ALBERT

Description: During inflammation, activated neutrophils go through the oxidative burst, releasing various oxidants, including superoxide radical, hydrogen peroxide, and hypochlorous acid (HOCl). Activated neutrophils also release myeloperoxidase (MPO), which generates HOCl from hydrogen peroxide and chloride ions. HOCl preferentially oxidizes cysteine and methionine residues to cysteine sulfenic acid and methionine sulfoxide, respectively, at rates ~100 times faster than it oxidizes tyrosine, another commonly oxidized amino acid. HOCl can also oxidize tyrosine to chlorotyrosine. Of great interest in this regard is the fact that the ADAMTS13 cleavage site in VWF, the Tyr1605–Met1606 peptide bond, contains two residues that are potential targets for myeloperoxidase-mediated oxidation. Given previous studies from our laboratory that VWF cleavage by ADAMTS13 is inhibited by oxidants, we hypothesized that neutrophil oxidants might oxidize either or both of these two amino acid residues and thereby potentially inhibit ADAMTS13-mediated cleavage. We tested our hypothesis using a peptide substrate for ADAMTS13 based on the VWF A2 sequence Leu1591–Arg1668. We incubated the VWF A2 peptide either without HOCl or with 25 or 75 μM HOCl, followed by quenching the oxidant with free methionine. The peptides were then incubated with purified recombinant ADAMTS13 and the reaction sampled every 15 min for one hour. We analyzed the cleavage reaction in two ways: by electrophoretic separation on a Tricine gel and densitometric quantification of the cleavage product, and by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) to determine the location and extent of oxidative modification and quantity of the cleavage product. We found that, after exposure to 75 μM HOCl, the A2 peptide contained methionine sulfoxide at position 1606 in 99% of the molecules in the sample, whereas only 0.3% contained both chlorotyrosine at position 1605 and methionine sulfoxide at 1606. The rate of substrate cleavage by ADAMTS13 was markedly reduced with oxidation, as measured by both assays, with the rate for the peptide treated with 75 μM HOCl being only 20% of that of the non-oxidized peptide. Taken together, these findings suggest that oxidants released by activated neutrophils during inflammation have a prothrombotic effect, mediated at least in part by inhibition of VWF cleavage by ADAMTS13.

Description: Abstract 3204 A high pressure circulatory system has two diametrically opposed requirements for its function: it must be able to rapidly gel to prevent blood loss when the integrity of the vasculature is compromised while simultaneously maintaining fluidity when the vasculature is intact. The endothelium is primarily responsible for maintaining blood fluidity, producing rapidly acting labile substances that inhibit both the clotting of blood and the adhesion and aggregation of platelets. Among these substances are the prostaglandins (PGE1, PGI2, PGD2), which bind platelet membrane receptors, raise concentrations of intracellular cyclic adenosine monophosphate (cAMP), and inhibit platelet functions. The major effector of increased cAMP is the serine/threonine kinase protein kinase A (PKA). Of the numerous targets for PKA, one of the most highly phosphorylated upon cAMP increase is glycoprotein (GP) Ibβ, a component of the GPIb-IX-V complex, the platelet receptor for VWF that mediates the initial adhesion of platelet to the vessel wall at sites of injury. The GPIb-IX-V complex consists of 4 type I transmembrane polypeptides, GPIbα, GPIbβ, GPV and GPIX. GPIbα and GPIbβ are disulfide linked in a 1:2 ratio, and the resulting GPIb is non-covalently associated with GPIX and GPV in a 2:2:1 ratio. The VWF-binding site resides within the N-terminal 300 amino acids of GPIbα 500 Å above the platelet surface. Although current data indicate that PKA phosphorylation of the GPIbβ cytoplasmic domain (at Ser166) inhibits the ability of GPIbα to bind VWF, the molecular mechanism(s) have yet to be elucidated. The cytoplasmic domain of GPIbβ associates with calmodulin (in the juxtamembrane 20 amino acids) in resting platelets; calmodulin dissociates upon platelet activation. With elevated cytosolic cAMP, GPIbβ Ser166 becomes phosphorylated and associates with 14-3-3ζ. An interesting feature of the cytoplasmic sequence N-terminal to Ser166 is its extreme cationic nature, containing 8 Arg residues in a stretch of 17 amino acids. Other cytosolic proteins with similar polybasic sequence (MARCKS, GAP43) function as organizers of the signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), and promote the formation of lipid rafts; we reasoned that the polybasic region of GPIbβ might function similarly, organizing rafts when unbound by protein, but not when occupied by calmodulin or 14-3-3ζ. Platelet activation increases raft-associated GPIb-IX-V two fold, with concomitant dissociation of calmodulin from GPIbβ. Here we present evidence that the cytoplasmic domain of GPIbβ plays a role in the localization of the GPIb-IX-V complex to lipid rafts. Treatment of platelets with agents that increase cAMP (PGI2 or forskolin) inhibited GPIb-IX-V-dependent platelet functions, including ristocetin-induced aggregation, shear-induced aggregation and adhesion to VWF under flow. This effect was prevented by the cell-permeable PKA-specific inhibitor H-89. Consistent with the functional importance of GPIb-IX-V localization to lipid rafts, PGI2 and forskolin reduced the raft content of GPIb-IX-V by 35%, and this effect was reversed by H-89. We have thus uncovered a mechanism for long-observed inhibition of platelet adhesion by agents that elevate cytosolic cAMP concentrations, which depends on modulating the quantity of GPIb-IX-V complexes associated with lipid rafts. “Resting” platelets ex vivo are relatively quiescent because calmodulin occupies the GPIbβ polybasic region. The situation changes rapidly when platelets are activated, with more of the complex assuming a ligand-competent state as calmodulin dissociates and the complex organizes rafts. Elevations of cAMP promote phosphorylation of GPIbβ, enabling 14-3-3ζ association, which also displaces the GPIbβ tail from the membrane, disrupting raft association and adhesive function. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Hemophilia A is characterized by a deficiency in factor VIII (FVIII) activity, resulting in failure of blood to clot properly, leading to traumatic and spontaneous bleeding, particularly into joints and soft tissues. Current treatment strategies rely on intravenous administration of FVIII, but require frequent dosing owing to the relatively short half-life of FVIII. We previously reported the development of a recombinant FVIIIFc fusion protein (rFVIIIFc), that shows an approximate 1.5-fold increase in half-life relative to otherwise unmodified FVIII, potentially enabling less frequent dosing compared to rFVIII. Both FVIII and rFVIIIFc exhibit comparable specific activities and similar affinities for von Willebrand factor (VWF). Furthermore, hydrogen-deuterium exchange (H/Dx) analysis established structural comparability of rFVIII and the FVIII component of rFVIIIFc, while small angle x-ray scattering (SAXS) results suggested that the Fc is flexibly tethered to the C-terminus of FVIII, leaving the regions of FVIII necessary for interaction with phospholipid, VWF, FIXa, and FX binding sterically accessible. Consistent with these findings, the crystal structure of rFVIIIFc shows that the FVIII structure is unaffected by the appended Fc domain, while the Fc domain itself is not visible, suggesting conformational freedom. Aims The goal of this study is to further compare the structure of rFVIII with that of the FVIII component of rFVIIIFc by evaluating the affinities of a panel of anti-FVIII antibodies having structurally distinct epitopes. Additionally, we assess the conformational flexibility of the Fc component of rFVIIIFc using negative stain electron microscopy. Methods Thirty-nine anti-FVIII mouse monoclonal antibodies, 14 of which are being reported here for the first time, were evaluated by surface plasmon resonance (SPR) to determine their affinities for both rFVIII and rFVIIIFc. Affinities were then compared to assess whether the respective epitopes, which collectively span the 5 structural domains of FVIII, are conformationally conserved between FVIII and rFVIIIFc. Subsequently, Fab fragments were generated for two of these antibodies, GMA-8015 and ESH8, and the structures of the complexes formed between these Fabs and FVIII were visualized by negative stain EM. The structure of rFVIIIFc was determined by negative stain EM to determine the positional variability of the Fc element with respect to the FVIII component of rFVIIIFc. Results and Conclusion The affinities of all 39 antibodies for rFVIII and rFVIIIFc, expressed in terms of KD, spanned the picomolar to nanomolar range and each showed comparable affinities for rFVIII and rFVIIIFc. Among these are 13 antibodies which are known to compete with VWF for FVIII binding, providing further evidence that the structural determinants of VWF binding are the same in both molecules. As expected, the GMA-8015 and ESH8 Fabs bound to the A2 and C2 domains of FVIII, respectively, and were clearly visible in EM images. For rFVIIIFc, particles were grouped into 100 classes, and the resulting class averages revealed a high degree of conformational freedom between FVIII and the appended Fc domain, while the gross morphology of the FVIII component remained unchanged. Notably, rFVIII and rFVIIIFc exhibited similar affinities for ESH8, despite the close proximity of the ESH8 epitope and the Fc fusion site in the C2 domain at the C-terminus of FVIII, indicating flexible tethering between FVIII and the Fc domain. These findings provide further mechanistic insight into the observed functional similarity of FVIII and rFVIIIFc with respect to cofactor activity and VWF binding. Disclosures: Leksa: Biogen Idec: Employment, Equity Ownership. Liu:Biogen Idec: Employment, Equity Ownership. Goodman:Biogen Idec: Employment, Equity Ownership. Chiu:Biogen Idec: Research Funding. Walz:Biogen Idec: Honoraria, Research Funding. Peters:Biogen Idec: Employment, Equity Ownership. Kulman:Biogen Idec: Employment, Equity Ownership.

Description: Abstract 2117 Regulation of the bond between platelet glycoprotein (GP) Ibα of the GPIb-IX-V complex, and the von Willebrand Factor (VWF) A1 domain is critical to the balance between hemostasis and thrombosis, particularly in high shear conditions. The GPIbα-A1 interaction is known to be activated by shear stress and inhibited by neighboring domains in VWF, but the role of neighboring domains in the shear-dependence remained unknown. Here it is shown that platelet aggregation required shear stress in the presence of VWF proteins that contain the neighboring D′D3 domain (Plus D′D3 or plasma VWF) but that platelets aggregate spontaneously with a protein that lacks this region (Delta D′D3). Moreover, platelets and microspheres coated with the N-terminal 300 amino acids of GPIbα (GC300) bind to immobilized VWF in a shear-enhanced manner for Plus D′D3 but not for Delta D′D3. In single-molecule force spectroscopy experiments, the D′D3 domain decreased the number of GPIbα-A1 bonds that formed, but did not alter bond rupture force, consistent with the hypothesis that D′D3 shields the A1 domain. By expressing recombinant VWF fragments that contain the A1 domain and various lengths of the N-terminal region, we determined that most of the inhibition by the D′D3 domain was conferred by 23 amino acids in the linker between the A1 domain and the D′D3 domain. By anchoring the fragments to the surface in an oriented manner, we demonstrated that binding was much stronger when force was applied between GPIbα and the A1 C-terminus, than when force was applied between GPIbα and the A1 N-terminus, similar to what has been observed for integrins. Based on these results, we propose the following model for regulation of VWF by mechanical force. When multimeric VWF is stretched in flow, the D′D3 domains are pulled away from the A1 domains, exposing the latter to bind platelets. When force is applied between GPIbα and the C-terminus of A1, it induces an activating conformational change that could be analogous to that seen in integrins. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4224 Del-1 is an extracellular matrix protein that is highly expressed during the embryonic period. It comprises, from N- to C-terminus, three epidermal growth factor (EGF) domains (containing an RGD motif in the second EGF domain) and two factor VIII-homologous C domains (C1C2 domain). After the embryonic period, hypoxia and vascular injury can increase Del-1 expression by endothelial cells and macrophages, promoting angiogenesis and phagocytosis of phosphatidylserine (PS)-exposing apoptotic bodies. This is possibly due to the capacity of Del-1 to bridge the interaction between apoptotic bodies and phagocytes. Del-1 binds to PS-exposing cells and membrane fragments through its C1C2 domains and to integrins of phagocytic cells through its RGD sequence, enhancing phagocytosis. We expressed in mammalian cells a Del-1 fragment composed of the C1 and C2 domains containing two human protein C tags and a biotin tag at the C-terminus. The recombinant fragment was purifed using monomeric avidin resin. The recombinant Del-1 C1C2 bound PS specifically on a commercial lipid array (PIP-strip, Echelon), a finding confirmed in a 96-well assay using immobilized lipids. The binding of C1C2 Del-1 to phosphatidylserine (PS) was consistent and stable when compared to the binding of commercially available annexin-V (biotinylated and PE-conjugated). We evaluated by flow cytometry the ability of Del-1 C1C2 to bind membrane-exposed PS. Apoptotic THP-1 cells were incubated with Del-1 C1C2 in the absence of calcium or with annexin V (2.5 mM calcium was present in all the buffers and through all washing steps). Protein binding was detected using a monoclonal anti-biotin-PE conjugated antibody. Neither the percentage of PS-positive cells nor the mean fluorescence intensity (MFI) was statistically different between Del-1 C1C2 and annexin V when samples were diluted with buffer (instead of washed) before flow cytometric analysis. However, both the percentage of PS-positive cells and MFI decreased by approximately 50% in the annexin V samples when the cells were washed after incubation. Cell-derived microparticles from outdated RBC units were also used to compare the utility of C1C2 Del-1 vs commercial PE-conjugated annexin V for the detection of microparticles by flow cytometry. We found a 1.4-fold average increase in the quantity of microparticles detected with C1C2 Del-1 when compared to annexin V. In addition, cell-derived microparticles can be isolated and concentrated from the Del-1 C1C2–treated sample using magnetic beads coupled to anti-protein C antibody. In conclusion, the C1C2 domain of Del-1 represents a powerful and sensitive tool for the detection and isolation of PS-exposing cells and microparticles. The calcium-independence of this reagent for PS simplifies the detection of apoptotic cells and microparticles in plasma. Moreover, the addition of multiple tags at the C-terminus allows the capture, labeling and enrichment of PS-specific microparticles by bridging C1C2-coated microparticles to magnetic beads. C1C2 Del-1 thus represents a powerful new tool for analysis in the fields of microparticle biology, thrombosis, and immunology. Disclosures: No relevant conflicts of interest to declare.

Description: Background: A variety of approaches have been used to extend the half-life of human factor IX (FIX) in the circulation, including PEGylation and recombinant fusion of either the Fc domain of IgG or albumin to the C-terminus of FIX. Introduction of XTENs, unstructured hydrophilic polypeptides of defined amino acid composition and low immunogenic potential, represents a promising alternative approach for generating FIX variants with improved pharmacokinetic properties. Moreover, the hydrophilic properties of XTEN have the potential to increase the solubility of FIX and thereby render it suitable for subcutaneous administration, which is currently an unmet need. Because XTEN is introduced into FIX by DNA recombination, the location, length, composition and number of XTEN modifications can be readily varied, and impact of these modifications on the activity and clearance of FIX can be evaluated. Aim: To identify sites in FIX that can accommodate the introduction of XTENs without abrogating FIX activity and apply this approach to both otherwise non-modified FIX and a recombinant FIX-Fc fusion protein. Methods: The highly active FIX Padua variant (R338L) was used as a scaffold to counter FIX activity loss due to reduced activity caused by the introduction of XTENs. XTEN insertion within the Gla domain was avoided due to the essential role of the Gla domain in anchoring FIX to phospholipid surfaces and subendothelial type IV collagen. XTEN insertion sites were selected by analysis of available FIX structures in the Protein Data Bank in conjunction with the following criteria: 1) calculated accessible surface area, 2) solvent accessibility assessed by hydrogen/deuterium exchange mass spectrometry (H/DX-MS), 3) exclusion of sites within defined secondary structural elements, 4) preference for positions with significant inter-species protein sequence variability, and 5) exclusion of sites proximal to known hemophilia B mutations. A 42-residue XTEN element (AE-42) was inserted at sites selected by using these criteria or fused at the C-terminus of FIX. FIX activities of these variants were evaluated in conditioned medium of transfected HEK293 cells. Longer XTENs (AE-72, -144 and -288) were then similarly tested at sites shown to be permissive for AE-42 insertion. Finally, based on results obtained with these single XTEN variants, FIX variants with multiple XTEN insertions of varying lengths and at representative sites were evaluated for FIX activity. Results: Based on the criteria described above, a total of 33 sites in FIX were selected and evaluated by insertion of AE-42. Of these, two in EGF domain 2 (EGF2), one in the EGF2-activation peptide (AP) linker region, four in the AP, and four in the catalytic domain, including the C terminus, were identified as permissive sites by FIX activity assay. Only sites in AP and sites at or close to the C-terminus of FIX tolerated longer XTENs (AE-144, AE-288 or AE-864). FIX activity detected in conditioned medium inversely correlated with the length of XTEN introduced. Four representative sites in FIX were selected to generate a combinatorial library of 79 FIX variants. Three groups, FIX with a single XTEN, FIX with dual XTENs and FIX-Fc with a single XTEN insertion, showed detectable activity, while combination of insertion/fusion at three or more sites abolished FIX activity. Conclusions: Several permissive sites for XTEN insertion are present in FIX and select combinations of XTEN insertions variants retain FIX activity. Active FIX-XTEN variants identified here are candidates for pharmacokinetic characterization in hemophilia B mice. Disclosures Liu: Biogen: Employment. van der Flier:Biogen: Employment. Bardan:Biogen: Employment. Ding:Amunix: Employment. Schellenberger:Amunix Operating Inc: Employment. Kulman:Biogen: Employment. Peters:Biogen: Employment.

Description: INTRODUCTION Prophylactic treatment for hemophilia B patients is the therapy of choice to improve quality of life and minimize annual bleeding rates and damage to joints. A new generation of extended half-life (EHL) FIX replacement products has been generated to improve patient care by reducing treatment burden. The factors include the currently marketed rFIXFc, as well as rFIX attached to PEG or albumin. The latter two are in clinical trials. All FIX preparations are administered by intravenous dosing, which can be particularly challenging for young patients and patients with limited venous access; these difficulties are reduced, but not eliminated, by the less frequent dosing achieved with EHL rFIX therapies. In the current study, we evaluate an XTEN-recombinant protein technology in order to develop EHL rFIX-XTEN molecules that are suitable for both acute treatment, as well as prophylactic subcutaneous dosing in hemophilia B, and could potentially further reduce the burden of treatment. XTEN are unstructured polypeptide sequences that consist of a limited set of natural amino acids (Pro, Ala, Gly, Glu, Ser, Thr). Fusion of XTEN to proteins alters its hydrodynamic properties and reduces the rate of clearance and degradation of the fusion protein. These XTEN fusion proteins are produced using recombinant technology, without the need for chemical modifications, and degraded by natural pathways. MATERIALS AND METHODS Recombinant FIX and FIXFc molecules were expressed as the natural Arg338Leu (R388L, Padua) variant with improved activity. XTEN polypeptides are fused to either the C-terminus of rFIX or inserted into the EGF2 domain or activation peptide (AP) domain of rFIX or rFIXFc. The fusion proteins were prepared by transient expression in human HEK293 cells followed by affinity purification. Hemophilia B (HemB) mice were dosed by either intravenous or subcutaneous injection with a single bolus of 50 or 200 IU/kg of the rFIX- or rFIXFc-fusion proteins. Plasma activity levels were determined over time using a FIX activated partial thromboplastin time assay (aPTT). PK parameters were determined using non-compartmental modeling with Phoenix WinNonlin 6.2.1 (Pharsight). RESULTS Insertion of XTEN sequences with increasing length (42, 72, 144 or 288 amino acids long) at either C-terminus of rFIX-R338L or in the AP domain showed a size dependent increase in plasma recovery up to 60% following intravenous bolus dosing. Combinations of XTEN insertions in the EGF2 or AP domain with Fc-mediated half-life extension in rFIXFc-R338L, extended the half-life as well as increased the plasma recovery. The AUC/D for rFIX-CT-XTEN.288 and rFIXFc-AP-XTEN.72 were 8.5 and 14.5 fold improved when compared to intravenously dosed rFIX, respectively. Following a subcutaneous dose of either rFIXFc-AP-XTEN.72 or rFIX-CT-XTEN.288, we observed 28 and 40-fold improved AUC/D; 15- and 30-fold improved Cmax/D and 3-fold increased bioavailability. When compared to rFIXFc the improvement in pharmacokinetic parameters was 6- and 9-fold improved AUC/D; 3- and 10-fold Cmax/D and 1.5- and 2-fold improved bioavailability for FIXFc-AP-XTEN.72 and rFIX-CT-XTEN.288, respectively. Taken together, subcutaneous dosing of rFIX-CT-XTEN.288 and rFIXFc-AP-XTEN.72 in HemB mice showed improved AUC/D when compared to intravenous dosing of rFIXFc. CONCLUSIONS rFIXFc-AP-XTEN.72 and rFIX-CT-XTEN.288 show greatly improved subcutaneous pharmacokinetics in HemB mice compared to both rFIX and rFIXFc. These promising preclinical subcutaneous dosing data in HemB mice suggests the potential of once weekly or every two weeks prophylactic subcutaneous dosing of FIX-XTEN molecules in patients. In addition, the molecules have potential for acute treatment by intravenous dosing. Further studies are ongoing to address the efficacy and allometric scaling in preclinical animal models. Disclosures van der Flier: Biogen: Employment. Liu:Biogen: Employment. Liu:Biogen: Employment, Equity Ownership, Honoraria, Research Funding. Mercury:Biogen: Employment. Ismail:Biogen: Employment. Seth-Chhabra:Biogen: Employment, Equity Ownership, Honoraria, Patents & Royalties, Research Funding. Kulman:Biogen: Employment. Schellenberger:Amunix Operating Inc: Employment. Light:Biogen: Employment, Equity Ownership. Peters:Biogen: Employment.

Description: Background: The purification of vitamin K-dependent clotting factors typically involves multiple chromatographic steps, including an ion exchange-based pseudo-affinity step to enrich for species with sufficiently high gamma-carboxyglutamic acid (Gla) content to achieve maximal specific activity. Variants of these factors have been engineered to improve their pharmacokinetic properties by appending or inserting a variety of elements, including the Fc domain of IgG, unstructured hydrophilic peptides of defined amino acid composition (XTEN), albumin, and polyethylene glycol (PEG). In most cases, however, such modification alters both the hydrodynamic and electrostatic properties of the resulting molecule relative to those of the predicate molecule, thereby complicating their purification, particularly with regard to Gla enrichment by pseudo-affinity chromatography. Factor IX (FIX)- and factor X (FX)-binding protein (FIX/X-bp) isolated from the venom of the Japanese Habu snake (T. flavoviridis) has been shown to bind with high affinity and specificity to both FIX and FX, and structural studies have demonstrated that FIX/X-bp binds to the highly carboxylated calcium-bound forms of the Gla domains of these proteins. We therefore reasoned that FIX/X-bp could serve as a novel affinity ligand for rapid and simple purification of variants of FIX and FX with high specific activity. Aims: To generate and purify recombinant FIX/X-bp (rFIX/X-bp) and assess its utility for the purification of FIX, FIX-XTEN, FIX-albumin, and FX with high Gla content. Methods: A two-chained rFIX/X-bp molecule in which a polyhistidine tag was appended to one chain was generated by stable co-transfection of Chinese hamster ovary (CHO) cells. Culture medium was concentrated by tangential-flow filtration (TFF), and rFIX/X-bp was purified by one of two methods: 1) immobilized metal ion affinity chromatography (IMAC), followed by anion-exchange chromatography, or 2) affinity chromatography on immobilized FIX in calcium-containing buffer and subsequent elution in EDTA-containing buffer. The potent anticoagulant activity of rFIX/X-bp was verified by prothrombin time (PT) and activated partial thromboplastin time (APTT) assays, and its ability to bind to human FIX, FX, factor VII (FVII), protein S, and prothrombin was evaluated by biolayer interferometry. The affinity of rFIX/X-bp for FIX and FX was determined by surface plasmon resonance (SPR). An affinity column was then generated by chemical conjugation of rFIX/X-bp to NHS-activated Sepharose. Recombinant FIX, FIX-albumin, and FIX-XTEN were first affinity purified on IXSelect resin from the culture medium of transiently transfected HEK293 cells, and the resulting protein preparations, which were heterogeneous with regard to Gla content, were then applied to the rFIX/X-bp affinity column in calcium- or magnesium-containing buffer and eluted with EDTA-containing buffer. Activity was assessed by APTT assay, and Gla content was determined by mass spectrometric peptide mapping. Recombinant FX was purified from the culture medium of transiently transfected HEK293 cells by sequential barium citrate adsorption, anion exchange chromatography, and affinity chromatography on a rFIX/X-bp column. Results: In the presence of calcium or magnesium ions, rFIX/X-bp binds to FIX and FX with high affinity (KD≈ 10 pM), to a lesser extent to protein S and prothrombin, but not to FVII. FIX and FIX-albumin that had been affinity purified on a rFIX/X-bp column had specific activities that were consistent with published data and greater than 11 Gla residues per molecule. The Gla content of FX that had been affinity purified on a rFIX/X-bp column was 10 Gla residues per molecule (out of 11 possible). Conclusions: rFIX/X-bp is a universal ligand for the purification of highly carboxylated FX and FIX variants, including FIX-albumin and FIX-XTEN. Disclosures Mercury: Biogen: Employment. Liu:Biogen: Employment. Ismail:Biogen: Employment. Zhang:Biogen: Employment. Lu:Biogen: Employment. Cameron:Biogen: Employment. Goodman:Biogen: Employment. Culyba:Biogen: Employment. Ravindran Nair:Biogen: Employment. Holthaus:Biogen: Employment. Kulman:Biogen: Employment. Peters:Biogen: Employment.