Albumin Fusion Technology Research Articles

Mouse Type-I Interferon-Mannosylated Albumin Fusion Protein for the Treatment of Chronic Hepatitis.

Although a lot of effort has been put into creating drugs and combination therapies against chronic hepatitis, no effective treatment has been established. Type-I interferon is a promising therapeutic for chronic hepatitis due to its excellent anti-inflammatory effects through interferon receptors on hepatic macrophages. To develop a type-I IFN equipped with the ability to target hepatic macrophages through the macrophage mannose receptor, the present study designed a mouse type-I interferon-mannosylated albumin fusion protein using site-specific mutagenesis and albumin fusion technology. This fusion protein exhibited the induction of anti-inflammatory molecules, such as IL-10, IL-1Ra, and PD-1, in RAW264.7 cells, or hepatoprotective effects on carbon tetrachloride-induced chronic hepatitis mice. As expected, such biological and hepatoprotective actions were significantly superior to those of human fusion proteins. Furthermore, the repeated administration of mouse fusion protein to carbon tetrachloride-induced chronic hepatitis mice clearly suppressed the area of liver fibrosis and hepatic hydroxyproline contents, not only with a reduction in the levels of inflammatory cytokine (TNF-α) and fibrosis-related genes (TGF-β, Fibronectin, Snail, and Collagen 1α2), but also with a shift in the hepatic macrophage phenotype from inflammatory to anti-inflammatory. Therefore, type-I interferon-mannosylated albumin fusion protein has the potential as a new therapeutic agent for chronic hepatitis.

Engineering of a Long-Acting Bone Morphogenetic Protein-7 by Fusion with Albumin for the Treatment of Renal Injury.

The bone morphogenetic protein-7 (BMP7) is capable of inhibiting TGF-β/Smad3 signaling, which subsequently results in protecting the kidney from renal fibrosis, but its lower blood retention and osteogenic activity are bottlenecks for its clinical application. We report herein on the fusion of carbohydrate-deficient human BMP7 and human serum albumin (HSA-BMP7) using albumin fusion technology and site-directed mutagenesis. When using mouse myoblast cells, no osteogenesis was observed in the glycosylated BMP7 derived from Chinese hamster ovary cells in the case of unglycosylated BMP7 derived from Escherichia coli and HSA-BMP7. On the contrary, the specific activity for the Smad1/5/8 phosphorylation of HSA-BMP7 was about 25~50-times lower than that for the glycosylated BMP7, but the phosphorylation activity of the HSA-BMP7 was retained. A pharmacokinetic profile showed that the plasma half-life of HSA-BMP7 was similar to that for HSA and was nearly 10 times longer than that of BMP7. In unilateral ureteral obstruction mice, weekly dosing of HSA-BMP7 significantly attenuated renal fibrosis, but the individual components, i.e., HSA or BMP7, did not. HSA-BMP7 also attenuated a cisplatin-induced acute kidney dysfunction model. The findings reported herein indicate that HSA-BMP7 has the potential for use in clinical applications for the treatment of renal injuries.

A versatile insertion point on albumin to accommodate peptides and maintain their activities

Genetic fusion of human serum albumin to peptides is an important strategy to enhance the plasma half-life of the peptide. An inherent challenge of such method is the reduction of specific activity of the cargo peptides upon connecting at N- or C-termini of albumin. Here, we report a finding that residue 363–364 of albumin can be inserted with a peptide while maintaining the peptide activities. We insert a peptide inhibitor into this site, and at the N-terminus of albumin, for comparison. The chimeric protein displays potent inhibition (IC50 value of 30 nM) to its target (uPAR), but not the N-terminally fused construct. We also study the chimera of HSA with a cyclic peptide inhibitor of murine urokinase-type plasminogen activator grafted at either the internal site or the N-terminus. The internally peptide-grafted protein possesses a much more potent inhibition compared to the N-terminally located fusion (IC50 value of 32 nM vs 19 μM). We further demonstrate that such internal fusion does not affect albumin expression, secondary structure, and inherent drug binding activity. Thus, this work identifies a versatile insertion point inside albumin for maintaining fusion peptide activity, and opens a new avenue to expand the applications of albumin fusion technology.

Production of potent long-lasting consensus interferon using albumin fusion technology in Pichia pastoris expression system

Consensus interferon (cIFN) is a wholly synthetic therapeutic protein which is used to treat hepatitis C/B and certain types of malignancies. It has short serum half-life, therefore, to maintain its therapeutic level in the human body it requires thrice-weekly administration. Various strategies like PEGylation and micro-encapsulation have been developed during the last few years to enhance the pharmacokinetics of small therapeutic peptides. This study executed the human albumin-fusion technology, a simple and flexible approach to extend the serum circulating half-life of cIFN, because human serum albumin (HSA) has long circulating half-life (19 days) and very minute immunological activities. We integrated the codon-optimized HSA-cIFN fusion gene into Pichia pastoris genome by homologous recombination. The selection of hyper-resistant P. pastoris clone against Zeocin™ achieved a high-level secretory expression (250 mg/L) of fusion protein. HSA-cIFN fusion protein was purified using one-step purification by affinity chromatography with 34% recovery. The SDS-PAGE and SEC-HPLC analysis confirmed the final purified product has molecular weight of 87 kDa with 98% purity. Western blot analysis using anti-IFN antibodies further verified the purified HSA-cIFN fusion protein. The specific biological activity was 2.1 × 106 IU/mg as assessed by cytopathic inhibition assay, and half-life of fusion protein was estimated by in vitro thermal and proteolytic stability studies. This work concludes that by using albumin fusion technology, codon optimization and one-step purification a high yield of 86 mg/L of biologically active protein with improved serum half-life was obtained.

Development of Kupffer cell targeting type-I interferon for the treatment of hepatitis via inducing anti-inflammatory and immunomodulatory actions

Because of its multifaceted anti-inflammatory and immunomodulatory effects, delivering type-I interferon to Kupffer cells has the potential to function as a novel type of therapy for the treatment of various types of hepatitis. We report herein on the preparation of a Kupffer cell targeting type-I interferon, an albumin-IFNα2b fusion protein that contains highly mannosylated N-linked oligosaccharide chains, Man-HSA(D494N)-IFNα2b, attached by combining albumin fusion technology and site-directed mutagenesis. The presence of this unique oligosaccharide permits the protein to be efficiently, rapidly and preferentially distributed to Kupffer cells. Likewise IFNα2b, Man-HSA(D494N)-IFNα2b caused a significant induction in the mRNA levels of IL-10, IL-1Ra, PD-L1 in RAW264.7 cells and mouse isolated Kupffer cells, and these inductions were largely inhibited by blocking the interferon receptor. These data indicate that Man-HSA(D494N)-IFNα2b retained the biological activities of type-I interferon. Man-HSA(D494N)-IFNα2b significantly inhibited liver injury in Concanavalin A (Con-A)-induced hepatitis model mice, and consequently improved their survival rate. Moreover, the post-administration of Man-HSA(D494N)-IFNα2b at 2 h after the Con-A challenge also exerted hepato-protective effects. In conclusion, this proof-of-concept study demonstrates the therapeutic effectiveness and utility of Kupffer cell targeting type-I interferon against hepatitis via its anti-inflammatory and immunomodulatory actions.

Overproduction, purification and characterization of human interferon alpha2a-human serum albumin fusion protein produced in methilotropic yeast Pichia pastoris

Human interferon alpha2a (hIFNα2a) is a therapeutic protein that used in cancer and hepatitis B/C therapy. The main problem of using hIFNα-2a is its short elimination half life due to its low molecular weight. Development of higher molecular weight protein by albumin fusion technology is a rational strategy to solve the problem. In our previous research we constructed an open reading frame (ORF) encoding hIFNα2a-human serum albumin (HSA) fusion protein that expressed in Pichia pastoris (P. pastoris) protease deficient strain SMD1168. This research was performed to overproduce, purify and characterize the fusion protein. To overproduce the protein, cultivation was performed in buffered complex medium containing glyserol (BMGY) for 24 h and protein overproduction was applied in buffered complex medium containing methanol (BMMY) for 48 hours at 30°C. The fusion protein was purified by blue sepharose affinity chromatography. Molecular weight characterization by SDS PAGE corresponds with its theoretical size, 85 kDa. Western blot analysis demonstrated that the fusion protein was recognized by anti hIFNα2 and anti HSA monoclonal antibody as well. Amino acid sequence of the fusion protein was determined by LC MS/MS2 mass spectrometry with trypsin as proteolitic enzyme. There were three fragments that identified as hIFNα2a and seven fragments that identified as HSA. Total identified amino acids were 150 residues with 20% coverage from total residues. To conclude, hIFNα2a-HSA fusion protein was overproduced, purified and characterized. Characterization based on molecular weight, antibody recognition and amino acid sequence confirmed that the fusion protein has correct identity as theoretically thought.

The effect of albumin fusion structure on the production and bioactivity of the somatostatin-28 fusion protein in Pichia pastoris

Somatostatin, a natural inhibitor of growth hormone (GH), and its analogs have been used in clinical settings for the treatment of acromegaly, gigantism, thyrotropinoma, and other carcinoid syndromes. However, natural somatostatin is limited for clinical usage because of its short half-life in vivo. Albumin fusion technology was used to construct long-acting fusion proteins and Pichia pastoris was used as an expression system. Three fusion proteins (SS28)(2)-HSA, (SS28)(3)-HSA, and HSA-(SS28)(2), were constructed with different fusion copies of somatostatin-28 and fusion orientations. The expression level of (SS28)(3)-HSA was much lower than (SS28)(2)-HSA and HSA-(SS28)(2) due to the additional fusion of the somatostatin-28 molecule. MALDI-TOF mass spectrometry revealed that severe degradation occurred in the fermentation process. Similar to the standard, somatostatin-14, all three fusion proteins were able to inhibit GH secretion in blood, with (SS28)(2)-HSA being the most effective one. A pharmacokinetics study showed that (SS28)(2)-HSA had a prolonged half-life of 2 h. These results showed that increasing the number of small protein copies fused to HSA may not be a suitable method for improving protein bioactivity.

Comparative Study of Somatostatin-Human Serum Albumin Fusion Proteins and Natural Somatostatin on Receptor Binding, Internalization and Activation

Albumin fusion technology, the combination of small molecular proteins or peptides with human serum albumin (HSA), is an effective method for improving the medicinal values of natural small molecular proteins or peptides. However, comparative studies between HSA-fusion proteins or peptides and the parent small molecules in biological and molecular mechanisms are less reported. In this study, we examined the binding property of two novel somatostatin-HSA fusion proteins, (SST14)2-HSA and (SST28)2-HSA, to human SSTRs in stably expressing SSTR1-5 HEK 293 cells; observed the regulation of receptor internalization and internalized receptor recycling; and detected the receptors activation of HSA fusion proteins in stably expressing SSTR2- and SSTR3-EGFP cells. We showed that both somatostatin-HSA fusion proteins had high affinity to all five SSTRs, stimulated the ERK1/2 phosphorylation and persistently inhibited the accumulation of forskolin-stimulated cAMP in SSTR2- and SSTR3-expressing cells; but were less potent than the synthetic somatostatin-14 (SST-14). Our experiments also showed that somatostatin-HSA fusion proteins did not induce the receptors internalization; rather, they accelerated the recycling of the internalized receptors induced by SST-14 to the plasma membrane. Our results indicated that somatostatin-HSA fusion proteins, different from SST-14, exhibit some particular properties in binding, regulating, and activating somatostatin receptors.

The Effect of Albumin Fusion Patterns on the Production and Bioactivity of the Somatostatin-14 Fusion Protein in Pichia pastoris

Somatostatin is a natural inhibitor of growth hormone, and its analogues are clinically used for the therapy of acromegaly, gigantism, thyrotropinoma, and other carcinoid syndrome. However, natural somatostatin is limited for clinical usage because of its short half-life in vivo. Albumin fusion technology was used to construct long-acting fusion proteins, and Pichia pastoris was used as an expression system. Three fusion proteins, (somatostatin (SS)14)2-human serum albumin (HSA), (SS14)3-HSA, and HSA-(SS14)3, were constructed with different fusion copies of somatostatin-14 and fusion orientations. The expression level of (SS14)3-HSA and HSA-(SS14)3 was much lower than (SS14)2-HSA due to the additional fusion of the somatostatin-14 molecule. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry revealed that severe degradation occurred in the fermentation process. Similar to the standard of somatostatin-14, all three fusion proteins were able to inhibit growth hormone secretion in the blood, with (SS14)2-HSA being the most effective one. On the whole, (SS14)2-HSA was the most effective protein in both production level and bioactivity, and increasing the number of small protein copies fused to HSA may not be a suitable method to improve the protein bioactivity.

Balugrastim: A long-acting, once-per-cycle, recombinant human albumin-fusion filgrastim.

e13551 Background: Balugrastim is a once-per-cycle fixed-dose genetic fusion protein composed of human serum albumin (HSA) and granulocyte colony-stimulating factor (G-CSF) in development for prevention of severe neutropenia in cancer patients receiving chemotherapy. Albumin fusion is a clinically validated technology that extends product half-life, allowing for infrequent dosing, better tolerability, lower cost, and improves drug design, potentially lowering immunogenicity risk. Here, we describe the technology used to produce balugrastim and summarize preclinical findings compared with pegfilgrastim (Neulasta). Methods: Design and production of balugrastim was described previously (Halpern et al. Pharmaceut Res 2002;19:1720−1729). Biologic activity of balugrastim was assessed in an NFS-60 cell line proliferation assay vs filgrastim and pegfilgrastim. PK and PD properties were studied in healthy and neutropenic animal models. Results: Albumin fusion produces a long-acting G-CSF with comparable pharmacologic properties to pegfilgrastim. In vitro, balugrastim had binding affinity and cell proliferation activity comparable to pegfilgrastim, and both were lower than non-PEGylated filgrastim on a molar basis. Overall increases in leukocytes, neutrophilic granulocytes, and monocytes were dose dependent and consistent with the effects expected for a long-acting G-CSF with some variation based on the specific animal model used. A single balugrastim dose in BDF1 mice elicited a dose-dependent increase in peripheral granulocytes and mobilized hematopoietic progenitor cells. In cynomolgus monkeys, balugrastim caused an increase in peripheral neutrophils similar to pegfilgrastim, with higher responses after 2nd, 3rd, and 4th doses. In mice, balugrastim had shorter terminal half-life and mean residence time, and faster clearance than pegfilgrastim. In monkeys, terminal half-life of balugrastim was slightly longer than pegfilgrastim. Conclusions: An albumin fusion technology platform was used to produce balugrastim – a novel, biologically active albumin G-CSF fusion protein with greater structural homogeneity and comparable pharmacologic properties to conventionally PEGylated G-CSFs.

Innovative coagulation factors: albumin fusion technology and recombinant single-chain factor VIII

Albumin fusion technology has been used to enhance the pharmacokinetic properties of recombinant coagulation factors. The goal of linking albumin to coagulation factors is to extend the half-life of the coagulation factor, thereby allowing for less frequent dosing for patients with bleeding disorders, such as hemophilia. The novel recombinant fusion proteins linking coagulation factors VIIa and IX with albumin (rVIIa-FP and rIX-FP, respectively) have a longer half-life and similar hemostatic efficacy compared with available recombinant coagulation factor products. Clinical evaluation of these fusion proteins is underway, and preliminary results with rIX-FP in patients with hemophilia B are encouraging. Other advances in coagulation factor therapy include a unique recombinant single-chain factor VIII (FVIII) protein, which has improved intrinsic stability and a higher affinity for von Willebrand factor (VWF), relative to other recombinant FVIIIs, and a recombinant VWF-albumin fusion protein (rVWF-FP), which has a significant longer half-life compared to available VWF products. Evaluation of these novel recombinant proteins continues and will help determine their potential to enhance the management of patients with bleeding disorders.

Extending the pharmacokinetic half-life of coagulation factors by fusion to recombinant albumin

The prophylactic treatment of haemophilia B and the management of haemophilia A or B with inhibitors demand frequent administrations of coagulation factors due to the suboptimal half-lives of the products commercially available and currently in use, e.g. recombinant factor IX (rFIX) and recombinant factor VIIa (rFVIIa), respectively. The extension of the half-lives of rFIX and rFVIIa could allow for longer intervals between infusions and could thereby improve adherence and clinical outcomes and may improve quality of life. Albumin fusion is one of a number of different techniques currently being examined to prolong the half-life of rFIX and rFVIIa. Results from a phase I clinical trial demonstrated that the recombinant fusion protein linking FIX to albumin (rIX-FP) has a five-times longer half-life than rFIX, and preclinical studies with the recombinant fusion protein linking FVIIa to albumin (rVIIa-FP) suggest that rVIIa-FP possesses a significantly extended half-life versus rFVIIa. In this review, we describe albumin fusion technology and examine the recent progress in the development of rIX-FP and rVIIa-FP.

Pre-Clinical Characteristics of a Recombinant Fusion Protein Linking Activated Coagulation Factor VII with Albumin (rVIIa-FP)

AbstractAbstract 1115Recombinant factor VIIa (rFVIIa) is approved to control bleeding in hemophilia A and B patients who have developed inhibitory antibodies to replacement therapy and in acquired hemophilia. rFVIIa is rapidly eliminated with a terminal half-life of approximately 2.5 hours in humans. This short half life necessitates frequent injections and considerably limits prophylactic use. A recombinant fusion protein linking activated factor VII with albumin (rVIIa-FP) was engineered to extend the half life of rFVIIa.The present studies were conducted to gather knowledge about the pharmacokinetic and pharmacodynamic (PK/PD) properties of rVIIa-FP in preclinical animals models to enable a more precise estimate of the procoagulant activity of rVIIa-FP for the future clinical use in haemophilia patients with inhibitors to FVIII and FIX. Single intravenous doses of either rVIIa-FP or the licensed rFVIIa comparator, NovoSeven® were administered to mice, rabbits and monkeys. Subsequently, plasma samples were harvested at different time-points after study drug administration and systemic levels of rVIIa-FP or NovoSeven® were assessed either by FVIIa activity measurements using the Staclot® assay system or by FVII antigen measurements using an enzyme linked immunoabsorbance assay (ELISA). When investigating dose responses or duration of hemostatic activity, rVIIa-FP and NovoSeven® were administered to mice or rabbits before tail clip or induction of venous stasis, respectively. Following a single intravenous bolus application the procoagulant activity of both study drugs was assessed in both models at different time points after administration up to 24 hours to proof prolonged procoagulant effects of rVIIa-FP compared to NovoSeven®.Intriguingly, in all animal species tested the systemic bioavailability, clearance and in addition the terminal half-life (t1/2β), of rVIIa-FP were significantly better in comparison to NovoSeven®. Based on FVIIa activity measurements in plasma samples derived from monkeys the 11 fold higher area under the curve (AUC) was accompanied with a 4-fold longer t1/2β, while in vivo recovery (IVR) of rVIIa-FP exceeded that of NovoSeven® by a 70 %. In rabbits the 20 fold higher AUC was associated with a 5-fold longer t1/2β and a 4-fold higher IVR when measuring specific FVIIa activity. In mice measurements of FVII antigen revealed a 11 fold higher AUC, while t1/2β was 4 times longer and IVR was found to be doubled in comparison to NovoSeven®. The assessment of pharmacodynamic activity after dosing based on specific FVIIa activity adjusted by the Staclot® assay revealed, that the procoagulant effect of rVIIa-FP was not different from NovoSeven®, when measuring total blood loss under acute bleeding conditions after tail clip. However, when assessing late time-points after treatment the prolonged systemic availability of rVIIa-FP translated into a longer and sustained hemostatic activity of rVIIa-FP compared to NovoSeven® in both animal models. Intriguingly, ex vivo studies in hemophilia A mice showed that the 4 fold prolonged systemic availability of rVIIa-FP in plasma translates into sustained FVIIa activity when recorded as thrombin generation activity compared to NovoSeven® at 16 hours after treatment. Furthermore in rabbits, the 5 fold longer half-life translated into a significantly greater procoagulant activity as measured by thrombus formation in both jugular veins after venous stasis.In conclusion, the recombinant albumin fusion technology was successfully applied to human recombinant FVIIa for a significantly improved of PK/PD profile as observed in different pre-clinical animal species. Future clinical studies can proof whether the observed improved PK/PD characteristics will also translate into a half-life extension and a longer hemostatic effect in hemophilia patients with inhibitors to FVIII and FIX.Allowed: The entire body of the abstract, including text and tables, must not exceed 3,800 characters. Spaces are not included in this number; title, authors' names, and affiliations are counted separately. Figures are not included in the character count. Disclosures:Zollner:CSL Behring GmbH: Employment. Schuermann:CSL Behring GmbH: Employment. Raquet:CSL Behring GmbH: Employment. Mueller-Cohrs:CSL Behring GmbH: Employment. Weimer:CSL Behring GmbH: Employment. Pragst:CSL Behring GmbH: Employment. Dickneite:CSL Behring GmbH: Employment. Schulte:CSL Behring GmbH: Employment.

Improved kinetics of rIX‐FP, a recombinant fusion protein linking factor IX with albumin, in cynomolgus monkeys and hemophilia B dogs

Prophylaxis of hemophilia B, at present, requires multiple infusions of human factor (F)IX concentrates per week. A FIX molecule with a prolonged half-life has the potential to greatly improve the convenience of, and adherence to, prophylaxis. The aim of our studies was to investigate the pharmacokinetic (PK) and pharmacodynamic (PD) profile of a recombinant fusion protein linking coagulation FIX with albumin (rIX-FP). Cynomolgus monkeys and hemophilia B dogs received single intravenous doses of rIX-FP (50-500 IU kg(-1)). rIX-FP plasma levels were determined by an activity-based assay (dogs only) and anti-FIX ELISA methods. Additionally, activated partial thromboplastin time (APTT) was determined in hemophilia B dogs. Data were compared with a direct study comparator (recombinant FIX [rFIX]) or previously published data. The terminal half-life of rIX-FP was prolonged in both species compared with FIX reference data. In hemophilia B dogs, human FIX antigen levels remained above 0.05 IU mL(-1) more than three times longer after rIX-FP (7.3 days) compared with rFIX (2.3 days), whereas respective calculations based on activity levels confirmed the observed superior profile. Prolonged PDs of rIX-FP were demonstrated with APTT<60 s sustained around four times longer with rIX-FP (5.9 days) than rFIX (1.5 days). These studies indicate that the recombinant albumin fusion technology successfully improves the PK profile of FIX. Clinical studies will test whether the improved kinetics result in a significant half-life extension in patients with hemophilia B.

Balancing the Pharmacokinetics and Pharmacodynamics of Interferon-α2b and Human Serum Albumin Fusion Protein by Proteolytic or Reductive Cleavage Increases Its in Vivo Therapeutic Efficacy

Human serum albumin (HSA) fusion (Albufusion) technology has evolved to be a general strategy to increase the in vivo half-lives of therapeutic proteins. However, because of the steric hindrance effect of HSA, conventional Albufusion technology improves the pharmacokinetics (PK) at the cost of pharmacodynamics (PD). To achieve balanced PK and PD of interferon-α2b (IFN-α2b) and HSA fusion protein, protease cleavage sites or disulfide linkage that enabled releasing of intact IFN-α2b with full activity was introduced between these two moieties. Nonreleasable and releasable fusion proteins showed similar cell surface receptor binding affinities; however, releasable fusion proteins exhibited release efficiency proportional increase of in vitro antiviral and antiproliferative activities. The release rate also had a profound impact on the in vivo pharmaceutical properties of fusion proteins. Releasable fusion proteins with intermediate release rate had the most balanced PK and PD, which translated into improved therapeutic efficacy in the HT29 human colon cancer xenograft model. Releasable Albufusion (rAlbufusion) allows tailored design of the PK/PD profile and greatly extends the utility of conventional Albufusion technology.

Genetic fusion to albumin improves the pharmacokinetic properties of factor IX

Haemophilia B is a X-chromosome linked disease characterised by a deficiency of functionally active coagulation Factor IX (FIX). Patients with severe haemophilia B at risk of recurrent bleeding are treated approximately twice a week in a prophylactic setting by application of FIX concentrates. To increase convenience and compliance of the therapy it is desirable to reduce the dosing frequency by improving the pharmacokinetic properties of FIX. Here a concept of rFIX (recombinant factor IX) albumin fusion proteins (rIX-FPs) with cleavable linker peptides derived from the FIX activation sequence is presented. Constructs of the genetic fusion of FIX to albumin via cleavable linkers were expressed in mammalian cells and characterised after purification. In vitro activation studies with FXIa demonstrated that cleavage of the linker and the activation peptide proceeded comparably well. In a clotting assay the rIX-FPs with cleavable linker showed a 10- to 30-fold increase in the molar specific clotting activity compared to fusion proteins with non-cleavable linkers. Furthermore, in-vivo recovery, terminal half-life and the AUC of rIX-FPs in rats and rabbits as determined by FIX antigen measurements were significantly increased compared to rFIX (BeneFIX). In FIX deficient (FIX(-/-)) mice the in-vivo recovery and the AUC were also significantly increased. The efficacy in reducing bleeding time was shown in FIX(-/-) mice by a tail tip bleeding model. The results suggest that rIX-FPs with a cleavable linker between FIX and albumin are a promising concept that may support the use of the albumin fusion technology to extend the half-life of FIX.

Use of albumin fusion technology to prolong the half-life of recombinant factor VIIa

A significant proportion of patients with haemophilia A develop inhibitors to administered factor VIII (FVIII) and require therapy with bypassing agents such as activated factor VII (FVIIa) or activated prothrombin complex concentrates. NovoSeven® is a commercially available recombinant FVIIa (rFVIIa) with a very short half-life of approximately 2.4 hours. As a result, patients generally require multiple, frequent infusions for the management of bleeding episodes. Thus, there is growing interest in extending the circulating half-life of coagulation factors through the use of innovative drug delivery and formulation technologies. One such approach uses albumin fusion technology in which human albumin is genetically fused to the C-terminus of rFVIIa via a flexible glycine serine linker. The properties of this rFVIIa fusion protein (rVIIa-FP) have recently been examined in pre-clinical studies. Results from these investigations demonstrate the feasibility of this approach, which successfully extended the half-life and biological activity of rFVIIa without compromising haemostatic efficacy. These data suggest that rVIIa-FP may be a promising therapy for the treatment of haemophilia patients with inhibitors and warrants further investigation in clinical trials.

Albumin-Interferon-α in the Treatment of Chronic Hepatitis C

Despite advances in treating chronic hepatitis C, newer treatments are required. Albumin fusion technology allows the enhanced pharmacokinetic properties of albumin to be combined with the pharmacodynamic properties of other agents. Albumin-interferon-α has an extended half-life that supports bimonthly or monthly dosing and may provide superior steady-state exposure to interferon. Preclinical studies show that albumin-interferon has better antiviral activity and pharmacokinetics than either standard or pegylated interferons. Phase I–II and Phase IIa studies that have explored a wide dose range (from 10 to 1200 μg) in interferon-experienced and -naïve patients have shown the drug to be safe and well tolerated, with no dose-limiting adverse events. Its half-life is 6–7 days and it is detectable 4 weeks after a single dose. The longer term safety and efficacy of albumin-interferon and ribavirin is being evaluated in both interferon-naïve patients and nonresponders to previous standard and pegylated interferon therapy.