Identification of novel glycosylation events on human serum-derived factor IX.

Human Factor IX is a highly post-translationally modified protein that is an important clotting factor in the blood coagulation cascade. Functional deficiencies in Factor IX result in the bleeding disorder haemophilia B, which is treated with plasma-derived or recombinant Factor IX concentrates. Here, we investigated the post-translational modifications of human serum-derived Factor IX and report previously undescribed O-linked monosaccharide compositions at serine 141 and a novel site of glycosylation. At serine 141 we observed two monosaccharide compositions, with HexNAc1Hex1NeuAc2 dominant and a low level of HexNAc1Hex1NeuAc1. This O-linked site lies N-terminal to the first cleavage site for the activation peptide, an important region of the protein that is removed to activate Factor IX. The novel site is an N-linked site in the serine protease domain with low occupancy in a non-canonical consensus motif at asparagine 258, observed with a HexNAc4Hex5NeuAc2 monosaccharide composition attached. This is the first reported instance of a site of modification in the serine protease domain. The description of these glycosylation events provides a basis for future functional studies and contributes to structural characterisation of native Factor IX for the production of effective therapeutic biosimilars and biobetters.

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

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

A novel method for separation of caseins from milk by phosphates precipitation.

Milk protein of farm animals is difficult to isolate because of the presence of casein micelles, which are hard to separate from whey by using centrifugation or filtration. Insoluble casein micelles also create an obstacle for purification instruments to operate efficiently. The conventional method, to precipitate caseins by lowering pH to 4.6 and then recover the whey fraction for further purification using chromatography techniques, is not applicable to proteins having an isoelectric point similar to caseins. In addition, the acid condition used for casein removal usually leads to significantly poor yields and reduced biological activities. In this study, a novel method of precipitating caseins under neutral or weak acidic conditions is presented. The method employs a phosphate salt and a freeze-thaw procedure to obtain a casein-free whey protein fraction. This fraction contains more than 90% yield with little loss of bioactivity of the target protein, and is readily available for further chromatographic purification. This method was successfully applied to purify recombinant human factor IX and recombinant hirudin from the milk of transgenic pigs in the presented study. It is an efficient pretreatment approach prior to chromatographic purification of milk protein from farm animals and particularly of great value to collect those recombinants secreted from transgenic livestock.

Virus removal capacity at varying ionic strength during nanofiltration of AlphaNine® SD.

Nanofiltration is incorporated into the manufacturing processes of many protein biopharmaceuticals to enhance safety by providing the capacity to retain pathogens while allowing protein drugs to pass through the filter. Retention is mainly a function of size; however, the shape of the pathogen may also influence retention. The ability of the Viresolve(®) Pro nanofilter to remove different sized viruses during the manufacture of a Coagulation Factor IX (Alphanine(®) SD) was studied at varying ionic strength, a process condition with the potential to affect virus shape and, hence, virus retention. Eight viruses were tested in a scale-down of the nanofiltration process. Five of the viruses (EMCV, Reo, BVDV, HIV, PRV) were nanofiltered at normal sodium processing conditions and three (PPV, HAV and WNV) were nanofiltered at higher and lower sodium. Representative Reduction Factors for all viruses were ≥4.50 logs and removal was consistent over a wide range of ionic strength.

Protein sieving characteristics of sub-20-nm pore size filters at varying ionic strength during nanofiltration of Coagulation Factor IX.

Nanofiltration assures that protein therapeutics are free of adventitious agents such as viruses. Nanofilter pores must allow passage of protein drugs but be small enough to retain viruses. Five nanofilters have been evaluated to identify those that can be used interchangeably to yield a high purity Coagulation Factor IX product. When product preparations prior to nanofiltration were analyzed using electrophoresis, Western blot, liquid chromatography - tandem mass spectrometry and size exclusion HPLC, factor IX, inter - α - trypsin inhibitor and C4b binding protein (C4BP) were observed. C4BP was removed from product by all five nanofilters when nanofiltration was performed at physiological ionic strength. However, at high ionic strength, C4BP was removed by only two nanofilters. HPLC indicated that the Stokes radius of C4BP was larger at low ionic strength than at high ionic strength. The results suggest that C4BP exists in an open conformation at physiological ionic strength and is removed by nanofiltration whereas, at high ionic strength, the protein collapses to an extent that allows passage through some nanofilters. Manufacturers should be aware that protein contaminants in other nanofiltered protein drugs could behave similarly and conditions of nanofiltration must be evaluated to ensure consistent product purity.

Stable and high-level production of recombinant Factor IX in human hepatic cell line.

Hemophilia B is a genetic disease of the coagulation system that affects one in 30,000 males worldwide. Recombinant human Factor IX (rhFIX) has been used for hemophilia B treatment, but the amount of active protein generated by these systems is inefficient, resulting in a high-cost production of rhFIX. In this study, we developed an alternative for rhFIX production. We used a retrovirus system to obtain two recombinant cell lines. We first tested rhFIX production in the human embryonic kidney 293 cells (293). Next, we tested a hepatic cell line (HepG2) because FIX is primarily expressed in the liver. Our results reveal that intracellular rhFIX expression was more efficient in HepG2/rhFIX (46%) than in 293/rhFIX (21%). The activated partial thromboplastin time test showed that HepG2/rhFIX expressed biologically active rhFIX 1.5 times higher than 293/rhFIX (P = 0.016). Recovery of rhFIX from the HepG2 by reversed-phase chromatography was straightforward. We found that rhFIX has a pharmacokinetic profile similar to that of FIX purified from human plasma when tested in hemophilic B model. HepG2/rhFIX cell line produced the highest levels of rhFIX, representing an efficient in vitro expression system. This work opens up the possibility of significantly reducing the costs of rhFIX production, with implications for expanding hemophilia B treatment in developing countries.

Coagulation factor IX analysis in bioreactor cell culture supernatant predicts quality of the purified product.

Coagulation factor IX (FIX) is a complex post-translationally modified human serum glycoprotein and high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete γ-carboxylation, is critical for rFIX clinical efficacy. Bioreactor operating conditions can impact rFIX production and post-translational modifications (PTMs). With the goal of optimizing rFIX production, we developed a suite of Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics methods and used these to investigate rFIX yield, γ-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. We detail the dynamics of site-specific PTM occupancy and structure on rFIX during production, which correlated with the efficiency of purification and the quality of the purified product. We identified new PTMs in rFIX near the GLA domain which could impact rFIX GLA-dependent purification and function. Our workflows are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses.

Gold nano-urchin integrated label-free amperometric aptasensing human blood clotting factor IX: A prognosticative approach for "Royal disease".

This article is clearly presenting the development of a biosensor for human factor IX (FIX) to diagnose the blood clotting deficiency, a so-called 'Royal disease' using an interdigitated electrode (IDE) with the zinc oxide surface modification. Gold nano-urchins (GNUs) with 60 nm in diameter was integrated into a streptavidin-biotinylated aptamer strategy to enhance the active surface area. Two different comparative studies have been done to validate the system to be practiced in the current work holds with a higher capability for the high-performance sense. Whereby, the presence and absence of GNUs in the aptasensing system for FIX interaction were investigated using the amperometric measurement, using a linear sweep voltage of 0-2 V at 0.01 V step voltage. The detection limit was 6 pM based on 3σ calculation when GNUs integrated aptamer assay was utilized for FIX detection, which shows 8 folds sensitivity enhancement comparing the condition in the absence of GNU and 50 folds higher than sensitive radio-isotope and surface plasmon resonance assays. Albeit, the surface and molecular characterizations were well demonstrated by scanning electron microscopy, atomic force microscopy, 3D nano-profilometry and further supports were rendered by UV-Vis spectroscopy and Enzyme-linked apta-sorbent assay (ELASA). Furthermore, the spiking experiment was done by FIX-spikes in human blood serum in order to demonstrate the stability with a higher non-fouling.

The carboxylation efficiency of the vitamin K-dependent clotting factors: studies with factor IX.

Haemophilia B is characterized by a deficiency of the gamma-carboxylated protein, factor IX (FIX). As a first step to optimize a gene therapy strategy to treat haemophilia B, we employed a previously described approach (Biochemistry 2000;39: 14322) of altering the propeptide of vitamin K-dependent proteins in vitro, to improve the carboxylation efficiency of FIX. Both native FIX and FIX with a prothrombin propeptide (proPT-FIX) produced recombinant FIX in vitro following transfection of their cDNAs into human embryonic kidney (HEK) 293 cells. Using hydroxyapatite chromatography to separate carboxylated from uncarboxylated FIX, we are able to show that >90% of FIX is gamma-carboxylated and that substituting the propeptide of prothrombin into FIX does not further increase the relative amounts of carboxylated material. These results demonstrate that the nature of the propeptide, per se is not the sole determinant of optimal carboxylation of FIX in our expression system in HEK 293 cells.

Improvement of virus safety of an antihemophilc factor IX by virus filtration process.

Viral safety is an important prerequisite for clinical preparations of plasma-derived pharmaceuticals. One potential way to increase the safety of therapeutic biological products is the use of a virus-retentive filter. In order to increase the viral safety of human antihemophilic factor IX, particularly in regard to non-enveloped viruses, virus removal process using a polyvinylidene fluoride membrane filter (Viresolve NFP) has been optimized. The most critical factor affecting the filtration efficiency was operating pH and the optimum pH was 6 or 7. Flow rate increased with increasing operating pressure and temperature. Recovery yield in the optimized production-scale process was 96%. No substantial changes were observed in the physical and biochemical characteristics of the filtered factor IX in comparison with those before filtration. A 47-mm disk membrane filter was used to simulate the process performance of the production-scale cartridges and to test if it could remove several experimental model viruses for human pathogenic viruses, including human hepatitis A virus (HAV), porcine parvovirus (PPV), murine encephalomyocarditis virus (EMCV), human immunodeficiency virus type 1 (HIV), bovine viral diarrhea virus (BVDV), and bovine herpes virus (BHV). Nonenveloped viruses (HAV, PPV, and EMCV) as well as enveloped viruses (HIV, BVDV, and BHV) were completely removed during filtration. The log reduction factors achieved were >or=6.12 for HAV, >or=4.28 for PPV, >or=5.33 for EMCV, >or=5.51 for HIV, >or=5.17 for BVDV, and >or=5.75 for BHV. These results indicate that the virus filtration process successfully improved the viral safety of factor IX.

Purification and characterization of an abnormal factor IX (Christmas factor) molecule. Factor IX Chapel Hill.

Human Factor IX (Christmas factor) was isolated from the plasma of a patient with mild hemophilia B. The patient's plasma contained 5% Factor IX clotting activity but 100% Factor IX antigenic activity as determined by immunological assays, which included inhibitor neutralization and a radioimmunoassay for Factor IX. This abnormal Factor IX is called Factor IX Chapel Hill (Factor IXCH). Both normal Factor IX and Factor IXCH have tyrosine as the NH2-terminal amino acid. The two proteins have a similar molecular weight, a similar amino acid analysis, the same number of gamma-carboxyglutamic acid residues (10 gamma-carboxyglutamic acid residues), and a similar carbohydrate content. Both exist as a single-chain glycoprotein in plasma. The major difference between normal Factor IX and Factor IXCH is that the latter exhibits delayed activation to Factor IXa in the presence of Factor XIa and Ca2+. Thus, Factor IXCH differs from other previously described abnormal Factor IX molecules.

Replacing the first epidermal growth factor-like domain of factor IX with that of factor VII enhances activity in vitro and in canine hemophilia B.

Using the techniques of molecular biology, we made a chimeric Factor IX by replacing the first epidermal growth factor-like domain with that of Factor VII. The resulting recombinant chimeric molecule, Factor IXVIIEGF1, had at least a twofold increase in functional activity in the one-stage clotting assay when compared to recombinant wild-type Factor IX. The increased activity was not due to contamination with activated Factor IX, nor was it due to an increased rate of activation by Factor VIIa-tissue factor or by Factor XIa. Rather, the increased activity was due to a higher affinity of Factor IXVIIEGF1 for Factor VIIIa with a Kd for Factor VIIIa about one order of magnitude lower than that of recombinant wild-type Factor IXa. In addition, results from animal studies show that this chimeric Factor IX, when infused into a dog with hemophilia B, exhibits a greater than threefold increase in clotting activity, and has a biological half-life equivalent to recombinant wild-type Factor IX.

An RNA aptamer that discriminates bovine factor IX from human factor IX.

An RNA aptamer has been selected by SELEX against bovine factor IX using an RNA pool containing 74-nucleotides randomized region. Selected RNA aptamer (Clone 5) could discriminate bovine factor IX effectively from human factor IX. Interestingly, the nucleotide regions 73-78 and 80-83 of the selected aptamer were determined to be important for bovine factor IX-binding using phosphate interference. Based on phosphate interference and binding studies the minimal motif for aptamer with discriminating ability is found with the nucleotide regions from 65 to 106. The discriminating ability of this mini aptamer is calculated as more than 1,000 fold. The equilibrium dissociation constant (K(d)) for the above complex was 10 nM as determined by surface plasmon resonance. Based on the available structural informations, probable binding site of aptamer on the target was predicted.

Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo.

We have shown previously that minicircle DNA vectors free of plasmid bacterial DNA sequences are capable of persistent high level of transgene expression in vivo. The minicircle is generated in bacteria from a parental plasmid containing an inducible phage oC31 integrase gene and a therapeutic expression cassette flanked with attB and attP sites. The oC31-mediated intramolecular recombination between attB and attP results in the formation of two circular DNA molecules, one containing the eukaryotic expression cassette (minicircle), and the other the plasmid bacterial DNA backbone (BB). Previously, the minicircle was purified away from the plasmid BB by a restriction enzyme digestion step and ultracentrifugation in cesium chloride. We have now included the endonuclease I-SceI gene together with its recognition site in the minicircle-producing plasmid to allow the linearization and degradation of the plasmid BB in bacteria. The minicircle can then be isolated by routine plasmid purification procedures such as a one-step affinity column. With additional modifications to our previous strategy, we can prepare a minicircle encoding a 4-kb human factor IX expression cassette, up to 1.8 mg of minicircle with 97% purity was prepared from a 1 liter bacterial culture. The high yield, simple purification, and robust and persistent transgene expression make these vectors viable for gene therapy applications.

Human plasma fractionation and the impact of new technologies on the use and quality of plasma-derived products.

Recent years brought several important changes in the domain of human plasma derived products. High purity and effective anti-viral treatment became a reality. This radically improved the quality of patient treatment. At the same time recent discoveries in molecular biology paved the way for the production of several crucial plasma components by recombinant technology. In the light of these developments the future possibilities for different plasma components production is widely discussed and the eventual benefit of more expensive technologies is being evaluated. This paper, analyzes and presents methods applied by different producers to obtain plasma derived components preparations. The impact of these technologies, the quality of the products and the future of the plasma industry is being discussed.

The manufacturing process for recombinant factor IX.

Advances in recombinant DNA manufacturing technology have now made possible the production of a highly purified and active recombinant factor IX (rFIX) product. Recombinant factor IX was developed by (1) stable insertion of the genes for both factor IX and PACE-SOL (a truncated, soluble serine protease needed to enhance the capacity of cells to remove the amino-terminal propeptide from rFIX) into Chinese hamster ovary cells; (2) selection of a cell line that was capable of expressing high amounts of active rFIX while growing in bioreactors containing a completely defined culture medium that does not contain blood or plasma products; and (3) inclusion of four independent chromatography steps, none of which require monoclonal antibodies. Furthermore, rFIX has been extensively tested to demonstrate similarity to plasma-derived factor IX and has been shown to be a consistent, high-purity product. For example, a high-specific-activity product (276+/-23 IU/mg) has been consistently produced throughout 65 consecutive batches from five consecutive manufacturing campaigns. Thus, rFIX offers a consistent and high-purity source of factor IX treatment for patients with hemophilia B.

Biochemical characterization of recombinant factor IX.

Mature human factor IX is a 55,000-d glycoprotein with a modular domain structure and numerous posttranslational modifications. A recombinant form of human factor IX (rFIX) has been produced from a Chinese hamster ovary cell line that was engineered for high-level protein processing and expression. To ensure that the recombinant molecule contains the requisite structural and functional features of the plasma-derived form, rFIX was subjected to detailed biochemical and biophysical characterization. The laboratory studies showed that the posttranslational modifications and primary, secondary, and tertiary structures of rFIX were similar to those of plasma-derived factor IX (pdFIX). In addition, rFIX displayed a high degree of purity and a product release specification for specific activity that is > or = 200 IU/mg.

Evaluation of CNBr, FMP and hydrazide resins for immunoaffinity purification of factor IX.

The American Red Cross has developed an immunoaffinity chromatography method to purify human coagulation Factor IX to high levels of purity for therapeutic treatment of hemophilia B. The resin currently used in this process is Sepharose CL2B, a cross-linked 2% agarose, which is activated with cyanogen bromide to immobilize an anti-Factor IX monoclonal antibody. This study evaluated two alternative resins and coupling chemistries, a synthetic polymer bead activated by 2-fluoro-1-methyl-pyridinium toluene 4-sulfonate (FMP) and a cross-linked 2% agarose bead with free hydrazide groups for site-specific coupling. The cyanogen bromide and FMP chemistries immobilize the monoclonal antibody in a random orientation. In hydrazide coupling, the monoclonal antibody is immobilized by the non-antigen-binding part of the molecule which, theoretically, should increase the amount of immobilized monoclonal antibody able to bind antigen. To examine this, the capacity of the resins to bind Factor IX and the purity and recovery of Factor IX eluted from the resins were measured. The FMP-activated resin exhibited the lowest capacity, binding only 2% of the Factor IX feed. Sepharose CL2B bound 87% of the loaded protein, while the hydrazide resin bound 43%. These results suggest that (a) hydrazide activation may be insufficient to orient monoclonal antibody and (b) other factors such as steric hindrances and diffusional resistances during immobilization may be important. Neither of the other resins tested demonstrated improved performance compared with cyanogen bromide-activated Sepharose CL2B for the immunoaffinity purification of Factor IX.

Immunopurification of human coagulation factor IX using monoclonal antibodies.

This study examines the suitability of four recently characterised monoclonal antibodies (MAbs) for the immunoaffinity purification of human coagulation factor IX (FIX) from plasma concentrates. Initial studies using 125I-FIX indicated that appreciable amounts of bound FIX could be eluted from immobilised MAbs with 0.2 M glycine, 50% ethanediol pH 10 (buffer N). Further studies with FIX concentrates showed that buffer N eluted FIX without compromising the activity of the zymogen. Although FIX was eluted from all four MAbs with this buffer, the best yield (82%) was obtained with MAb ESN-3. ESN-3 bound 40 to 60 iu FIX per mg MAb when immobilised on Sepharose 4B. After washing, column elution with buffer N yielded FIX at 100-200 iu/mg. The purity of the product was confirmed by sodium-dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blotting. The product contained no detectable mouse IgG (less than 3%) and less than 1% FII, X, or protein C.

Purification and properties of human factor IXa.

Human factor IXa was purified 5,000-fold from serum by ion exchange chromatography. The preparation was free from other clotting factors. Both pH sensitivity and heat stability of purified factor IXa appeared to be different from those of factor IX in the plasma. The molecular weight of human factor IXa is 80,000 as estimated from gel-filtration experiments. Modification of seryl or histidyl side chains abolished the activity of factor IXa.