High variability in Factor IX one-stage assay in samples spiked with nonacog beta pegol among different pairs of reagent/detection system.

INTRODUCTION: Haemophilia B (HB) is an X-linked hereditary bleeding disorder characterized by coagulation factor IX (FIX) deficiency. To improve the quality of life of patients and adherence to treatment, recombinant factor concentrates modified to extend their half-life have been developed, called extended half-life factors (EHL: extended half-life). Nonacog beta pegol (N9-GP) is a glycopegylated recombinant human FIX molecule that has a half-life of 93 h with a single dose and has shown a higher recovery percentage than other molecules. To diagnose and monitor the treatment of haemophiliac patients, FIX activity is determined with the one-stage clotting assay (OSA) and/or the chromogenic assay. The objective of this work, carried out in three centres, was to measure the recovery of N9-PG with 10 different activated partial thromboplastin time (APTT) reagents on three platforms, in samples spiked in vitro with N9-GP, at four different concentration levels. METHODS: It was measured the recovery of N9-GP with 10 different APTT reagents (polyphenol, ellagic acid, silice dioxide, colloidal silica as APTT activator on three platforms, in sample spiked in vitro with N9-GP. RESULTS: The results show heterogeneity in the activity of N9-GP measured by OSA with the different APTT reagents when the calibrations were performed with the specific calibrator of each coagulometer. A recovery percentage between 87% and 108% was obtained only with polyphenol and ellagic acid as activator in the three platforms evaluated. The other reagents studied overestimate or underestimate, with no clear profile. When a calibration curve was performed with a calibrator prepared from the N9-GP vial, all APTT reagents met the established recovery requirement. CONCLUSION: APTT reagents with polyphenol or ellagic acid as activator would be the only ones appropriate when using the commercially available OSA with specific calibrator to monitor patients treated with N9-GP.

Alta variabilidad en la determinación del Factor IX en muestras de plasma adicionadas con nonacog beta pegol entre distintos pares de reactivos/sistemas de detección / High variability in the determination of Factor IX in plasma samples spiked with nonacog beta pegol between different reagents/detection system pairs / Alta variabilidade na determinação do Fator IX em amostras de plasma enriquecidas com nonacog beta pegol entre diferentes pares de reagentes/ sistema de detecção

Resumen La hemofilia B es un trastorno hemorrágico hereditario, ligado al cromosoma X, que se caracteriza por el déficit del factor IX (FIX) de la coagulación. Para mejorar la calidad de vida de los pacientes y la adherencia al tratamiento se han desarrollado concentrados de factores recombinantes modificados para extender su vida media, denominados factores de vida media extendida (EHL: extended half life concentrates). El nonacog beta pegol (N9-GP) es una molécula de FIX humano recombinante glicopegilada que tiene una vida media de 93 h con una sola dosis y ha mostrado un porcentaje de recuperación mayor que otras moléculas. Para diagnosticar y monitorear el tratamiento del paciente hemofílico se determina la actividad del FIX con el ensayo coagulable en una etapa (OSA: one stage assay) y/o en el ensayo cromogénico. El objetivo de este trabajo, realizado en tres centros, fue medir la recuperación de N9-GP con 10 reactivos de APTT diferentes en tres plataformas, en muestras deficientes en FIX adicionadas in vitro con N9-GP, en cuatro niveles de concentración diferentes. Los resultados muestran una heterogeneidad en la actividad de N9-GP medidos por OSA con los diferentes reactivos de APTT cuando se realizaron las calibraciones con el estándar específico de cada coagulómetro. Se obtuvo un porcentaje de recuperación mayor de 92% con Cephascreen, Actin FSL y APTTest elágico en las tres plataformas evaluadas. Estos reactivos serían los únicos apropiados cuando se usa el OSA calibrado con plasma comercial para monitorear el tratamiento de los pacientes que reciben N9-GP.

Abstract Hemophilia B (HB) is an X-linked hereditary bleeding disorder characterised by coagulation factor IX (FIX) deficiency. To improve the quality of life of patients and adherence to treatment, recombinant factor concentrates glicomodified to extend their half-life have been developed. These are called extended half-life factors (EHL: extended half-life concentrates). Nonacog beta pegol (N9-GP) is a glycopegylated recombinant human FIX molecule that has a half-life of 93 h with a single dose and has shown a higher recovery percentage than other molecules. For diagnosis and monitoring the treatment of hemophiliac patients, FIX activity is determined with the One Stage Clotting Assay (OSA) and/or the chromogenic assay. The objective of this work, carried out in three centres, was to measure the recovery of N9-GP with 10 different APTT reagents on three platforms, in FIX deficient samples spiked in vitro with N9-GP, at four different concentration levels. The results show a heterogeneity in the activity of N9-GP measured by OSA with the different APTT reagents when the calibrations were performed with the specific standard of each coagulometer. A recovery percentage greater than 92% was obtained with Cephascreen, Actin FSL and APTTest ellagic in the three platforms evaluated. These reagents would be the only ones appropriate when using the commercial plasma-calibrated OSA to monitor the treatment of patients treated with N9-GP.

Resumo A hemofilia B é uma doença hemorrágica hereditária ligada ao cromossomo X caracterizada pela deficiência do fator de coagulação IX (FIX). Para melhorar a qualidade de vida dos pacientes e a adesão ao tratamento, foram desenvolvidos concentrados de fatores recombinantes modificados para prolongar sua meia-vida, chamados de fatores de meia-vida estendida (EHL: extended half life concentrates). Nonacog beta pegol (N9-GP) é uma molécula de FIX humano recombinante glicopeguilada que tem meia-vida de 93 h com uma dose única e mostrou uma porcentagem de recuperação maior do que outras moléculas. Para diagnosticar e monitorar o tratamento de pacientes hemofílicos, a atividade do FIX é determinada com o ensaio coagulável em um estágio (OSA: One Stage Assay) e/ou o ensaio cromogênico. O objetivo deste trabalho, realizado em três centros, foi medir a recuperação de N9-GP com 10 reagentes de APTT diferentes em três plataformas, em amostras deficiente de fator IX adicionadas in vitro com N9-GP, em quatro níveis de concentração diferentes. Os resultados mostram uma heterogeneidade na atividade de N9-GP medidos por OSA com os diferentes reagentes de APTT quando as calibragens foram realizadas com o padrão específico de cada coagulômetro. Uma porcentagem de recuperação superior a 92% foi obtida com Cephascreen, Actin FSL e APTTest elágico nas três plataformas avaliadas. Esses reagentes seriam os únicos apropriados ao usar o OSA calibrado com plasma comercial para monitorar o tratamento de pacientes tratados com N9-GP.

Recombinant Expression of Complex Proteins in Human Cell Lines.

Coagulation factors, as factor VII, VIII, and IX, are complex proteins which are very difficult to express. Blood coagulation factor IX is a vitamin K-dependent protein, and it has become a valuable biopharmaceutical in the treatment of hemophilia B. Here, we describe the techniques used to generate human cell lines producing human recombinant factor IX, as an example of complex protein, as well as in vitro characterization of this coagulation factor.To produce the FIX human adherent 293T SK-Hep-1 cells were used and stably modified by a lentiviral vector carrying the hFIX and the eGFP genes. The eGFP was employed as a reporter protein.

Gene therapy for hemophilia: looking beyond factor expression.

Hemophilia A (factor VIII [FVIII] deficiency) and hemophilia B (factor IX [FIX] deficiency) are the X-linked recessive bleeding disorders that clinically manifest with recurrent bleeding, predominantly into muscles and joints. In its severe presentation, when factor activity is less than 1% of normal, hemophilia presents with spontaneous musculoskeletal bleeds and may progress to debilitating chronic arthropathy. Management of hemophilia has changed profoundly in the past decades. From on-demand to prophylactic factor concentrate replacement, the treatment goal shifted from controlling bleeds to preventing bleeds and improving quality of life. In this new scenario, gene therapy has arisen as a paradigm-changing therapeutic option, a one-time treatment with the potential to achieve sustained coagulation FVIII or FIX expression even within the normal range. This review discusses the critical impact of adeno-associated virus (AAV) gene transfer in hemophilia care, including the recent clinical outcomes, changes in disease perceptions, and its treatment burden. We also discuss the challenging scenario of the AAV-directed immune response in the clinical setting and potential strategies to improve the long-lasting efficacy of hemophilia gene therapy efficacy.

Generation of hyperfunctional recombinant human factor IX variants expressed in human cell line SK-Hep-1.

OBJECTIVE: To develop recombinant factor IX (FIX) variants with augmented clotting activity. RESULTS: We generated three new variants, FIX-YKALW, FIX-ALL and FIX-LLW, expressed in SK-Hep-1 cells and characterized in vitro and in vivo. FIX-YKALW showed the highest antigen expression level among the variants (2.17 µg-mL), followed by FIX-LLW (1.5 µg-mL) and FIX-ALL (0.9 µg-mL). The expression level of FIX variants was two-five fold lower than FIX-wild-type (FIX-WT) (4.37 µg-mL). However, the biological activities of FIX variants were 15-31 times greater than FIX-WT in the chromogenic assay. Moreover, the new variants FIX-YKALW, FIX-LLW and FIX-ALL also presented higher specific activity than FIX-WT (17, 20 and 29-fold higher, respectively). FIX variants demonstrated a better clotting time than FIX-WT. In hemophilia B mice, we observed that FIX-YKALW promoted hemostatic protection. CONCLUSION: We have developed three improved FIX proteins with potential for use in protein replacement therapy for hemophilia B.

Molecular characterization of hemophilia B patients in Colombia.

BACKGROUND: Hemophilia B (HB) is a coagulation disorder with an X-linked recessive inheritance pattern, caused by plasma FIX deficiency. In Colombia, HB is considered a rare and high-cost disease, with 362 males reported in 2017. METHODS: Here, we characterized 20 HB apparently unrelated families by PCR amplification and Sanger sequencing. RESULTS: Fourteen unique variants were identified: seven missense, three nonsense, one variant in the 3' UTR region, two large deletions >50 bp, and one intronic substitution that affects splicing c.520+13A>G that was present in 7/20 patients (35%). All these variants have been previously reported in the literature, except for exons 3 and 4, deletions, present in one patient. The genotype-phenotype association correlates with the reported in the literature, with the exception of one patient. CONCLUSION: This molecular analysis allowed us to establish the causal variant of HB in 100% of patients, to provide the appropriate genetic counseling to each of the families, and to propose a more cost-effective carrier analysis. Here, we reported the first variants in Colombian population with Hemophilia B, finding a new variant and one intron recurrent variant present in 35% of patients.

Hemofilia B o enfermedad de Christmas / Hemophilia B or Christmas disease

La hemofilia B o enfermedad de Christmas se diferenció por primera vez de la hemofilia A en 1947. Su forma clásica consiste en un trastorno hereditario de la coagulación causado por mutaciones en el gen F9, que codifica para el factor IX de la coagulación. Su herencia está ligada al cromosoma X; las mujeres son portadoras, pero se manifiesta clínicamente en hombres, aunque se han descrito casos de mujeres portadoras sintomáticas. El factor IX activado es una proteína dependiente de vitamina K, sintetizada en el hígado, que forma parte del complejo tenasa, cuya función es formar la mayor cantidad de trombina en el nuevo modelo de la coagulación basado en células. De acuerdo a la actividad del factor IX, su deficiencia se puede clasificar en leve (5% a 40%), moderada (1% a 5%), o severa (<1%). Su diagnóstico se realiza con la presencia de un TPT alargado que corrige con plasma normal y con la determinación del nivel funcional del factor IX, y se confirma con el estudio molecular que demuestra la mutación en el gen F9. Su diagnóstico diferencial incluye otras patologías como la hemofilia A. El tratamiento con factor IX recombinante es el más utilizado en la actualidad, pero se vienen desarrollando nuevas terapias con virus adeno-asociados recombinantes que prometen mejorar la calidad de vida para algunos pacientes afectados. La profilaxis juega un papel fundamental, en particular en los casos de enfermedad moderada y severa.

Hemophilia B or Christmas disease was first differentiated from hemophilia A in 1947. Its classic form consists of an inherited bleeding disorder caused by mutations in the F9 gene, which codes for coagulation factor IX. Its inheritance is linked to the X chromosome; women are carriers, but it manifests clinically in men, although cases of symptomatic women carriers have been described. Factor IX activates a vitamin K-dependent protein, synthesized in the liver, which is part of the tenase complex whose function is to form the largest amount of thrombin (factor IIa) in the new model of cell-based coagulation. According to factor IX activity, its deficiency can be classified as mild (5% to 40%), moderate (1% to 5%), and severe (<1%). The diagnosis is made when there is a prolonged TPT that corrects with normal plasma, and by assessing the functional level of factor IX. The diagnosis is confirmed by molecular analysis that demonstrates the F9 gene mutation. Its differential diagnosis includes disorders such as hemophilia A. Treatment with recombinant factor IX is widely used, but also new therapies are being developed with recombinant adeno-associated viruses that promise to improve the quality of life for some of these patients. Prophylaxis plays an important role in cases of moderate and severe disease

Intermediate purification of CHO-derived recombinant human Factor IX using hydrophobic interaction membrane-based chromatography and its comparison to a sulfated resin.

This work investigated the use of hydrophobic interaction membrane chromatography for intermediate purification of recombinant human Factor IX (rFIX) produced by CHO cells. The first purification step was based on a strong anion exchange monolith, thus forming a purification process fully based on convective media, which allow operation at high flow rates and low pressure drops, as well as modular scale-up. Although the starting material was challenging (CHO cell culture supernatant harvested at 70% cell viability), the two-step purification process showed promising results, with a global purification factor of 298, a global recovery of 69%, and DNA and endotoxin levels close to regulatory limits. Final host cell DNA (68.8 ng per dose of 500 IU), endotoxins (60 EU per dose of 500 IU) and activated FIX (FIXa/FIX = 2.33%) were in levels close to those recommended by regulatory authorities. HCP removal was of 99.98%, decreasing from 9 424 358 ppm in the supernatant to a final HCP value of 2071 ppm. The use of a supernatant harvested at higher viability and/or the addition of a third polishing step focusing on HCP removal could allow meeting the desired HCP range of 50-100 ppm, as well as the regulatory requirements for the other critical contaminants.

Investigations into, and development of, a lyophilized and formulated recombinant human factor IX produced from CHO cells.

OBJECTIVES: To develop a recombinant human factor IX (rFIX) formulation equivalent to commercially available products in terms of cake appearance, residual moisture, proportion of soluble aggregates and activity maintenance for 3 months at 4-8 °C. RESULTS: NaCl and low bulking agent/cryoprotectant mass ratio had a negative impact on cake quality upon lyophilisation for a wide range of formulations tested. Particular devised formulations maintained rFIX activity after lyophilization with a similar performance when compared with the rFIX formulated using the excipients reported for a commercially available FIX formulation (Benefix). rFIX remained active after 3 months when stored at 4 °C, though this was not the case with samples stored at 40 °C. Interestingly, particular formulations had an increase in residual moisture after 3 months storage, but not above a 3% threshold. All four formulations tested were equivalent to the Benefix formulation in terms of particle size distribution and cake appearance. CONCLUSIONS: Three specific formulations, consisting of surfactant polysorbate-80, sucrose or trehalose as cryoprotectant, mannitol or glycine as bulking agent, L-histidine as buffering agent, and NaCl added in the reconstitution liquid at 0.234% (w/v) were suitable for use with a CHO cell-derived recombinant FIX.

Production of recombinant coagulation factors: Are humans the best host cells?

The main treatment option for Hemophilia A/B patients involves the administration of recombinant coagulation factors on-demand or in a prophylactic approach. Despite the safety and efficacy of this replacement therapy, the development of antibodies against the coagulation factor infused, which neutralize the procoagulant activity, is a severe complication. The production of recombinant coagulation factors in human cell lines is an efficient approach to avoid such complication. Human cell lines can produce recombinant proteins with post translation modifications more similar to their natural counterpart, reducing potential immunogenic reactions. This review provides a brief overview of the most important characteristics of recombinant FVIII and FIX products available on the market and the improvements that have recently been achieved by the production using human cell lines.

Evaluation of a web-based registry of inherited bleeding disorders: a descriptive study of the Brazilian experience with HEMOVIDAweb Coagulopatias.

BACKGROUND: Inherited bleeding disorders (IBD) consist of a group of rare heterogeneous diseases, which require treatment for life. Management of these disorders is complex and costly. Therefore, good quality data of the affected population is crucial to guide policy planning. The aim of this manuscript is to describe the impact of a national, web-based registry - the Hemovidaweb Coagulopatias (HWC) - in the management of the IBD in Brazil. METHODS: The system was developed in PHP 5.0 language and is available on the internet at http://coagulopatiasweb.datasus.gov.br . The system was validated in September 2008 and launched nationally with input from January 1, 2009. HWC collects variables related to socio-demographic, clinical, laboratory and treatment data of patients with IBD. RESULTS: Within 7 years, there was an increment of 90.8% on the diagnosis of IBD altogether, which increased from 11,040 in December 2007 to 21,066 in December 2014. This is now the fourth and third largest world population of patients with haemophilia and von Willebrand's disease (vWD), respectively, according to the most recent (2015) Annual Global Survey of the World Federation of Hemophilia. The data collected provided the basis for planning and implementing home therapy, prophylaxis and immune tolerance induction (ITI), recently initiated in Brazil. CONCLUSION: HWC was an effective tool in the increment of registration of patients with IBD in Brazil. Furthermore, it was essential to support policy planning, monitoring, evaluation and treatment. Future development should focus on surveillance, health outcomes and research. Every country should implement a national registry on IBD.

Características clínicas y epidemiológicas de pacientes con hemofilia del eje cafetero, Colombia / Clinical and epidemiological characteristics of patients with Haemophilia from coffee region, Colombia

INTRODUCCIÓN: La hemofilia es un trastorno hemorrágico hereditario, ligado al cromosoma X, con una prevalencia estimada de 8,4 casos de hemofilia A y 2,7 casos de hemofilia B por cada 100 000 hombres. OBJETIVO: Describir características sociodemográficas, clínicas y percepción del efecto de la patología en el paciente. MATERIALES Y MÉTODOS: Se realizó un estudio transversal, en pacientes con diagnóstico de hemofilia. La fuente de información fueron las historias clínicas, además de una encuesta aplicada para actualizar la caracterización clínica y epidemiológica, el análisis estadístico fue realizado con el software Stata versión 14®. RESULTADOS: 48 pacientes fueron incluidos en el estudio, el 97,9% presenta hemofilia tipo A, de los cuales el 12,5%, 43,8% y 41,7% son leve, moderada y severa respectivamente. El 35% de los pacientes con hemofilia severa desarrollaron anticuerpos contra el factor VIII. El 66% de los pacientes ha presentado alguna manifestación de la patología en los últimos seis meses y el 61,7% refieren que ha tenido un impacto negativo en algún aspecto de su vida. CONCLUSIONES: Los factores asociados a un requerimiento mayor de dosis del factor para el control de la patología fueron antecedente de sangrado, mayor edad y el desarrollo de inhibidores. Los pacientes con hemofilia severa presentaron desarrollo de inhibidores en un 30%, frente a la moderada y leve con un 3 y 0,3% respectivamente.

INTRODUCTION: Hemophilia is an inherited bleeding disorder, linked to the X chromosome, with an estimated prevalence of 8,4 cases of hemophilia A and 2.7 cases of hemophilia B per 100 000 men. OBJETIVE: To describe sociodemographic, clinical characteristics and perception of the effect of the pathology in the patient. MATERIAL AND METHODS: A descriptive cross-sectional study was conducted with patients diagnosed with hemophilia. The required information was extracted from each patient's medical history, additionally, each patient completed a questionnaire to update the clinical and epidemiological information. The analysis was realized in Stata version 14®. RESULTS: From the 48 patients included in the study, 97,9% have type A hemophilia, of whom 12,5%, 43,8% and 41,7% are mild, moderate and severe respectively. 35% of patients with severe hemophilia have developed antibodies against factor VIII. 66% of patients presented some manifestation of the disease in the last six months and 61,7% had a negative impact on some aspect of their life. CONCLUSION: Factors associated with a higher dose requirement of factor for pathology control were antecedent bleeding, increased age and inhibitor development. Patients with severe hemophilia had development inhibitors in 30%, compared to moderate and mild with 3 and 0.3%, respectively.

Approaches for recombinant human factor IX production in serum-free suspension cultures.

OBJECTIVE: To establish a serum-free suspension process for production of recombinant human factor IX (rhFIX) based on the human cell line HEK 293T by evaluating two approaches: (1) serum-free suspension adaptation of previously genetic modified cells (293T-FIX); and (2) genetic modification of cells already adapted to such conditions (293T/SF-FIX). RESULTS: After 10 months, 293T-FIX cells had become adapted to FreeStyle 293 serum-free medium (SFM) in Erlenmeyer flasks. After 48 and 72 h of culture, 2.1 µg rhFIX/ml and 3.3 µg rhFIX/ml were produced, respectively. However, no biological activity was detected. In the second approach, wild-type 293T cells were adapted to the same SFM (adaptation process took only 2 months) and then genetically modified for rhFIX production. After 48 h of culture, rhFIX reached 1.5 µg/ml with a biological activity of 0.2 IU/ml, while after 72 h, the production was 2.4 µg/ml with a biological activity of 0.3 IU/ml. CONCLUSION: The findings demonstrate that the best approach to establish an rhFIX production process in suspension SFM involves the genetic modification of cells already adapted to the final conditions. This approach is time saving and may better ensure the quality of the produced protein.

Importancia del diagnóstico de portadoras en familias con antecedentes de hemofilia / Importance of the diagnosis of carriers in families with history of hemophilia

La hemofilia es una enfermedad hemorrágica hereditaria ligada al cromosoma X que se presenta debido a mutaciones en los genes del factor VIII (hemofilia A) y el factor IX (hemofilia B), que ocasiona una disminución o deficiencia funcional de estas proteínas en plasma. Sus frecuencias son de 1 en 5 000 y 1 en 30 000 varones recién nacidos vivos, respectivamente. Afecta casi exclusivamente a los varones y las mujeres portadoras presentan un riesgo del 50 por ciento de transmitir la enfermedad a sus hijos, por lo que es importante en las familias con antecedentes de hemofilia identificar las portadoras a través de las consultas de asesoramiento genético, en el cual se brinda información acerca de la enfermedad, la confección del árbol genealógico, el cálculo del riesgo de recurrencia, el diagnóstico molecular y la posibilidad de diagnóstico prenatal en gestantes portadoras. Es imprescindible que el asesoramiento genético constituya un proceso educativo e informativo, nunca impositivo

Hemophilia is a hereditary bleeding disorderX-linked that a rises dueto mutations in the genes offactor VIII(hemophiliaA)andfactor IX(hemophiliaB), which causes a decrease orfunctional deficiency of these proteins inplasma.Their frequencies are 1 in5 000 males and1 in30 000 live births, respectively. It affects males almost exclusively, and female carriers have a50 percent risk of transmitting the disease to their children. So, it is important in families with history of hemophilia that carriers are identified through genetic counseling, which provides information about the disease, making pedigree,calculation of the risk of recurrence, molecular diagnosis and possibility of prenatal diagnosis in pregnant carriers.It is imperative that genetic counselingconstitutes an educational and informative process,never as an imposition

[Hemophilia.] / Hemophilia.

Hemophilia is a genetic disease in which the clinical manifestation is mainly the presence of hemorrhage. There are two known types of hemophilia: hemophilia A and B, which have a deficiency of factor VIII or IX clotting, respectively. The intensity of bleeding in hemophilia depends on the plasma levels of factor VIII or IX and has traditionally been classified as mild (> 5 % activity), moderate (1-5 % activity) and severe (< 1 % activity). In laboratory tests, isolated prolongation of activated partial thromboplastin time (aPTT) can be found, but it is necessary to determine the plasma levels of factor VIII or IX to establish the diagnosis of hemophilia A or B. The treating of this disease involves replacing exogenous factor VIII or IX concentrates. Gene therapy could be an option in the future to achieve the cure of the disease. Complications of hemophilia are the risk of transfusion-associated infections, pseudotumor hemophilic, hemophilic arthropathy and the presence of serum inhibitors.

La hemofilia es una enfermedad genética en la que las manifestaciones clínicas consisten básicamente en la presencia de hemorragias. Se conocen dos tipos de hemofilia: A y B, las cuales se originan por la deficiencia de los factores VIII y IX de la coagulación, respectivamente. La intensidad de la hemorragia en la hemofilia depende de los niveles plasmáticos del factor VIII o IX y tradicionalmente se ha clasificado como leve (> 5 % de actividad), moderada (de 1 a 5 % de actividad) y severa (< 1 % de actividad). En las pruebas se identifica prolongación aislada del tiempo de tromboplastina parcial activada (TTPa), pero es necesario determinar los niveles plasmáticos del factor VIII o IX para establecer el diagnóstico de hemofilia A o B. El tratamiento de esta enfermedad consiste en la reposición exógena con concentrados del factor VIII o IX. En el futuro, la terapia genética podría ser una opción para lograr la curación de la enfermedad. Las complicaciones de la hemofilia son el riesgo de infecciones relacionadas con las transfusiones, el pseudotumor hemofílico, la artropatía hemofílica y la presencia de inhibidores.

Anion-exchange purification of recombinant factor IX from cell culture supernatant using different chromatography supports.

Both recombinant and plasma-derived factor IX concentrates are used in replacement therapies for the treatment of haemophilia B. In the present work, the capture step for a recombinant FIX (rFIX) purification process was investigated. Different strong anion-exchange chromatography media (the resins Q Sepharose(®) FF and Fractogel(®) TMAE, the monolith CIM(®) QA and the membrane adsorber Sartobind(®) Q) were tested for their rFIX binding capacity under dynamic conditions. In these experiments, crude supernatant from CHO cells was used, thus in the presence of supernatant contaminants and mimicking process conditions. The highest dynamic binding capacity was obtained for the monolith, which was then further investigated. To study pseudoaffinity elution of functional rFIX with Ca(2+) ions, a design of experiments to evaluate the effects of pH, NaCl and CaCl2 on yield and purification factor was carried out. The effect of pH was not statistically significant, and a combination of no NaCl and 45mM CaCl2 yielded a good purification factor combined with a high yield of active rFIX. Under these conditions, activity yield of rFIX was higher than the mass yield, confirming selective elution of functional, γ-carboxylated rFIX. Scaling-up of this process 8 fold resulted in very similar process performance. Monitoring of the undesired activated FIX (FIXa) revealed that the FIXa/FIX ratio (1.94%) was higher in the eluate than in the loaded sample, but was still within an acceptable range. HCP and DNA clearances were high (1256 and 7182 fold, respectively), indicating that the proposed process is adequate for the intended rFIX capture step.

Hemofilia II. Aspectos moleculares y de genética poblacional / Hemophilia II. Molecular and population genetics

La prevalencia de la hemofilia esporádica fue estimada hace más de 40 años y se demostró que aproximadamente un tercio de los casos son de novo. La mayoría de las mutaciones que ocurren en la hemofilia se producen durante la espermatogénesis masculina; en otros casos, los cambios ocurren en los estadios tempranos de desarrollo del embrión o una mutación germinal en la madre. El proceso de inactivación del cromosoma X es al azar. Extensos estudios han evidenciado que son más frecuentes las mutaciones en las meiosis masculinas que en las femeninas, con una proporción global de 3,5/1, especialmente las inversiones de los intrones 22 y 1. Se revisaron aspectos moleculares y bioquímicos de los factores VIII y IX. Destacamos la importancia del dominio B del factor VIII que contribuye a múltiples funciones esenciales, como el control de la calidad de la síntesis, la secreción, la unión con los fosfolípidos plaquetarios, la inactivación y el aclaramiento de la molécula completa

The sporadic hemophilia prevalence was estimated more than 40 years ago and it was shown that approximately a third of the cases are novo. Most of the mutations that occur in hemophilia are produced during the male spermatogenesis; in other cases, they occur in early stages of the embrión development or in the mother a germinal mutation. The X-cromosoma- inactivation process is at random. Extended studies have shown that male meiosis are more frequent than female ones, with a global proportion of 3,5/1, specially introns inversions 22 and 1. There were revised molecular and biochemical aspects of factors VIII and IX. We ruled out the importance of B domain in factor VIII, which contributes to multiple essential functions, as the quality control of synthesis, secretion, union with platelet phospholipids, inactivation and complete clearance of the molecule

Síndrome de anticuerpos antifosfolípidos asociado a la presencia de inhibidor adquirido del factor IX en una paciente sin hemofilia / Antiphospholipid antibody syndrome associated with acquired factor IX inhibitor in a non-hemophilic patient

Se presenta el caso de una paciente de 23 años de edad, con diagnóstico de síndrome de anticuerpos antifosfolípidos, con un cuadro típico de trombosis venosa profunda recurrente y pérdidas fetales, en presencia de anticoagulante lúpico y anticuerpos anticardiolipina positivos. En el transcurso de su enfermedad presenta manifestaciones hemorrágicas, no explicadas por el uso de anticoagulantes orales. Los estudios de hematología permitieron documentar una disminución de los factores IX y VIII, con evidencia de inhibidor específico del factor IX. La deficiencia de factores de la coagulación mejoró gracias al uso de esteroides y ciclofosfamida. (Acta Med Colomb 2011; 36: 196-199).

We report the case of a 23-year-old female patient with an antiphospholipid syndrome characterized by recurrent deep venous thromboses and fetal losses, with positive lupus anticoagulant and anticardiolipin antibodies. During the course of the disease she had hemorrhagic manifestations not attributable to oral anticoagulants. Hematological analyses documented reduced levels of factors IX and VIII, with evidence of a specific inhibitor of factor IX. The deficiency of clotting factors improved with steroids and cyclophosphamide. (Acta Med Colomb 2011; 36: 196-199).

Estudio familiar de las hemofilias A y B: 5 años de experiencia en la detección de portadoras / Family study of hemophilia A and B: 5 years of experience in carrier detection

La hemofilia se caracteriza por ser una enfermedad congénita del trastorno de la coagulación y constituye un desorden recesivo ligado al cromosoma X. El estudio molecular se realiza por estudios indirectos por ser causada por mutaciones heterogéneas en los genes del FVIII y FIX. Se realizó el estudio de 40 familias afectadas con hemofilia A (HA) y 10 hemofilia B (HB). La extracción de ADN se realizó por el método de precipitación salina a 293 muestras de sangre y 19 de líquido amniótico, y se hizo el análisis de los polimorfismos St14, Bcl I y Hind III para la HA y Taq I, Xmn I y Dde I para la HB. Se usó la técnica de PCR. En el caso de la HA se obtuvo el 35 por ciento de informatividad para St14 y Hind III y 32,5 para Bcl 1. El polimorfismo Dde I fue el más informativo para la HB con el 33 por ciento; mientras que Taq I representó el 10 por ciento de informatividad y XmnI el 0 por ciento. Se comprobó que de las 40 familias analizadas con HA, 23 fueron informativas. Por otra parte, fueron informativas 4 familias de las afectadas con HB. Se realizaron 19 diagnósticos prenatales con previa determinación del sexo fetal, incluidos 3 varones enfermos

Hemophilia is a congenital disease of coagulation disorder and it is a recessive disorder linked to X-chromosome. The molecular study is conducted by indirect studies due to it is caused by heterogeneous mutations in gen of FVIII and FIX in 40 families with hemophilia A (HA) and 10 with hemophilia B (HB). DNA extraction was carried out by saline precipitation method in 293 blood samples and 19 samples of amniotic fluid, as well as the analysis of St14, Bcl I and Hind III polymorphism for the AH and Taq I, Xmn I and Dde I for BH. The PCR technique was used. In the caser of AH it was possible to achieve a 35 percent of information for St14 and Hind III and a 32.5 percent for Bcl. Dde polymorphism supplied more information for BH for a 33 percent; whereas the Taq I represented the 10 percent of information and Xmn I the 0 percent. We verified that from the families analyzed with HA, in 23 of them we there was information. Besides, in 4 families affected by HB there was information. A total of 19 prenatal diagnoses were made with a previous determination of fetus sex, including 3 males ill

Purificación del factor IX recombinante expresado en leche de cerdas transgénicas / Purification of recombinant factor IX expressed in milk of transgenic sows

Se purificó a partir de la leche de cerdas transgénicas, factor IX recombinante, y se obtuvieron rendimientos entre 1 a 2 g de esta proteína por litro, lo que resulta una nueva vía para la obtención de este producto con una alta eficiencia, ya que su expresión es 1 000 veces superior a la del factor IX plasmático humano. Mediante la combinación de 2 pasos cromatográficos: intercambio iónico en DEAE-Shephadex A-50 y cromatografía de afinidad con heparina, se realizó la purificación del factor IX, con esta leche como material de partida. Se estudiaron diferentes métodos para la eliminación de las caseínas, contaminante principal del proceso, y se escogió finalmente la ultracentrifugación, por las numerosas ventajas que presenta con respecto a la precipitación isoeléctrica y la precipitación por sales. El factor IX puede ser purificado de la leche transgénica con una alta pureza utilizando métodos cromatográficos que no usan inmunoafinidad y son finalmente escalables en la producción industrial, lo cual proporciona nuevas perspectivas para el tratamiento de la hemofilia B mediante la preparación de posibles formulaciones orales.

Recombinant factor IX was purified from milk of transgenic sows, and yieldings between 1 and 2 g of this protein per liter were obtained. This is a new way to get this product with a high efficiency, since its expression is 1 000 times higher than of the human plasmatic factor IX. By combining 2 chromatographic steps (ion exchange in DEAE-Shephadex A-50 and affinity chromatography with heparin), the factor IX was purified, with this milk as a starting material. Different methods were studied to eliminate caseins, the main pollutant of the process, and ultracentrifugation was selected due to its numerous advantages over the isoelectric precipitation and salt precipitation. Factor IX may be purified from transgenic milk with an elevated purity by chromatographic methods that do not use immunoaffinity and are finally scalable in industrial production, which provides new perspectives for treating hemophilia B by preparing new oral formulations.