Recombinant Coagulation Factor Research Articles

Complications of haemophilia in babies (first two years of life): a report from the Centers for Disease Control and Prevention Universal Data Collection System.

To describe the prevalence and complications in babies ≤2 years with haemophilia. We used a standardized collection tool to obtain consented data on eligible babies aged ≤2 years with haemophilia enrolled in the Centers for Disease Control and Prevention Universal Data Collection System surveillance project at US Hemophilia Treatment Centers (HTCs). Of 547 babies, 82% had haemophilia A, and 70% were diagnosed within one month of birth. Diagnosis was prompted by known maternal carrier status (40%), positive family history (23%), bleeding (35%) and unknown 2%; 81% bled during the first two years. The most common events were bleeding (circumcision, soft tissue, oral bleeding) and head injury. There were 46 episodes of intracranial haemorrhage (ICH) in 37 babies (7%): 18 spontaneous, 14 delivery related, 11 traumatic, 2 procedure related and 1 unknown cause. Of the 176 central venous access devices (CVADs) in 148 (27%) babies, there were 137 ports, 22 surgically inserted central catheters and 20 peripherally inserted central catheters. Ports had the lowest complication rates. Inhibitors occurred in 109 (20%) babies who experienced higher rates of ICH (14% vs. 5%; P = 0.002), CVAD placement (61% vs. 19%; P < 0.001) and CVAD complications (44% vs. 26%; P < 0.001). The most common replacement therapy was recombinant clotting factor concentrates. Bleeding events in haemophilic babies ≤2 years were common; no detectable difference in the rates of ICH by the mode of delivery was noted. Neonatal factor exposure did not affect the inhibitor rates. Minor head trauma, soft tissue and oropharyngeal bleeding were the leading indications for treatment.

Population pharmacokinetics of a new long‐acting recombinant coagulation factor IX albumin fusion protein for patients with severe hemophilia B

Essentials The new recombinant factor IX (FIX) albumin fusion protein (rIX-FP) has a prolonged half-life. A population pharmacokinetic (PK) model was based on FIX activity levels of hemophilia B patients. The model was used to simulate different dosing scenarios of rIX-FP to help guide dosing. The population PK model supported prolonged dosing of rIX-FP with intervals of up to 2 weeks. Click to hear Prof.Makris's presentation on new treatments in hemophilia SUMMARY: Background The recombinant fusion protein linking recombinant coagulation factor IX with recombinant albumin (rIX-FP; Idelvion® ) exhibits a longer half-life than plasma-derived factor IX (FIX) and the commercially available recombinant FIX products. Objectives (i) Characterize the population pharmacokinetics (PK) of rIX-FP in hemophilia B patients, (ii) identify covariates that are potential determinants of rIX-FP PK variability and (iii) simulate different dosing scenarios of rIX-FP following single and steady-state dosing. Patients/Methods A population PK model was developed based on FIX activity levels of 104 patients who had received treatment with rIX-FP. Patients were aged 1-65 years with FIX activity ≤ 2 IU dL-1 . PK sampling was performed for up to 14 days (336 h). Results Simulation of a single intravenous infusion of rIX-FP (25-75 IU kg-1 ) predicted that the median trough exogenous FIX activity levels would remain > 5 IU dL-1 for up to 16 days in adolescents/adults aged ≥ 12 years, up to 12 days in children aged 6 to < 12 years, and up to 9.5 days in children aged < 6 years. For steady-state dosing, the median trough exogenous FIX activity levels were maintained at > 5 IU dL-1 for the duration of the dosing interval for the 25, 35 and 40 IU kg-1 weekly regimens and for 75 IU kg-1 every 14 days in adolescents/adults, and for the 35 and 40 IU kg-1 weekly regimens in children. Conclusion The population PK model developed here correlates well with observed clinical data and supports prolonged dosing of rIX-FP with intervals of up to 2 weeks.

688. Supplemental Immune Suppression Is Required for AAV-F8 ITI in Hemophilia A Mice with Pre-Existing Inhibitors

Hemophilia is an inherited coagulation disorder resulting in the loss of functional clotting factors VIII (hemophilia A) or IX (hemophilia B). Patients with the severe hemophilia (less than 1% residual clotting factor activity) develop spontaneous bleeds into the joints and closed spaces and have a high incidence of morbidity if not treated. Current treatment involves frequent infusion of short-lived exogenous plasma derived or recombinant clotting factors. A major complication with protein therapy is the development of inhibitory antibodies in 25-30% hemophilia A and 3-5% hemophilia B patients that prevent normalization of hemostasis with continued protein therapy. Inhibitor patients are treated with either bypassing agents or immune tolerance induction (ITI) therapy requiring daily infusions (months to years) of supraphysiological levels of clotting factor protein. ITI therapy is effective in up to 70% of hemophilia A patients, while it often fails in hemophilia B patients due to the development of pathogenic antibodies that induce anaphylaxis and nephrotic syndrome. Both treatment options for inhibitor patients have high associated financial and health related costs for the patient. We have previously shown that hemophilia B mice with pathogenic inhibitory antibodies are effectively treated with a novel immune tolerance induction protocol using a liver directed adeno-associated virus expressing the gene encoding for human factor IX protein (AAV8-F9). AAV8-F9 ITI rapidly eliminates pre-existing inhibitors and provided sustained therapeutic levels of FIX protein even following repeated exposure to recombinant FIX protein. Based on our success with hemophilia B, we sought to develop a complementary protocol for hemophilia A mice with pre-existing inhibitors. Our initial studies were conducted using a codon-optimized F8 (coF8) gene, which has been shown to enhance the levels of transcribed FVIII protein. In the first set of experiments we tested two different vector doses (1×1011 and 1×1012 vg) of an AAV8-cohF8 vector for factor VIII expression levels and tolerance induction in hemophilia A mice on two different genetic backgrounds (BALB/c-F8−/Y and 129/BL6-F8−/Y). Since BALB/c-F8−/Y mice develop lower titer FVIII inhibitors following intravenous hFVIII protein challenge compared to 129/BL6-F8−/Y mice we expected better outcomes with AAV8-cohF8 vector treated BALB/c-F8−/Y mice. Surprisingly BALB/c-F8−/Y mice spontaneously developed FVIII inhibitors approximately 4 weeks following vector delivery. In contrast,129/BL6-F8−/Y mice expressed vector dose dependent therapeutic levels FVIII protein without inhibitors and 75% of the mice remained inhibitor free following four weekly IV FVIII protein challenges. Subsequent studies for AAV8-cohF8 ITI were conducted in 129/BL6-F8−/Y mice. We generated a group of inhibitor mice and treated a subset of these mice with 1×1012 vg AAV8-coF8. AAV8-cohF8 ITI alone was not sufficient to eliminate inhibitors. Therefore we designed two additional studies to determine if 1×1012 vg AAV8-coF8 ITI would be more effective if combined with transient immune suppression using oral rapamycin (4mg/kg) or anti-CD20 (10mg/kg) mediated B cell depletion. While rapamycin adjunct therapy failed to lower inhibitors, preliminary data show that anti-CD20 combined with AAV8-coF8 ITI leads to a sharp reduction in high titer inhibitors. Given that FVIII protein invokes a stronger immune response in mice and patients, our data suggests that successful gene therapy for hemophilia A may require supraphysiological levels of FVIII protein to prevent inhibitor formation and transient immune suppression to be effective as an ITI therapy.

Gene Therapy for Coagulation Disorders

Molecular genetic details of the human coagulation system were among the first successes of the genetic revolution in the 1980s. This information led to new molecular diagnostic strategies for inherited disorders of hemostasis and the development of recombinant clotting factors for the treatment of the common inherited bleeding disorders. A longer term goal of this knowledge has been the establishment of gene transfer to provide continuing access to missing or defective hemostatic proteins. Because of the relative infrequency of inherited coagulation factor disorders and the availability of safe and effective alternative means of management, the application of gene therapy for these conditions has been slow to realize clinical application. Nevertheless, the tools for effective and safe gene transfer are now much improved, and we have started to see examples of clinical gene therapy successes. Leading the way has been the use of adeno-associated virus-based strategies for factor IX gene transfer in hemophilia B. Several small phase 1/2 clinical studies using this approach have shown prolonged expression of therapeutically beneficial levels of factor IX. Nevertheless, before the application of gene therapy for coagulation disorders becomes widespread, several obstacles need to be overcome. Immunologic responses to the vector and transgenic protein need to be mitigated, and production strategies for clinical grade vectors require enhancements. There is little doubt that with the development of more efficient and facile strategies for genome editing and the application of other nucleic acid-based approaches to influence the coagulation system, the future of genetic therapies for hemostasis is bright.

Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium

ABSTRACTSignal peptides play an important role in directing and efficiently transporting secretory proteins to their proper locations in the endoplasmic reticulum of mammalian cells. The aim of this study was to enhance the expression of recombinant coagulation factor VII (rFVII) in CHO cells by optimizing the signal peptides and type of fed-batch culture medium used. Five sub-clones (O2, I3, H3, G2 and M3) with different signal peptide were selected by western blot (WB) analysis and used for suspension culture. We compared rFVII expression levels of 5 sub-clones and found that the highest rFVII expression level was obtained with the IgK signal peptide instead of Ori, the native signal peptide of rFVII. The high protein expression of rFVII with signal peptide IgK was mirrored by a high transcription level during suspension culture. After analyzing culture and feed media, the combination of M4 and F4 media yielded the highest rFVII expression of 20 mg/L during a 10-day suspension culture. After analyzing cell density and cell cycle, CHO cells feeding by F4 had a similar percentage of cells in G0/G1 and a higher cell density compared to F2 and F3. This may be the reason for high rFVII expression in M4+F4. In summary, rFVII expression was successfully enhanced by optimizing the signal peptide and fed-batch medium used in CHO suspension culture. Our data may be used to improve the production of other therapeutic proteins in fed-batch culture.

Long-acting recombinant coagulation factor IX albumin fusion protein (rIX-FP) in hemophilia B: results of a phase 3 trial

AbstractA global phase 3 study evaluated the pharmacokinetics, efficacy, and safety of recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 63 previously treated male patients (12-61 years) with severe hemophilia B (factor IX [FIX] activity ≤2%). The study included 2 groups: group 1 patients received routine prophylaxis once every 7 days for 26 weeks, followed by either 7-, 10-, or 14-day prophylaxis regimen for a mean of 50, 38, or 51 weeks, respectively; group 2 patients received on-demand treatment of bleeding episodes for 26 weeks and then switched to a 7-day prophylaxis regimen for a mean of 45 weeks. The mean terminal half-life of rIX-FP was 102 hours, 4.3-fold longer than previous FIX treatment. Patients maintained a mean trough of 20 and 12 IU/dL FIX activity on prophylaxis with rIX-FP 40 IU/kg weekly and 75 IU/kg every 2 weeks, respectively. There was 100% reduction in median annualized spontaneous bleeding rate (AsBR) and 100% resolution of target joints when subjects switched from on-demand to prophylaxis treatment with rIX-FP (P < .0001). The median AsBR was 0.00 for all prophylaxis regimens. Overall, 98.6% of bleeding episodes were treated successfully, including 93.6% that were treated with a single injection. No patient developed an inhibitor, and no safety concerns were identified. These results indicate rIX-FP is safe and effective for preventing and treating bleeding episodes in patients with hemophilia B at dosing regimens of 40 IU/kg weekly and 75 IU/kg every 2 weeks. This trial was registered at www.clinicaltrials.gov as #NCT0101496274.

Antibody response to recombinant human coagulation factor VIII in a new rat model of severe hemophilia A

Neutralizing antibodies toward FVIII replacement therapy (inhibitors) are the most serious treatment-related complication in hemophilia A (HA). A rat model of severe HA (F8(-/-) ) has recently been developed, but an immunological characterization is needed to determine the value of using the model for research into inhibitor development. Characterize the antibody response towards recombinant human coagulation factor VIII (rhFVIII) in the HA rat, following a human prophylactic dosing regimen. Two identical studies were performed, which included a total of 17 homozygous HA rats (F8(-/-) , 0% FVIII activity), 12 heterozygous rats (F8(+/-) ), and 12 wild-type (F8(+/+) ) rats. All rats received intravenous injections of rhFVIII at 50 IU kg(-1) twice weekly for 4 weeks. Predosing blood samples were analyzed for binding and neutralizing anti-rhFVIII antibodies at weeks 1-7. In both studies, antibodies developed after 4-6 administrations of rhFVIII, and neutralizing antibodies reached levels similar to human patients (range 1-111 BU, median 6.0 BU) at the end of the study. There was no significant difference between the two studies or between genotypes in time to response or levels reached for binding and neutralizing antibodies. Interestingly, early spontaneous bleeds were associated with a faster antibody response. Following intravenous administration of human FVIII, according to a clinical prophylaxis regimen, a robust and reproducible antibody response is seen in this HA rat model, suggesting that the model is useful for intervention studies with the aim of suppressing, delaying, or preventing the inhibitor response. Also, bleeds seem to have an adjuvant effect on the immune response.

Public expenditure for plasma-derived and recombinant medicinal products in Italy.

In Italy, the supply of plasma-derived medicinal products funded by the National Health Service can be through public healthcare facilities, accredited pharmacies or toll fractionation agreements between Regions and the manufacturer. Pharmaceutical public expenditure includes the supply related to the first two channels and costs can significantly vary because of channel-specific price reductions. This paper describes 2011 public expenditure for plasma-derived medicinal products purchased on the market, as well as the cost analysis per active substance. Analysis of the usage of plasma-derived medicinal products and of the related expenditure in public facilities has been carried out using medicinal product traceability data. The analysis related to the accredited pharmacies channel has been carried out using quantities for every medicinal package recorded by Pharmacy Associations and applying reference prices in force on March 1(st), 2012 as well as discounts for the accredited pharmaceutical expenditure imposed by law. At national and regional level, total and total per capita expenditures on plasma-derived medicinal products by market channel and funded by the National Health Service are shown. Analysis was conducted considering the active substances in three groups: substances included in toll fractionation agreements, recombinant coagulation factors, and other substances not included in toll fractionation agreements. In 2011, the national expenditure estimate for plasma-derived and recombinant medicinal product acquisition on the market was about € 535 million. The purchased volumes and mean purchased prices per unit of each substance have a significant influence on the observed regional variability of the pharmaceutical public expenditure. A strategy of regional comparison aimed at both sharing a national range of reference for purchase prices and evaluating modalities for centralised purchasing is desirable.

Plasma-derived versus recombinant factor VIII concentrates for the treatment of haemophilia A: plasma-derived is better.

In the last two decades, great advances in our understanding of the genetic and molecular bases of haemophilia, along with the results of extensive clinical research, have led to dramatic improvements in the management of people with this inherited bleeding disorder. The period from the 1990s onward has been characterised by an increase in the availability and quality of recombinant coagulation factors and the broad implementation of prophylactic treatment regimens1. In parallel, progressive improvement of virus-inactivation methods in plasma products, as well as of methods used to screen viruses in blood donations and plasma pools (i.e., polymerase chain reaction testing) led to an impressive increase in the safety of plasma-derived factor concentrates, as clearly documented by the fact that no blood-borne transmission of hepatitis viruses or human immunodeficiency virus has occurred in the last 20 years2. As a consequence, the quality of life of haemophiliacs has dramatically improved and their life expectancy has progressively approached that of their non-haemophilic peers3. In this context, the most challenging complication of current therapy has become the development of inhibitory alloantibodies, which renders replacement therapies ineffective, limit the access of patients to a safe and effective standard of care and predispose them to an unacceptably high risk of morbidity and mortality4.

Evaluation of Nonacog Beta Pegol Long-term Safety in the Immune-deficient Rowett Nude Rat (Crl:NIH-Foxn1rnu).

Nonacog beta pegol is a 40-kDa polyethylene glycosylated (PEGylated) human recombinant coagulation factor IX, intended for the treatment of hemophilia B. Human coagulation factors are immunogenic in animals; therefore, to evaluate the long-term toxicity of nonacog beta pegol, an immune-deficient, athymic rat (Rowett nude; Crl:NIH-Foxn1(rnu)) was used. Rats (n = 216) were given intravenous nonacog beta pegol 0, 40, 150, 600, or 1,200 IU/kg every 5th day for 26 weeks. To avoid infections, the animals were housed in a full-barrier environment with sterilized food and bedding. Standard toxicity end points were unaffected by treatment. All treated animals were exposed to nonacog beta pegol throughout the study, and no animals developed antidrug antibodies. Immunohistochemical staining revealed PEG in choroid plexus epithelial cells in a dose-dependent manner. Transmission electron microscopy showed that PEG was distributed in cytoplasmic vesicles of these cells, with no apparent effect on cellular organelle structures. Fourteen (6.5%) animals were euthanized or died prematurely due to nontreatment-related infections in the urogenital system and skin. In conclusion, the athymic rat is a suitable model for testing chronic toxicity of human proteins that are immunogenic in animals. Nonacog beta pegol was generally well tolerated, with no adverse effect of PEG on choroid plexus epithelial cells.

遺伝子組換え型活性型第VII因子製剤投与により慢性硬膜下血腫に対する穿頭血腫灌流除去術中の出血制御に成功した後天性血友病Ａの１例

後天性血友病A (AHA) は凝固第VIII因子 (FVIII) に対する自己抗体により発症する比較的まれな凝固異常症である. 今回我々は慢性硬膜下血腫の穿頭血腫灌流除去術を遺伝子組換え型活性型第VII因子製剤 (ノボセブン®HI: rFVIIa) 投与下に施行し, 良好な止血管理が出来たAHA症例を経験したので報告する. 症例は, 54歳男性. 心原性脳塞栓にて前医入院. 尿道カテーテルによる尿道損傷後, 血尿が持続, APTT延長が認められたため, 精査加療目的に当院転院となった. 転院時, FVIII活性8.0％と低下, FVIII inhibitor力価2.0 BU/mlと高値のためAHAと診断され, プレドニゾロン (PSL) (0.5 mg/kg) による免疫抑制療法が開始された. 治療開始後第9病日頭部CTで著明な正中偏位と脳の圧排を伴う左慢性硬膜下血腫を発症, rFVIIa投与下に緊急で穿頭血腫灌流除去術が施行され, 重篤な出血なく止血管理に成功した. 第12病日に検出感度以下へのinhibitor低下, 第19病日にFVIII活性正常化したため寛解と判定された. 転院後PSL漸減中にAHAが再発したが, PSLの再増量にて再度寛解となった.

Emerging genetic and pharmacologic therapies for controlling hemostasis: beyond recombinant clotting factors.

For more than 3 decades, the scientific community has pursued gene correction of hemophilia, with the goal that an individual with congenitally deficient factor VIII or factor IX might synthesize adequate endogenous clotting factor to be relieved of burdensome repeated clotting factor infusions, as well as the emotional weight of continuous hemorrhage risk. Recent reports of successful factor IX gene therapy and partial correction of the bleeding phenotype have raised the bar for success for a robust crop of new clinical gene therapy efforts for both hemophilia A and B. At the same time that gene therapy is gaining momentum, suggesting the possibility of relief from regular intravenous coagulation protein replacement, a number of innovative technologies that enhance hemostatic potential independently of replacement factor administration are demonstrating success in human clinical application. Human clinical trial progress is reviewed regarding a recombinant bispecific IgG antibody to factors IXa and X that mimics factor VIII cofactor activity, as well as monoclonal antibody and short interfering RNA strategies that demonstrate hemostatic efficacy via opposing inhibitors of coagulation. These strategies, associated with prolonged hemostatic potential following subcutaneous (ACE910, ALN-AT3, Concizumab) or single administration (eg, gene therapy) make it possible to imagine a day when recombinant clotting factor administration, rather than being a daily preoccupation, is relegated to an adjunctive role in supporting more novel standard of care therapies.

Efficacy and Safety Results of Prolong-9FP Clinical Program of Recombinant Fusion Protein Linking Coagulation Factor IX with Albumin (rIX-FP) in Previously Treated Patients with Hemophilia B

▪A fusion protein genetically linking recombinant human coagulation FIX with recombinant human albumin (rIX-FP) has been developed with an improved PK profile, thus improving hemophilia B treatment by allowing less frequent dosing.Two Phase 3 studies (CSL654-3001 and CSL654-3002) were completed. CSL654-3001 study evaluated safety and efficacy of rIX-FP for prophylaxis treatment (PT) of every 7-, 10- and 14-day and on-demand (ODT) of bleeding episodes in 63 previously treated patients (PTP), 12-61 years of age with hemophilia B (FIX ≤ 2%). Subjects in the on-demand arm received only ODT for 6 months and then switched to every 7-day PT. Subjects in the prophylaxis arm received every 7-day PT, and eligible subjects switched to every 10- or 14-day PT for approximately 12-18 months. Annualized spontaneous bleeding rates (AsBR) were compared between ODT and PT periods (in on-demand arm), and between 7-day PT and 10- or 14-day PT (in prophylaxis arm). CSL654-3002 study evaluated safety and efficacy of rIX-FP for weekly prophylaxis regimen in 27 previously treated patients younger than 12 years with hemophilia B (FIX ≤ 2%) for approximately 12 months. Annualized spontaneous bleeding rates (AsBR) were calculated. The median annualized spontaneous bleeding rate were all 0.00 for all treatment interval (7-day, 10-day or 14-day) and in both studies age groups (1-11 years and 12-65 years) during the two completed phase 3 studies.Seventy-six subjects from both studies continued their prophylaxis regimen in the on-going extension study. In addition, subjects (including children), switched to longer treatment intervals of 10-day, 3 times per month or 14-day or lowered their weekly prophylaxis dose. Nine subjects switched to 21-day treatment interval with 100 IU/kg rIX-FP. As of 28 July 2015, at least 50 subjects (PTP) had achieved 100 EDs without developing an inhibitor to FIX or antibodies to rIX-FP. The long term safety and efficacy of rIX-FP will be presented. This presentation includes the new information regarding the change to longer than 7-day treatment regimens in the extension study, among those subjects (1-61 years of age) that previously participated in the lead in studies.Conclusion: The Prolong - 9FP clinical programdemonstrated the clinical efficacy of rIX-FP for routine prophylaxis every 7-, 10- and 14-day treatment intervals. Routine prophylaxis once every 21 days may be effective in preventing bleeding episodes in a selected patient population. In addition, rIX-FP demonstrated favorable long-term safety and tolerability. DisclosuresSantagostino:Novo Nordisk: Speakers Bureau; Bayer: Speakers Bureau; CSL Behring: Speakers Bureau; Baxter/Baxalta: Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Biogen/Sobi: Speakers Bureau; Biotest: Speakers Bureau; Kedrion: Speakers Bureau; Octapharma: Speakers Bureau; Roche: Speakers Bureau. Voigt:CSL Behring: Employment. Wolko:CSL Behring: Employment. Cole:CSL Behring: Employment. Li:CSL Behring: Employment. Jacobs:CSL Behring: Employment.

Efficacy and Safety of Recombinant Fusion Protein Linking Coagulation Factor IX with Albumin (rIX-FP) in Previously Treated Patients with Hemophilia B Undergoing Major Orthopedic Surgeries

A fusion protein genetically linking recombinant human coagulation FIX with recombinant human albumin (rIX-FP) has been developed with an improved PK profile, improving hemophilia B treatment by allowing less frequent dosing and FIX consumption for perioperative management. A surgical sub-study was included in 3 Phase III studies, including an on-going Phase IIIb extension study in subjects with hemophilia B, as part of the global PROLONG-9FP clinical program. This report will focus on the use of rIX-FP for perioperative prophylaxis.Efficacy was evaluated by Investigator assessment (4-point scale), predicted and estimated actual blood loss, and consumption of rIX-FP during the 14-day perioperative period. Safety, including inhibitors to FIX, was assessed. The pre-operative dose of rIX-FP was based on individual incremental recovery, targeting 80-100% FIX activity. FIX activity was monitored at local laboratories to determine repeat doses during the surgical period.Five major orthopedic surgeries have been assessed to date. Hemostatic response was rated by investigators as excellent or good for all surgeries. A single dose of rIX-FP prior to surgery was sufficient to maintain hemostasis during surgery. Estimated actual blood loss was consistent with predicted blood loss in patients without hemophilia B. The total number of rIX-FP injections ranged from 6-7 over the 14 day post-operative period. The median rIX-FP consumption prior to surgery, Days 1-3, Days 1-7 and Days 8-14 post surgery were 84, 33, 81 and 107 IU/kg, respectively. There were no inhibitors to FIX or antibodies to rIX-FP during the study. The extension study is still ongoing, and additional orthopedic surgeries data may be presented.These results support the efficacy and safety of rIX-FP for perioperative management in patients with hemophilia B, with prolonged dosing intervals and lower FIX consumption than conventional therapies. DisclosuresNegrier:CSL Behring: Speakers Bureau. Wolko:CSL Behring: Employment. Voigt:CSL Behring: Employment. Jacobs:CSL Behring: Employment. Santagostino:CSL Behring: Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Baxter/Baxalta: Speakers Bureau; Biotest: Speakers Bureau; Kedrion: Speakers Bureau; Roche: Speakers Bureau; Octapharma: Speakers Bureau; Biogen/Sobi: Speakers Bureau; Novo Nordisk: Speakers Bureau; Bayer: Speakers Bureau.

Prevalence of Hepatitis C Infection and Virus Clearance in Patients with Hemophilia

IntroductionHemophilia A and B are hereditary genetic disorders caused by deficiency of clotting factor VIII and IX respectively. Infusions of clotting factors constitute the mainstay of treatment. Before availability of recombinant factor VIII and IX, patients with hemophilia received products derived from human plasma and were at risk of blood-borne infections exposure. Since then, ongoing advances have yielded not only safer recombinant clotting factors but also effective treatment of infections. Hepatitis C infection and its complications constitute a common cause of morbidity and mortality in older hemophilia patients. In the last few years, FDA approved various antiviral medications which allow achievement of sustained virologic response and even cure hepatitis C infection. Goal of our study is to show prevalence of hepatitis C infection and virus clearance at our hemophilia treatment center.MethodsWe performed retrospective chart review of patients followed at a single hemophilia treatment center in the United States. We included 59 patients with factor VIII or IX deficiency, age 30 and older, followed in clinic between 2005 and 2014. Patients with acquired hemophilia were excluded from study. Data was collected from physician notes and laboratory tests results which included hepatitis C antibodies and viral load.Results39 (66.1%) patients had history of hepatitis C infection. 21 (35.6%) patients had detectable hepatitis C viral load [95% CI 0.24-0.49] while 18 (30.5%) patients cleared the virus [95% CI 0.19-0.44].ConclusionsOur study showed that 66.1% of hemophilia patients followed at our institution had current or past hepatitis C infection. More than half of them were virus carriers during their last viral load check. Many of them had refused interferon-based treatment due to the requirement of being on it for long duration and concerns about side effects. Others had received the treatment but failed it. This constituted as much as 35.6% of hemophilia patients at our hemophilia treatment center. Availability of the newer antiviral agents that are better tolerated and yield high cure rates provide an opportunity to further reduce the disease burden in these patients. DisclosuresNo relevant conflicts of interest to declare.

Differences in N-glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells.

Background & MethodsRecombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and also with those of plasma derived FVII (pdFVII), using various analytical methods. rFVII was purified from selected production clones derived from BHK, CHO, and HEK293 cells after stable transfection, and rFVII isolates were analyzed for protein activity, impurities and post-translational modifications.Results & DiscussionThe analytical results showed no apparent gross differences between the various FVII proteins, except in their N-linked glycosylation pattern. Most N-glycans found on rFVII produced in HEK293 cells were not detected on rFVII from CHO and BHK cells, or, somewhat unexpectedly, on pdFVII; all other protein features were similar. HEK293rFVII glycans were mainly characterized by a higher structural variety and a lower degree of terminal sialylation, and a high amount of terminal N-acetyl galactosamines (GalNAc). All HEK293rFVII oligosaccharides contained one or more fucoses (Fuc), as well as hybrid and high mannose (Man) structures.ConclusionsFrom all rFVII isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal GalNAc, and CHO cells were therefore assumed to be the best option for the production of rFVII.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0205-1) contains supplementary material, which is available to authorized users.

Development, Validation, and Application of a Novel Ligand-Binding Assay to Selectively Measure PEGylated Recombinant Human Coagulation Factor VIII (BAX 855).

BAX 855 is a PEGylated recombinant factor VIII preparation that showed prolonged circulatory half-life in nonclinical and clinical studies. This paper describes the development, validation, and application of a novel ligand-binding assay (LBA) to selectively measure BAX 855 in plasma. The LBA is based on PEG-specific capture of BAX 855, followed by immunological factor VIII (FVIII)-specific detection of the antibody-bound BAX 855. This assay principle enabled sensitive measurement of BAX 855 down to the low nanomolar range without interference from non-PEGylated FVIII as demonstrated by validation data for plasma from animals typically used for nonclinical characterization of FVIII. The selectivity of an in-house-developed anti-PEG and a commercially available preparation, shown by competition studies to primarily target the terminating methoxy group of PEG, also allowed assessment of the intactness of the attached PEG chains. Altogether, this new LBA adds to the group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex biological matrices. The feasibility and convenience of using this method was demonstrated during extensive nonclinical characterization of BAX 855.

Оценка влияния на бюджет применения рекомбинантных факторов свертывания крови VIII у пациентов с гемофилией А

Оценка влияния на бюджет применения рекомбинантных факторов свертывания крови VIII у пациентов с гемофилией А

Absence of Vitamin K-Dependent γ-Carboxylation in Human Periostin Extracted from Fibrotic Lung or Secreted from a Cell Line Engineered to Optimize γ-Carboxylation.

Periostin (PN, gene name POSTN) is an extracellular matrix protein that is up-regulated in bronchial epithelial cells and lung fibroblasts by TH-2 cytokines. Its paralog, TGF-β-induced protein (βig-h3, gene name TGFBI), is also expressed in the lung and up-regulated in bronchial myofibroblasts by TGF-β. PN and βig-h3 contain fasciclin 1 modules that harbor putative recognition sequences for γ-glutamyl carboxylase and are annotated in UniProt as undergoing vitamin K-dependent γ-carboxylation of multiple glutamic acid residues. γ-carboxylation profoundly alters activities of other proteins subject to the modification, e.g., blood coagulation factors, and would be expected to alter the structure and function of PN and βig-h3. To analyze for the presence of γ-carboxylation, proteins extracted from fibrotic lung were reacted with monoclonal antibodies specific for PN, βig-h3, or modification with γ-carboxyglutamic acid (Gla). In Western blots of 1-dimensional gels, bands stained with anti-PN or -βig-h3 did not match those stained with anti-Gla. In 2-dimensional gels, anti-PN-positive spots had pIs of 7.0 to >8, as expected for the unmodified protein, and there was no overlap between anti-PN-positive and anti-Gla-positive spots. Recombinant PN and blood coagulation factor VII were produced in HEK293 cells that had been transfected with vitamin K 2, 3-epoxide reductase C1 to optimize γ-carboxylation. Recombinant PN secreted from these cells did not react with anti-Gla antibody and had pIs similar to that found in extracts of fibrotic lung whereas secreted factor VII reacted strongly with anti-Gla antibody. Over 67% coverage of recombinant PN was achieved by mass spectrometry, including peptides with 19 of the 24 glutamates considered targets of γ-carboxylation, but analysis revealed no modification. Over 86% sequence coverage and three modified glutamic acid residues were identified in recombinant fVII. These data indicate that PN and βig-h3 are not subject to vitamin K-dependent γ-carboxylation.

Recombinant Human Coagulation Factor IX Fc Fusion Protein Effective in Kids B-LONG Study

The Kids B-LONG study was conducted to evaluate the pharmacokinetics, safety, and efficacy of recombinant human coagulation factor IX Fc fusion protein (rFIXFc) in pediatric subjects with hemophilia B. rFIXFc is considered safe and efficacious for prophylaxis and the control of acute bleeding in children age < 12 years.