient plasmas. These results support the concept of novel therapeutic approaches that address specific APS disease mechanisms.
Assuntos
Anexina A5/metabolismo , Anticorpos Antifosfolipídeos/imunologia , Anticoagulantes/metabolismo , Antimaláricos/farmacologia , Hidroxicloroquina/farmacologia , Anexina A5/ultraestrutura , Antimaláricos/metabolismo , Síndrome Antifosfolipídica/sangue , Coagulação Sanguínea/efeitos dos fármacos , Células Cultivadas , Cristalização , Humanos , Hidroxicloroquina/metabolismo , Bicamadas Lipídicas/metabolismo , Microscopia de Força Atômica , Ligação Proteica/efeitos dos fármacosRESUMO
Treatment with the antimalarial drug hydroxychloroquine (HCQ) has been associated with reduced risk of thrombosis in the antiphospholipid (aPL) syndrome (APS) and, in an animal model of APS, with reduction of experimentally induced thrombosis. Recognition of beta2-glycoprotein I (beta2GPI) by aPL antibodies appears to play a major role in the disease process. We therefore used the techniques of ellipsometry and atomic force microscopy (AFM) to investigate whether HCQ directly affects the formation of aPL IgG-beta2GPI complexes on phospholipid bilayers. HCQ, at concentrations of 1 mug/mL and greater, significantly reduced the binding of aPL-beta2GPI complexes to phospholipid surfaces and THP-1 (human acute monocytic leukemia cell line) monocytes. The drug also reduced the binding of the individual proteins to bilayers. This HCQ-mediated reduction of binding was completely reversed when the HCQ-protein solutions were dialyzed against buffer. HCQ also caused modest, but statistically significant, reductions of clinical antiphospholipid assays. In conclusion, HCQ reduces the formation of aPL-beta2GPI complexes to phospholipid bilayers and cells. This effect appears to be due to reversible interactions between HCQ and the proteins and may contribute to the observed reduction of thrombosis in human and experimental APS. These results support the possibility that HCQ, or analogous molecules, may offer novel nonanticoagulant therapeutic strategies for treating APS.
Assuntos
Anticorpos Antifosfolipídeos/metabolismo , Hidroxicloroquina/farmacologia , Fosfolipídeos/metabolismo , beta 2-Glicoproteína I/metabolismo , Anticorpos Antifosfolipídeos/sangue , Anticorpos Antifosfolipídeos/química , Anticoagulantes/farmacologia , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/efeitos dos fármacos , Complexo Antígeno-Anticorpo/metabolismo , Antimaláricos/farmacologia , Síndrome Antifosfolipídica/sangue , Síndrome Antifosfolipídica/tratamento farmacológico , Linhagem Celular , Humanos , Técnicas In Vitro , Bicamadas Lipídicas/metabolismo , Microscopia de Força Atômica , Complexos Multiproteicos/química , Complexos Multiproteicos/efeitos dos fármacos , Complexos Multiproteicos/metabolismo , Ligação Proteica/efeitos dos fármacos , beta 2-Glicoproteína I/químicaRESUMO
The activation of coagulation factor X by tissue factor (TF) and coagulation factor VIIa (VIIa) on a phospholipid surface is thought to be the key step in the initiation of blood coagulation. In this reaction, the product, fXa, is transiently and reversibly bound to the TF-VIIa enzyme complex. This in effect leads to a probabilistic inhibition of subsequent fX activations; a new fX substrate molecule cannot be activated until the old fXa molecule leaves. In this study, we demonstrate that benzamidine and soybean trypsin inhibitor-conjugated Sepharose beads, which bind fXa and sequester it away from the reaction, serve to enhance fX activation by the TF-VIIa complex. Thus, removal of fXa from the reactive zone, by either flow, fXa sequestration, or binding to distant lipid surfaces, can serve to enhance the levels of TF-VIIa activity. Using resonance energy transfer, we found the dissociation constants of fX and fXa for 100 nm diameter phospholipid vesicles to be on the order of 30-60 nM, consistent with previous measurements employing planar lipid surfaces. On the basis of the measurements of binding of fXa to phospholipid surfaces, we demonstrate that the rates of fX activation by the TF-VIIa complex under a variety of experimental conditions depend inversely on the amount of product (fXa) bound to the TF-phospholipid surface. These data support an inhibitory role for the reaction product, fXa, and indicate that models previously employed in understanding this initial coagulation reaction must now be re-evaluated to account for both the product occupancy of the phospholipid surface and the binding of the product to the enzyme. Moreover, the inhibitory properties of fXa can be described on the basis of the estimated surface density of fXa molecules on the TF-phospholipid surface.
Assuntos
Fator Xa/química , Fator Xa/fisiologia , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Tromboplastina/antagonistas & inibidores , Tromboplastina/metabolismo , Benzamidinas/química , Benzamidinas/metabolismo , Regulação para Baixo/fisiologia , Fator VIIa/química , Fator VIIa/metabolismo , Fator Xa/metabolismo , Humanos , Microesferas , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Ligação Proteica , Especificidade por Substrato , Tromboplastina/química , Fatores de Tempo , Inibidor da Tripsina de Soja de Kunitz/química , Inibidor da Tripsina de Soja de Kunitz/metabolismo , Regulação para Cima/fisiologiaRESUMO
Thrombosis occurs in a dynamic rheological field that constantly changes as the thrombus grows to occlusive dimensions. In the initiation of thrombosis, flow conditions near the vessel wall regulate how quickly reactive components are delivered to the injured site and how rapidly the reaction products are disseminated. Whereas the delivery and removal of soluble coagulation factors to the vessel is thought to occur via classic convection-diffusion phenomena, the movement of cells and platelets to the injured wall is strongly augmented by flow-dependent cell-cell collisions that enhance their ability to interact with the wall. In addition, increased shear conditions have been shown to activate platelets, alter the cellular localization of proteins such as tissue factor (TF) and TF pathway inhibitor, and regulate gene production. In the absence of high shearing forces, red cells, leukocytes, and platelets can form stable aggregates with each other or cells lining the vessel wall, which, in addition to altering the biochemical makeup of the aggregate or vessel wall, effectively increases the local blood viscosity. Thus, hemodynamic forces not only regulate the predilection of specific anatomic sites to thrombosis, but they strongly influence the biochemical makeup of thrombi and the reaction pathways involved in thrombus formation.
Assuntos
Circulação Sanguínea , Coagulação Sanguínea , Trombose/etiologia , Trombose/fisiopatologia , Animais , HumanosRESUMO
For many years, the essential role of tissue factor (TF) in coagulation and thrombogenesis has been recognized. The catalytic complex of TF and VIIa (TF:VIIa) is membrane-bound whereas its substrate, factor X (FX), is distributed between a phospholipid-bound fraction and one that is in true solution in 3-dimensional space. This complicates analytical solutions for the kinetic mechanisms describing this reaction because dimensionality must be preserved. We believe that, at the time of activation, FX is simultaneously bound to TF:VIIa and the phospholipid surface. The hydrolysis of a peptide bond activates FX and the product, Xa, is yet bound to the catalytic complex in a manner such that it must leave before a new molecule of X encounters the complex. This means that, in principle, the classically defined Vmax does not apply because on a surface, infinite substrate and its attendant infinite collision frequency do not apply. We show that increasing the lipid surface area available to each TF:VIIa increases the apparent k(cat) and that it approaches a maximum asymptotically, exhibiting a K(1/2) at a 40 nm lipid radius. Notably, this is of the same order as transient confinement zones that have been identified on the surface of living cells. We believe the increased lipid surface area allows the Xa to easily diffuse away from the enzyme complex along the 2D lipid surface, thereby allowing new substrate to approach the enzyme and minimizing collisions between the product and the enzyme complex (product inhibition). Thus, after Xa leaves the vicinity of the enzyme, a new FX molecule is able to bind TF:VIIa and the rate at which this complex forms cannot exceed the leaving rate of Xa from the TF:VIIa and phospholipid sites. Thus, this parameter is of critical interest. Starting with the off-rate of Xa from appropriate phospholipid surfaces, we note that the literature values differ by a factor of approximately 500. Using energy transfer techniques between 30% phosphatidylserine/70% phosphatidylcholine vesicles and human F.Xa, we measured this off rate and found it agrees closely with the Biacore generated data. We have determined the binding parameters of Xa to vesicles and a continuous supported bilayer. Our data are in excellent agreement with the data derived using a lipid coated Biacore chip.
Assuntos
Fator VIIa/metabolismo , Fosfolipídeos/metabolismo , Tromboplastina/metabolismo , Catálise , Difusão , Fator X/metabolismo , Humanos , Cinética , Modelos Biológicos , Transporte Proteico , Propriedades de SuperfícieRESUMO
Although the phospholipid requirement for tissue factor (TF) activity has been well-established, the mechanism by which the surface regulates enzymatic activity remains unclear. We added phospholipid vesicles to already relipidated TF (30/70 PS/PC) and found that added lipid can both enhance and inhibit the rate of factor X (F.X) activation. Using active-site-inhibited F.Xa we demonstrate that F.Xa is a more potent inhibitor of TF/VIIa at lower lipid concentrations, and that this inhibition is attributable to high surface occupancy by F.Xa near the enzyme. We also find that exactly twice as many F.Xa molecules are bound to a lipid surface at saturation as F.X, and that a dimer model of F.Xa binding to the lipid can account for the experimentally observed, preferential binding of F.Xa (compared to F.X) to phospholipid surfaces. We manipulated the amount of phospholipid available to each TF molecule by controlling vesicle size and the number of TF molecules per vesicle and found that, as the 2D radius of phospholipid available to each TF molecule was increased, the observed k(cat) increased hyperbolically toward a maximum or "true k(cat)". At a 2D lipid radius of approximately 37 nm, the observed k(cat) was 50% of the "true k(cat)". Thus, phospholipid surface serves as a conduit for F.X presentation and F.Xa removal, and the rate at which F.Xa leaves the vicinity of the enzyme, either by lateral diffusion or desorption from the surface, regulates the rate of F.X activation. We argue that these findings require reevaluation of existing models of coagulation.
Assuntos
Fator VIIa/química , Fator X/metabolismo , Fator Xa/metabolismo , Modelos Químicos , Fosfolipídeos/química , Fosfolipídeos/fisiologia , Tromboplastina/química , Ligação Competitiva/fisiologia , Catálise , Dimerização , Ativação Enzimática/fisiologia , Fator VIIa/antagonistas & inibidores , Fator VIIa/metabolismo , Fator Xa/química , Inibidores do Fator Xa , Humanos , Cinética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fosfatidilcolinas/química , Fosfatidilserinas/química , Fosfolipídeos/metabolismo , Ligação Proteica/fisiologia , Especificidade por Substrato/fisiologia , Tromboplastina/antagonistas & inibidores , Tromboplastina/metabolismo , TitulometriaRESUMO
Upon plaque rupture or vascular injury, tissue factor (TF) protein in the vessel wall becomes exposed to flowing blood, initiating a cascade of reactions resulting in the deposition of fibrin and platelets on the injured site. Paradoxically, the growing thrombus may act as a barrier, restricting the convective and diffusive exchange of substrates and coagulation products between the blood and reactive vessel wall, thus limiting the role TF plays in thrombus growth. In this study, various in vitro, platelet-fibrin clots were prepared on TF:VIIa-coated surfaces and the rate at which factor (F) X in the well-mixed clot supernatant permeates the clot and is converted to X(a) was monitored over several hours. The apparent diffusion coefficients of FX((a)) in fibrin and platelet-fibrin clots at 37 degrees C was 2.3 x 10(-7) and 5.3 x 10(-10) cm(2)/second, respectively, indicating that the mean time required for FX((a)), and likely FIX((a)), to diffuse 1 mm in a fibrin clot is 4 hours, and in the presence of platelets, 3.6 months. As complete human thrombotic occlusion has been observed within 10 minutes, an alternative source of procoagulant activity that can localize to the outer surface of growing thrombi, such as platelet factor XI or blood-borne TF, appears essential for rapid thrombus growth.
Assuntos
Coagulação Sanguínea/fisiologia , Plaquetas/fisiologia , Fator Xa/metabolismo , Tromboplastina/antagonistas & inibidores , Trombose/metabolismo , Plaquetas/química , Plaquetas/patologia , Difusão , Fator VIIa/química , Fator VIIa/genética , Fator VIIa/fisiologia , Fator Xa/farmacocinética , Fibrina/química , Fibrina/fisiologia , Humanos , Microscopia Confocal , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tromboplastina/química , Tromboplastina/genética , Tromboplastina/metabolismoRESUMO
It is well established that tissue factor (TF) is abundantly present in various extravascular tissues, in the adventitia of blood vessels, and in atheroma. Thus, in the event of plaque rupture or damage to the blood vessel wall, TF is readily exposed to flowing blood, allowing it to form a complex with circulating factor VIIa (FVIIa) in order to activate factor X (FX) both directly, and indirectly via factor IX (FIX). Platelets quickly adhere to the injured site, facilitating localized thrombin formation and subsequent fibrin production. With each new layer of platelets and fibrin that adheres to the injured surface, the exposed TF on the vessel wall, along with the localized circulating factors IX (FIXa) and X (FXa) that it generates, becomes increasingly isolated from the events near the surface of the growing thrombus. The physical blanketing of an injured surface by platelets and fibrin in addition to the release of platelet tissue factor pathway inhibitor (TFPI), prevents FIXa and FXa from diffusing more than a few tens of microns away from the vessel wall, far short of the 3 mm thickness needed for occlusive thrombosis. Thus an alternative FXa-generating mechanism must be involved that allows for the formation of prothrombinase activity far away from the vessel wall near the front of a growing thrombus.
Assuntos
Coagulação Sanguínea/fisiologia , Tromboplastina/metabolismo , Trombose/metabolismo , Processamento Alternativo , Animais , Transporte Biológico , Fatores de Coagulação Sanguínea/metabolismo , Humanos , Tromboplastina/genética , Trombose/etiologia , Trombose/genéticaRESUMO
Tissue factor (TF) is an essential enzyme activator that forms a catalytic complex with FVII(a) and initiates coagulation by activating FIX and FX, ultimately resulting in thrombin formation. TF is found in adventitia of blood vessels and the lipid core of atherosclerotic plaques. In unstable coronary syndromes, plaque rupture initiates coagulation by exposing TF to blood. Biologically active TF has been detected in vessel walls and circulating blood. Elevated intravascular TF has been reported in diverse pro-thrombotic syndromes such as myocardial infarction, sepsis, anti-phospholipid syndrome and sickle-cell disease. It is unclear how TF circulates, although it may be present in pro-coagulant microparticles. We now report identification of a form of human TF generated by alternative splicing. Our studies indicate that alternatively spliced human tissue factor (asHTF) contains most of the extracellular domain of TF but lacks a transmembrane domain and terminates with a unique peptide sequence. asHTF is soluble, circulates in blood, exhibits pro-coagulant activity when exposed to phospholipids, and is incorporated into thrombi. We propose that binding of asHTF to the edge of thrombi contributes to thrombus growth by creating a surface that both initiates and propagates coagulation.
Assuntos
Processamento Alternativo , Tromboplastina/isolamento & purificação , Anticorpos/análise , Coagulação Sanguínea , Plaquetas/metabolismo , Eletroforese , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Fosfolipídeos/farmacologia , Proteínas Recombinantes/farmacologia , Tromboembolia/etiologia , Tromboplastina/química , Tromboplastina/genética , Tromboplastina/metabolismoRESUMO
BACKGROUND: Several studies suggest a role for an increased circulating pool of tissue factor (TF) in atherothrombotic diseases. Furthermore, certain cardiovascular risk factors, such as diabetes, hyperlipemia, and smoking, are associated with a higher incidence of thrombotic complications. We hypothesized that the observed increased blood thrombogenicity (BT) observed in patients with type 2 diabetes mellitus may be mediated via an increased circulating tissue factor activity. We have extended our study to smokers and hyperlipidemic subjects. METHODS AND RESULTS: Poorly controlled patients with type 2 diabetes mellitus (n=36), smokers (n=10), and untreated hyperlipidemic subjects (n=10) were studied. Circulating TF was immunocaptured from plasma, relipidated, and quantified by factor Xa (FXa) generation in the presence of factor VIIa. BT was assessed as thrombus formation on the Badimon perfusion chamber. Patients with improvement in glycemic control showed a reduction in circulating TF (362+/-135 versus 243+/-74 pmol/L per min FXa, P=0.0001). A similar effect was observed in BT (15 445+/-1130 versus 12 072+/-596 microm/mm2, P=0.01). Two hours after smoking 2 cigarettes, TF was increased (217+/-72 versus 283+/-106 pmol/L per min FXa, P=0.003). Hyperlipidemic subjects showed higher TF (237+/-63 versus 195+/-44 pmol/L per min FXa, P=0.035) than healthy volunteers. CONCLUSIONS: These findings suggest that high levels of circulating TF may be the mechanism of action responsible for the increased thrombotic complications associated with the presence of these cardiovascular risk factors. These observations strongly emphasize the usefulness of the management of the patients based on their global risk assessment.